Multidrug resistant

Clin Infect Dis, 2004 Sep 15, 39(6), e53 - 5 Epub 2004 Aug 27.
Prolonged survival of an HIV-infected patient with multidrug-resistant Mycobacterium bovis infection treated with surgical resection; Ramos A et al.; We describe a case of multidrug-resistant tuberculosis due to Mycobacterium bovis in a human immunodeficiency virus-infected woman with good immunologic status . The patient presented with a hard mass measuring 10 cm in diameter on the lower left ribs and a lung nodule measuring 3 cm in diameter in the left superior lobe . No adequate pharmacological treatment was available . Both lesions were surgically resected . The patient has remained asymptomatic (without fever, cough, lymphadenopathy, or cutaneous masses) for 20 months, after discharge from the hospital.

J Clin Microbiol, 2004 Oct, 42(10), 4432 - 7
Microscopic observation drug susceptibility assay, a rapid, reliable diagnostic test for multidrug-resistant tuberculosis suitable for use in resource-poor settings; Moore DA et al.; There is an urgent need for new tools to improve our ability to diagnose tuberculosis (TB) and multidrug-resistant TB (MDR-TB) in resource-poor settings . In a retrospective analysis undertaken in a region with a high incidence of TB, we evaluated the performance of the microscopic observation drug susceptibility assay (MODS), a novel assay developed in Peru which uses an inverted light microscope and culture in Middlebrook 7H9 broth to detect mycobacterial growth . MODS detected 94.0% of 1,908 positive sputum cultures, whereas Lowenstein-Jensen (LJ) culture detected only 86.9% (P < 0.001) . The median time to culture positivity was 8 days (compared to 16 days for the same 208 samples by LJ culture; P < 0.001, Wilcoxon signed rank test) . The results obtained by direct susceptibility testing using MODS demonstrated excellent concordance for isoniazid and rifampin and the detection of multidrug resistance with those obtained by indirect colorimetric methods: the microplate Alamar Blue assay (MABA) and the tetrazolium microplate assay (TEMA) (agreement, 95, 98, and 94%; kappa values, 0.8, 0.7, and 0.7, respectively) . The concordance of the susceptibility testing results for ethambutol and streptomycin was poor . MODS is a novel assay which can detect the organisms responsible for TB and MDR-TB directly from sputum inexpensively, rapidly, and effectively . A comprehensive prospective evaluation of MODS is under way in Peru, and independent validation in nonresearch laboratories should be undertaken at the earliest opportunity.

Biochim Biophys Acta, 2004 Oct 11, 1665(1-2), 111 - 7
Impact of the growth phase on the activity of multidrug resistance pumps and membrane potential of S . cerevisiae: effect of pump overproduction and carbon source; Cadek R et al.; The potentiometric fluorescence probe diS-C3(3) is expelled from S . cerevisiae by ABC pumps Pdr5 and Snq2 and can conveniently be used for studying their performance . The activity of these pumps in a strain with wild-type PDR1 allele was shown to drop sharply on glucose depletion from the medium and then again at the end of the diauxic shift when the cells are adapted to growth on respiratory substrates . The presence of the PDR1-3 allele causing pump overproduction prevented this second drop and the pump activity typical for diauxic cells was largely retained . Growth phase-dependent changes of membrane potential measured by the same probe in pump-free mutants included a Deltapsi drop in the late exponential and diauxic growth phase, indicating lowered activity of H+ -ATPase . Suppression of activity of both ABC pumps and H+ -ATPase obviously signifies cell transition to an energy-saving mode . Challenging respiration-adapted cells with glucose showed a novel feature of yeast ABC pumps--a strong dependence of pump activity on the type of the carbon source.

Wien Klin Wochenschr, 2004 Aug 31, 116(15-16), 561 - 4
Highly refractory acute myeloid leukemia; Fureder W et al.; In this study we evaluated 103 patients suffering from acute myeloid leukemia (AML) who did not respond to induction chemotherapy and defined a sub-group of patients with highly refractory disease characterized by a persistence of more than 1 G/L blast cells in the peripheral blood between days 12 and 16 of the first induction cycle . Only seven patients (one female, six males) met these criteria . Their median age was 65 years (range 41-82 years) . Four had de novo AML and three secondary AML . Cytogenetic analysis was performed in six patients: complex aberrations were detected in four patients and, unexpectedly, normal karyotypes were found in the other two . Analysis of multidrug-resistance factors revealed high co-expression of P-glycoprotein (P-gp) and lung resistance protein (LRP) in all four patients with highly refractory disease tested a finding in only 6% of patients with refractory disease and 3% of patients who achieved complete remission (CR) of disease . Furthermore, patients with highly refractory AML had substantially higher leukocyte counts than patients with refractory AML or CR, although this was not significant statistically . Overall, patients with highly refractory AML are characterized by a high incidence of complex cytogenetic aberrations and marked expression of drug transporters.

Proc Natl Acad Sci U S A, 2004 Oct 12, 101(41), 14925 - 30 Epub 2004 Oct 04.
The complete genomic sequence of Nocardia farcinica IFM 10152; Ishikawa J et al.; We determined the genomic sequence of Nocardia farcinica IFM 10152, a clinical isolate, and revealed the molecular basis of its versatility . The genome consists of a single circular chromosome of 6,021,225 bp with an average G+C content of 70.8% and two plasmids of 184,027 (pNF1) and 87,093 (pNF2) bp with average G+C contents of 67.2% and 68.4%, respectively . The chromosome encoded 5,674 putative protein-coding sequences, including many candidate genes for virulence and multidrug resistance as well as secondary metabolism . Analyses of paralogous protein families suggest that gene duplications have resulted in a bacterium that can survive not only in soil environments but also in animal tissues, resulting in disease.

Drug Metab Dispos, 2005 Jan, 33(1), 55 - 9 Epub 2004 Oct 04.
Bisphenol a glucuronidation and excretion in liver of pregnant and nonpregnant female rats; Inoue H et al.; In male rats challenged with the environmental estrogen bisphenol A, the compound is highly glucuronidated in the liver and is excreted largely into the bile . Given that in pregnancy the microsomal glucuronidation toward bisphenol A is attenuated, we hypothesized that elimination of bisphenol A from the liver may be reduced in pregnancy . This study was conducted to trace the elimination of bisphenol A in female rats, especially in pregnancy . In Sprague-Dawley rats, 1.5 mumol of bisphenol A was perfused into the liver via the portal vein . In both the male and the nonpregnant female, the infused bisphenol A was glucuronidated, then the resultant glucuronide was excreted mainly into the bile . In pregnant rats, however, bilious excretion of bisphenol A glucuronide was 60% of that observed in nonpregnant rats, and venous excretion increased reciprocally . During 1-h perfusion, total excretion of the glucuronide from the liver of male, nonpregnant female, and pregnant rats was 889.5 +/- 69.6, 1256.7 +/- 54.8, and 1038.8 +/- 33.3 nmoles, respectively . In Eisai hyperbilirubinemic rats (EHBR), perfusion of the liver with bisphenol A enabled us to determine that multidrug resistance-associated protein (MRP)2-mediating transport is the mechanism behind excretion of the glucuronide into the bile . The expression of MRP2 has been reported to be noticeably reduced in pregnancy . These results suggest that bisphenol A elimination by hepatic glucuronidation is slightly less in pregnancy than in non-pregnancy and that in pregnancy, more bisphenol A glucuronide is eliminated to the vein because of reduced MRP2 expression.

Cancer Res, 2004 Oct 1, 64(19), 7130 - 8
Celastraceae sesquiterpenes as a new class of modulators that bind specifically to human P-glycoprotein and reverse cellular multidrug resistance; Munoz-Martinez F et al.; Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer . Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains . However, their molecular mechanism of reversion has not been characterized . In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action . Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine . Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters . Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of {(3)H}colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target . Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein . Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations . Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential applications in cancer chemotherapy because of their MDR reversal potency and specificity for P-glycoprotein.

Drug Metab Dispos, 2005 Jan, 33(1), 1 - 9 Epub 2004 Oct 01.
The roles of transporters and enzymes in hepatic drug processing; Liu L et al.; A simple, physiological model was used to illustrate the competing nature of transporters and metabolic enzymes in hepatic drug processing . Enalapril, a drug whose basolateral influx and canalicular efflux are mediated by rat organic anion-transporting polypeptide 1 (Oatp1) and rat multidrug resistance-associated protein 2 (Mrp2), respectively, and metabolism by the carboxylesterases, was enlisted as the example to illustrate how the transport and intrinsic clearances are inter-related in the estimation of the hepatic and metabolic, and excretion clearances . Moreover, simulations were performed to explore the effects of inhibitors or inducers of transporters/enzymes to unravel the compensatory changes of alternate pathways . Generally speaking, inhibition of one pathway led to an apparent increase in the alternate (competing) pathway and total hepatic clearance was decreased; induction would lead to an apparent decrease in the alternate pathway and an increase in total hepatic clearance . A reduction in influx clearance brought about parallel decreases in the biliary and metabolic clearances, whereas a reduction in efflux basolateral clearance evoked similar increases in biliary and metabolic clearances . However, the steady-state tissue concentration (C(L,ss)) or area under the tissue concentration-time curve (AUC(L)) was reliant only on the unbound fraction in liver, and the secretory and metabolic intrinsic clearances and not the influx and efflux clearances . Variations in the influx and efflux intrinsic clearances evoked temporal changes in the tissue concentration-time profile but not the AUC(L) or C(L,ss) . The pharmacokinetic theory developed offers data interpretation from literature reports on P-glycoprotein and cytochrome P450 substrates with mdr1a/1b knockout versus wild-type mice, and rat liver perfusion studies, with and without the use of inhibitors . In some cases, critiques on data interpretation were made.

Bioorg Med Chem, 2004 Nov 1, 12(21), 5553 - 62
Synthesis of new 6-alkylvinyl/arylalkylvinyl substituted 1,2,4-trioxanes active against multidrug-resistant malaria in mice; Singh C et al.; 3-Alkyl/arylalkyl substituted 2-butenols 9, 10, 23a-d undergo regiospecific photooxygenation to furnish beta-hydroxyhydroperoxides 11, 12, 24a-d, respectively, in reasonable yields . Acid catalyzed condensation of 11, 12, 24a-d with various ketones furnish new 1,2,4-trioxanes 13-18, 25a-d, 26a-d, 27a-d in good yields . Several of these trioxanes show promising antimalarial activity against multidrug-resistant Plasmodium yoelii in mice by oral and intramuscular routes.

Biochem Biophys Res Commun, 2004 Nov 5, 324(1), 365 - 71
Stable suppression of MDR1 gene expression and function by RNAi in Caco-2 cells; Celius T et al.; Vector-based RNAi was used to establish a stable Caco-2 cell line with a persistent knockdown of multidrug resistant gene 1 (MDR1) and P-glycoprotein (P-gp) . Several positive clones were collected, many of which showed significantly reduced levels of MDR1 mRNA and P-gp compared to wt Caco-2 cells . Selected clones were sub-cultivated for six passages and real-time PCR showed that MDR1 expression remained significantly reduced (up to 96%) over this period of time . RNAi-MDR1 clones frozen long term also kept their low MDR1 expression levels when re-cultured . Permeability studies were performed across RNAi-MDR1 clone cell monolayers, and the efflux of cyclosporine A, digoxin, vinblastine, and vincristine showed 58%, 61%, 91%, and 78% decrease in active transport, respectively, compared to wt Caco-2 cells . This stably modified Caco-2 cell line provides a novel tool for studies on MDR1 and other ABC transporter protein gene cellular functions.

Parasitol Today, 1991, 7(4), 70 - 6
The P-glycoprotein homologues of Plasmodium falciparum: Are they involved in chloroquine resistance?
Cowman AF.
Chloroquine has been the mainstay of antimalarial chemotherapy but the rapid spread of resistance to this important drug has now compromised its efficacy . The mechanism of chloroquine resistance has not been known but recent evidence from Plasmodium falciparum, the causative agent of the most severe form of human malaria, suggested similarities to the multidrug resistance phenotype (MDR) of mammalian tumour cells which is mediated by a protein molecule termed P-glycoprotein . Two mdr genes (pfmdr1 and pfmdr2) encoding P-glycoprotein homologues have been identified in P . falciparum and one of these (pfmdr1) has several alleles that have been linked to the chloroquine resistance phenotype . In contrast analysis of a genetic cross between chloroquine-resistant and -sensitive P . falciparum has suggested that the genes encoding the known P-glycoprotein homologues are not linked . This review outlines the similarities of the chloroquine resistance phenotype with the MDR phenotype of mammalian tumour cells and explores the possible role of the pfmdr genes.

Parasitol Today, 1986 Sep, 2(9), 250 - 3
Current approaches to malaria chemotherapy and prophylaxis; Wernsdorfer WH; Malaria continues to be one of the most serious and widespread parasitic diseases, still occurring in over 100 countries despite concentrated efforts to eradicate it from many regions . Sixty-one countries now report their malaria cases to the WHO, and the latest analysis of these figures' shows little improvement in the overall problem during the last 15 years . Some countries, notably India and China, continue to report downward trends, but the problem continues to deteriorate in rural areas where intense economic development is taking place, particularly in Asia and the Americas . In 1984, 5.3 million cases of malaria were reported to the WHO . This is believed to represent but a small fraction of the total number because, for example, 38 of the tropical African countries do not report their malaria cases . Estimates based on the degree of malaria endemicity suggest a total incidence o f around 100 million cases annually . Chloroquine-resistant falciporum malaria has been confirmed in more than 40 countries, often showing cross-resistance to other drugs, and attempts to combat resistance using combination drugs have led to disturbing reports of side-effects as well as multidrug resistance . Vector control is also impaired in many areas due to insecticide resistance . Faced with these problems, we asked Dr Walther Wernsdorfer, head of the WHO Malaria Action Programme, what is the current WHO philosophy of malaria chemotherapy and prophylaxis?

J Liposome Res, 2004, 14(1-2), 61 - 76
Electron cryo-microscopy reveals mechanism of action of propranolol on artificial membranes; De Carlo S et al.; The pharmacological activity of several amphiphilic drugs is often related to their ability to interact with biological membranes . Propranolol is an efficient multidrug resistance (MDR) modulator; it is a nonselective beta-blocker and is thought to reduce hypertension by decreasing the cardiac frequency and thus blood pressure . It is used in drug delivery studies in order to treat systemic hypertension . We are interested in the interaction of propranolol with artificial membranes, as liposomes of controllable size are used as biocompatible and protective structures to encapsulate labile molecules, such as proteins, nucleic acids or drugs, for pharmaceutical, cosmetic or chemical applications . We present here a study of the interaction of propranolol, a cationic surfactant, with pure egg phosphatidylcholine (EPC) vesicles . The gradual transition from liposome to micelle of EPC vesicles in the presence of propranolol was monitored by time-resolved electron cryo-microscopy (cryo-EM) under different experimental conditions . The liposome-drug interaction was studied with varying drug/lipid (D/L) ratios and different stages were captured by direct thin-film vitrification . The time-series cryo-EM data clearly illustrate the mechanism of action of propranolol on the liposome structure: the drug disrupts the lipid bilayer by perturbing the local organization of the phospholipids . This is followed by the formation of thread-like micelles, also called worm-like micelles (WLM), and ends with the formation of spherical (globular) micelles . The overall reaction is slow, with the process taking almost two hours to be completed . The effect of a monovalent salt was also investigated by repeating the lipid-surfactant interaction experiments in the presence of KCl as an additive to the lipid/drug suspension . When KCl was added in the presence of propranolol the overall reaction was the same but with slower kinetics, suggesting that this monovalent salt affects the general lipid-to-micelle transition by stabilizing the membrane, presumably by binding to the carbonyl chains of the phosphatidylcholine.

J Med Invest, 2004 Aug, 51(3-4), 194 - 201
Reduction of expression of the multidrug resistance protein (MRP)1 in glioma cells by antisense phosphorothioate oligonucleotides; Matsumoto Y et al.; The tumor cells' acquisition of resistance to multiple drugs due to overexpression of the multidrug resistance protein (MPRP)1 gene is one of major obstacles in cancer chemotherapy . We have attempted to reverse the multidrug resistance (MDR) phenotype by treating etoposide resistant glioma cell lines (T98G-VP and Gli36-VP) with RP1 antisense oligonucleotides . 20-mer phosphorothioate oligodeoxynucleotide (0.3 microM), complementary to the coding region in the MRP cDNA sequence, could significantly inhibit the growth of multidrug resistant cell lines, T98G-VP and Gli36-VP, cultured in etoposide containing medium . No such effect was observed for the parental T98G and Gli36 cell lines . Further investigations by the reverse transcription-polymerase chain reaction and immunoblotting revealed that antisense oligomer could result in a reduction in the level of MRP1 mRNA, probably through hindering MRP1 gene transcription . This study demonstrates that the antisense oligonucleotides can increase the sensitivity of the tumor cells to the anticancer drug by decreasing the expression of the MRP gene . This strategy may be applicable to cure cancer patients with MRP mediated MDR phenotype.

Hum Mutat, 2004 Nov, 24(5), 438 - 9
ABCC6 mutations in Italian families affected by pseudoxanthoma elasticum (PXE); Gheduzzi D et al.; Pseudoxanthoma elasticum (PXE) is a genetic disorder, characterized by cutaneous, ocular and cardiovascular clinical symptoms, caused by mutations in a gene (ABCC6) that encodes for MRP6 (Multidrug Resistance associated Protein 6), an ATP-binding cassette membrane transporter . The ABCC6 gene was sequenced in 38 unrelated PXE Italian families . The mutation detection rate was 82.9% . Mutant alleles occurred in homozygous, compound heterozygous and heterozygous forms, however the great majority of patients were compound heterozygotes . Twenty-three different mutations were identified, among which 11 were new . Fourteen were missense (61%); five were nonsense (22%); two were frameshift (8.5%) and two were putative splice site mutations (8.5%) . The great majority of mutations were located from exon 24 to 30, exon 24 being the most affected . Among the others, exons 9 and 12 were particularly involved . Almost all mutations were located in the intracellular site of MRP6 . A positive correlation was observed between patient's age and severity of the disorder, especially for eye alterations . The relevant heterogeneity in clinical manifestations between patients with identical ABCC6 mutations, even within the same family, seems to indicate that, apart from PXE causative mutations, other genes and/or metabolic pathways might influence the clinical expression of the disorder .

J Biol Chem, 2004 Dec 17, 279(51), 53571 - 83 Epub 2004 Sep 30.
Identification and characterization of functionally important elements in the multidrug resistance protein 1 COOH-terminal region; Westlake CJ et al.; The ATP binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), transports a broad spectrum of conjugated and unconjugated compounds, including natural product chemotherapeutic agents . In this study, we have investigated the importance of the COOH-terminal region of MRP1 for transport activity and basolateral plasma membrane trafficking . The COOH-terminal regions of some ABCC proteins have been implicated in protein trafficking, but the function of this region of MRP1 has not been defined . In contrast to results obtained with other ABCC proteins, we found that the COOH-proximal 30 amino acids of MRP1 can be removed without affecting trafficking to basolateral membranes . However, the truncated protein is inactive . Furthermore, removal of as few as 4 COOH-terminal amino acids profoundly decreases transport activity . Although amino acid sequence conservation of the COOH-terminal regions of ABC proteins is low, secondary structure predictions indicate that they consist of a broadly conserved helix-sheet-sheet-helix-helix structure . Consistent with a conservation of secondary and tertiary structure, MRP1 hybrids containing the COOH-terminal regions of either the homologous MRP2 or the distantly related P-glycoprotein were fully active and trafficked normally . Using mutated proteins, we have identified structural elements containing five conserved hydrophobic amino acids that are required for activity . We show that these are important for binding and hydrolysis of ATP by nucleotide binding domain 2 . Based on crystal structures of several ABC proteins, we suggest that the conserved amino acids may stabilize a helical bundle formed by the COOH-terminal three helices and may contribute to interactions between the COOH-terminal region and the protein's two nucleotide binding domains.

Biofizika, 2004 Jul-Aug, 49(4), 685 - 91
{Changes in lipid asymmetry and transport of glutathione-S-conjugates under the influence of calcium ions in human erythrocytes}; Nude mice model of human hepatocellular carcinoma via orthotopic implantation of histologically intact tissue; Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, ChinaAIM: To establish a nude mice model of human hepatocellular carcinoma (HCC) via orthotopic implantation of histologically intact tissue, in order to study biologic features of HCC in vivo and to direct clinical treatment respectively . METHODS: Histologically intact fresh specimens of HCC were orthotopically implanted in nude mice (BALB/c, nu/nu) . Survival rate and growth curve were investigated with B-ultrasound . Morphological characteristics of pathology and spontaneous metastatic rates were detected with microscopy . Expression of multidrug resistance genes studied with immunohistochemical method and RT-PCR, and other biologic features of implanted tumor were observed and compared with human HCC specimens . RESULTS: Out of the specimens from two patients with HCC, only one specimen survived in nude mice . The orthotopic implantation tumor survival rate, spontaneous intrahepatic metastatic rate, pulmonary metastatic rate and bone metastases rate were 100%, 75.0%, 37.5% and 37.5% respectively in the first passage . AFP was kept on secreting and increasing with the size of the tumor . The morphological characteristics and biologic features were similar to the donor's, the protein and mRNA of MDR1 and LRP were expressed in tumors of the model and the donor, and there was no significant difference between them (P>0.05) . CONCLUSION: The model of nude mice with orthotopic implantation of histologically intact HCC tissue is an ideal model to study biologic features of HCC in vivo and to direct clinical treatment.

J Thorac Cardiovasc Surg, 2004 Oct, 128(4), 523 - 8
Resectional surgery combined with chemotherapy remains the treatment of choice for multidrug-resistant tuberculosis; Shiraishi Y et al.; OBJECTIVE: Multidrug-resistant tuberculosis remains a significant health problem . The best available treatment for multidrug-resistant tuberculosis is the combination of pulmonary resection and antituberculous chemotherapy . We herein report the results of pulmonary resection combined with chemotherapy for multidrug-resistant tuberculosis at our institution during the years 2000 through 2002 . METHODS: Between 1983 and 2002, 87 patients underwent 95 pulmonary resections for multidrug-resistant tuberculosis . Of these, the 30 (34%) patients operated on from January 1, 2000, to December 31, 2002, are reviewed in the present study . All patients were maintained on multidrug regimens preoperatively and postoperatively . Indications for surgical intervention included persistently positive sputum and a high risk of relapse . Thirty-three pulmonary resections were performed, consisting of pneumonectomy (n = 12), lobectomy (n = 17), and segmentectomy (n = 4) . The bronchial stump was reinforced with a latissimus dorsi muscle flap in 29 resections . RESULTS: There was no operative mortality . Bronchopleural fistulas occurred in 2 patients . Five patients had a space problem . All patients attained sputum-negative status after the operation . Relapse occurred in 3 patients: 2 had a relapse at the bronchial stump, and the remaining patient had a relapse in the postlobectomy space . One late death occurred . Of the 29 survivors, 27 (93%) were free from disease, with a median follow-up of 24 months (range, 8-47 months) . CONCLUSIONS: An increasing number of patients with multidrug-resistant tuberculosis are requiring resectional surgery in the 21st century . Pulmonary resection combined with chemotherapy achieves high cure rates with acceptable morbidity and remains the treatment of choice for multidrug-resistant tuberculosis.

Antivir Ther, 2004 Aug, 9(4), 519 - 28
ATP binding cassette multidrug transporters limit the anti-HIV activity of zidovudine and indinavir in infected human macrophages; Jorajuria S et al.; OBJECTIVES: To investigate whether P-glycoprotein (P-gp) and multidrug resistance proteins (MRPs), which limit the bioavailability of HIV protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs), modulate the anti-HIV activity of NRTIs, non-NRTIs and PIs in vitro . DESIGN: We used primary cultures of major HIV target cells: human monocyte-derived macrophages (MDMs) and lymphocytes . METHODS: P-gp and MRP expression in response to long-term zidovudine (3'-azido-3'-deoxythymidine; AZT) or indinavir treatment was quantified by RT-PCR . MDM and lymphocytes were infected in vitro with HIV-1/Ba-L and HIV-1-LAI, respectively, and treated with antiretroviral drugs . We evaluated the activity of these drugs in combination with PSC833, a P-gp inhibitor, and/or probenecid, an MRP1 inhibitor . Intracellular AZT triphosphate derivative (AZT-TP) was quantified by HPLC-MSMS . P-gp ATPase activity was measured with inside-out native membrane vesicles enriched in P-gp . RESULTS: Levels of MDR1, mrp4 and mrp5 mRNA were high following AZT treatment . In infected MDM, PSC833 and probenecid increased the anti-HIV activity of AZT and indinavir . AZT (5 nM) decreased HIV replication by 34% alone and by 72% in combination with P-gp/MRP inhibitors . Indinavir (10 nM) gave 14% inhibition alone and 81% in combination . The increase in anti-HIV activity of AZT was correlated with an increase in intracellular AZT-TP concentration . However, unlike PIs, neither AZT nor its metabolites interacted with P-gp . CONCLUSION: AZT increases the expression of multidrug transporters, thereby decreasing its pharmacological activity . The cellular efflux of AZT probably involves MRP4 or MRP5 . In contrast, increases in indinavir anti-HIV activity require the inhibition of both P-gp and MRP1.

Int J Tuberc Lung Dis, 2004 Sep, 8(9), 1114 - 9
Simple, phage-based (FASTPplaque) technology to determine rifampicin resistance of Mycobacterium tuberculosis directly from sputum; Albert H et al.; SETTING: Cape Town, South Africa . OBJECTIVE: To evaluate the performance of a simple, manual, phage-based test for determining rifampicin (RMP) resistance of Mycobacterium tuberculosis directly from smear-positive sputum specimens . DESIGN: A comparative study of the performance of the FASTPlaque (phage amplification) technology to determine RMP resistance directly from smear-positive sputum compared with isolation and the conventional indirect Middlebrook 7H11 agar proportion method . RESULTS: The FASTPlaque direct RMP test achieved sensitivity, specificity and overall accuracy of 100% (11/11), 100% (134/134) and 100% (145/145), respectively, compared with the conventional indirect susceptibility test method (resolved data) . The FASTPlaque direct RMP test reported results within 2 days from receipt of the specimen, while the conventional method took between 27 and 103 days (mean +/- SD 33.2 +/- 7.2 days) . CONCLUSION: FASTPlaque technology applied directly to smear-positive sputum offers performance comparable to conventional methods, with results available in 2 days instead of weeks to months . The test may form a useful part of DOTS-Plus programmes to combat multidrug-resistant tuberculosis, improving patient prognosis and reducing ongoing transmission of disease . It does not require specialised equipment, making it appropriate for high-burden countries.

Int J Tuberc Lung Dis, 2004 Sep, 8(9), 1107 - 13
The Beijing genotype is emerging among multidrug-resistant Mycobacterium tuberculosis strains from Germany; Kubica T et al.; SETTING: Germany, 1995 to 2001 . OBJECTIVE: To determine the genetic relationship of 451 multidrug-resistant (MDR) Mycobacterium tuberculosis strains from Germany and to identify strains of the Beijing genotype . DESIGN: All strains were analysed using IS6110 fingerprinting and a cluster analysis was performed . Clustering of isolates was used as a measure for recent transmission . RESULTS: Two hundred and fourteen of 433 strains (49.4%) with more than four IS6110 copies formed 46 fingerprint clusters comprising two to 32 patients . Transmission links based on classical epidemiological data could be established for 39 cases (18.2%) and in 14 clusters (30.4%), and included three cases of exogenous reinfection with MDR strains . One hundred and seventy-five strains (38.8%) were of the Beijing genotype with an increasing annual proportion from 19.2% in 1995 to 58.3% in 2001 . About 70% of these patients had an indication of foreign birth, mainly the former Soviet Union . CONCLUSION: Transmission of MDR strains seems to be contributing to the spread of MDR-TB in Germany, and exogenous reinfection with MDR strains must be considered as a possible cause of treatment failure . A high proportion of these MDR strains is probably carried over from the former Soviet Union, and strains of the Beijing genotype represent an increasing cause of MDR-TB in Germany.

Cancer Chemother Pharmacol, 2005 Feb, 55(2), 159 - 69 Epub 2004 Sep 29.
Absorption and metabolism of genistein and its five isoflavone analogs in the human intestinal Caco-2 model; Chen J et al.; The purposes of this study were to determine the effect of structural change on the intestinal disposition of isoflavones and to elucidate the mechanisms responsible for transport of phase II isoflavone conjugates . Transport and metabolism of six isoflavones (i.e., genistein, daidzein, glycitein, formononetin, biochanin A, and prunetin) were studied in the human intestinal Caco-2 model and mature Caco-2 cell lysate . Glucuronides were the main metabolites in intact Caco-2 cells for all isoflavones except prunetin, which was mainly sulfated . In addition, the 7-hydroxy group was the main site for glucuronidation whereas the 4'-hydroxy group was only one of the possible sites for sulfation . Glucuronidated isoflavones (except biochanin A) were preferably excreted to the basolateral side, whereas sulfated metabolites (except genistein and glycitein) were mainly excreted into the apical side . Polarized excretion of most isoflavone conjugates was inhibited by the multidrug resistance-related protein (MRP) inhibitor leukotriene C(4) (0.1 micro M) and the organic anion transporter (OAT) inhibitor estrone sulfate (10 micro M) . When formation and excretion rates of isoflavones were determined simultaneously, the results showed that formation served as the rate-limiting step for all isoflavone conjugates (both glucuronides and sulfates) except for genistein glucuronide, which had comparable excretion and formation rates . In conclusion, the intestinal disposition of isoflavones was structurally dependent, polarized, and mediated by MRP and OAT . Formation generally served as the rate-limiting step in the cellular excretion of conjugated isoflavones in the Caco-2 cell culture model.

Cancer Biother Radiopharm, 2004 Aug, 19(4), 457 - 65
Tracers to monitor the response to chemotherapy: in vitro screening of four radiopharmaceuticals; de Geus-Oei LF et al.; OBJECTIVES: It has been postulated that radiopharmaceuticals can be used to predict the therapeutic response to (chemo)therapy, which could lead to individualized treatment regimens . In this study, 18F-deoxyglucose, 99mTc-tetrofosmin, 125I-deoxyuridineribose, and 125I-methyltyrosine were tested for this purpose . METHODS: The uterine sarcoma cell line MES-SA (MDR-) and its multidrug resistant variant, MES-SA/Dx5 (MDR+), were used . The MDR+ cells express high levels of P-glycoprotein, which makes them relatively resistant to various chemotherapeutic agents . Cells were cultured in the presence of escalating concentrations of doxorubicin, and the cellular uptake of the radiopharmaceuticals was determined . RESULTS: Decreasing 18F-deoxyglucose uptake at escalating doxorubicin concentrations reflected the chemosensitivity of the cells: 18F-deoxyglucose uptake in the MDR- cells was reduced to 40% of the baseline level in the presence of 1 microM of doxorubicin, compared to 74% in the MDR+ cells . The 125I-deoxyuridineribose uptake in MDR- cells was reduced to 2% of the baseline level when cultured at a concentration of 1 microM of doxorubicin, while this was 79% in the MDR+ cells . The same trend was observed with 125I-methyltyrosine . The enhanced doxorubicin chemosensitivity of MDR+ cells in the presence of verapamil, a modulator of P-glycoprotein, was reflected by the reduced uptake of 18F-deoxyglucose, 125I-deoxyuridineribose, and 125I-methyltyrosine . Furthermore, baseline 99mTc-tetrofosmin uptake in MDR+ cells was more than six-fold lower than in MDR- cells . CONCLUSION: In the presence of doxorubicin, the uptake of 18F-deoxyglucose, 125I-deoxyuridineribose and, to a lesser extent, 125I-methyltyrosine is more pronouncedly reduced in MDR- cells than in MDR+ cells . The reversal of doxorubicin-resistance of MDR+ cells by verapamil was also reflected by the uptake of 18F-deoxyglucose, 125I-deoxyuridineribose, and 125I-methyltyrosine . 99mTc-tetrofosmin uptake reflected P-glycoprotein expression without exposure to doxorubicin . Copyright Mary Ann Liebert, Inc.

Cancer Biother Radiopharm, 2004 Aug, 19(4), 411 - 21
Influence of glutathione depletion on plasma membrane cholesterol esterification and on Tc-99m-sestamibi and Tc-99m-tetrofosmin uptakes: a comparative study in sensitive U-87-MG and multidrug-resistant MRP1 human glioma cells; Le Jeune N et al.; In our previous studies, we demonstrated a possible effect of cellular glutathione (GSH) depletion on plasma-membrane permeability and fluidity in glioma-cell lines . We therefore investigated the effect of GSH modulation on accumulation of two radiotracers, Tc-99m-sestamibi (MIBI) and Tc-99m-tetrofosmin (TFOS), and on plasma-membrane cholesterol content in sensitive U-87-MG and resistant U-87-MG-CIS and U-87-MG-MEL (MRP1 positive) human glioma-cell lines . GSH depletion was mediated by BSO pretreatment and addition of N-acetylcysteine reversed the effect . MIBI and TFOS uptakes, total cholesterol, and cholesteryl-ester contents were evaluated under each condition . In contrast with TFOS, MIBI accumulation was inversely proportional to the cell multidrug resistance phenotype . Similar cholesterol contents were observed in all cell lines, demonstrating that MRP1 did not modify lipid membrane composition . A decrease of intracellular GSH allows an increase of plasma-membrane cholesterol and a decrease of cholesteryl-ester content, which in turn results in spectacular TFOS uptake . The GSH status of the cells plays an important role in the plasma membrane cholesterol composition and TFOS uptake, which appears to be particularly sensitive to this modification . In contrast with MIBI, TFOS is not an MRP1 probe in glioma cells, and therefore appears to be a suitable tracer in this indication . Copyright Mary Ann Liebert, Inc.

Neurology, 2004 Sep 28, 63(6), 1090 - 2
Failure to confirm association of a polymorphism in ABCB1 with multidrug-resistant epilepsy; Tan NC et al.; Alteration of ATP-binding cassette subfamily B member 1 transporter (ABCB1) can plausibly cause drug-resistant epilepsy as it influences brain penetration of drugs . The CC genotype at the ABCB1 C3435T polymorphism was reported to be associated with multidrug resistance . A replication study in 401 drug-resistant and 208 drug-responsive subjects with epilepsy showed no significant association between the CC genotype and drug-resistant epilepsy . The authors suggest the initial association may have arisen by chance.

Nucleic Acids Res, 2004 Sep 27, 32(17), 5066 - 75 Print 2004.
Pdr3 is required for DNA damage induction of MAG1 and DDI1 via a bi-directional promoter element; Zhu Y et al.; In order to understand how gene regulation is achieved in eukaryotes in response to DNA damage, we used budding yeast as a model lower eukaryotic organism and investigated the molecular events leading to the expression of two closely clustered damage-inducible genes, MAG1 and DDI1 . MAG1 and DDI1 are co-activated by a shared 8 bp repeat sequence, UAS(DM) . In this study, we screened a yeast genomic library, identified Pdr3 as the transcriptional activator and demonstrated in vivo and in vitro that Pdr3 binds UAS(DM) . Pdr3 is required for the activation of a number of genes encoding membrane efflux pumps and deletion of PDR3 results in reduced basal-level expression and loss of DNA damage induction of MAG1 and DDI1 . Interestingly, Pdr1, another transcriptional activator homologous to Pdr3 that is also required for the activation of multidrug-resistance genes, is not involved in the regulation of MAG1 and DDI1 expression, although it may also bind to UAS(DM) . Deletion of PDR3 does not affect the expression of other well-documented DNA damage-inducible genes; hence, yeast DNA damage-inducible genes appear to have distinct effectors although to a certain extent they share a common regulatory pathway mediated by DNA damage checkpoints.

Eur J Pharm Sci, 2004 Oct, 23(2), 181 - 8
Substrates and inhibitors of efflux proteins interfere with the MTT assay in cells and may lead to underestimation of drug toxicity; Vellonen KS et al.; The MTT (3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide) assay is a widely used method in assessment of cytotoxicity and cell viability, and also in anti-cancer drug studies with tumour cells . These cells often express efflux proteins, such as P-glycoprotein (MDR1) or multidrug resistance (MDR) protein 1 (MRP1) . MDCKII cells that overexpress these proteins (MDCKII-MDR1 or MDCKII-MRP1) and normal cells (MDCKII-wt) were used to investigate the effects of efflux pump activity on the results of MTT assay . Efflux protein activity was confirmed with calcein-AM efflux assay, and MTT assay was compared to another cytotoxicity test, the LDH release assay . Inhibition of MRP and MDR1 efflux proteins in MDCKII cell lines was associated paradoxically with increased reduction of MTT, implying an apparent increase in cell viability . This effect was seen when MK 571 (MRP1 and MRP2 inhibitor) or verapamil (MRP1 and MDR1 inhibitor) were used to block efflux protein activity . The calcein-AM efflux assay also showed that the MTT reagent inhibits the function of MDR1 in the MDCKII-MDR1 cell line . This study shows that MDR1 and possibly MRP proteins interfere with the MTT assay . Due to wide substrate specificity of efflux proteins and popularity of the MTT assay this interference is not trivial . Presence of any efflux protein substrate may therefore lead to underestimated results in MTT assay, thereby causing potential bias and erroneous conclusions in cytotoxicity studies.

Biochem Pharmacol, 2004 Nov 1, 68(9), 1815 - 23
The role of structural factors in the kinetics of cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines: implications for overcoming multidrug resistance towards leukaemia K562/DOX cells; Tarasiuk J et al.; The appearance of multidrug resistance (MDR) of tumour cells to a wide array of antitumour drugs, structurally diverse and having different mechanisms of action, constitutes the major obstacle to the successful treatment of cancer . Our approach to search for non-cross resistant antitumour agents is based on the rational design of derivatives, which have a high kinetics of passive cellular uptake rendering their active efflux by MDR exporting pumps inefficient . Recently, two families of acridine cytotoxic agents were obtained, pyrazoloacridines (PACs) and pyrazolopyrimidoacridines (PPACs) . The aim of this study was to examine molecular basis of the reported differences in retaining cytotoxic activity of these derivatives at cellular level against resistant erythroleukaemia K562/DOX (overexpressing P-glycoprotein) cell line . The study was performed using a spectrofluorometric method, which allows continuous monitoring of the uptake and efflux of fluorescent molecules by living cells . It was demonstrated that the presence of two additional rings, pyrazole and pyrimidine, fused to the acridine chromophore structure (PPAC) favoured more rapid cellular diffusion than the presence of only one additional pyrazole ring (PAC) . The presence of hydrophobic substituent OCH3 markedly favoured the cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines while compounds having hydrophilic substituent OH exhibited very low kinetics of cellular uptake . In contrast, it was found that neither structure of the ring system nor the hydrophobic/hydrophilic character of examined substituents determined the rate of active efflux of these compounds by P-glycoprotein . Our data showed that a nearly linear relation exists between the resistance factor (RF) and lnV+ reflecting the impact of the cellular uptake rate (V+) on the ability of these compounds to overcome MDR.

Gastric Cancer, 2004, 7(3), 160 - 6
Regulation of drug sensitivity of gastric cancer cells by human calcyclin-binding protein (CacyBP); Shi Y et al.; BACKGROUND: Calcyclin-binding protein (CacyBP) was previously identified as an upregulated gene in a multidrug-resistant gastric cancer cell line, SGC7901/ADR, compared to its parental cells, SGC7901, by subtractive hybridization . The aim of this study was to explore the role of CacyBP in multidrug resistance (MDR) in gastric cancer cells . METHODS: The cDNA encoding CacyBP was generated by reverse-transcription-polymerase chain reaction (RT-PCR), and mouse antisera against CacyBP was raised using recombinant CacyBP as the immunogen . The expression of CacyBP in gastric cancer cells was determined by Northern and Western blots . Sense and antisense vectors for CacyBP were introduced into SGC7901 and SGC7901/ADR cells, respectively . The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was performed to evaluate the drug sensitivity of gastric cancer cells . Flow cytometry was employed to determine adriamycin accumulation and retention in gastric cancer cells . RESULTS: Northern and Western blots demonstrated upregulation of CacyBP in SGC7901/ADR cells compared to SGC7901 cells . SGC7901-CacyBP and SGC7901/ADR-anCacyBP cells were prepared, in which CacyBP was genetically increased and decreased, respectively . As compared with SGC7901, SGC7901-CacyBP cells exhibited significantly increased ( P < 0.01) IC(50) values for vincristine, adriamycin, and 5-fluorouracil . Meanwhile, as compared with SGC7901/ADR, SGC7901/ADR-anCacyBP cells exhibited significantly decreased ( P < 0.01) IC(50) values for these three drugs . SGC7901-CacyBP and SGC7901/ADR-anCacyBP cells displayed no obvious difference ( P > 0.05) in intracellular adriamycin content compared to their corresponding parental cells . CONCLUSIONS: Upregulation of CacyBP is associated with MDR in gastric cancer cells . CacyBP could regulate the responses of gastric cancer cells to chemotherapy . But the underlying mechanisms of CacyBP-related MDR need further identification.

Eur J Nucl Med Mol Imaging, 2004 Nov, 31(11), 1523 - 1529 Epub 2004 Sep 21.
In vitro detection of mdr1 mRNA in murine leukemia cells with 111In-labeled oligonucleotide; Bai J et al.; PURPOSE: The feasibility of intracellular mdr1 mRNA expression detection with radiolabeled antisense oligonucleotide (ODN) was investigated in the murine leukemia cell line, P388/S, and its subclonal, adriamycin-resistant cell line, P388/R . METHODS: The expression level of mdr1 mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) . Existence of the multidrug resistance (MDR) phenomenon was assessed via cellular uptake of 99mTc-sestamibi (MIBI), a known substrate for P-glycoprotein . A 15-mer phosphorothioate antisense ODN complementary to the sequences located at -1 to 14 of mdr1 mRNA and its corresponding sense ODN were conjugated with the cyclic anhydride of diethylene triamine penta-acetic acid (cDTPA) via an amino group linked to the terminal phosphate at the 5' end at pH 8-9 . The DTPA-ODN complexes at concentrations of 0.1-17.4 microM were reacted with 111InCl3 at pH 5 for 1 h . The hybridization affinity of labeled ODN was evaluated with size-exclusion high-performance liquid chromatography following incubation with the complementary sequence . Cellular uptake of labeled ODN was examined in vitro . Furthermore, enhancing effects of synthetic lipid carriers (Transfast) on transmembrane delivery of ODN were assessed . RESULTS: P388/R cells displayed intense mdr1 mRNA expression in comparison with P388/S cells . 99mTc-MIBI uptake in P388/S cells was higher than that in P388/R cells . Specific radioactivity up to 1,634 MBq/nmol was achieved via elevation of added radioactivity relative to ODN molar amount . The hybridization affinity of antisense 111In-ODN was preserved at approximately 85% irrespective of specific activity . Cellular uptake of antisense 111In-ODN did not differ from that of sense 111In-ODN in either P388/S cells or P388/R cells . However, lipid carrier incorporation significantly increased transmembrane delivery of 111In-ODN; moreover, specific uptake of antisense 111In-ODN was demonstrated in P388/R cells . CONCLUSION: Radiolabeling of ODN at high specific radioactivity and specific uptake of antisense 111In-ODN in drug-resistant cells may facilitate future gene imaging of mdr1 mRNA.

Pathol Oncol Res, 2004, 10(3), 133 - 41 Epub 2004 Sep 25.
Human osteosarcoma xenografts and their sensitivity to chemotherapy; Bruheim S et al.; Despite the increased survival rates of osteosarcoma patients attributed to adjuvant chemotherapy, at least one third of the patients still die due to their disease . Further improvements in the management of osteosarcoma may rely on a more individualised treatment strategy, as well as on the introduction of new drugs . To aid in the preclinical evaluation of new candidate substances against osteosarcoma, we have established 11 human osteosarcoma xenograft lines and characterised them with regard to response to five different reference drugs . Doxorubicin, cisplatin methotrexate, ifosfamide and lomustine were effective in 3/11, 3/11, 1/10, 5/11 and 4/11 of the xenografts, respectively . Five xenografts were resistant to all compounds tested . We also assessed the mRNA expression levels of the xenografts for the O(6)-Methylguanine DNA Methyltransferase (MGMT), DNA topoisomerase II- (Topo II)-alpha, Gluthathione-S-transferase (GST)-pi, Multidrug-resistance related protein (MRP) 1 and Multidrug-resistance (MDR) 1 genes . There was an inverse correlation between the transcript levels of GST-pi and doxorubicin growth inhibition (r=-0.66; p<0.05), and between the transcript levels of MGMT and the effect of lomustine (r=-0.72; p<0.01), whereas the expression of MRP1 and cisplatin growth inhibition was positively correlated (r=0.82; p<0.005) . This panel of xenografts should constitute a good tool for pharmacological and molecular studies in osteosarcoma.

J Cell Physiol, 2004 Dec, 201(3), 409 - 19
FGF6 mediated expansion of a resident subset of cells with SP phenotype in the C2C12 myogenic line; Israeli D et al.; Fibroblast growth factor 6 (FGF6) is selectively expressed during muscle development and regeneration . We examined its effect on muscle precursor cells (mpc) by forcing stable FGF6 expression in C2C12 cells in vitro . FGF6 produced in genetically engineered mpc was active, inducing strong morphological changes, altering cell adhesion and compromising their ability to differentiate into myotubes . Expression of MyoD and myogenin, but not of Myf5, was abrogated in FGF6 engineered mpc . These effects were reversed by FGF inhibitors . Ectopic expression of MyoD also restored fiber formation indicating that FGF6 interferes with the myogenic differentiation pathway upstream of MyoD . We also report that in the presence of FGF6, the minor (0.5-2%) subpopulation of cells actively excluding Hoechst 33342 in a verapamil-dependent manner (SP phenotype) was increased to 15-20% and the expression of the mdr1a gene (but not mdr1b) was upregulated by 400-fold . Our data establish a previously undescribed link between FGF6--a muscle specific growth factor--and a multidrug resistance gene expressed in stem cells, and suggest a role for FGF6 in the maintenance of a reserve pool of progenitor cells in the skeletal muscle.

Int J Cancer, 2005 Jan 10, 113(2), 229 - 40
Regulation of the multidrug resistance transporter P-glycoprotein in multicellular prostate tumor spheroids by hyperthermia and reactive oxygen species; Wartenberg M et al.; Hyperthermia is an important component of many cancer treatment protocols . In our study the regulation of the multidrug resistance (MDR) transporter P-glycoprotein by hyperthermia was studied in multicellular prostate tumor spheroids . Hyperthermia treatment of small (50-100 microm) tumor spheroids significantly increased P-glycoprotein and mdr-1 mRNA expression with a maximum effect at 42 degrees C, whereas only moderate elevation of P-glycoprotein was found in large (350-450 microm) tumor spheroids . Hyperthermia caused an elevation of intracellular reactive oxygen species (ROS) . Inhibition of ROS generation with NADPH-oxidase inhibitors diphenylen iodonium (DPI) and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) abolished P-glycoprotein expression but did not affect its transcript levels following heat treatment . This indicates that P-glycoprotein levels are controlled by regulating its translation rate or stability . Hyperthermia incubation resulted in a differential activation of p38 mitogen-activated protein kinase (MAPK), extracellular regulated kinase 1,2 (ERK1,2), and c-jun N-terminal kinase (JNK) immediately, 4 hr and 24 hr after treatment . Furthermore, upregulation of hypoxia-inducible factor 1alpha (HIF-1alpha) was observed . Elevation of HIF-1alpha and P-glycoprotein expression following hyperthermia treatment were abolished upon coadministration of the p38 inhibitor SB203580 . In contrast the JNK inhibitor SP600125 and the ERK1,2 inhibitor UO126 resulted in increase of HIF-1alpha and P-glycoprotein in the control as well as the hyperthermia-treated samples, indicating negative regulation of intrinsic HIF-1alpha and P-glycoprotein expression by ERK1,2 and JNK signaling cascades . In summary our data demonstrate that hyperthermia-induced upregulation of P-glycoprotein and HIF-1alpha is mediated by activation of p38, whereas ERK1,2 and JNK are involved in repression of P-glycoprotein and HIF-1alpha under control conditions.

Can J Physiol Pharmacol, 2004 Jul, 82(7), 431 - 7
Rh2, a compound extracted from ginseng, hypersensitizes multidrug-resistant tumor cells to chemotherapy; Jia WW et al.; Rh2 is a ginsenoside extracted from ginseng that has drawn attention in a few laboratories in Asian countries because of its potential tumor-inhibitory effect . In the present study, we tested Rh2 on many tumor-cell lines for its effects on cell proliferation, induction of apoptosis, and potential interaction with conventional chemotherapy agents . Our results showed that Rh2 inhibited cell growth by G1 arrest at low concentrations and induced apoptosis at high concentrations in a variety of tumor-cell lines, possibly through activation of caspases . The growth arrest and apoptosis may be mediated by 2 separate mechanisms . Apoptosis is not dependent on expression of the wild-type p53 nor the caspase 3 . In addition, the apoptosis induced by Rh2 was mediated through glucocorticoid receptors . Most interestingly, Rh2 can act either additively or synergistically with chemotherapy drugs on cancer cells . Particularly, it hypersensitized multidrug-resistant breast cancer cells to paclitaxel . These results suggest that Rh2 possesses strong tumor-inhibiting properties, and potentially can be used in treatments for multidrug-resistant cancers, especially when it is used in combination with conventional chemotherapy agents.

Cells Tissues Organs, 2004, 177(3), 151 - 9
Topical colchicine selection of keratinocytes transduced with the multidrug resistance gene (MDR1) can sustain and enhance transgene expression in vivo; Pfutzner W et al.; In gene therapy, a clinically relevant therapeutic effect requires long-term expression of the desired gene at a level sufficient to correct or at least alleviate the underlying gene defect . One approach to achieve persistent as well as high-level transgene expression in a significant percentage of target cells would be to select cells expressing both the desired transgene and a linked selectable gene--such as the human multi-drug resistance (MDR1) gene--in a bicistronic vector . Because of its accessibility, the skin is a very attractive target tissue to select genetically modified cells, allowing topical application of a selecting agent, thus minimizing potential toxic side effects . Among the potential selecting drugs, agents that block cell division, such as colchicine, are of particular interest because the use of anti-mitotic drugs takes advantage of the rapid keratinocyte (KC) turnover in the epidermis and the need for continued proliferation to substitute the KC lost due to selection . Before assessing the therapeutic benefit of such an approach, several key questions need to be answered in preclinical models: (1) Does topical colchicine application achieve the desired in vivo effect by blocking KC mitosis without eliciting unwanted toxic side effects? (2) Are MDR-transduced (MDR+) human KC still able to proliferate and differentiate when treated with colchicine? (3) Can MDR+ KC be enriched by topical selection? (4) Does topical selection result in persistent transgene expression by selecting KC stem cells expressing MDR? To answer these questions and to test the feasibility of such an approach both an in vitro skin equivalent and an in vivo human skin graft model were developed in which MDR+ KC were treated with different dosages of colchicine . Quantitative and qualitative analyses of MDR expression in human KC showed that topical colchicine treatment selects high-level transgene expression in a high percentage of KC . Moreover, determination of transgene copy numbers demonstrated that MDR+ KC progenitor cells were enriched by topical selection resulting in long-term expression of the transgene in the skin . Thus, in summary, these models demonstrate that topical selection of MDR+ KC is a safe approach to efficiently enhance long-term gene expression in the skin and holds future promise for clinical gene therapy applications .

Antimicrob Agents Chemother, 2004 Oct, 48(10), 3758 - 64
Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum beta-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada; Boyd DA et al.; A major outbreak involving an Escherichia coli strain that was resistant to expanded-spectrum cephalosporins occurred in Toronto and surrounding regions in 2000 to 2002 . We report the complete sequence of a plasmid, pC15-1a, that was found associated with the outbreak strain . Plasmid pC15-1a is a circular molecule of 92,353 bp consisting of two distinct regions . The first is a 64-kb region that is essentially homologous to the non-R-determinant region of plasmid R100 except for several point mutations, a few small insertions and deletions, and the absence of Tn10 . The second is a 28.4-kb multidrug resistance region (MDR) that has replaced the R-determinant region of the R100 progenitor and consists mostly of transposons or partial transposons and five copies of the insertion element IS26 . All drug resistance genes found in pC15-1a, including the beta-lactamase genes bla(CTX-M-15), bla(OXA-1), and bla(TEM-1), the tetracycline resistance gene tetA, and aminoglycoside resistance genes aac(6')-Ib and aac(3)-II, are located in the MDR . The bla(CTX-M-15) gene was found downstream of ISEcp1as part of a transposition unit, as determined from the surrounding sequence . Examination of the plasmids from CTX-M-15-harboring strains isolated from hospitals across Canada showed that pC15-1a was found in several strains isolated from a site in western Canada . Comparison of pC15-1a and pCTX15, found in an E . coli strain isolated in India in 1999, revealed that the plasmids had several features in common, including an R100 backbone and several of the resistance genes, including bla(CTX-M-15), bla(TEM-1), bla(OXA-1), tetA, and aac(6')-Ib.

Int J Cancer, 2005 Jan 10, 113(2), 213 - 20
Overexpression and significance of prion protein in gastric cancer and multidrug-resistant gastric carcinoma cell line SGC7901/ADR; Du J et al.; In our previous work, cellular prion protein (PrPc) was identified as an upregulated gene in adriamycin-resistant gastric carcinoma cell line SGC7901/ADR compared to its parental cell line SGC7901 . Here we investigate the expression of PrPc in gastric cancer and whether it was involved in multidrug resistance (MDR) of gastric cancer . We demonstrated that PrPc was ubiquitously expressed in gastric cancer cell lines and tissues . PrPc conferred resistance of both P-glycoprotein (P-gp)-related and P-gp-nonrelated drugs on SGC7901, which was accompanied by decreased accumulation and increased releasing amount of adriamycin in PrPc-overexpressing cell line . Inhibition of PrPc expression by antisense or RNAi technology could partially reverse multidrug-resistant phenotype of SGC7901/ADR . PrPc significantly upregulated the expression of the classical MDR-related molecule P-gp but not multidrug resistance associated protein and glutathione S-transferase pi . The PrPc-induced MDR could be partially reversed by P-gp inhibitor verapamil . PrPc could also suppress adriamycin-induced apoptosis and alter the expression of Bcl-2 and Bax, which might be another pathway contributing to PrPc-related MDR . The further study of the biological functions of PrPc may be helpful for understanding the mechanisms of occurrence and development of clinical gastric carcinoma and PrPc-related MDR and developing possible strategies to treat gastric cancer.

Int J Cancer, 2005 Jan 1, 113(1), 158 - 65
Inhibition of leukemic cell growth by a novel anti-cancer drug (GUT-70) from calophyllum brasiliense that acts by induction of apoptosis; Kimura S et al.; During our search for cancer chemopreventing compounds derived from plant sources, we discovered that the natural product GUT-70, isolated from the stem bark of Calophyllum brasiliense collected in Brazil, significantly inhibits the growth of leukemic cells . GUT-70, characterized as a tricyclic coumarin, 5-methoxy-2,2-dimethyl-6-(2-methyl-1-oxo-2-butenyl) -10-propyl-2H,8H-benzo{1,2-b;3,4-b'}dipyran-8-one (C(23)H(26)O(5)), inhibited all 6 human leukemic cell lines evaluated, including the P-glycoprotein overexpressing cell line, in a concentration and time-dependent manner with IC(50) values from 2-5 microM . Furthermore, GUT-70 did not inhibit colony formation by normal hematopoietic progenitors up to 30 microM and also did not inhibit the proliferation of normal human hepatocytes up to 30 microM . GUT-70 activated the caspase 2, 3, 8 and 9, and induced the apoptosis in leukemic cells, which was inhibited by caspase inhibitors . GUT-70 induced anti-leukemic effects independent of the p53-p2l(WAFl/CIP1) pathway and increased the overall expression of p27(KIP1) and p57(KIP2), to stop the cell cycle at the G(1)/S transition . Thus, a novel anti-cancer drug, GUT-70 isolated from the stem bark of C . brasiliense induces caspase-mediated and p53-independent apoptosis to overcome multidrug resistance and may become a potent leukemia therapeutics.

Int J Cancer, 2004 Dec 20, 112(6), 934 - 42
RLIP76 (RALBP1)-mediated transport of leukotriene C4 (LTC4) in cancer cells: implications in drug resistance; Sharma R et al.; Increased active transport of LTC(4) observed frequently in multidrug-resistant cancer cells have been attributed to ABC-transporter proteins particularly, MRP1 . We have demonstrated recently that a novel non-ABC transporter, RLIP76 (RALBP1) can also mediate ATP-dependent transport of GSH-conjugates (GS-E) as well as doxorubicin (DOX) . We demonstrate RLIP76 reconstituted in artificial liposomes can catalyze ATP-dependent transport of LTC(4), which can be modulated by PKC-alpha . The ATPase activity of E . coli expressed homogenous RLIP76 was stimulated in a saturable fashion by LTC(4) with half maximal stimulation at 130 nM . Proteoliposomes reconstituted with RLIP76 catalyzed temperature and osmolar sensitive ATP-dependent transport of LTC(4) with K(m) values of 5.1 mM and 210 nM for ATP and LTC(4), respectively . V(max) for transport was found to be 3.2 nmol/min/mg . Colchicine inhibited LTC(4) transport to 50% at 5.8 microM . PKC-alpha catalyzed phosphorylation of RLIP76 and increased its transport activity by 2-3-fold . Membrane vesicles prepared from the small (SCLC) and non-small (NSCLC) lung cancer cell lines as well as HL-60 (leukemia) and U937 (lymphoma) cell lines exhibited ATP-dependent transport of LTC(4), which was inhibited by anti-RLIP76 antibodies . The rate of transport of LTC(4) in SCLC (H69, H378) was half of that observed in NSCLC cell lines but after transfection with RLIP76, the transport rate of LTC(4) in H69 became comparable to that in NSCLC cell lines . Anti-RLIP76 antibodies inhibited LTC(4) transport by 67-81% in all 8 cell lines examined, whereas N-19 anti-MRP1 antibodies inhibited transport of LTC(4) by only 11-26% . These results suggest that RLIP76 is the major LTC(4) transporter in cancer cells and that its transport activity is regulated by PKC-alpha-mediated phosphorylation . (c) 2004 Wiley-Liss, Inc

Mol Pharmacol, 2004 Oct, 66(4), 1004 - 10
Modulation of multidrug resistance-associated protein 2 (Mrp2) and Mrp3 expression and function with small interfering RNA in sandwich-cultured rat hepatocytes; Tian X et al.; Canalicular multidrug resistance-associated protein 2 (Mrp2) and basolateral Mrp3 mediate the excretion of organic anions, including conjugated and unconjugated xenobiotics and bile acids, from the liver . The utility of RNA interference to specifically knock down the expression and function of transport proteins was demonstrated in sandwich-cultured rat hepatocytes, which exhibit functional and properly localized Mrp2 and Mrp3 over time in culture . Specific knockdown of Mrp2 (approximately 50% decrease in expression) resulted in an approximately 45% decrease in the biliary excretion index of carboxydichlorofluorescein (CDF) (9.3% versus 16.5%), but did not affect Mrp3 or radixin expression . Specific Mrp3 knockdown (approximately 50% decrease in expression) resulted in significantly higher accumulation of CDF in cells + bile canaliculi (32.3 +/- 2.5 versus 24.4 +/- 4.3 pmol/mg of protein/10 min), but no change in cellular accumulation (13.7 +/- 2.2 versus 15.6 +/- 4.0 pmol/mg of protein/10 min), consistent with an approximately 60% increase in the biliary excretion index of carboxydichlorofluorescein . The extent of protein knockdown was in good agreement with changes in carboxydichlorofluorescein disposition . In conclusion, modulation of drug transporters in sandwich-cultured rat hepatocytes by small interfering RNA treatment is a feasible in vitro approach to study the expression and function of drug transport proteins.

Nucl Med Commun, 2004 Oct, 25(10), 1039 - 48
Optimization of technetium-99m Sestamibi single-photon emission tomography to define multidrug resistance with confidence; Moorin RE et al.; BACKGROUND: The efflux rate of technetium-99m Sestamibi (99mTc-Sestamibi) is a kinetic phenomenon related to the response of cancer cells to chemotherapy, and may be used to determine drug resistance . Measurement of the efflux rate requires accurate quantitative single-photon emission tomography (SPET) imaging within the time constraints imposed by the kinetics of the process . METHODS: A phantom study, at activity concentrations typically found with 99mTc-Sestamibi in vivo, was undertaken to optimize the SPET parameters and, in particular, to determine whether 180 degrees acquisition arcs with heads in 'L' configuration could be used for accurate quantification . Following the development of the most appropriate SPET protocol, a small patient pilot study was undertaken . RESULTS: Studies designed to evaluate statistical uncertainty (noise), contrast restitution and spatial resolution of the data sets, using different acquisition and reconstruction parameters, showed that 180 degrees SPET using a 64 x 64 matrix, 6 degrees angular sampling and iterative reconstruction was optimal . Finer linear and/or angular sampling afforded negligible improvement in resolution, but markedly increased the statistical uncertainty . Comparison of 360 degrees and 180 degrees acquisitions, utilizing conventional filtered backprojection and iterative reconstruction algorithms, demonstrated that the statistical uncertainty was reduced to a greater extent for 180 degrees data collection . For 360 degrees (64 x 64) data acquisition, statistical uncertainty decreased from 15% to 11% using the iterative algorithm, whilst the 180 degrees (64 x 64) data showed a reduction from 20% to 7%, and approached values obtained by planar imaging . The efflux measurements obtained in the patient pilot study were consistent with the observed chemotherapy response . CONCLUSION: Our study shows that 180 degrees acquisition arcs are a practical option for accurate quantitative SPET kinetic imaging for potential studies of chemotherapy response in patients with lung cancer.

Adv Drug Deliv Rev, 2004 Oct 14, 56(12), 1793 - 809
Potential role of ABC transporters as a detoxification system at the blood-CSF barrier; de Lange EC; Exchange of compounds between blood and brain occurs at two barriers, the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) . The barrier function is mainly a result of the functionality of the cerebral endothelial cells and choroidal epithelial cells, respectively . These cell types have restricted permeability due to the presence of tight junctions between the cells . Furthermore, these cells express a broad range of transporters . So far, the BBB has been viewed as the most important barrier, especially as its surface is about 3 orders of magnitude larger than that of the BCSFB . Today, there is a shift in the appreciation of the contribution of the BCSFB . In a few recent studies, it has been shown that the BCSFB expresses two types of ATP-binding cassette (ABC) transporters, being the multidrug transporters P-glycoprotein (P-gp) and the multidrug resistance-related protein 1 (MRP1) . The knowledge on the function of these transporters in the BCSFB is relatively scarce, but in general, it seems that MRP1 transport is directed towards the blood side, which makes this transporter helpful in elimination of harmful compounds from the CSF . Thereby MRP1 potentially contributes to detoxification of the brain, as a whole, as it is also expressed at the level of the BBB . P-gp, however, while also functional as an efflux pump at the BBB, has an opposite transport direction at the level of the BCSFB, towards the CSF . P-gp may therefore raise the concentration of neurotoxic P-gp substrates in the CSF . Whether this will have a significant contribution to the toxicity in the regions directly exposed to the CSF (periventricular organs) remains to be determined . Specifically, in the epithelial cells of the choroid plexus of the BCSFB, P-gp and MRP1 together serve a protective role by preventing the accumulation of their overlapping and often toxic substrates . A concerted action of P-gp and MRP1 at the choroid plexus might contribute to the maintenance of the role of the BCSFB in brain homeostasis.

Epilepsy Res, 2004 Jul-Aug, 60(2-3), 203 - 13
Selective and persistent upregulation of mdr1b mRNA and P-glycoprotein in the parahippocampal cortex of chronic epileptic rats; van Vliet E et al.; There is recent evidence that increased expression of multidrug transporters, such as P-glycoprotein (P-gp), may lead to reduced antiepileptic drug (AED) concentrations in the brain, shortly after status epilepticus (SE), thereby suggesting a possible mechanism for drug-resistance . To get insights on whether increased P-gp expression is a consequence of the initial insult, or evolves more gradually as a result of recurrent spontaneous seizures, we used a rat model of temporal lobe epilepsy in which spontaneous seizures develop after an electrically induced SE . We investigated the temporal and region-specific expression of two isoforms of the multidrug resistance gene (mdr1a and mdr1b, both encoding for P-gp) in two regions within the temporal lobe (the dentate gyrus (DG) and the parahippocampal cortex (PHC)) . Using real-time PCR, we found that the mdr1b isoform was increased in the temporal lobe, 1 week after SE; however, this increase was reversible in dentate gyrus while it persisted in the parahippocampal cortex of chronic epileptic rats . Mdr1b upregulation was related to the occurrence of spontaneous seizures, since this isoform was unchanged in rats that were stimulated, but that did not develop SE (non-SE) . The mdr1a isoform was transiently upregulated in the dentate gyrus . P-gp immunostaining was enhanced in endothelial and glia-like cells, 1 week after SE . In chronic epileptic rats, the number of strongly P-gp positive glia-like cells was much lower than 1 week after SE, and it was mainly present in the most ventral part of the temporal lobe . These cells were in close apposition to strongly stained blood vessels . These findings show that both mdr1a and mdr1b are induced by SE, although the increase in mdr1b isoform was more persistent . More importantly, increased P-gp expression is still present in chronic epileptic rats.

Int J Antimicrob Agents, 2004 Oct, 24(4), 386 - 92
Yeast strains designed for screening of reversal agents and genetic suppressors of multidrug resistance; Kozovska Z et al.; Multidrug resistance in yeast results from over-expression of drug efflux transporter genes due to gain-of-function mutations in transcription factors . To suppress multidrug resistance at the level of gene expression, we have developed a yeast-based screening system for the detection of compounds down-regulating the major multidrug ABC transporter Pdr5p expressed under the control of Pdr3p transcription factor . Here, we report the construction and properties of the improved set of yeast strains designed along with such screening also for a global analysis of genetic suppressors of multidrug resistance . The basic components of this system, the P(GAL1)-PDR3 and P(PDR5)-pma1(D378N) fusion genes, were individually or simultaneously integrated into corresponding chromosomes of a hypersensitive S . cerevisiae strain deleted in the PDR1 and PDR3 genes . This resulted in increased mitotic stability of a set of new test strains compared with the original prototrophic strain ZK11-1 developed previously . In addition, some of the strains designed are auxotrophic for leucine, uracil and histidine allowing them to be used in genetic screens for positive selection of multicopy or loss-of-function genetic suppressors of multidrug resistance.

Int J Urol, 2004 Sep, 11(9), 721 - 7
Increased expression of lung resistance-related protein in lower grade urothelial carcinoma of the renal pelvis and ureter; Kong CZ et al.; BACKGROUND: Lung resistance-related protein (LRP), like multidrug resistance gene 1 (MDR1) and multidrug resistance-associated proteins (MRP), has been associated with intrinsic therapeutic resistance in various malignancies . To date, there has been no study on the expression of LRP in urothelial carcinomas of the renal pelvis and ureter . We investigated the protein and mRNA expression levels of LRP, MDR1 and MRP1 in this malignancy and the clinical significance of their expression was evaluated . METHODS: Forty urothelial carcinomas of the renal pelvis and ureter and 31 normal upper urothelial samples were examined by immunohistochemistry and reverse transcription polymerase chain reaction to determine the protein and mRNA levels of the multidrug resistance-related genes, respectively . RESULTS: The positive staining rates and mRNA levels of LRP were the highest among these multidrug resistance-related genes in both normal urothelium and carcinoma examinations . In contrast to the up-regulated expression of MDR1, the expression of LRP tended to be down-regulated in carcinomas . Moreover, the expression of LRP inversely correlated with tumor grades, but this correlation was not found for the other two genes . However, there was no correlation among the expression of the three genes observed . CONCLUSION: Lung resistance-related protein was strongly expressed in urothelial carcinomas of the renal pelvis and ureter, particularly in well-differentiated carcinomas.

Curr Pharm Des, 2004, 10(24), 2965 - 79
Imaging drug resistance with radiolabeled molecules; Vaidyanathan G et al.; A major obstacle to successful cancer chemotherapy is drug resistance . Multidrug resistance (MDR) is often seen with chemotherapeutic agents such as anthracycline derivatives, vinca alkaloids and taxanes . Multiple aspects of cellular biochemistry have been implicated in the MDR process . Cellular mechanisms of resistance are due to the presence of efflux pumps, P-glycoprotein (P-gp) and multiple resistance-associated protein (MRP), which belong to the ATP-binding cassette (ABC) family of transporters . Another form of drug resistance is involved in the chemotherapy of cancers with alkylating agents such as nitrosourea derivatives and nitrogen mustards . The cytotoxicity of these agents is primarily due to alkylation of the DNA guanine residues at their O6-position, which leads, via a cascade of events, to DNA strand breaks . The DNA repair protein, alkylguanine-DNA alkyl transferase (AGT) removes the alkyl groups from the lesions stoichiometrically to a cysteine in its active site . This process is irreversible and results in the degradation of the protein and its recovery is entirely from de novo synthesis . Noninvasive methodologies for monitoring the transport activity of these efflux pumps and determining tumor content of AGT could serve as critical tools for optimizing chemotherapeutic protocols on a patient-specific basis and gaining an understanding of the dynamics of resistance in living patients . In this review, we will describe the efforts made to date to synthesize radioactive probes of chemotherapy resistance and their use to quantitate these transporters and DNA repair protein by radionuclide imaging.

Curr Top Med Chem, 2004, 4(13), 1385 - 98
Pharmacogenetics of drug transporters and its impact on the pharmacotherapy; Sakaeda T et al.; Most drug responses are determined by the interplay of several gene products that influence pharmacokinetics and pharmacodynamics, i.e., drug metabolizing enzymes, drug transporters, and drug targets . With the sequencing of the human genome, it has been estimated that approximately 500-1200 genes code for drug transporters . Concerning the effects of genetic polymorphisms on pharmacotherapy, the best characterized drug transporter is the multidrug resistant transporter P-glycoprotein/MDR1, the gene product of MDR1 . Little such information is available on other drug transporters . MDR1 is a glycosylated membrane protein of 170 kDa, belonging to the ATP-binding cassette superfamily, and is expressed mainly in intestines, liver, kidneys and brain . A number of various types of structurally unrelated drugs are substrates for MDR1, and their intestinal absorption, hepatobiliary secretion, renal secretion and brain transport are regulated by MDR1 . The first investigation on the effects of MDR1 genotypes on pharmacotherapy was reported in 2000: a silent single nucleotide polymorphism (SNP), C3435T in exon 26, was found to be associated with the duodenal expression of MDR1, and thereby the plasma concentration of digoxin after oral administration . At present, a total of 28 SNPs have been found at 27 positions on the MDR1 gene . Clinical investigations on the association of MDR1 genotypes with the expression and function of MDR1 in tissues, and with pharmacokinetics and pharmacodynamics have mainly focused on C3435T; however, there are still discrepancies in the results, suggesting that the haplotype of the gene should be analyzed instead of a SNP . C3435T is also reported to be a risk factor for a certain class of diseases including the inflammatory bowel diseases, Parkinson's disease and renal epithelial tumor, and this also might be explained by the effects on MDR1 expression and function . In this review, the latest reports on the effects of genetic polymorphisms of MDR1 on pharmacotherapy are summarized, and the pharmacogenetics of other transporters is briefly introduced.

Biochemistry, 2004 Sep 28, 43(38), 12081 - 9
The drug-binding pocket of the human multidrug resistance P-glycoprotein is accessible to the aqueous medium; Loo TW et al.; P-Glycoprotein (P-gp) is an ATP-dependent drug pump that transports a broad range of compounds out of the cell . Cross-linking studies have shown that the drug-binding pocket is at the interface between the transmembrane (TM) domains and can simultaneously bind two different drug substrates . Here, we determined whether cysteine residues within the drug-binding pocket were accessible to the aqueous medium . Cysteine mutants were tested for their reactivity with the charged thiol-reactive compounds sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) and {2-(trimethylammonium)ethyl)}methanethiosulfonate (MTSET) . Residue Ile-306(TM5) is close to the verapamil-binding site . It was changed to cysteine, reacted with MTSES or MTSET, and assayed for verapamil-stimulated ATPase activity . Reaction of mutant I306C(TM5) with either compound reduced its affinity for verapamil . We confirmed that the reduced affinity for verapamil was indeed due to introduction of a charge at position 306 by demonstrating that similar effects were observed when Ile-306 was replaced with arginine or glutamic acid . Mutant I306R showed a 50-fold reduction in affinity for verapamil and very little change in the affinity for rhodamine B or colchicine . MTSES or MTSET modification also affected the cross-linking pattern between pairs of cysteines in the drug-binding pocket . For example, both MTSES and MTSET inhibited cross-linking between I306C(TM5) and I868C(TM10) . Inhibition was enhanced by ATP hydrolysis . By contrast, cross-linking of cysteine residues located outside the drug-binding pocket (such as G300C(TM5)/F770C(TM8)) was not affected by MTSES or MTSET . These results indicate that the drug-binding pocket is accessible to water.

Probl Tuberk Bolezn Legk, 2004, (7), 32 - 5
{Changes in drug resistance of Mycobacteria in the simultaneous use of chemotherapy and intravenous infusions of dissolved ozone}; Belianin II et al.; The outcomes of treatment were analyzed in 56 patients with ever-progressive multidrug-resistant pulmonary tuberculosis who had been long isolating Mycobacterium tuberculosis (MBT) . The patients were divided into 2 groups . In the study group (n = 36), 75% isolated MBT resistant to streptomycin (S), isoniazid (I), rifampicin (R), and kanamycin (K) . In this connection, 41.7% of them received only 2 second-line antituberculous drugs and 27.8% took 3 drugs . The control group (n = 20) was comparable with the study group in the rate of bacterial isolation and in the drug resistance of the causative agent . In addition to chemotherapy (CT), dissolved ozone (pO3) was intravenously injected to the patients of the study group twice a week . They received a total of 12 to 55 infusions . Four-month addition of pO3 infusions to CT eliminated the resistance of isolated MBT to I and/or R . MBT became susceptible to I in 38.9% of the patients, R in 16.7%, and to K in 11.2% . By month 4, the isolated MBT became susceptible to I, R, and K in 47.2% . The mechanisms responsible for lowering drug resistance in MBT are discussed . The clinical example shows that patients with multidrug-resistant tuberculosis may be treated with first-line drugs provided that systemic intravenous injection of pO3 is performed.

World J Gastroenterol, 2004 Oct 15, 10(20), 3062 - 4
Multidrug resistance reversal in human gastric carcinoma cells by neferine; Cao JG et al.; AIM: To investigate the reversal effect of neferine on multidrug resistance in human gastric carcinoma cell line . METHODS: Cells of a human gastric cancer cells line, SGC7901, and its vincristine (VCR) -resistant variant, SGC7901/VCR, were cultivated with or without neferine and/or VCR . The cytotoxic effect of VCR was evaluated by the MTT assay . Cell apoptosis induced by VCR was determined by flow cytometry (FCM) . The expression of P-glycoprotein (P-gp) and a multidrug-resistance-associated protein (MRP) in cells was examined by immunofluorescence and FCM . RESULTS: Neferine at the concentration from 2.5 micromol/L to 10 micromol/L had no cytotoxicity to SGC7901 cells, and its variant SGC7901/VCR cells . The IC50 of VCR against SGC7901 and SGC7901/VCR cells was 0.059 microg/mL and 2.32 microg/mL, respectively, indicating that SGC7901/VCR cells were 39 times more resistant to VCR than its parent SGC7901 cells . After treatment with neferine at concentrations of 2.5, 5 and 10 micromol/L, the IC(50) of VCR to SGC7901/VCR cell line decreased to 0.340, 0.128 and 0.053 microg/mL, respectively, thus, increased the chemosensitivity by 6.8-, 18.1- and 43.8-fold, respectively . SGC7901/VCR cells were apoptosis resistant to VCR . Neferine (2.5, 5 and 10 micromol/L) promoted the VCR-induced apoptosis of SGC7901/VCR cells in a dose-dependent manner . The expressions of P-gp and MRP were strongly positive in SGC7901/VCR cells, which were significantly down-regulated after treatment with neferine (10 micromol/L) for 24 h . CONCLUSION: Neferine reverses multidrug resistance of human gastric carcinoma SGC7901/VCR cells, which may be associated with the down-regulations of P-gp and MRP expression in SGC701/VCR cells.

J Infect Dis, 2004 Oct 15, 190(8), 1456 - 63 Epub 2004 Sep 20.
Efficacy of monthly tafenoquine for prophylaxis of Plasmodium vivax and multidrug-resistant P . falciparum malaria; Walsh DS et al.; We assessed monthly doses of tafenoquine for preventing Plasmodium vivax and multidrug-resistant P . falciparum malaria . In a randomized, double-blind, placebo-controlled study, 205 Thai soldiers received either a loading dose of tafenoquine 400 mg (base) daily for 3 days, followed by single monthly 400-mg doses (n = 104), or placebo (n = 101), for up to 5 consecutive months . In volunteers completing follow-up (96 tafenoquine and 91 placebo recipients), there were 22 P . vivax, 8 P . falciparum, and 1 mixed infection . All infections except 1 P . vivax occurred in placebo recipients, giving tafenoquine a protective efficacy of 97% for all malaria (95% confidence interval {CI}, 82%-99%), 96% for P . vivax malaria (95% CI, 76%-99%), and 100% for P . falciparum malaria (95% CI, 60%-100%) . Monthly tafenoquine was safe, well tolerated, and highly effective in preventing P . vivax and multidrug-resistant P . falciparum malaria in Thai soldiers during 6 months of prophylaxis .

Cancer Chemother Pharmacol, 2005 Feb, 55(2), 179 - 88 Epub 2004 Sep 16.
Reversal of multidrug resistance of cancer through inhibition of P-glycoprotein by 5-bromotetrandrine; Jin J et al.; PURPOSE: The present study aimed to evaluate the MDR reversal activity of bromotetrandrine (BrTet), a bromized derivative of tetrandrine (Tet), in vitro and in vivo . METHODS: Drug sensitivity was determined using the MTT assay . The in vivo effect of Tet was investigated using nude mice grafted with sensitive and resistant KB human epidermoid cancer cells . Doxorubicin (Dox) accumulation was analyzed by fluorospectrophotometry and the protein and mRNA levels of P-glycoprotein (P-gp) were determined by immunocytochemistry and RT-PCR, respectively . RESULTS: BrTet at 0.25, 0.5 and 1 micro M reversed Dox resistance in MDR human breast cancer MCF-7/Dox cells dose-dependently and its potency was greater than that of Tet at the same concentrations . BrTet reversed vincristine (VCR), Dox and paclitaxel resistance in MDR human oral epidermoid carcinoma KBv200 cells as well as innate VCR and Dox resistance in human hepatocellular carcinoma Bel(7402) cells . However, BrTet showed no effect on the IC(50) values of the above-mentioned anticancer drugs in sensitive MCF-7 and KB cells . No reversal effect of BrTet on the cytotoxicity of 5-fluorouracil and cisplatin, non-P-gp substrates, was observed . In nude mice bearing KBv200 xenografts on the left flank and KB xenografts on the right flank, i.p . injection of 5 mg/kg and 10 mg/kg BrTet significantly enhanced the antitumor activity of Dox against KBv200 xenografts with inhibitory rates of 33.0% and 39.2%, while Dox alone inhibited the growth of KBv200 xenografts by only 11.6% . No enhancement by BrTet was seen in KB xenografts . Moreover, BrTet at 5 mg/kg reversed paclitaxel resistance in KBv200 xenografts . Fluorospectrophotometric assay showed that BrTet significantly increased the intracellular accumulation of Dox in MCF-7/Dox cells in a dose-dependent manner . BrTet also inhibited the overexpression of P-gp in MCF-7/Dox cells, but had no effect on mdr1 expression . CONCLUSIONS: BrTet showed significant MDR reversal activity in vitro and in vivo . Its activity may be related to the inhibition of P-gp overexpression and the increase in intracellular accumulation of anticancer drugs . BrTet may be a promising MDR modulator for eventual assessment in the clinic.

Nat Med, 2004 Oct, 10(10), 1117 - 21 Epub 2004 Sep 19.
Modeling epidemics of multidrug-resistant M . tuberculosis of heterogeneous fitness; Cohen T et al.; Mathematical models have recently been used to predict the future burden of multidrug-resistant tuberculosis (MDRTB) . These models suggest the threat of multidrug resistance to TB control will depend on the relative 'fitness' of MDR strains and imply that if the average fitness of MDR strains is considerably less than that of drug-sensitive strains, the emergence of resistance will not jeopardize the success of tuberculosis control efforts . Multidrug resistance in M . tuberculosis is conferred by the sequential acquisition of a number of different single-locus mutations that have been shown to have heterogeneous phenotypic effects . Here we model the impact of initial fitness estimates on the emergence of MDRTB assuming that the relative fitness of MDR strains is heterogeneous . We find that even when the average relative fitness of MDR strains is low and a well-functioning control program is in place, a small subpopulation of a relatively fit MDR strain may eventually outcompete both the drug-sensitive strains and the less fit MDR strains . These results imply that current epidemiological measures and short-term trends in the burden of MDRTB do not provide evidence that MDRTB strains can be contained in the absence of specific efforts to limit transmission from those with MDR disease.

Nat Med, 2004 Oct, 10(10), 1111 - 6 Epub 2004 Sep 19.
Modeling the emergence of the 'hot zones': tuberculosis and the amplification dynamics of drug resistance; Blower SM et al.; 'Hot zones' are areas that have >5% prevalence (or incidence) of multidrug-resistant tuberculosis (MDRTB) . We present a new mathematical model (the amplifier model) that tracks the emergence and evolution of multiple (pre-MDR, MDR and post-MDR) strains of drug-resistant Mycobacterium tuberculosis . We reconstruct possible evolutionary trajectories that generated hot zones over the past three decades, and identify the key causal factors . Results are consistent with recently reported World Health Organization (WHO) data . Our analyses yield three important insights . First, paradoxically we found that areas with programs that successfully reduced wild-type pansensitive strains often evolved into hot zones . Second, some hot zones emerged even when MDR strains were substantially less fit (and thus less transmissible) than wild-type pansensitive strains . Third, levels of MDR are driven by case-finding rates, cure rates and amplification probabilities . To effectively control MDRTB in the hot zones, it is essential that the WHO specify a goal for minimizing the amplification probability.

Chin Med J (Engl), 2004 Sep, 117(9), 1358 - 63
Expression of multidrug resistance-related markers in primary neuroblastoma; Lu QJ et al.; BACKGROUND: Multidrug resistance is associated with a poor prognosis in various human cancers . However, the clinical significance of the expression of multidrug resistance-related markers in neuroblastoma is still on debate . In this study, the effect of the expression of p-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and lung resistance protein (LRP) in neuroblastoma was evaluated . METHODS: The streptavidin-biotin immunoperoxidase (SP) technique was used to evaluate the expression of P-gp, MRP, and LRP in 70 cases of untreated primary neuroblastoma . RESULTS: The frequencies of the expression of P-gp, MRP, and LRP were 61.4%, 38.6%, and 24.3%, respectively . A significant positive correlation was observed between P-gp and MRP expression (P=0.001), as well as between LRP and MRP expression (P=0.01) . The rates of expression of P-gp and MRP were higher in tumors from patients aged greater than one year old than in tumors from patients aged less than 1 year old at time of diagnosis (P=0.01 and 0.018, respectively) . MRP expression in tumors that had metastasized was significantly more frequent than in tumors that had not metastasized (P=0.015) . The expression of all tested proteins showed a significant relationship with whether or not the tumor had differentiated (P=0.006, 0.000 or 0.001, respectively) . MRP expression was significantly associated with a reduction in both median survival time and 2-year cumulative survival (P=0.02) . By contrast, P-gp and MRP expression did not correlate with survival . According to Cox regression analysis, only the co-expression of P-gp and MRP had significant prognostic value (relative hazard, 3.513, P=0.033) . CONCLUSIONS: The intrinsic, multidrug resistance of neuroblastoma involves the combined effects of P-gp, MRP, and LRP . MRP expression may be an important factor determining prognosis in neuroblastoma.

Chin Med J (Engl), 2004 Sep, 117(9), 1348 - 52
Cytokine-induced killer cells showing multidrug resistance and remaining cytotoxic activity to tumor cells after transfected with mdr1 cDNA; Li HF et al.; BACKGROUND: Routine treatment of cancer such as surgery, radiation or chemotherapy is sometimes unable to eradicate metastatic malignant cells . So we tried a new method and increased the adoptive immunotherapy of Cytokine-induced killer (CIK) cells in tumor patients and the multidrug resistance (mdr1) cDNA was transfected into CIK cells . METHODS: CIK cells were obtained from peripheral blood and induced by IFN-gamma, anti-CD3 monoclonal antibody, IL-2 and IL-1 . CIK cells were transfected with plasmid PHaMDR containing human mdr1 cDNA by electroporation . RT-PCR was used to detect mdr1 mRNA in transfected CIK cells . P-glycoprotein (P-gp) expressed on surface of CIK cells was assayed by FITC-conjugated anti-P-gp monoclonal antibody and flow cytometry . Multidrug resistance to doxorubicin and colchicine and cytotoxic activity to human breast cancer cell line MCF7 were performed using MTT method . RESULTS: mdr1 mRNA was detected in transfected CIK cells . P-gp was expressed on the surface of the transfected CIK cells, and the P-gp positive cells reached 21% - 37% of the total CIK cells after transfection . The IC50 to doxorubicin increased to 22.3 - 45.8 times, and that to colchicines to 6.7 - 11.35 times, as compared to those of untransfected CIK cells . However, the cytotoxic activity to MCF7 cell line remained unaltered . CONCLUSIONS: CIK cells were successfully transfected with mdr1 cDNA by using electroporation . The transfected CIK cells had the characteristics of multidrug resistance without change in their cytotoxic activity to tumor cells.

Int J Oncol, 2004 Oct, 25(4), 1031 - 7
Transcription factor NF-Y regulates mdr1 expression through binding to inverted CCAAT sequence in drug-resistant human squamous carcinoma cells; Okamura H et al.; In this study, the expression and transcriptional regulation of the multidrug resistance-1 (MDR1) gene in multidrug-resistant SCCTF cells and -sensitive SCCKN cells derived from human squamous carcinoma were investigated . RT-PCR revealed that mdr1 mRNA was highly expressed in SCCTF cells while it was under the limit of detection in SCCKN cells . With an electrophoretic mobility shift assay using the mdr1 promoter region, a DNA-protein complex was detected strongly in SCCTF cells, but weakly in SCCKN cells . Incubation of the DNA-protein complex with an anti-NF-Y antibody caused a supershift in the migration to a position near the origin of the gel . Chromatin immunoprecipitation assay with an anti-NF-Y antibody showed that NF-Y binds to mdr1 promoter in SCCTF cells . The mdr1 promoter region including its NF-Y binding sequence was cloned into the luciferase reporter plasmid pGL3-basic vector, and this vector was used to transfect SCCTF and SCCKN cells . The luciferase assay showed that the inverted CCAAT sequence in the mdr1 promoter region is involved in the positive regulation of mdr1 promoter activity . NF-YA protein was expressed at higher levels in SCCTF cells than that in SCCKN cells . Hoechst dye staining also showed that MDR1 protein acts more effectively as an efflux pump in SCCTF cells than that in SCCKN cells.

Cancer Gene Ther, 2004 Nov, 11(11), 699 - 706
Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference; Stege A et al.; Multidrug resistance (MDR) is the major cause of failure of effective chemotherapeutic treatment of disseminated neoplasms . The "classical" MDR phenotype of human malignancies is mediated by drug extrusion by the adenosine triphosphate binding cassette (ABC)-transporter P-glycoprotein (MDR1/P-gp) . For stable reversal of "classical" MDR by RNA interference (RNAi) technology, an H1-RNA gene promoter-driven expression vector encoding anti-MDR1/P-gp short hairpin RNA (shRNA) molecules was constructed . By introduction of anti-MDR1/P-gp shRNA expression vectors into the extremely high drug-resistant human gastric carcinoma cell line EPG85-257RDB, the MDR phenotype was completely reversed . The reversal of MDR was accompanied by a complete suppression of MDR1/P-gp expression on mRNA and protein level, and by a considerable increased intracellular anthracyline accumulation in the anti-MDR1/P-gp shRNA-treated cells . The data indicate that stable shRNA-mediated RNAi can be tremendously effective in reversing MDR1/P-gp-mediated MDR and is therefore a promising strategy for overcoming MDR by gene therapeutic applications.

Cancer Gene Ther, 2004 Nov, 11(11), 707 - 12
Reversal of P-glycoprotein-mediated multidrug resistance with small interference RNA (siRNA) in leukemia cells; Peng Z et al.; The multidrug resistance (MDR) mediated by P-glycoprotein (P-gp), the MDR1 gene product, is one of the major obstacles in leukemia treatment . The present study was designed to explore a MDR1-targeted small interfering RNA (si-MDR1) approach for reversal of P-gp-mediated MDR in the MDR human leukemia cell line k562/A02 . It was found that si-MDR1 significantly inhibited MDR1 expression at both mRNA and protein levels . Depletion of MDR1 by si-MDR1 correlated with the increased sensitivity of the cells to cytotoxic agents and with the enhanced intracellular retention of daunorubicin (DNR) . One base-pair mutated control (si-MDR1-Mut) lost the effect of si-MDR1 on both the degradation of mdr1 mRNA and the reduction of P-gp expression . These findings indicate that siRNA specifically and efficiently interferes with the expression of mdr1 and could be used as a molecularly defined therapeutic approach for MDR in the treatment of leukemia.

J Antimicrob Chemother, 2004 Oct, 54(4), 809 - 17 Epub 2004 Sep 16.
Prospective audit of bacteraemia management in a university hospital ICU using a general strategy of short-course monotherapy; Corona A et al.; OBJECTIVE: As optimal antibiotic therapy for bacteraemia remains unknown, different strategies have evolved . Routine practice in the University College London Hospitals intensive care unit (ICU) is to use short-course (5-6 days) monotherapy, unless specifically indicated (e.g . endocarditis, osteomyelitis) . We decided to assess this approach for treating community-, hospital-, and ICU-acquired bacteraemia by monitoring clinical response, relapse rate and patient outcome . DESIGN: Six-month prospective observational study from February to July 2000 . SETTING: Mixed medical-surgical tertiary referral ICU . PATIENTS: All 713 patients admitted to the ICU over the study period . MEASUREMENTS AND RESULTS: In total, 102 bacteraemic episodes occurred in 84 patients . Eight (57%) of 14 community-acquired bacteraemias, 22 (79%) of 28 hospital-acquired bacteraemias, and 48 (80%) of 60 ICU-acquired bacteraemias (in 49 patients) were treated with short-course monotherapy . Compared with previous reported studies, these patients had a low rate (23.8%) of death directly attributable to the bacteraemia and a satisfactory clinical response in 72% . Of six relapses (all Gram-negative), four had received combination therapy for severe deep-seated infections . ICU-acquired multidrug-resistant Gram-negative bacteraemias (6.5%) and fungaemias (3%) were also uncommon . No patient discharged from ICU subsequently developed a new bacteraemia relapse, or any long-term complication such as osteomyelitis . CONCLUSIONS: Our general strategy of short-course antibiotic monotherapy for treating bacteraemia in the critically ill appears to provide a satisfactory clinical response, low relapse rate and no long-term complications in a well-defined group of patients . Multicentre studies are warranted to compare short versus long course therapy, and monotherapy versus combination therapy.

Am J Physiol Gastrointest Liver Physiol, 2005 Feb, 288(2), G327 - 36 Epub 2004 Sep 16.
Role of microtubules in estradiol-17{beta}-D-glucuronide-induced alteration of canalicular Mrp2 localization and activity; Mottino AD et al.; Estradiol-17beta-d-glucuronide (E(2)-17G) induces a marked but reversible inhibition of bile flow in the rat together with endocytic retrieval of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane to intracellular structures . We analyzed the effect of pretreatment (100 min) with the microtubule inhibitor colchicine or lumicholchicine, its inactive isomer (1 mumol/kg iv), on changes in bile flow and localization and function of Mrp2 induced by E(2)-17G (15 mumol/kg iv) . Bile flow and biliary excretion of bilirubin, an endogenous Mrp2 substrate, were measured throughout, whereas Mrp2 localization was examined at 20 and 120 min after E(2)-17G by confocal immunofluorescence microscopy and Western analysis . Colchicine pretreatment alone did not affect bile flow or Mrp2 localization and activity over the short time scale examined (3-4 h) . Administration of E(2)-17G to colchicine-pretreated rats induced a marked decrease (85%) in bile flow and biliary excretion of bilirubin as well as internalization of Mrp2 at 20 min . These alterations were of a similar magnitude as in rats pretreated with lumicolchicine followed by E(2)-17G . Bile flow and Mrp2 localization and activity were restored to control levels within 120 min of E(2)-17G in animals pretreated with lumicolchicine . In contrast, in colchicine-pretreated rats followed by E(2)-17G, bile flow and Mrp2 activity remained significantly inhibited by 60%, and confocal and Western studies revealed sustained internalization of Mrp2 120 min after E(2)-17G . We conclude that recovery from E(2)-17G cholestasis, associated with exocytic insertion of Mrp2 in the canalicular membrane, but not its initial E(2)-17G-induced endocytosis, is a microtubule-dependent process.

Insect Mol Biol, 2004 Oct, 13(5), 539 - 48
The dMRP/CG6214 gene of Drosophila is evolutionarily and functionally related to the human multidrug resistance-associated protein family; Tarnay JN et al.; ATP-binding cassette (ABC) transporters are involved in the transport of substrates across biological membranes and are essential for many cellular processes . Of the fifty-six Drosophila ABC transporter genes only white, brown, scarlet, E23 and Atet have been studied in detail . Phylogenetic analyses identify the Drosophila gene dMRP/CG6214 as an orthologue to the human multidrug-resistance associated proteins MRP1, MRP2, MRP3 and MRP6 . To study evolutionarily conserved roles of MRPs we have initiated a characterization of dMRP . In situ hybridization and Northern analysis indicate that dMRP is expressed throughout development and appears to be head enriched in adults . Functional studies indicate that DMRP is capable of transporting a known MRP1 substrate and establishes DMRP as a high capacity ATP-dependent, vanadate-sensitive organic anion transporter.

Cas Lek Cesk, 2004, 143(7), 471 - 5
{Multidrug resistance markers monitoring in acute myeloid leukemia cells}; Jankovicova K et al.; BACKGROUND: P-gp, MRP, LRP, Bcl-2, and Bax proteins play an important role in the multidrug resistance of leukemic cells . P-gp, MRP, and LRP proteins decrease the intracellular drug concentration, Bcl-2 and Bax proteins influence the apoptosis process . The overexpression of some of these proteins is associated with poor prognosis of leukemia . The aim of this study was to find the relationship between some multidrug resistance markers and some clinical and laboratory parameters . We compare also P-gp expression between acute myeloid leukemia (AML) FAB subgroups M0-M6 . METHODS AND RESULTS: With use of flow cytometry we measured the expression of P-gp, MRP, LRP, Bcl-2, and Bax proteins in acute myeloid leukemia patients cells . The study proved the association between blast percentage and P-gp protein expression (p=0.015) and the association between blast percentage and Bcl-2 protein expression (p=0.041) . It was also shown the tendency of the LRP protein expression to associate with higher age of patients (p=0.062) . Another correlation between MRP and Bax expression (p=0.006), as well as between LRP and Bax expression was found (p=0.034) . In AML M0 FAB subgroup of patients the trend to higher P-gp protein expression was observed . CONCLUSIONS: The laboratory methodology to multidrug resistance markers detection was introduced . Some relations between multidrug resistance markers playing role in acute myeloid leukemia patients prognosis were suggested.

Cell Oncol, 2004, 26(1-2), 3 - 11
Bodipy-FL-verapamil: a fluorescent probe for the study of multidrug resistance proteins; Rosati A et al.; Most of the substances used as fluorescent probes to study drug transport and the effect of efflux blockers in multidrug resistant cells have many drawbacks, such as toxicity, unspecific background, accumulation in mitochondria . New fluorescent compounds, among which Bodipy-FL-verapamil (BV), have been therefore proposed as more useful tools . The uptake of BV has been evaluated by cytofluorimetry and fluorescence microscopy using cell lines that overexpress P-glycoprotein (P388/ADR and LLC-PK(1)/ADR) or MRP (multidrug resistance-related protein) (PANC-1) and clinical specimens from patients . The effect of specific inhibitors for P-glycoprotein (verapamil and vinblastine) or MRP (MK571 and probenecid) has been also studied . BV intracellular concentrations were significantly lower in the two P-glycoprotein overexpressing cell lines in comparison with the parental lines . In addition, verapamil and vinblastine increased the intracellular concentrations of the dye; MK571 and probenecid, two MRP inhibitors, increased BV levels in PANC-1 cells, that express this protein . These findings were confirmed in clinical specimens from patients . Fluorescence microscopy revealed a faint fluorescence emission in P-glycoprotein or MRP expressing cell lines; however, treatment with specific inhibitors significantly increased the fluorescence . BV is a useful tool for studying multidrug resistance proteins with different techniques such as cytofluorimetry and fluorescence microscopy, but does not discriminate between P-glycoprotein and MRP . In comparison with other classic fluorescent probes, the assay with this dye is extremely rapid, simple, not toxic for cells, devoid of fluorescent background, and can be useful in the clinical settings.

Proc Natl Acad Sci U S A, 2004 Sep 28, 101(39), 14073 - 8 Epub 2004 Sep 15.
Alkalitolerance: a biological function for a multidrug transporter in pH homeostasis; Lewinson O et al.; MdfA is an Escherichia coli multidrug-resistance transporter . Cells expressing MdfA from a multicopy plasmid exhibit multidrug resistance against a diverse group of toxic compounds . In this article, we show that, in addition to its role in multidrug resistance, MdfA confers extreme alkaline pH resistance and allows the growth of transformed cells under conditions that are close to those used normally by alkaliphiles (up to pH 10) by maintaining a physiological internal pH . MdfA-deleted E . coli cells are sensitive even to mild alkaline conditions, and the wild-type phenotype is restored fully by MdfA expressed from a plasmid . This activity of MdfA requires Na(+) or K(+) . Fluorescence studies with inverted membrane vesicles demonstrate that MdfA catalyzes Na(+)- or K(+)-dependent proton transport, and experiments with reconstituted proteoliposomes confirm that MdfA is solely responsible for this phenomenon . Studies with multidrug resistance-defective MdfA mutants and competitive transport assays suggest that these activities of MdfA are related . Together, the results demonstrate that a single protein has an unprecedented capacity to turn E . coli from an obligatory neutrophile into an alkalitolerant bacterium, and they suggest a previously uncharacterized physiological role for MdfA in pH homeostasis.

J Biol Chem, 2004 Nov 19, 279(47), 48787 - 93 Epub 2004 Sep 15.
EmrE, a multidrug transporter from Escherichia coli, transports monovalent and divalent substrates with the same stoichiometry; Rotem D et al.; Multidrug transporters recognize and transport substrates with apparently little common structural features . At times these substrates are neutral, negatively, or positively charged, and only limited information is available as to how these proteins deal with the energetic consequences of transport of substrates with different charges . Multidrug transporters and drug-specific efflux systems are responsible for clinically significant resistance to chemotherapeutic agents in pathogenic bacteria, fungi, parasites, and human cancer cells . Understanding how these efflux systems handle different substrates may also have practical implications in the development of strategies to overcome the resistance mechanisms mediated by these proteins . Here, we compare transport of monovalent and divalent substrates by EmrE, a multidrug transporter from Escherichia coli, in intact cells and in proteoliposomes reconstituted with the purified protein . The results demonstrated that whereas the transport of monovalent substrates involves charge movement (i.e . electrogenic), the transport of divalent substrate does not (i.e . electroneutral) . Together with previous results, these findings suggest that an EmrE dimer exchanges two protons per substrate molecule during each transport cycle . In intact cells, under conditions where the only driving force is the electrical potential, EmrE confers resistance to monovalent substrates but not to divalent ones . In the presence of proton gradients, resistance to both types of substrates is detected . The finding that under some conditions EmrE does not remove certain types of drugs points out the importance of an in-depth understanding of mechanisms of action of multidrug transporters to devise strategies for coping with the problem of multidrug resistance.

IUBMB Life, 2004 Jun, 56(6), 355 - 9
Induction of drug-metabolizing enzymes and transporters in human bronchial epithelial cells by beclomethasone dipropionate; Kuzuya Y et al.; Inhaled steroids are the most potent anti-inflammatory therapy commonly used in bronchial asthma . There are, however, a small number of asthmatic patients who do not respond to inhaled steroid-treatment . The stimulation of metabolism and excretion of inhaled drugs at bronchial tissues might lead to a decrease in the effect of the drugs, although the molecular mechanism of this resistance is unclear . In this study, we found that beclomethasone dipropionate (BDP) stimulated the expression of mRNAs for uridine 5'-diphosphate glucuronosyl transferase 2B4 and 2B11, and transporters such as multidrug resistance P-glycoprotein, multidrug resistance-associated protein 1 and 2 in cultured bronchial epithelial cells . It is possible that the individual differences of expression of drug metabolizing enzymes and transporters and their enhancement with BDP are implicated in the individual differences of reactivity over steroid medical treatment.

Mol Cancer Ther, 2004 Sep, 3(9), 1061 - 7
Biological evaluation of cryptophycin 52 fragment A analogues: effect of the multidrug resistance ATP binding cassette transporters on antitumor activity; Al-Awar RS et al.; Cryptophycin 52 (LY355703) is a potent antiproliferative analogue of the marine natural product cryptophycin 1 . It has been shown to have a broad range of antitumor activity against human tumor xenografts and murine tumors including tumors resistant to Taxol and Adriamycin . Its mechanism of action involves arresting cells in the G2-M phase of the cell cycle by binding to microtubules and suppressing their dynamics . This 16-membered depsipeptide can be divided into four major subunits or fragments (A-D) . We reported previously on our synthetic efforts around fragment A and discovered that this region of the molecule was amenable to a structure-activity relationship study that resulted in highly active antiproliferative agents when evaluated in the CEM leukemia cell line . The synthetic analogues were designed to help improve the efficacy and aqueous solubility of the parent compound; therefore, many in this series contained ionizable functional groups such as an amino group, a hydroxy group, or a carboxylic acid . Although several of these analogues showed improvements in potency over cryptophycin 52 in drug-sensitive tumor xenograft models, many lost their activity against Adriamycin-resistant tumor lines . It was discovered on additional in vitro evaluation that these analogues became good substrates of the multidrug resistance transporter P-glycoprotein.

J Biol Chem, 2004 Nov 19, 279(47), 48855 - 64 Epub 2004 Sep 09.
Multidrug resistance protein 4 (ABCC4)-mediated ATP hydrolysis: effect of transport substrates and characterization of the post-hydrolysis transition state; Sauna ZE et al.; Multidrug resistance protein 4 (MRP4/ABCC4), transports cyclic nucleoside monophosphates, nucleoside analog drugs, chemotherapeutic agents, and prostaglandins . In this study we characterize ATP hydrolysis by human MRP4 expressed in insect cells . MRP4 hydrolyzes ATP (Km, 0.62 mm), which is inhibited by orthovanadate and beryllium fluoride . However, unlike ATPase activity of P-glycoprotein, which is equally sensitive to both inhibitors, MRP4-ATPase is more sensitive to beryllium fluoride than to orthovanadate . 8-Azido{alpha-32P}ATP binds to MRP4 (concentration for half-maximal binding approximately 3 microm) and is displaced by ATP or by its non-hydrolyzable analog AMPPNP (concentrations for half-maximal inhibition of 13.3 and 308 microm) . MRP4 substrates, the prostaglandins E1 and E2, stimulate ATP hydrolysis 2- to 3-fold but do not affect the Km for ATP . Several other substrates, azidothymidine, 9-(2-phosphonylmethoxyethyl)adenine, and methotrexate do not stimulate ATP hydrolysis but inhibit prostaglandin E2-stimulated ATP hydrolysis . Although both post-hydrolysis transition states MRP4.8-azido{alpha-32P}ADP.Vi and MRP4.8-azido{alpha-32P}ADP.beryllium fluoride can be generated, nucleotide trapping is approximately 4-fold higher with beryllium fluoride . The divalent cations Mg2+ and Mn2+ support comparable levels of nucleotide binding, hydrolysis, and trapping . However, Co2+ increases 8-azido{alpha-32P}ATP binding and beryllium fluoride-induced 8-azido{alpha-32P}ADP trapping but does not support steady-state ATP hydrolysis . ADP inhibits basal and prostaglandin E2-stimulated ATP hydrolysis (concentrations for half-maximal inhibition 0.19 and 0.25 mm, respectively) and beryllium fluoride-induced 8-azido{alpha-32P}ADP trapping, whereas Pi has no effect up to 20 mm . In aggregate, our results demonstrate that MRP4 exhibits substrate-stimulated ATP hydrolysis, and we propose a kinetic scheme suggesting that ADP release from the post-hydrolysis transition state may be the rate-limiting step during the catalytic cycle.

Exp Parasitol, 2004 Jul-Aug, 107(3-4), 115 - 9
Resistance reversal action of ketoconazole against mefloquine resistance of Plasmodium yoelii nigeriensis; Awasthi A et al.; Ketoconazole at 200 mg/kg dose has been found to exert marginal antimalarial action against multidrug resistant (MDR) Plasmodium yoelii nigeriensis (P . yoelii nigeriensis) in Swiss mice with 25% protection (2/8 mice) while at lower Ketoconazole dose i.e., 75-100 mg/kg, 14.28% mice were protected . Mefloquine (MFQ) (at 8 and 16 mg/kg) exerted suppressive action against MDR P . yoelii nigeriensis resulting in 25 and 14.28% protection of mice respectively . Combined treatment with Ketoconazole and mefloquine resulted in protection of 5/6 mice (83.33%) at MFQ 4 mg/kg + Ketoconazole 100 mg/kg dose, 7/8 (87.5%) mice at MFQ 8 mg/kg + Ketoconazole 20 mg/kg dose and 5/7 (71.42%) mice at MFQ 16 mg/kg + Ketoconazole 25 mg/kg dose and 5/6 (83.33%) mice at MFQ 16 mg/kg + Ketoconazole 100 mg/kg dose . Ketoconazole has been found to enhance the protective effect of mefloquine against MFQ resistant P . yoelii nigeriensis resulting in 66-88% protection of the mice treated with the appropriate combinations . The combination also increased suppression of parasitaemia at different times . The Ketoconazole combination with MFQ significantly increased the mean survival time of the treated mice compared to individual drugs alone . The study shows that Ketoconazole when administered with MFQ exerts bio-enhancing action against mefloquine resistance of MDR P . yoelii nigeriensis.

Zhonghua Wai Ke Za Zhi, 2004 Jul 7, 42(13), 795 - 8
{Reversal of multidrug resistance of MCF-7/ADR in nude mice by grape seed polyphenol}; Zhang CJ et al.; OBJECTIVE: To study the reversing effect of Grape seed polyphenol (GSP) on multidrug resistance of MCF-7/ADR cell in vivo . METHODS: The transplantable breast carcinoma cell line MCF-7/ADR model was established in BALB/C-nu/nu mice by subcutaneous implantation . Flow cytometry (FCM) was used to investigate the changes of Pgp expression and apoptosis rate after different drug treatment . RESULTS: GSP has some effect on inhibition of tumor growth (the rate of inhibition was 18.35%), and combined with adriamycin can significantly inhibit tumor growth in nude mice, 20 mg/kg GSP can effectively reverse the resistance of MCF-7/ADR cells to ADR in vivo, and the rate of inhibition was 54.64% . FCM results showed that the expression of Pgp was significantly decreased (32.03 +/- 2.09) After administration of GSP and ADR, there was distinct difference between it and control (55.13 +/- 2.12) . The mean rate of apoptosis was 15.12% +/- 1.04%, and was significantly increased compared with control (9.07% +/- 0.43%), P < 0.05 . CONCLUSION: GSP can effectively reversed the resistance of MCF-7/ADR cells in nude mice, the mechanism may be correlated with inhibition of Pgp expression and apoptosis.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2004 Aug, 12(4), 431 - 5
{Construction and analysis of subtractive cDNA library associated with multidrug resistance of acute leukemia}; Ji L et al.; The study was aimed to construct subtractive cDNA library associated with multidrug resistance (MDR) of acute leukemia for screening genes related to MDR in leukemia . The improved PCR-based subtractive hybridization was performed to clone differential genes between HL-60/VCR and HL-60 cell line . The mRNA of HL-60/VCR and HL-60 cell line were isolated . Then the mRNA of HL-60/VCR group was reversely transcribed into cDNA by Cap-Finder method, and the mRNA of HL-60 was reversely transcribed into cDNA by ordinary method to be marked by biotin for the hybridization next with HL-60/VCR cDNA . After hybridizing, filtrating through the sephacryl S-400 column, absorbing by the magnetic beads, and amplifying by PCR method, the fragments were cloned by T-A method and the cDNA library was constructed . Then the quality of cDNA library was identified by dot-blotting hybridization method . The results showed that after constriction, the library demonstrated its good quality . There was a high proportion of large fragments in this library . From small amount of samples a large amount of candidate fragments could be screened rapidly at once by dot-blotting hybridization . It is concluded that a differentially-expressed subtractive cDNA library in MDR of leukemia with high quality and larger fragments can be efficiently constructed by improving subtractive hybridization and selective PCR method.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2004 Aug, 12(4), 411 - 5
{Clinical studies on expressions of Fas and mdr-1 in acute leukemia and their correlations}; Ye F et al.; To explore the Fas and mdr-1 expression and their correlation in multidrug resistance (MDR), Fas and mdr-1 expressions of bone marrow from 59 patients with newly diagnosed AL before therapy and after complete remission were detected by direct immunofluorescence with flow cytometry and semi-quantitative RT-PCR, respectively . The results showed that in newly diagnosed AL patients, Fas expression in AML was higher than in ALL (P < 0.05), mdr-1 expression in AML and ALL had no difference (P > 0.05), the expressions of Fas and mdr-1 had correlation (r = -0.282, P < 0.05) in AL; the results of simple-variable and multivariable COX survival factor model analysis suggested that Fas and mdr-1 expressions were prognostic factors for the effect of therapy and survival . Log rank test, comparing the groups of Fas(+) with Fas(-), mdr-1(+) with mdr-1(-), demonstrated that the CR rates and median remission time of every two groups had significant difference . It is concluded that in AL, Fas and mdr-1 expressions have high correlation with the effect of treatment, Fas expression probably is one of the favorable prognostic factors, mdr-1 is an unfavorable prognostic and less effective factor.

J Formos Med Assoc, 2004 Sep, 103(9), 671 - 7
Drug resistance pattern of Mycobacterium tuberculosis in a university hospital in Taiwan, 1998-2002; Liaw YS et al.; BACKGROUND AND PURPOSE: During 1998-2002, most specialized tuberculosis (TB) hospitals in Taiwan were closed; as a result, more TB patients are being managed in general hospital settings . This study investigated the prevalence, patterns and risk factors of drug-resistant Mycobacterium tuberculosis at a university hospital in the 5 years after decentralization of the TB administrative and clinical control infrastructure which occurred during the implementation of the national health insurance system in Taiwan . METHODS: A total of 1411 initial isolates of M . tuberculosis from specimens collected during January 1998 through December 2002 were tested for drug susceptibility of first-line anti-TB drugs using the agar proportional method . RESULTS: The numbers of newly diagnosed culture-positive TB patients increased from 139 in 1998 to 380 in 2002 . The drug resistance pattern of M . tuberculosis among these isolates was as follows: 268 (19.0%) strains were resistant to isoniazid, 86 (6.1%) to rifampin, 221 (15.7%) to ethambutol, 141 (10.0%) to streptomycin, and 430 (30.5%) to 1 of these 4 drugs . Multidrug resistance (MDR), i.e., resistance to at least rifampin and isoniazid, was observed in 72 isolates (5.1%) . Of the 1411 patients, isolates from patients with age < 65 years had a higher multiple drug resistance rate than those from patients with age > or = 65 years (57/781, 7.3% vs 15/630, 2.4%; p < 0.001) . In the analysis of risk factors for MDR, patients with MDR isolates had a significantly higher incidence of previous TB history, anti-TB therapy, longer duration of symptoms, cavitary lesions in chest X-ray, and mortality . CONCLUSION: A dramatic increase in cases of TB among patients treated at this university hospital was seen after the decentralization of the TB control infrastructure in Taiwan . The prevalence of drug resistance in isolates from culture-positive TB patients was 30.5% and the prevalence of MDR was 5.1%.

Medinfo, 2004, 2004, 202 - 6
Implementation and Initial Evaluation of a Web-based Nurse Order Entry System for Multidrug-resistant Tuberculosis Patients in Peru; Choi SS et al.; Socios En Salud uses directly observed therapy to treat a majority of the multidrug-resistant tuberculosis in Peru . The nurses play an important role in this community-based model as the patients' primary care givers . Since nurses, rather than physicians, are involved in patients' daily care, we developed a nurse-order entry system to test whether such a system would improve the accuracy and quality of medication data . We compared regimen information from patient electronic medical records, paper charts and pharmacy records . After a two-month training period on the new system, we conducted the trial for 52 days in two of Lima's six geographic treatment areas, and re-reviewed the three sources of medication data . We measured the error rates after the trial period and found there was no significant difference in the control group's (Lima Este), error rate (8.6% vs . 6.9%, P=0.66) after the trial . The intervention group (Lima Callao), however, showed a significant drop in the error rate (17.4% vs . 3.1%, P=0.0074) after the same time interval . Additionally, the nurse expressed satisfaction with the order entry system and its ease of use . The decrease in error rates and user satisfaction regarding the system are promising measures of our order entry system's success.

Acta Oncol, 2004, 43(5), 443 - 52
Differential effects of toremifene on doxorubicin, vinblastine and Tc-99m-sestamibi in P-glycoprotein-expressing breast and head and neck cancer cell lines; Mubashar M et al.; The effect of toremifene on P-glycoprotein-mediated multidrug resistance (MDR) in breast and head and neck cancer cell lines was measured in vitro and in vivo . Pgp expression was low and high, respectively, in drug-sensitive (MCF7-S, KB) and drug-resistant (MCF7-R, MCF7-R1, KBV1) cell lines . Toremifene (7.5 microM) significantly enhanced cytoplasmic and nuclear accumulation of doxorubicin in drug-resistant cells . Toremifene (10 microM) increased the in vitro cytotoxicity of doxorubicin in drug-resistant breast cancer cells (13-fold and 21-fold for MCF7-R and MCF7-R1, respectively) without affecting the sensitivity of MCF7-S cells . Similarly, toremifene (10 microM) caused a 12-fold increase in the sensitivity of KBV1 cells to vinblastine . In contrast, toremifene (5 microM) reduced the net uptake of the radiolabelled Pgp substrate, Tc-99m-sestamibi, in the Pgp-overexpressing cell lines by factors of 0.32 and 0.42 for MCF7-R1 and KBV1 cells, respectively (p < 0.01), and, to a lesser extent, by corresponding factors of 0.89 and 0.86 in the drug-sensitive cell lines (p < 0.05 and p > 0.05, respectively) . In nude mice bearing both KB and KBV1 xenograft tumours, significantly higher tumour levels of Tc-99m-sestamibi were recorded in KB tumours compared with KBV1 tumours . After 3 days of treatment with intraperitoneal toremifene (25 mg/kg), tumour levels of Tc-99m-sestamibi were reduced in KB and KBV1 tumours but only statistically significantly for KB tumours . Toremifene is a potent MDR modulating agent with respect to chemotherapeutic agents but has the opposite effect with respect to Tc-99m-sestamibi . This finding is of importance in view of the widespread use of Tc-99m-sestamibi as an imaging surrogate for a chemotherapeutic agent.

Pharm Res, 2004 Aug, 21(8), 1398 - 404
Transport characteristics of fexofenadine in the Caco-2 cell model; Petri N et al.; PURPOSE: To investigate the membrane transport mechanisms of fexofenadine in the Caco-2 model . METHODS: Transport studies were performed in Caco-2 cell monolayers 21-25 days after seeding . The apparent permeability (Papp) of fexofenadine was determined in the concentration range 10-1000 microM in the basolateral-to-apical (b-a) and 50-1000 microM in the apical-to-basolateral (a-b) direction . The concentration-dependent effects of various inhibitors of P-glycoprotein (P-gp) (GF120918, ketoconazole, verapamil, erythromycin), multidrug resistant associated protein (MRP) (indomethacin, probenecid), and organic anion transporting polypeptide (OATP) (rifamycin SV) on the bidirectional transport of 150 microM fexofenadine were also examined . RESULTS: Fexofenadine displayed polarized transport, with the Pappb-a being 28- to 85-fold higher than the Papp(a-b) . The Papp(a-b) was independent of the concentration applied, whereas Pappb-a decreased with increasing concentration (Vmax = 5.21 nmol cm(-2)s(-1) and K(M) = 150 microM), suggesting saturation of an apical efflux transporter . All four P-gp inhibitors had a strong, concentration-dependent effect on the Papp of fexofenadine in both directions, with GF 120918 being the most specific among them . The IC50 of verapamil was 8.44 microM on the P-gp-mediated secretion of fexofenadine . The inhibitors of OATP or MRP appeared not to affect the Papp(a-b) of fexofenadine in the Caco-2 model . CONCLUSIONS: This study clearly indicates that P-gp was the main transport protein of fexofenadine in the Caco-2 model . Even though P-gp was completely inhibited, fexofenadine was predicted to have a low fraction dose absorbed in humans due to poor intestinal permeability, and low passive diffusion seems to be the major absorption mechanism.

Pharm Res, 2004 Aug, 21(8), 1313 - 30
Functional expression and localization of P-glycoprotein in the central nervous system: relevance to the pathogenesis and treatment of neurological disorders; Lee G et al.; The expression of membrane drug transport systems in the central nervous system plays an important role in the brain disposition and efficacy of many pharmacological agents used in the treatment of neurological disorders such as neoplasia, epilepsy, and HIV-associated dementia . Of particular interest is P-glycoprotein, a membrane-associated, energy-dependent, efflux transporter that confers the multidrug resistance phenotype to many cells by extruding a broad range of xenobiotics from the cell, resulting in poor clinical outcomes . In addition, the expression pattern of P-glycoprotein has recently been suggested to play a key role in the etiology and pathogenesis of certain diseases such as Alzheimer's and Parkinson's diseases . This review will focus on the cellular localization, molecular expression, and functional activity of P-glycoprotein in several compartments of the central nervous system and address its relevance in the pathogenesis and pharmacological treatment of neurological disorders.

Mayo Clin Proc, 2004 Sep, 79(9), 1119 - 23
Incidence of tuberculosis in Olmsted County, Minnesota, 1990-2001; Jump SM et al.; OBJECTIVE: To describe and compare the incidence and clinical characteristics of tuberculosis in Olmsted County, Minnesota, among US-born and foreign-born persons . PATIENTS AND METHODS: We performed a retrospective cohort study at the Mayo Clinic in Rochester, Minn, of all residents of Olmsted County (2000 population: 124,277) diagnosed as having tuberculosis between January 1, 1990, and December 31, 2001 . Potential cases were identified with use of a computerized diagnostic coding database and microbiological laboratory data; all identified medical records were abstracted . Definite cases were those in which Mycobacterium tuberculosis was recovered in culture . Probable cases were those that met predefined clinical or radiographic evidence of tuberculosis and other criteria . Age-specific, sex-specific, and country of origin-specific incidence rates were calculated with use of Olmsted County census data . Variables were compared among risk groups using the Fisher exact test . RESULTS: During a 12-year period, 71 cases of tuberculosis (53 definite, 18 probable) were identified, for an incidence of 5.3 per 100,000 person-years . Of these cases, 54 (76%) occurred during the second half of the study (incidence: 7.7 per 100,000 person-years) . The incidence among US-born persons was similar throughout the study period; however, the Incidence among foreign-born persons increased more than 3-fold during the second half of the study period . Twenty-five patients (35%) were former refugees . All isoniazid-resistant infections (12% of isolates) and multidrug-resistant infections (6% of isolates) occurred among foreign-born persons . CONCLUSION: The incidence of tuberculosis increased substantially in Olmsted County between 1990 and 2001, primarily because of an increase in the number of cases among foreign-born persons.

J Biol Chem, 2004 Nov 19, 279(47), 49517 - 22 Epub 2004 Sep 08.
The constitutive androstane receptor and pregnane X receptor function coordinately to prevent bile acid-induced hepatotoxicity; Zhang J et al.; A double null mouse line (2XENKO) lacking the xenobiotic receptors CAR (constitutive androstane receptor) (NR1I3) and PXR (pregnane X receptor) (NR1I2) was generated to study their functions in response to potentially toxic xenobiotic and endobiotic stimuli . Like the single knockouts, the 2XENKO mice are viable and fertile and show no overt phenotypes under normal conditions . As expected, they are completely insensitive to broad range xenobiotic inducers able to activate both receptors, such as clotrimazole and dieldrin . Comparisons of the single and double knockouts reveal specific roles for the two receptors . Thus, PXR does not contribute to the process of acetaminophen hepatotoxicity mediated by CAR, but both receptors contribute to the protective response to the hydrophobic bile acid lithocholic acid (LCA) . As previously observed with PXR (Xie, W., Radominska-Pandya, A., Shi, Y., Simon, C . M., Nelson, M . C., Ong, E . S., Waxman, D . J., and Evans, R . M . (2001) Proc . Natl . Acad . Sci . U . S . A . 98, 3375-3380), pharmacologic activation of CAR induces multiple LCA detoxifying enzymes and provides strong protection against LCA toxicity . Comparison of their responses to LCA treatment demonstrates that CAR predominantly mediates induction of the cytochrome p450 CYP3A11 and the multidrug resistance-associated protein 3 transporter, whereas PXR is the major regulator of the Na+-dependent organic anion transporter 2 . These differential responses may account for the significant sensitivity of the CAR knockouts, but not the PXR knockouts, to an acute LCA dose . Because this sensitivity is not further increased in the 2XENKO mice, CAR may play a primary role in acute responses to this toxic endobiotic . These results define a central role for CAR in LCA detoxification and show that CAR and PXR function coordinately to regulate both xenobiotic and bile acid metabolism.

Bioorg Med Chem, 2004 Sep 1, 12(17), 4625 - 31
Analogues of tetramethylrosamine as transport molecules for and inhibitors of P-glycoprotein-mediated multidrug resistance; Gibson SL et al.; Tetramethylrosamine and its thio- and seleno- analogues (TMR-O, TMR-S, and TMR-Se, respectively) were examined for their ability to be transported by Pgp into chemo-resistant CR1R12 cells . Verapamil (7 x 10(-6)M) enhanced the uptake of TMR-O and TMR-S into CR1R12 cells compared to those cultures not previously exposed to verapamil . The uptake of TMR-O and TMR-S in CR1R12 cells in the presence of 7 x 10(-6)M verapamil was equivalent to its uptake in the chemo-sensitive parent cell line AUXB1 in the absence or presence of verapamil . None of the TMR analogues were effective alone as photosensitizers of CR1R12 cells . However, when either TMR-S or TMR-Se was added to CR1R12 cells after 7 x 10(-6)M verapamil exposure for 2h, irradiation of cultures with 5.0J cm(-2) of 350-750 nm light caused significant phototoxicity . TMR-O showed no significant phototoxicity in the presence of verapamil . Chemo-sensitive AUXB1 cells are equally susceptible to phototoxicity using TMR-Se with or without previous exposure to verapamil . The Pgp modulators verapamil and CsA increased the uptake of CAM into CR1R12 . Exposure of CR1R12 cells to TMR-S or TMR-Se for 2h in the dark resulted in no significant change in the intracellular accumulation of CAM . However, 1h of light exposure after incubation of cells with TMR-S or TMR-Se resulted in an up to 2-fold increase in CAM uptake.

Biochem Biophys Res Commun, 2004 Aug 13, 321(1), 197 - 201
Plasmodium falciparum expresses a multidrug resistance-associated protein; Klokouzas A et al.; Plasmodium falciparum proteins that efflux toxic metabolic products such as oxidised glutathione (GSSG) are possible targets for anti-malarial drug development . Proteins capable of transporting GSSG and glutathione conjugates include the multidrug resistance-associated transporters (MRPs) . A gene, PFA0590w, encoding a MRP homologue, has been identified in P . falciparum . Here we show the presence of full-length mRNA (5.5 kb) of this PfMRP in trophozoites by RT-PCR and Northern blotting . A polyclonal anti-PfMRP antibody generated against two unique, hydrophilic peptides in the predicted sequence produced a strong immunoreactive protein band of 210-215 kDa on Western blots of schizonts of chloroquine-sensitive and chloroquine-resistant strains, confirming expression of PfMRP protein . Using confocal microscopy the protein was seen to be localised at the edge of the schizonts with no obvious staining of the food vacuole . We suggest that PfMRP may act as the GSSG transporter in the parasite plasma membrane.

Bioorg Med Chem Lett, 2004 Sep 6, 14(17), 4487 - 9
Cytotoxic effects and reversal of multidrug resistance by ibogan and related indole alkaloids; Kam TS et al.; A series of indole alkaloids of the ibogan-type was assessed for their cytotoxic effects as well as their potential in reversing MDR in vincristine-resistant KB cells . Of a total of 25 compounds tested, 3(S)-cyanocoronaridine, 3(S)-cyanoisovoacangine, 3(S)-cyanovoacangine, and 10,11-demethoxychippiine were found to show appreciable cytotoxicity toward KB cells, while coronaridine, heyneanine, 19-epi-heyneanine, dippinine B, and dippinine C, were found to reverse MDR in vincristine-resistant KB cells.

Pigment Cell Res, 2004 Oct, 17(5), 471 - 9
Stimulation of cyclic GMP efflux in human melanocytes by hypergravity generated by centrifugal acceleration; Ivanova K et al.; Gravity alteration (micro- and hypergravity) is known to influence cell functions . As guanosine 3',5'-cyclic monophosphate (cGMP) plays an important role in human melanocyte functions and different guanylyl cyclase isoforms are responsible for cGMP synthesis in human non-metastatic and metastatic melanoma cells, we investigated the effects of hypergravity on the regulation of cGMP levels in cultured human melanocytes and in melanoma cell lines with different metastatic potentials . Hypergravity was produced by horizontal centrifugal acceleration . Here we report that long-term application of hypergravity (up to 5 g for 24 h) stimulated cGMP efflux in cultured melanocytes and in non-metastatic melanoma cells in the presence of 0.1 mM 3-isobutyl-1-methylxanthine (IBMX), a non-selective phosphodiesterase (PDE) inhibitor . Under these conditions, cAMP synthesis and melanin production were up-regulated in pigmented melanocytes and non-metastatic melanoma cells . Hypergravity also stimulated cGMP transport in the presence of 1 microM trequinsin, an inhibitor of cGMP-binding PDE (PDE5) and of transport by multidrug resistance proteins MRP4/5, whereas 50 microM trequinsin partially inhibited cGMP transport . Transport was further inhibited by probenecid, an inhibitor of endogenous non-selective transporters as well as of MRP4/5 and by cycloheximide as an inhibitor of de novo protein synthesis . In contrast, hypergravity did not affect cGMP efflux in metastatic melanoma cells, which might be related to an up-regulated cGMP efflux at 1 g . The results of the present study indicate that hypergravity may stimulate cGMP efflux in melanocytes and in non-metastatic melanoma cells most probably by an enhanced expression of endogenous transporters and/or MRP4/5 . Thus, an altered acceleration vector may induce signaling events in melanocytic cells.

Drugs R D, 2004, 5(5), 297 - 304
Mismatched double-stranded RNA: polyI:polyC12U; Mutation of the aromatic amino acid interacting with adenine moiety of ATP to a polar residue alters the properties of multidrug resistance protein 1; Mayo Clinic College of Medicine, Mayo Clinic, Scottsdale, Arizona 85259, USAStructural analyses of several bacterial ATP-binding cassette (ABC) transporters indicate that an aromatic amino acid residue in a nucleotide-binding domain (NBD) interacts with the adenine ring of the bound ATP and contributes to the ATP binding . Substitution of this aromatic residue with a polar serine residue in bacterial histidine transporter completely abolished both ATP binding and ATP-dependent histidine transport . However, substitution of the aromatic amino acid residue in the human cystic fibrosis transmembrane conductance regulator with a polar cysteine residue did not have any effect on the ATP-dependent chloride channel function of the protein . To determine whether the other eucaryotic ABC transporters use the strategy analogous to that in some bacterial ABC transporters, the aromatic Trp653 residue in NBD1 and the Tyr1302 residue in NBD2 of human multidrug resistance-associated protein 1 (MRP1) was mutated to either a different aromatic residue or a polar cysteine residue . Substitution of the aromatic residue with a different aromatic amino acid, such as W653Y or Y1302W, did not affect ATP-dependent leukotriene C4 (LTC4) transport . In contrast, substitution of the aromatic residue with a polar cysteine residue, such as W653C or Y1302C, decreased the affinity for ATP, resulting in greatly increased Kd values for ATP binding or Km values for ATP in ATP-dependent LTC4 transport . Interestingly, although substitution of the aromatic Trp653 in NBD1 of MRP1 with a polar cysteine residue greatly decreases the affinity for ATP, the ATP-dependent LTC4 transport activities are much higher than that of wild-type MRP1, supporting our hypothesis that the increased release rate of the bound ATP from the mutated NBD1 facilitates the protein to start a new cycle of ATP-dependent solute transport.

Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi, 2004 Aug, 22(4), 264 - 6
{Toxic effect of arsenite on the expression of liver multidrug resistance-associated protein 2 in rat}; Li GX et al.; OBJECTIVE: To investigate the role of multidrug resistance-associated protein 2 (MRP2) in the hepatic cell membrane of rats . METHODS: Thirty healthy Wistar rats were divided randomly into six groups based on time of administration (2 w, 4 w, 6 w) of 20 mg/kg of sodium arsenite, and their corresponding control groups . Animals were administered every other day . Arsenic content in blood and bile were detected by atomic absorption spectroscopy (AAS), and the expression of MRP2 in the membrane of hepatocyte by Western blotting was determined . RESULTS: Total arsenic levels (including organic arsenic and inorganic arsenic) in blood and bile were significantly higher than control groups (P < 0.05) at all three different time points, especially in 2 w and 4 w group (16.8 and 13.8 fold greater than that in control) . The expression of MRP2 increased 36.61%, 32.36%, 12.73% more respectively in 2 w, 4 w, 6 w groups than those in control groups (P < 0.05) . The expression of MRP2 was correlated with total arsenic content in bile (r = 0.713, P < 0.05) . CONCLUSIONS: Bile is one of the major routes for the excretion of arsenite and its metabolites, and the overexpression of MRP2 may play an important role in the bile excretion of them at early stage.

Curr Opin Infect Dis, 2004 Oct, 17(5), 397 - 403
Recent tuberculosis advances in Latin America; Pelly T et al.; PURPOSE OF REVIEW: Tuberculosis kills more people than any other infection . Despite advances in diagnostic methods and greater understanding of the reasons for treatment failure, tuberculosis remains common throughout Latin America . RECENT FINDINGS: The impact of HIV and multidrug resistance on tuberculosis control has been enormous . HIV-positive patients may be at 10 times greater risk of multidrug resistant tuberculosis than HIV-negative patients . Hopefully, improved diagnostic techniques will allow more rapid diagnosis of tuberculosis and new colorimetric systems are being developed that will enable expedited drug-sensitivity testing . However, in alarming reports, only 58% of patients were treated with the recommended treatment regime in a Brazilian study, and dropout from treatment in parts of Bolivia was common . Many failings could be combated by rigorous education of patients and physicians . In an encouraging advance, multidrug resistant tuberculosis was successfully treated in a community-based programme, saving an estimated 90% of the cost of hospital-based treatment . An opportunity to identify treatment failure earlier is demonstrated by the finding that 2 months after the initiation of therapy, positive smears were found in only 3% of those whose treatment was successful, but 74% of those whose treatment failed . SUMMARY: The importance of inexpensive and widely available drugs to treat HIV and multidrug resistant tuberculosis in Latin America is clear . The need for rapid, affordable tests for tuberculosis diagnosis, and for easy drug sensitivity testing is also evident . Finally, improving treatment success is achievable even in the resource poor setting.

Cytometry, 2004 Sep, 61B(1), 1 - 8
Coexpression of p53 protein and MDR functional phenotype in leukemias: the predominant association in chronic myeloid leukemia; Cavalcanti GB Jr et al.; BACKGROUND: One of the best characterized resistance mechanisms of leukemias is multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) and multidrug-resistant related protein (MRP) . In addition to Pgp and MRP, p53 mutation or inactivation might play a relevant role in therapeutic failure . Some studies have demonstrated that Pgp and MRP may be activated in association with overexpression of mutant or inactivated p53 protein . The aim of this study was to investigate the association between p53 expression and MDR functional phenotype analyzed by flow cytometry (FCM) . METHODS: Rhodamine-123 assay analyzed by FCM was used to detect the MDR phenotype that was positive in 18 out of 41 (43.9%) cases of chronic myeloid leukemia (CML), 16 out of 28 (57.1%) chronic lymphoid leukemia (CLL) cases, 11 out of 28 (39.3%) acute myeloid leukemia (AML) cases, and four out of 22 (18.2%) acute lymphoid leukemia (ALL) cases . RESULTS: Variable levels of p53 expression were observed in leukemic cells: 12 out of 41 (29.2%) in CML, nine out of 28 (32.1%) in CLL, 15 out of 28 (53.6%) in AML, and eight out of 22 (36.4%) in ALL samples . CONCLUSIONS: In our study, no significant association between p53 expression and MDR functional phenotype was observed in ALL, CLL, and AML . On the other hand, a significant association (P = 0.0003) of the coexpression was observed in CML . The p53 overexpression was more frequently seen in the accelerated phase and the blastic phase of this disease . Our results suggest that an MDR functional phenotype could be associated with p53 mutation in the advanced stage of leukemias .

Cancer Detect Prev, 2004, 28(4), 283 - 93
The activity of latent benzoperimidine esters to inhibit P-glycoprotein and multidrug resistance-associated protein 1 dependent efflux of pirarubicin from several lines of multidrug resistant tumor cells; Glowacka-Rogacka D et al.; Multidrug resistance of tumor cells is associated with the presence of membrane proteins responsible for the cytostatics export . Recently, we have synthesized a new family of benzoperimidines causing the futile cycle of MDR pumps . In this study, biological data for benzoperimidine esters are presented for selected cell lines: sensitive (HL-60, GLC4, K562), P-gp resistant (HL-60/VINC, K562/DX), MRP1 resistant (HL-60/DX) and MRP1/LRP resistant (GLC4/DX) . Their ability to inhibit the efflux of anthracycline antitumor drug, pirarubicin and to restore its accumulation in MDR cells was studied using a spectrofluorometric method which allows to follow the uptake and efflux of fluorescent molecules by living cells . Benzoperimidine esters had high effectiveness in inhibiting pirarubicin efflux and in restoring its accumulation in resistant cells . In contrast, examined esters were less active in vitro in restoration of pirarubicin cytotoxicity towards resistant cells because an enzymatic cleavage of esters occurs in presence of serum esterases.

Expert Rev Mol Diagn, 2004 Sep, 4(5), 705 - 13
Assessment of drug resistance in acute myeloid leukemia; Funato T et al.; A major problem in the treatment of leukemia is the development of resistance to chemotherapeutic agents . Assessing the drug resistance of leukemic cells is therefore an important aspect of treatment . One of the main mechanisms of resistance is rapid drug efflux mediated by various members of the ATP-binding cassette transporter superfamily, such as multidrug resistance gene 1 (MDR1), which encodes P-glycoprotein, multidrug resistance-associated protein (MRP) 1 and lung resistance protein . To quantify the degree of acquisition of resistance, several techniques, including drug-sensitivity studies, flow cytometry assay and quantitative gene analysis, have been developed to detect MDR1 and MRP1 gene expression in leukemic cells . However, a significant number of patients may relapse in spite of low expression of MDR1 or MRP1, suggesting the involvement of other intracellular mechanisms, possibly related to cytarabine resistance . This review focuses on the methods aimed at the assessment of drug resistance in acute myeloid leukemia.

Ann Rheum Dis . 2004 Sep 2; {Epub ahead of print}
Expression of resistance markers to methotrexate predicts clinical improvement in patients with rheumatoid arthritis; Wolf J et al.; OBJECTIVE: MTX is transported into the cell by the reduced folate carrier (RFC) and out of the cell by members of the multidrug resistance protein family (MRP) . To determine the potential influence of transport proteins on the therapeutic efficacy of methotrexate (MTX) in patients with rheumatoid arthritis (RA), potential benefit of the presence of RFC and the absence of functional MRP was investigated . METHODS: 163 patients (116 female and 47 male; mean age 59.5 years) on MTX (mean weekly dose 12.2 mg) were enrolled into the study . RFC was determined using reverse transcriptase polymerase chain reaction and MRP function by flow cytometry using a calcein acetoxymethylesther/probenecid assay . Clinical response to MTX was evaluated by the EULAR response criteria and the American College of Rheumatology improvement criteria . The clinical data were obtained at the beginning of MTX therapy and at the time of blood sampling during ongoing therapy . Patients were divided into four groups according to the presence (+) and/or absence (-) of RFC and functional (f) MRP . RESULTS: fMRP+ RFC+ and fMRP- RFC- patients had significantly more frequent good EULAR response rates (60%; p= 0.014 and 53%; p=0.035, respectively) in comparison to the fMRP- RFC+ group (29%) . Also fMRP+ RFC- patients had a low frequency of good responses in disease activity . CONCLUSION: The absence of fMRP in conjunction with the presence of RFC did not prove to be related to beneficial effects of MTX, while the lack or the presence of both functional MRP and RFC led to a significantly better therapeutic outcome . Determination of these markers may predict responsiveness to MTX.

Am J Physiol Regul Integr Comp Physiol, 2004 Dec, 287(6), R1505 - 16 Epub 2004 Sep 02.
Temporal expression profiles of organic anion transport proteins in placenta and fetal liver of the rat; St-Pierre MV et al.; Physiological cholestasis linked to immature hepatobiliary transport systems for organic anions occurs in rat and human neonates . In utero, the placenta facilitates vectorial transfer of certain fetal-derived solutes to the maternal circulation for elimination . We compared the ontogenesis of organic anion transporters in the placenta and the fetal liver of the rat to assess their relative abundance throughout gestation and to determine whether the placenta compensates for the late maturation of transporters in the developing liver . The mRNA of members of the organic anion transporting polypeptide (Oatp) superfamily, the multidrug resistance protein (Mrp) family, one organic anion transporter (OAT), and the bile acid carriers Na(+)-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) was quantified by real-time PCR . The most abundant placental transporters were Oatp4a1, whose mRNA increased 10-fold during gestation, and Mrp1 . Mrp1 immunolocalized predominantly to epithelial cells of the endoplacental yolk sac, suggesting an excretory role that sequesters fetal-derived solutes in the yolk sac cavity, and faintly to the basal syncytiotrophoblast surface . The mRNA levels of Oatp2b1, Mrp3, and Bsep in the placenta exceeded those in the fetal liver until day 20 of gestation, suggesting that the fetus relies on placental clearance of substrates when expression in the developing liver is low . Mrp3 immunolocalized to the epithelium of the endoplacental yolk sac and less abundantly in the labyrinth zone and endothelium of the maternal arteries . The placental expression of Oatp1a1, Oatp1a4, Oatp1a5, Oatp1b2, Oat, Ntcp, Mrp2, and Mrp6 was low.

Biochem Pharmacol, 2004 Oct 1, 68(7), 1363 - 70
Induction of cellular resistance to nucleoside reverse transcriptase inhibitors by the wild-type breast cancer resistance protein; Wang X et al.; Breast cancer resistance protein (BCRP/ABCG2) is a novel member of ATP-binding cassette transporters, which induce multidrug resistance in cancer cells . We previously reported that a high level of BCRP expression in CD4(+) T cells conferred cellular resistance to nucleoside reverse transcriptase inhibitors (NRTIs) of human immunodeficiency virus type 1 (HIV-1) . However, this BCRP was found to have a mutation of Arg to Met at position 482 (BCRP(R482M)) . The present study demonstrated that the wild-type BCRP (BCRP(WT)) also conferred cellular resistance to NRTIs . MT-4 cells (a CD4(+) T-cell line) highly expressing BCRP(WT) (MT-4/BCRP) were generated and the expression of BCRP(WT) was confirmed by genotypic and phenotypic analyses . Compared to the parental MT-4 cells, MT-4/BCRP cells displayed resistance to zidovudine (AZT) in terms of antiviral activity as well as drug cytotoxicity . In addition, other NRTIs were also less inhibitory to HIV-1 replication in MT-4/BCRP cells than in MT-4 cells . Significant reduction of intracellular AZT accumulation was observed in MT-4/BCRP cells . An analysis for intracellular metabolism of AZT suggested that the resistance was attributed to the increased efflux of AZT and its metabolites in MT-4/BCRP cells . Furthermore, the BCRP-specific inhibitor fumitremorgin C completely restored the reduction of AZT in MT-4/BCRP cells . These results indicate that, like BCRP(R482M), BCRP(WT) also plays an important role in cellular resistance to NRTIs.

Mol Pharmacol, 2004 Dec, 66(6), 1397 - 405 Epub 2004 Dec.
Analysis of ATP-binding cassette transporter expression in drug-selected cell lines by a microarray dedicated to multidrug resistance; Annereau JP et al.; Discovery of the multidrug resistance protein 1 (MDR1), an ATP-binding cassette (ABC) transporter able to transport many anticancer drugs, was a clinically relevant breakthrough in multidrug resistance research . Although the overexpression of ABC transporters such as P-glycoprotein/ABCB1, MRP1/ABCC1, and MXR/ABCG2 seems to be a major cause of failure in the treatment of cancer, acquired resistance to multiple anticancer drugs may also be multifactorial, involving alteration of detoxification processes, apoptosis, DNA repair, drug uptake, and overexpression of other ABC transporters . As a tool for the study of such phenomena, we designed and created a microarray platform, the ABC-ToxChip, to evaluate relative levels of transcriptional activation among genes involved in the various mechanisms of resistance . In the ABC-ToxChip, a comprehensive set of genes important in toxicological responses (represented by 2200 cDNA probes) is complemented with probes specifically matching ABC transporters as well as oligonucleotides representing 18,000 unique human genes . By comparing the transcriptional profiles of KB-3-1 and DU-145 parental cells with resistant derivatives selected in colchicine (KB-8-5), and 9-nitro-camptothecin (RCO.1), respectively, we demonstrate that ABC transporters (ABCB1/MDR1 and ABCC2/MRP2, respectively) show dramatic overexpression, whereas the glutathione S-transferase gene GST-Pi shows the strongest decrease in expression among the 20,000 genes studied . The results were confirmed by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry . The custom-designed ABC-Tox microarray presented here will be helpful to elucidate mechanisms leading to anticancer drug resistance.

Cancer Res, 2004 Sep 1, 64(17), 6214 - 24
Enhancement of the efficacy of chemotherapy for lung cancer by simultaneous suppression of multidrug resistance and antiapoptotic cellular defense: novel multicomponent delivery system; Pakunlu RI et al.; The efficacy of chemotherapy of lung cancer is limited by the development of resistance in cancer cells during treatment . In most lung cancers, this resistance is associated with the overexpression of (a) multidrug resistance-associated protein (MRP) responsible for drug efflux from the cancer cells (pump resistance) and (b) BCL2 protein that activates antiapoptotic cellular defense (nonpump resistance) . A novel liposomal proapoptotic anticancer drug delivery system was developed to enhance anticancer efficacy of the well-established drug doxorubicin (DOX) . This multicomponent drug delivery system was tested on multidrug-sensitive and -resistant human small-cell lung cancer cells . The drug delivery system includes four components: (a) liposome as a carrier, (b) DOX as an inductor of apoptosis, (c) antisense oligonucleotides (ASOs) targeted to MRP1 mRNA as a suppressor of pump resistance, and (d) ASOs targeted to BCL2 mRNA as a suppressor of nonpump resistance . Intracellular internalization of ASOs and DOX; the influence of the proposed system on the expression of genes and proteins involved in the multidrug resistance, cytotoxicity, and apoptosis induction and antiapoptotic defense; and the activity of caspases were studied . It was found that the proposed liposomal delivery system successfully delivered ASOs and DOX to cell nuclei, inhibited MRP1 and BCL2 protein synthesis, and substantially increased the anticancer action of DOX by stimulating the caspase-dependent pathway of apoptosis in multidrug-resistant human lung cancer cells.

Cancer Res, 2004 Sep 1, 64(17), 5956 - 62
CpG hypermethylation of MDR1 gene contributes to the pathogenesis and progression of human prostate cancer; Enokida H et al.; Multidrug resistance 1 (MDR1) gene encodes for P-glycoprotein (P-gp), a Mr 170,000 transmembrane calcium-dependent efflux pump that is inactivated in prostate cancer . We hypothesize that inactivation of the MDR1 gene through CpG methylation contributes to the pathogenesis and progression of prostate cancer . To test this hypothesis, CpG methylation status of the MDR1 promoter and its correlation with clinicopathological findings were evaluated in 177 prostate cancer samples and 69 benign prostate hypertrophy (BPH) samples . Cellular proliferation index and apoptotic index were determined by proliferating cell nuclear antigen (PCNA) and single-strand DNA immunostaining, respectively . After 5-aza-2'-deoxycytidine treatment, increased expression of MDR1 mRNA transcript was found in prostate cancer cell lines (DU145, DuPro, and ND1) . MDR1 methylation frequency was significantly higher in prostate cancer samples compared with BPH samples (54.8 versus 11.6%, respectively, P < 0.001) . Logistic regression analysis revealed that PC patients are 11.5 times more likely to have MDR1 methylation than BPH patients (95% confidence interval 4.87-27.0) and that MDR1 methylation is independent of the age . Significant correlation of MDR1 methylation was observed with high pT category (P < 0.001), high Gleason sum (P = 0.008), high preoperative prostate-specific antigen (P = 0.01), and advancing pathological features . In addition, PCNA-labeling index were significantly higher in methylation-specific PCR (MSP)-positive than in MSP-negative prostate cancer samples (P = 0.048) . In contrast, no significant difference in apoptotic index was found between MSP-positive and -negative prostate cancer samples . These findings suggest that CpG hypermethylation of MDR1 promoter is a frequent event in prostate cancer and is related to disease progression via increased cell proliferation in prostate cancer cells.

J Mol Biol, 2004 Sep 17, 342(3), 697 - 702
Structure of the ligand-blocked periplasmic entrance of the bacterial multidrug efflux protein TolC; Higgins MK et al.; The trimeric TolC protein of Escherichia coli comprises an outer membrane beta-barrel and a contiguous alpha-helical barrel projecting across the periplasm . This provides a single 140 A long pore for multidrug efflux and protein export . We have previously reported that trivalent cations such as hexammine cobalt can severely inhibit the conductivity of the TolC pore reconstituted in planar lipid bilayers . Here, isothermal calorimetry shows that Co(NH(3))(6)(3+) binds to TolC with an affinity of 20 nM . The crystal structure of the TolC-Co(NH(3))(6)(3+) complex was determined to 2.75 A resolution, and showed no significant difference in the protein when compared with unliganded TolC . An electron density difference map revealed that a single ligand molecule binds at the centre of the periplasmic entrance, the sole constriction of TolC . The octahedral symmetry of the ligand and the three-fold rotational symmetry of the TolC entrance determine a binding site in which the ligand forms hydrogen bonds with the Asp(374) residue of each monomer . When Asp(374) was substituted by alanine, high affinity ligand binding was abolished and inhibition of TolC pore conductivity in lipid bilayers was alleviated . Comparable effects followed independent substitution of the neighbouring Asp(371), indicating that this aspartate ring also contributes to the high affinity ligand binding site . As the electronegative entrance is widely conserved in the TolC family, it may be a useful target for the development of inhibitors against multidrug resistant pathogenic bacteria.

Eur J Cancer, 2004 Sep, 40(14), 2064 - 70
The distribution of drug-efflux pumps, P-gp, BCRP, MRP1 and MRP2, in the normal blood-testis barrier and in primary testicular tumours; Bart J et al.; The drug-efflux pumps P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) are present in the blood-testis barrier (BTB) and may hamper the delivery of cytotoxic drugs to the testis . The precise localisation of P-gp and MRP1 in testicular tissue and the presence of the efflux pumps MRP2 and breast cancer resistance protein (BCRP) in the BTB are unknown . We therefore studied the localisation of these pumps in the BTB in normal testis (n = 12), in non-seminoma (n = 10) seminoma (n = 10), and testicular lymphoma (n = 9) . Slides were scored semi-quantitatively for P-gp, MRP1, MRP2 and BCRP and blood vessels with factor VIII antibody . In normal testis, P-gp and BCRP were strongly expressed by myoid cells and luminal capillary endothelial wall and P-gp also by Leydig cells . MRP1 was observed at the basal side of Sertoli cells and on Leydig cells . MRP2 was only weakly expressed by myoid cells . Seminomas and non-seminomas expressed P-gp and/or BCRP and/or MRP1, lymphomas strongly expressed P-gp, weakly expressed BCRP and did not or showed weak expression of MRP1 . There was very little staining for MRP2 in the tumours . Newly formed vessels in all tumours only expressed P-gp and BCRP . P-gp, BCRP and MRP1 are present in different cell layers of the normal testis, suggesting the optimal protection of spermatogenesis . In germ cell tumours, this expression pattern may explain the chemoresistance observed to P-gp, BCRP and MRP1 substrates . In germ cell tumours and testicular lymphomas, P-gp and BCRP expression by tumour cells and by newly formed vessels may also contribute to chemoresistance . These findings underscore the importance of removing the affected testis in cases of primary germ cell tumours and testicular lymphomas, irrespective of whether the patient has already undergone chemotherapy.

Zhong Xi Yi Jie He Xue Bao, 2003 Sep, 1(3), 221 - 5
{Study on tumor cells' multidrug resistance and its reversion by Chinese herbs}; Chen XY et al.; Multidrug resistance (MDR) is an important biological behavior of tumor cells in chemotherapy . And it is also one of the major causes of clinical chemotherapy failure . According to the literature at home and abroad, and combining with the results of authors' investigations, this paper mainly discusses the mechanism of tumor cells' MDR and its reversion by Chinese herbs.

Yao Xue Xue Bao, 2004 May, 39(5), 333 - 7
{Reversal of multidrug resistance by lomerizine in K562/ADM cells}; Zhu HJ et al.; AIM: To study the effect of lomerizine (Lom) on the reversal of multidrug resistance (MDR) in K562/ADM cells and its mechanism . METHODS: MTT assay was used to determine the influence of Lom on the cytotoxicity of adriamycin (ADM) . The effect of Lom on the apoptosis induced by ADM and vincristine (VCR) in K562/ADM cells was detected using flow cytometry . Intracellular accumulation of ADM was measured by fluorescence spectrophotometry . Flow cytometry was used to investigate the efflux of rhodamine 123 (Rh123) and the expression of P-glycoprotein (P-gp) in K562/ADM cells . RESULTS: Lom increased the cytotoxicity of ADM and the apoptosis induced by ADM or VCR in K562/ADM cells . At the concentration of 3, 10 and 30 micromol x L(-1), Lom reduced the IC50 value of ADM from 79.03 micromol x L(-1) to 28.14, 8.16 and 3.16 micromol x L(-1), respectively . Lom increased the intracellular accumulation of ADM and inhibited the efflux of Rh123 in K562/ ADM cells . No change in P-gp expression was observed after the treatment of Lom for 72 h . CONCLUSION: Lom had strong reversal effect on MDR in K562/ADM cells by inhibiting P-gp function.

J Biol Chem, 2004 Nov 5, 279(45), 46706 - 14 Epub 2004 Aug 26.
Induction of stem cell factor/c-Kit/slug signal transduction in multidrug-resistant malignant mesothelioma cells; Catalano A et al.; Malignant mesothelioma (MM) is strongly resistant to conventional chemotherapy by unclear mechanisms . We and others have previously reported that cytokine- and growth factor-mediated signal transduction is involved in the growth and progression of MM . Here, we identified a pathway that involves stem cell factor (SCF)/c-Kit/Slug in mediating multidrug resistance of MM cells . When we compared gene expression profiles between five MM cells and their multidrug-resistant (MM DX) sublines, we found that MM DX cells expressed both SCF and c-Kit and had higher mRNA levels of Slug . Knockdown of c-Kit or Slug expression with their respective small interfering RNA sensitized MM DX cells to the induction of apoptosis by different chemotherapeutic agents, including doxorubicin, paclitaxel, and vincristine . Transfection of c-Kit in parental MM cells in the presence of SCF up-regulated Slug and increased resistance to the chemotherapeutic agents . Moreover, MM cells expressing Slug showed a similar increased resistance to the chemotherapeutic agents . These results indicate that induction of Slug by autocrine production of SCF and c-Kit activation plays a key role in conferring a broad spectrum chemoresistance on MM cells and reveal a novel signal transduction pathway for pharmacological or genetic intervention of MM patients.

Bioorg Med Chem, 2004 Sep 15, 12(18), 4963 - 8
Multidrug-resistant cancer cell susceptibility to cytotoxic quassinoids, and cancer chemopreventive effects of quassinoids and canthin alkaloids; Murakami C et al.; Twenty-three quassinoids (1-23), which were isolated previously from Simaroubaceous plants, were evaluated for cytotoxicity against three multidrug-resistant cancer cell lines, KB-VIN, KB-7d, and KB-CPT . Nine compounds (2-7 and 9-11) showed significant cytotoxicity in all three cell lines . Compounds 1, 12-14, 17, and 20 demonstrated significant activity against the KB-7d and KB-CPT cell lines, and compounds 18, 19, and 23 revealed notable activity only against KB-7d cells . Structure-activity relationships were drawn based on these data . In addition, six quassinoid derivatives (24-29) and four canthin alkaloids (30-33), which were isolated from Brucea antidysenterica, were examined for their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA) induced Epstein-Barr virus early antigen (EBV-EA) activation as cancer chemopreventive agents . All of these compounds demonstrated significant inhibitory effects against EBV-EA activation.

Biotechniques, 2004 Aug, 37(2), 270 - 5
Transduction of multiple cell types using improved conditions for gene delivery and expression of SV40 pseudovirions packaged in vitro; Kimchi-Sarfaty C et al.; This comprehensive study demonstrates highly efficient transduction of a wide variety of human, murine, and monkey cell lines, using a procedure for in vitro packaging of plasmid DNA in recombinant simian virus 40 (SV40) capsid proteins to form pseudovirions . The pseudovirions are encapsidated by the VP1 major capsid protein, with no SV40 sequence requirement, and are able to carry up to 17.7 kb of supercoiled plasmid DNA . We developed a procedure to scale-up production of SV40 pseudovirions, as well as an efficient protocol to concentrate the virions with no loss of activity . We also developed a method that allows transduction of 10 times more cells than the original protocol . This protocol was tested using supercoiled in vitro-packaged plasmid carrying the human multidrug-resistance gene (MDR1 encoding P-glycoprotein; P-gp), or the enhanced green fluorescent protein reporter gene (EGFP) in .45 human lymphoblastoid cells and in K562 human erythroleukemia cells . Multiple transductions at 24-h intervals were shown to increase expression using the EGFP reporter gene . The protocols developed in this study establish in vitro-packaged SV40 pseudovirions as one of the most efficient gene delivery systems.

Rev Panam Salud Publica, 2004 Jul, 16(1), 68 - 73
{Multiple drug resistance: a threat for tuberculosis control}; Cardoso EM; Drug-resistant tuberculosis (TB) was reported soon after the introduction of streptomycin, although it did not receive major attention until recently . It was not considered a major issue in the industrialized world until outbreaks of multidrug-resistant TB (MDR-TB) were reported among HIV infected people . Administration of standard short-course chemotherapy (SSCC) with first-line drugs under directly observed therapy (DOT) is the cornerstone of modern TB control . Unfortunately, data available on the treatment outcome of MDR-TB cases under routine programmatic conditions suggest that patients with MDR-TB respond poorly to SSCC with first-line drugs . Since 1994, the World Health Organization and the International Union Against Tuberculosis and Lung Disease (IU-ATLD) have conducted anti-TB drug resistance surveys through a network of subregional laboratories and researchers . Drug resistance was present in almost all settings surveyed, and prevalence varied widely across regions . High prevalence of MDR-TB is widespread in the Russian Federation and areas of the former Soviet Union (Estonia, Kazakhstan, Latvia, and Lithuania) as well as Israel, Liaoning and Henan Provinces in China, and Ecuador . The Global Project has surveyed areas representing over one third of notified TB cases . However, enormous gaps still exist in the most crucial areas . The most effective strategy to prevent the emergence of drug resistance is through implementation of the directly observed treatment short (DOTS) strategy . Effective implementation of the DOTS strategy saves lives through decreased TB transmission, decreased risk of emergence of drug-resistance, and decreased risk for individual TB patients of treatment failure, TB relapse, and death . The World Bank recognizes the DOTS strategy as one of the most cost-effective health interventions, and recommends that effective TB treatment be a part of the essential clinical services package available in primary health care settings . Governments are responsible for ensuring the provision of effective TB control through the DOTS strategy . WHO and its international partners have formed the DOTS-Plus Working Group, which is attempting to determine the best possible strategy to manage MDR-TB . One of the goals of DOTS-Plus is to increase access to expensive second-line anti-TB drugs for WHO-approved TB control programmes in low- and middle-income countries.

Rev Panam Salud Publica, 2004 Jul, 16(1), 23 - 34
Tuberculosis along the United States-Mexico border, 1993-2001; Schneider E et al.; OBJECTIVES: Tuberculosis (TB) is a leading public health problem and a recognized priority for the federal Governments of both Mexico and the United States of America . The objectives of this research, primarily for the four states in the United States that are along the border with Mexico, were to: (1) describe the epidemiological situation of TB, (2) identify TB risk factors, and (3) discuss tuberculosis program strategies . METHODS: We analyzed tuberculosis case reports collected from 1993 through 2001 by the tuberculosis surveillance system of the United States . We used those data to compare TB cases mainly among three groups: (1) Mexican-born persons in the four United States border states (Arizona, California, New Mexico, and Texas), (2) persons in those four border states who had been born in the United States, and (3) Mexican-born persons in the 46 other states of the United States, which do not border Mexico . RESULTS: For the period from 1993 through 2001, of the 16 223 TB cases reported for Mexican-born persons in the United States, 12 450 of them (76.7%) were reported by Arizona, California, New Mexico, and Texas . In those four border states overall in 2001, tuberculosis case rates for Mexican-born persons were 5.0 times as high as the rates for persons born in the United States; those four states have 23 counties that directly border on Mexico, and the ratio in those counties was 5.8 . HIV seropositivity, drug and alcohol use, unemployment, and incarceration were significantly less likely to be reported in Mexican-born TB patients from the four border states and the nonborder states than in patients born in the United States from the four border states (P < 0.001) . Multivariate analysis revealed that among pulmonary tuberculosis patients who were 18-64 years of age and residing in the four border states, the Mexican-born patients were 3.6 times as likely as the United States-born patients were to have resistance to at least isoniazid and rifampin (i . e., to have multidrug-resistant TB) and twice as likely to have isoniazid resistance . Mexican-born TB patients from the four border states and the nonborder states were significantly more likely to have moved or to be lost to follow-up than were the TB patients born in the United States from the four border states (P < 0.001) . CONCLUSIONS: Increased collaborative tuberculosis control efforts by the federal Governments of both Mexico and the United States along the border that they share are needed if tuberculosis is to be eliminated in the United States.

J Nat Prod, 2004 Aug, 67(8), 1368 - 73
Briarane diterpenes from two species of octocorals, Ellisella sp . and Pteroeides sp; Tanaka C et al.; Eight new briarane diterpenes (1-4, 7-10) have been isolated from two species of octocorals and the structures elucidated by spectroscopic analysis . Two diterpenes (2, 3) from the gorgonian Ellisella sp . inhibited cytokinesis, causing multinuclei formation on NBT-II cells, while a known briarane (12) from the sea pen Pteroeides sp . showed reversal of multidrug resistance.

J Pharmacol Exp Ther, 2005 Jan, 312(1), 281 - 9 Epub 2004 Aug 26.
Intrinsic and Acquired Forms of Resistance against the Anticancer Ruthenium Compound KP1019 {Indazolium trans-{tetrachlorobis(1H-indazole)ruthenate (III)} (FFC14A); Heffeter P et al.; KP1019 {indazolium trans-{tetrachlorobis(1H-indazole)ruthenate (III)} (FFC14A) is a metal complex with promising anticancer activity . Since chemoresistance is a major obstacle in chemotherapy, this study investigated the influence of several drug resistance mechanisms on the anticancer activity of KP1019 . Here we demonstrate that the cytotoxic effects of KP1019 are neither substantially hampered by overexpression of the drug resistance proteins multidrug resistance-related protein 1, breast cancer resistance protein, and lung resistance protein nor the transferrin receptor and only marginally by the cellular p53 status . In contrast, P-glycoprotein overexpression weakly but significantly (up to 2-fold) reduced KP1019 activity . P-glycoprotein-related resistance was based on reduced intracellular KP1019 accumulation and reversible by known P-glycoprotein modulators . KP1019 dose dependently inhibited ATPase activity of P-glycoprotein with a K(i) of approximately 31 muM . Furthermore, it potently blocked P-glycoprotein-mediated rhodamine 123 efflux under serum-free conditions (EC(50), approximately 8 muM), however, with reduced activity at increased serum concentrations (EC(50) at 10% serum, approximately 35 muM) . Moreover, P-glycoprotein-mediated daunomycin resistance could only be marginally restored by KP1019 in serum-containing medium, also indicating an influence of serum proteins on the interaction between KP1019 and P-glycoprotein . Acquired KP1019 resistance was investigated by selecting KB-3-1 cells against KP1019 for more than 1 year . Only an approximately 2-fold KP1019 resistance could be induced, which unexpectedly was not due to overexpression of P-glycoprotein or other efflux pumps . Accordingly, KP1019-resistant cells did not display reduced drug accumulation . Their unique cross-resistance pattern confirmed an ABC transporter-independent resistance phenotype . In summary, the likeliness of acquiring insensitivity to KP1019 during therapy is expected to be low, and resistance should not be based on overexpression of drug efflux transporters.

Anticancer Res, 2004 Jul-Aug, 24(4), 2231 - 5
Effective knock down of very high ABCG2 expression by a hammerhead ribozyme; Kowalski P et al.; BACKGROUND: Ribozymes are an effective tool to reduce the mRNA levels of specific target genes . Overexpression of the drug transport protein, ABCG2, has been associated with multidrug resistance in cancer cells . MATERIALS AND METHODS: An expression plasmid encoding a hammerhead ribozyme against the ABCG2 gene was stably transfected into multidrug-resistant MCF7/MX cells that express very high levels of the ABCG2 protein . The effect of the ribozyme was determined by quantitative real-time RT-PCR, Western blot and cytotoxicity assays . RESULTS: The ribozyme reduced ABCG2 mRNA levels to less than 10% of control values, which resulted in the concomitant reduction of ABCG2 protein levels and sensitization of the cells to mitoxantrone and methotrexate . CONCLUSION: The ribozyme used was highly effective in reducing the expression of its target gene, ABCG2, and was able to modulate the associated multidrug-resistant phenotype.

Antimicrob Agents Chemother, 2004 Sep, 48(9), 3412 - 8
Genes required for intrinsic multidrug resistance in Mycobacterium avium; Philalay JS et al.; Genes required for intrinsic multidrug resistance by Mycobacterium avium were identified by screening a library of transposon insertion mutants for the inability to grow in the presence of ciprofloxacin, clarithromycin, and penicillin at subinhibitory concentrations . Two genes, pks12 and Maa2520, were disrupted in multiple drug-susceptible mutants . The pks12 gene (Maa1979), which may be cotranscribed with a downstream gene (Maa1980), is widely conserved in the actinomycetes . Its ortholog in Mycobacterium tuberculosis is a polyketide synthase required for the synthesis of dimycocerosyl phthiocerol, a major cell wall lipid . Mutants of M . avium with insertions into pks12 exhibited altered colony morphology and were drug susceptible, but they grew as well as the wild type did in vitro and intracellularly within THP-1 cells . A pks12 mutant of M . tuberculosis was moderately more susceptible to clarithromycin than was its parent strain; however, susceptibility to ciprofloxacin and penicillin was not altered . M . avium complex (MAC) and M . tuberculosis appear to have different genetic mechanisms for resisting the effects of these antibiotics, with pks12 playing a relatively more significant role in MAC . The second genetic locus identified in this study, Maa2520, is a conserved hypothetical gene with orthologs in M . tuberculosis and Mycobacterium leprae . It is immediately upstream of Maa2521, which may code for an exported protein . Mutants with insertions at this locus were susceptible to multiple antibiotics and slow growing in vitro and were unable to survive intracellularly within THP-1 cells . Like pks12 mutants, they exhibited increased Congo red binding, an indirect indication of cell wall modifications . Maa2520 and pks12 are the first genes to be linked by mutation to intrinsic drug resistance in MAC.

Brain Res, 2004 Sep 17, 1021(1), 32 - 40
Expression of P-glycoprotein (ABCB1) and Mrp1 (ABCC1) in adult rat brain: focus on astrocytes; Mercier C et al.; P-glycoprotein (P-gp, ABCB1) and the multidrug resistance-associated protein 1 (Mrp1, ABCC1) are two ATP-driven pumps that mediate the export of organic anions from cells and may confer cellular resistance to many cytotoxic hydrophobic drugs . Immunohistochemistry has shown that P-gp is expressed in rat brain capillary vessels forming the blood-brain barrier (BBB) . Mrp1 mRNAs have been detected by RT-PCR in rat brain isolated capillaries . Although many studies have been published in this field, very little information is available on the expression, distribution and physiological functions of the two pumps in rat brain . To characterize the cerebral expression of both P-gp and Mrp1 transporters, we studied immunoreactions of rat brain sections with the two most commonly used antibodies: the monoclonal C219 (anti-P-gp) and the polyclonal 6KQ (anti-Mrp1) . Immunological analyses revealed heterogeneity of the P-gp and Mrp1 expressions in rat brain . Indeed, choroidal and ependymal cells expressed Mrp1 rather than P-gp . However, tanycytes lining the third ventricle were strongly immunoreactive with both antibodies, suggesting a particular role for these cells in drug efflux mechanisms . Because of the detection of a 70-kDa component with 6KQ antibodies, immunoreactions obtained in rats were compared with these obtained in wild type and mrp1(-/-) mice . It showed that a positive reaction at the apical surface of the ependymal layer remained obvious, showing that 6KQ antibodies recognize an ependymal molecule, differing from the Mrp1 . In addition, a continuous expression of C219-labeled epitopes, similar to endothelial labeling, was detected at the blood-brain barrier, whereas a discontinuous labeling, co-localized with glial fibrillary acidic protein (GFAP) immunostaining, was obtained with 6KQ antibodies . We showed that P-gp was preferentially expressed in the endothelial component and Mrp1 in the astroglial component of the blood-brain barrier . Moreover, Mrp1 was rather expressed than P-gp in parenchyma astrocytes and in glia limitans lining the meninges . These findings provide new insights into the cerebral distribution of two ABC transporters linked to multidrug resistance (MDR).

Cancer Biol Ther, 2004 Sep, 3(9), 819 - 24 Epub 2004 Sep 23.
MDR1, Chemotherapy and Chromatin Remodeling; Baker EK et al.; The development of multidrug resistance (MDR) in cancer can severely impede the efficacy of chemotherapy treatment . P-glycoprotein (Pgp) overexpression, encoded by the MDR1 gene, is a well-established mediator of MDR . MDR1 expression is rapidly upregulated by chemotherapeutic drugs and a number of other exogenous stimuli, however the mechanisms underlying its transcriptional regulation remain unclear . In recent years, research has indicated that chromatin accessibility, or epigenetic modifications, will play a large role in controlling the endogenous MDR1 expression state, and its response to activation stimuli . This review examines some of these studies, and discusses how new developments from the greatly expanding epigenetics field may extend to MDR1 transcriptional research.

Cancer Biol Ther . 2004 Oct 27;3(10) {Epub ahead of print}
Role of Caveolin-1 in Etoposide Resistance Development in A549 Lung Cancer Cells; Belanger MM et al.; Caveolin 1 expression is downregulated in various cancer cell lines . Interestingly, in several drug-resistant cancer cells, a strong induction of caveolin 1 expression has been reported suggesting a role for caveolin 1 in the acquisition and/or the maintenance of multidrug resistance phenotype . In addition, it was reported that p-glycoprotein localized to caveolin-rich membrane domains in these cells . In this study, we progressively exposed A549 lung adenocarcinoma cells to increasing doses of etoposide . Both R1 and R2 cell lines had greatly increased levels of p-glycoprotein expression while mrp expression levels were moderately increased but only R2 cells had raised caveolin levels compared to control A549 cells . Both caveolin-1 and p-glycoprotein colocalize in Triton-insoluble membrane domains in all our cell lines but only caveolins-1 was solubilized by the addition of octylglucoside at 4C suggesting that these two proteins are located in different membrane domains . Using an anti-caveolin-1 antibody, we did not succeed to immunoprecipitate p-glycoprotein . Interestingly, total cellular cholesterol (the major lipid component of caveolae and triton-insoluble domains) was greatly increased in both R1 and R2 cell lines compared to naive A549 cells.

Bioorg Med Chem Lett, 2004 Sep 20, 14(18), 4767 - 70
Synthesis and biological evaluation of taxinine analogues as orally active multidrug resistance reversal agents in cancer; Zhao X et al.; Three novel taxinine analogues were prepared and tested for their activity as multidrug resistance (MDR) reversal agents in comparison with verapamil . In vitro testing demonstrated that compounds 8-10 possess MDR-reversal activity in the KB/V cell line . Half-hour after treatment with 5, 10, and 20 micromol/L compound 9, the intracellular rhodamine123 concentration increased 2.3, 2.9, and 3.2-fold, respectively, higher than 1.88-fold of 10 micromol/L verapamil in KB/V cell line . In vivo studies with VCR-resistant KB/V tumor xenografts showed that compound 9 in combination with VCR significantly inhibited tumor growth . Treatment with VCR or 9 alone did not result in growth inhibition . These results reveal that three taxinine analogues are good modifiers of MDR in tumor cells.

Cancer Cell, 2004 Aug, 6(2), 129 - 37
Predicting drug sensitivity and resistance: profiling ABC transporter genes in cancer cells; Szakacs G et al.; For analysis of multidrug resistance, a major barrier to effective cancer chemotherapy, we profiled mRNA expression of the 48 known human ABC transporters in 60 diverse cancer cell lines (the NCI-60) used by the National Cancer Institute to screen for anticancer activity . The use of real-time RT-PCR avoided artifacts commonly encountered with microarray technologies . By correlating the results with the growth inhibitory profiles of 1,429 candidate anticancer drugs tested against the cells, we identified which transporters are more likely than others to confer resistance to which agents . Unexpectedly, we also found and validated compounds whose activity is potentiated, rather than antagonized, by the MDR1 multidrug transporter . Such compounds may serve as leads for development.

J Pharm Pharmacol, 2004 Sep, 56(9), 1067 - 81
A review of selected anti-tumour therapeutic agents and reasons for multidrug resistance occurrence; Sawicka M et al.; It is assumed that proteins from the ABC family (i.e., glycoprotein P (Pgp)) and a multidrug resistance associated protein (MRP) play a main role in the occurrence of multidrug resistance (MDR) in tumour cells . Other factors that influence the rise of MDR are mechanisms connected with change in the effectiveness of the glutathione cycle and with decrease in expression of topoisomerases I and II . The aim of this review is to characterize drugs applied in anti-tumour therapy and to describe the present state of knowledge concerning the mechanisms of MDR occurrence, as well as the pharmacological agents applied in reducing this phenomenon.

J Eur Acad Dermatol Venereol, 2004 Sep, 18(5), 546 - 51
Cutaneous tuberculosis in Indian children: the importance of screening for involvement of internal organs; Pandhi D et al.; AIMS AND OBJECTIVES: Resurgence of skin tuberculosis especially with drug-resistant strains has been well documented in recent years, but this problem has not received much attention in the paediatric age group . Hence, we carried out the present study to analyse the clinical and therapeutic aspects of cutaneous tuberculosis in children . MATERIALS AND METHODS: A detailed clinical examination, investigations, such as haemogram, serology for HIV, Mantoux test, chest X-ray, cytology, culture and histopathology were carried out in all children . They were treated with antitubercular therapy (WHO regimen), and the clinical response was followed up . RESULTS: Of 142 patients with cutaneous tuberculosis, 68 were children (40 females, 28 males) . These children were aged from 9 months to 14 years . The duration of the disease varied from 1 month to 6 years . Family history of tuberculosis was present in 28 (41.2%) of the patients . Scrofuloderma was the most common presentation encountered in 30 (44.1%) patients with preferential involvement of the cervical (56.2%) and inguinal (20%) regions . Fifteen (22.1%) patients had lupus vulgaris, of which the keratotic type was the most common (46.7%), 16 had lichen scrofulosorum, three had tuberculosis verrucosa cutis, and four had more than one type of tuberculosis . Involvement of the lung in 14 (20.6%), bone in seven (10.2%), and both in four (5.9%) was found . Histopathology corroborated the clinical diagnosis in 54 (80.6%), culture was positive in six (8.8%) . Fifty (73.5%) patients completed the treatment with an excellent response, no multidrug resistant cases were seen . CONCLUSIONS: Cutaneous tuberculosis in children continues to be an important cause of morbidity, there is a high likelihood of internal involvement, especially in patients with scrofuloderma . A search is required for more sensitive, economic diagnostic tools . Response to treatment at 4 weeks often helps in substantiating the diagnosis of tuberculosis in doubtful cases .

AIDS, 2004 Jun, 18 Suppl 3, S9 - 13
Primary HIV-1 drug resistance in Brazil; Soares MA et al.; In this report we reviewed primary HIV-1 drug resistance in Brazil and compared it with that of other developed countries . An extensive survey was conducted in published studies on primary HIV-1 drug resistance in Brazil and in several developed countries in North America and Western Europe . Overall and genomic region-specific (protease or reverse transcriptase) rates were compared between countries and over time in some countries (whenever available) to detect their trend over time and in different groups of individuals (acutely or chronically infected patients) . Brazil has shown primary drug resistance rates that were on average lower than in most developed countries analysed . There were no reports in Brazil showing the occurrence of multidrug-resistant HIV-1 strains circulating in drug-naive patients, in contrast to some countries . Rates of protease secondary mutations observed in Rio de Janeiro (the second largest city affected by the HIV/AIDS epidemic in Brazil) did not show evidence of increase, but rather mutation-specific steady state equilibria . Despite the universal access to antiretroviral treatment in the country, rates of primary drug resistance are still low when compared with those of developed nations, arguing against the need for genotyping in patients before initiating therapy . The lower rates of primary drug resistance reported show that resistance should not be of concern in promoting expanded access to antiretroviral treatment in developing settings.

Mol Pharmacol, 2004 Sep, 66(3), 450 - 9
Prediction of in vivo biliary clearance from the in vitro transcellular transport of organic anions across a double-transfected Madin-Darby canine kidney II monolayer expressing both rat organic anion transporting polypeptide 4 and multidrug resistance associated protein 2; Sasaki M et al.; We have proposed previously that the evaluation of transcellular transport across the double-transfected Madin-Darby canine kidney II (MDCK II) monolayer that expresses both human organic anion transporting polypeptide 4 (OATP2/SLC21A6) and multidrug resistance associated protein 2 (MRP2/ABCC2) on the basal and apical membranes, respectively, may be useful in characterizing human biliary excretion (J Biol Chem 277: 6497-6503, 2002) . However, to demonstrate that this in vitro system represents in vivo biliary excretion, it is essential to compare in vitro data with in vivo biliary excretion . The problem is that we cannot determine the human biliary excretion for many ligands . In the present study, we have established a double-transfected MDCK II monolayer that expresses both rat Oatp4/Slc21a10 and Mrp2/Abcc2 on the basal and apical membranes, respectively, for the purpose of quantitatively comparing the clearance for transcellular transport with that for in vivo biliary excretion . The basal-to-apical transport of 17beta-estradiol-17beta-d-glucuronide, pravastatin, leukotriene C(4), cyclo-{D-Asp-Pro-d-Val-Leu-d-Trp} (BQ123), temocaprilat, and taurolithocholate 3-sulfate was significantly higher than that in the opposite direction in the double transfectant . Kinetic analysis suggested that that the rate-determining step of these compounds is the uptake process . The extent of the transcellular transport across the rat double-transfectant correlated well with that across the double-transfectant for human OATP2/SLC21A6 and MRP2/ABCC2 . Moreover, considering the scaling factor, the clearance values for in vitro transcellular transport correlated well with those for in vivo biliary clearance . The double-transfected MDCK II monolayer may be useful in analyzing the hepatic vectorial transport of organic anions and in predicting in vivo biliary clearance.

Mol Pharmacol, 2004 Sep, 66(3), 395 - 403
Regulation of the stability of P-glycoprotein by ubiquitination; Zhang Z et al.; Ubiquitination plays a crucial role in regulating protein turnover . Here we show that ubiquitination regulates the stability of the MDR1 gene product, P-glycoprotein, thereby affecting the functions of this membrane transporter that mediates multidrug resistance . We found that P-glycoprotein was constitutively ubiquitinated in drug-resistant cancer cells . Transfection of multidrug-resistant cells with wild-type ubiquitin or treatment with an N-glycosylation inhibitor increased the ubiquitination of P-glycoprotein and increased P-glycoprotein degradation . Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132), a proteasome inhibitor, induced accumulation of ubiquitinated P-glycoprotein, suggesting the involvement of the proteasome in the turnover of the transporter . Treatment of multidrug-resistant cells with 12-O-tetradecanoylphorbol-13-acetate, a phorbol ester that increases the phosphorylation of P-glycoprotein through activation of protein kinase C, or substituting phosphorylation sites of P-glycoprotein by nonphosphorylatable residues did not affect the ubiquitination of the transporter . Enhanced ubiquitination of P-glycoprotein resulted in a decrease of the function of the transporter, as demonstrated by increased intracellular drug accumulation and increased cellular sensitivity to drugs transported by P-glycoprotein . Our results indicate that the stability and function of P-glycoprotein can be regulated by the ubiquitin-proteasome pathway and suggest that modulating the ubiquitination of P-glycoprotein might be a novel approach to the reversal of drug resistance.

Drug Metab Dispos, 2004 Sep, 32(9), 953 - 8
Kinetic characterization of P-glycoprotein-mediated efflux of rhodamine 6G in the intact rabbit lung; Roerig DL et al.; P-Glycoprotein (P-gp) is an ATP-dependent drug efflux transporter involved in multidrug resistance and drug disposition in many organ systems . A majority of P-gp substrates are lipophilic amine drugs which also exhibit rapid extensive accumulation in lung tissue . P-gp is expressed in lung tissue, and the very nature of this drug efflux mechanism suggests a moderating role in pulmonary drug disposition . Little is known about P-gp-mediated efflux out of lung tissue or its kinetic characteristics as they may relate to the impact of P-gp on pulmonary drug accumulation . The present study develops an experimental and kinetic model to characterize the kinetics of P-gp-mediated efflux of rhodamine 6G dye (R6G) out of the intact rabbit lung . The perfusate concentration of R6G with time during recirculation through an isolated perfused rabbit lung was measured, and 66.6 +/- 2.6% (S.E.) of the perfusate R6G was taken up by the lung . In the presence of P-gp inhibitors, R6G uptake increased significantly to 87.5 +/- 1.1% (P < 0.002), indicating a functional pulmonary P-gp efflux transporter . Fractional lung accumulation of R6G increased with increasing R6G perfusate concentration, a result consistent with saturation of an efflux transporter . A parsimonious three-compartment kinetic model of R6G pulmonary disposition was used to interpret data sets from experiments with different perfusion variables and to estimate parameters descriptive of the dominant kinetic processes involved in R6G pulmonary accumulation . The estimated value of the kinetic parameter, k(pgp), rate constant for P-gp-mediated R6G efflux, indicates that this transporter plays a significant role in moderating R6G pulmonary disposition.

Drug Metab Dispos, 2004 Sep, 32(9), 909 - 14
Differences in the pharmacokinetics of peroxisome proliferator-activated receptor agonists in genetically obese Zucker and sprague-dawley rats: implications of decreased glucuronidation in obese Zucker rats; Kim MS et al.; Genetically obese Zucker rats exhibit symptoms similar to those of obese patients with insulin-resistance or Type II diabetes; therefore, they have been used as a genetic model to study obesity, as well as a pharmacological model for the discovery of new drugs for the treatment of Type II diabetes and hyperlipidemia . In the present study, we compared the pharmacokinetics of two novel peroxisome proliferator-activated receptor (PPAR) agonists, MRL-I {(2R)-7-{3-{2-chloro-4-(4-fluorophenoxy)phenoxy}propoxy}-2-ethyl-3,4-dihydro-2H-benzopyran-2-carboxylic acid} and MRL-II {(2R)-7-{3-{2-chloro-4-(2,2,2-trifluoroethoxy)phenoxy}propoxy}-3,4-dihydro-2-methyl-2H-benzopyran-2-carboxylic acid}, in obese Zucker and lean Sprague-Dawley rats following a single intravenous administration . The plasma clearance of both MRL-I and MRL-II was significantly lower in obese Zucker rats (4- and 2-fold, respectively) compared with Sprague-Dawley rats, but without any significant change in the volume of distribution, which resulted in a dramatic increase in the half-life (7- and 3-fold, respectively) . The reversible in vitro plasma protein binding of {(14)C}MRL-I and {(14)C}MRL-II was comparable in the two strains, approximately 96% bound . The expression levels of uridine diphosphate-glucuronosyltransferases 1A1, 1A6, 2B1, and CYP2C11 and 3A1 mRNA in liver were lower (30-50%) in Zucker compared with Sprague-Dawley rats, as were the liver glutathione S-transferases (70%), quinone reductase (30%), organic anion-transporting protein 2 (80%), and multidrug resistance-associated protein 2 (Mrp2) (50%) mRNA levels . However, Mrp3 mRNA levels were similar in both strains . Consistent with these observations, the intrinsic clearance (CL(int)), calculated from the V(max)/K(m) of glucuronidation of {(14)C}MRL-I and {(14)C}MRL-II in liver microsomes, was approximately 2-fold lower in obese Zucker rats; the K(m) values were comparable in the two strains for both compounds . In conclusion, differences in the pharmacokinetics of two novel PPAR agonists, both cleared, predominantly, by conjugation, were evident in genetically obese Zucker rats compared with Sprague-Dawley rats . These differences were consistent with changes in the mRNA levels of hepatic drug-metabolizing enzymes and transporters . This information should be considered when comparing pharmacokinetic and efficacious doses in the obese Zucker rats, used as a pharmacological model, with those in Sprague-Dawley rats, which are used widely for drug metabolism and toxicology studies.

J Med Chem, 2004 Aug 26, 47(18), 4627 - 30
Substituted pyridazino{3,4-b}{1,5}benzoxazepin-5(6H)ones as multidrug-resistance modulating agents; Ott I et al.; Pyridazino{3,4-b}{1,5}benzoxazepin-5(6H)ones substituted with propylene-linked basic side chains were synthesized and investigated for the ability to reverse multidrug resistance (MDR) at vincristine-pretreated HeLa-MDR1 cells . The substances were found to be effective chemosensitizers with activity comparable to that of the known MDR modulator verapamil . The observed antiproliferative effects were not caused by direct drug cytotoxicity.

Cell Mol Life Sci, 2004 Aug, 61(16), 2071 - 82
beta2-integrins mediate a novel form of chemoresistance in cycloheximide-induced U937 apoptosis; Wu RC et al.; In this study with cycloheximide (CHX, an inhibitor of protein synthesis) and the human leukaemic cell line U937, a novel form of chemoresistance, which we termed sudden drug resistance (SDR), was identified using Hoechst33258 staining, Western blot and DNA Ladder . CHXhigh (10-100 microg/ml)-induced apoptosis can spontaneously subside after 4-6 h or can be inhibited by short-term preincubation with CHXlow (2.5 microg/ml) . Unlike typical multidrug resistance, SDR is not caused by reduced drug accumulation or altered protein expression, and may be associated with a non-P-glycoprotein mechanism . To uncover this underlying mechanism, we focused on U937 cell aggregation promoted by CHX, because cell adhesion has been suggested to influence cell survival and prevent apoptosis . EDTA, or anti-CD18 monoclonal antibody, but not EGTA, acetylsalicylic acid or RGDS tetrapeptide, abrogated this homotypic aggregation and greatly increased CHX-induced apoptosis in a time-dependent manner, while fibrinogen and soluble intercellular adhesion molecule-1 exerted opposite effects . These results establish that beta2-integrin engagement is a key mediator of SDR, although it may be non-exclusive . This finding supplements the classical basis of chemoresistance and may provide another opportunity for improved leukemia therapy.

Transplantation, 2004 Aug 15, 78(3), 311 - 5
Pharmacogenetics in solid organ transplantation: present knowledge and future perspectives; Anglicheau D et al.; The promise of pharmacogenetics is to elucidate the inherited basis of differences between individual responses to drugs, in order to identify the right drug and dose for each patient . Genetic polymorphisms are implicated in the interindividual variability of the pharmacokinetic or pharmacodynamic characteristics of immunosuppressive drugs . The first pharmacogenetic trait identified was monogenic, and concerned the prototypic example of thiopurine methyltransferase (TPMT) implicated in azathioprine metabolism . Individuals with low TPMT activity, inherited in an autosomal codominant fashion, are at risk of drug-induced myelosuppression . TPMT activity determination and DNA-based tests are now used in clinical practice . It has been also demonstrated that there is a link between the polymorphisms of the cytochrome P450 3A5, 3A4 and the multidrug resistance-1 (MDR1) genes, and the daily dose necessary to achieve adequate blood tacrolimus levels . Analysis of MDR1 haplotypes or using the association of the different genes might further improve predictions . Since genotyping methods improve rapidly, it will soon be easy to test for thousands of single nucleotide polymorphisms in one assay . Present challenges are to determine the genes of interest and to validate such determination prospectively in clinical practice.

J Huazhong Univ Sci Technolog Med Sci, 2004, 24(2), 151 - 3
Difference in expression of Bcl-2 and Bcl-xl genes in cisplatin-sensitive and cisplatin-resistant human in ovarian cancer cell lines; Yu L et al.; To investigate the expression of Bcl-2 and Bcl-xl gene in sensitive (A2780) and drug-resistance (AD6) human ovarian cancer cell lines and explore the molecular mechanism of multidrug resistance, A2780 and AD6 were detected by using DNA gel electrophoresis, flow cytometry and RT-PCR . Our results showed that (1) "DNA ladder" was observed in A2780 and AD6 after cisplatin treatment; (2) after 3.0, 6.0, 9.9 microg/ml of cisplatin treatment, a significant difference was noted in the rate of apoptosis between in A2780 and AD6 (P<0.05); (3) Bcl-2 and Bcl-xl genes were overexpressed in AD6 . After cisplatin treatment, the expression of Bcl-2 and Bcl-xl genes was down-regulated in A2780 and AD6 . It is concluded that cisplatin could induce the apoptosis of ovarian cancer cells, and the over-expression of Bcl-2 and Bcl-xl genes may contribute to apoptotic inhibition and the development of multidrug-resistance of human ovarian cancer.

Mol Cell Biol, 2004 Sep, 24(17), 7612 - 21
Mrp4 confers resistance to topotecan and protects the brain from chemotherapy; Leggas M et al.; The role of the multidrug resistance protein MRP4/ABCC4 in vivo remains undefined . To explore this role, we generated Mrp4-deficient mice . Unexpectedly, these mice showed enhanced accumulation of the anticancer agent topotecan in brain tissue and cerebrospinal fluid (CSF) . Further studies demonstrated that topotecan was an Mrp4 substrate and that cells overexpressing Mrp4 were resistant to its cytotoxic effects . We then used new antibodies to discover that Mrp4 is unique among the anionic ATP-dependent transporters in its dual localization at the basolateral membrane of the choroid plexus epithelium and in the apical membrane of the endothelial cells of the brain capillaries . Microdialysis sampling of ventricular CSF demonstrated that localization of Mrp4 at the choroid epithelium is integral to its function in limiting drug penetration into the CSF . The topotecan resistance of cells overexpressing Mrp4 and the polarized expression of Mrp4 in the choroid plexus and brain capillary endothelial cells indicate that Mrp4 has a dual role in protecting the brain from cytotoxins and suggest that the therapeutic efficacy of central nervous system-directed drugs that are Mrp4 substrates may be improved by developing Mrp4 inhibitors .

Cancer Res, 2004 Aug 15, 64(16), 5804 - 11
Mechanism of the pharmacokinetic interaction between methotrexate and benzimidazoles: potential role for breast cancer resistance protein in clinical drug-drug interactions; Breedveld P et al.; The antifolate drug methotrexate (MTX) is transported by breast cancer resistance protein (BCRP; ABCG2) and multidrug resistance-associated protein1-4 (MRP1-4; ABCC1-4) . In cancer patients, coadministration of benzimidazoles and MTX can result in profound MTX-induced toxicity coinciding with an increase in the serum concentrations of MTX and its main metabolite 7-hydroxymethotrexate . We hypothesized that benzimidazoles interfere with the clearance of MTX and/or 7-hydroxymethotrexate by inhibition of the ATP-binding cassette drug transporters BCRP and/or MRP2, two transporters known to transport MTX and located in apical membranes of epithelia involved in drug disposition . First, we investigated the mechanism of interaction between benzimidazoles (pantoprazole and omeprazole) and MTX in vitro in membrane vesicles from Sf9 cells infected with a baculovirus containing human BCRP or human MRP2 cDNA . In Sf9-BCRP vesicles, pantoprazole and omeprazole inhibited MTX transport (IC50 13 microm and 36 microm, respectively) . In Sf9-MRP2 vesicles, pantoprazole did not inhibit MTX transport and at high concentrations (1 mm), it even stimulated MTX transport 1.6-fold . Secondly, we studied the transport of pantoprazole in MDCKII monolayers transfected with mouse Bcrp1 or human MRP2 . Pantoprazole was actively transported by Bcrp1 but not by MRP2 . Finally, the mechanism of the interaction was studied in vivo using Bcrp1-/- mice and wild-type mice . Both in wild-type mice pretreated with pantoprazole to inhibit Bcrp1 and in Bcrp1-/- mice that lack Bcrp1, the clearance of i.v . MTX was decreased significantly 1.8- to 1.9-fold compared with the clearance of i.v . MTX in wild-type mice . The conclusion is as follows: benzimidazoles differentially affect transport of MTX mediated by BCRP and MRP2 . Competition for BCRP may explain the clinical interaction between MTX and benzimidazoles.

Neurochem Int, 2004 Nov, 45(6), 865 - 73
Cyclic GMP transporters; Sager G; The biokinetics of guanosine 3',5'-cyclic monophosphate (cGMP) is characterized by three distinct processes: synthesis by guanylate cyclases (GCs), conversion of cGMP to GMP by cyclic nucleotide phosphodiesterases (PDEs) and the excretion of unchanged cGMP by transport proteins in the cell membrane . Efflux is observed in virtually all cell types including cells which originate from brain . Studies of intact cells, in which metabolic inhibitors and probenecid reduced extrusion of cGMP and wherein cGMP was extruded against concentration gradients, indicated the existence of ATP requiring organic anion transport system(s) . Functional studies of inside-out vesicles have revealed cGMP transport systems wherein translocation is coupled to hydrolysis of ATP . The extrusion of cGMP is inhibited by a number of unrelated compounds and this indicates that cGMP is substrate for multispecific transporters . Recent transfection studies suggest that members of the MRP (multidrug resistance protein) family; MRP4, MRP5 and MRP8 translocate cGMP across the cell membrane . Many of the MRPs have been detected in brain . In addition tertiary active transport by the organic anion transporter family has also been identified . At least one member (OAT1) shows relative high affinity for cGMP and is also expressed in brain . The biological significance of cGMP transporters has to be clarified . Their role in cGMP biokinetics, being responsible for one of the cellular elimination pathways, is well established . However, there is growing evidence that extracellular cGMP has effects on cell physiology and pathophysiology by an auto- or paracrine mechanism.

Zhonghua Zhong Liu Za Zhi, 2004 Apr, 26(4), 201 - 4
{Multidrug resistant effect of alternative splicing form of MAD2 gene-MAD2beta on human gastric cancer cell}; Yin F et al.; OBJECTIVE: To study the effect of alternative splicing form -MAD2beta of mitotic arrest deficient protein 2 (MAD2) on the formation of multidrug resistance in human gastric adenocarcinoma cell SGC7901 . METHODS: RNA was extracted from a multidrug resistance cell line SGC7901/ADR . The full-length MAD2beta cDNA was obtained by RT-PCR and cloned into the pUCm-T vector, and then recombined into the eukaryotic expression vector pcDNA3.1 in forward direction . Subsequently, pcDNA3.1/MAD2beta vectors were then transfected into SGC7901 cells by lipofectamine . Sensitivity to drug was detected by MTT assay . Cell cycle alteration and intracellular fluorescence intensity were determined by FACS . RESULTS: A fragment of 0.53 Kb was obtained and confirmed by DNA sequencing which was a new alternative splicing form of MAD2 named as MAD2beta . pcDNA3.1/MAD2beta transfected SGC7901 cells (SGC7901/MAD2beta) were more resistant to ADR, VCR and MMC than the control cells (SGC7901/pcDNA3.1), and also ADR fluorescence intensity of SGC7901/MAD2beta cells was lower (P < 0.05) than that of SGC7901/pcDNA3.1 cells . CONCLUSION: MAD2beta could increase the multidrug resistance of SGC7901 cell line.

Zhonghua Zhong Liu Za Zhi, 2004 Jun, 26(6), 353 - 5
{Correlation between uptake of 99Tcm-MIBI and expression of multidrug resistant protein in breast cancer}; Zhang XM et al.; OBJECTIVE: To assess the correlation between uptake of (99)Tc(m)-MIBI and expression level of multidrug resistant protein in breast cancer . METHODS: Thirty pathologically confirmed patients with primary invasive ductal carcinoma were examined by (99)Tc(m)-MIBI scintigraphy at 15 min and 90 min after injecting the tracer . The uptake of (99)Tc(m)-MIBI at the region of interest (ROI) was evaluated as tumor to normal background (T/N) ratio . Retention index (RI) was calculated from the early uptake ratio (EUR) and updelayed take ratio (DUR) . The expression of P-glycoprotein (P-gp) and multidrug resistant-associated protein (MRP) was detected by immunohistochemistry and the results were expressed as optical density (A) . The data were analyzed by t test, Pearson correlation and partial correlation analysis . RESULTS: The expression of P-gp and MRP in tumor tissue was 0.1183 +/- 0.0700 and 0.1195 +/- 0.0522, respectively . In group with RI < 0 . the expression of P-gp and MRP was 0.2181 +/- 0.0384 and 0.1718 +/- 0.0479, respectively . In group with RI >/= 0, the expression of P-gp and MRP was 0.1057 +/- 0.0217 and 0.0967 +/- 0.0362, respectively . There were significant differences between the two groups (t = 6.61, P = 0.0001; t = 5.01, P = 0.002) . There was positive correlation between P-gp and RI (r = -0.919, P = 0.001), DUR (r = -0.675, P = 0.001), MRP(r = 0.549, P = 0.001), respectively, but no correlation between P-gp and EUR (r = -0.097, P = 0.610) . There was positive correlation between MRP and RI (r = -0.547, P = 0.002), but no correlation between MRP and EUR (r = 0.292, P = 0.117) or DUR (r = -0.173, P = 0.361) . Partial correlation analysis showed that there was high positive correlation between P-gp and RI (r = -0.8847, P = 0.001), but no significant correlation between MRP and RI (r = -0.1296, P = 0.512) . CONCLUSION: The study suggests that increased level of P-gp expression may contribute to a low accumulation of (99)Tc(m)-MIBI in breast cancer, but no correlation with the expression level of MRP . Thus (99)Tc(m)-MIBI scintigraphy may predict the MDR development associated with P-gp expression in breast carcinoma.

Zhonghua Zhong Liu Za Zhi, 2004 Jun, 26(6), 328 - 32
{Identification of differentially expressed genes involved in multidrug resistance in K562/A02 by cDNA microarray}; Tan YH et al.; OBJECTIVE: To study the molecular mechanism underlying multidrug resistance (MDR) and identify unknown genes that might be involved in drug resistance development in K562/A02 cells . METHODS: K562/A02 was induced by gradually increasing the ADM concentration in culture medium of K562 cells, the differential expression of associated genes between K562 and K562/A02 was determined with cDNA microarray . Overexpression of neurofilament protein NF-H gene in K562/A02 cells was confirmed with RT-PCR and immunocytochemistry . Anti-sense oligodeoxynucleotides were transfected into K562/A02 cells by lipofectamine in order to further analyze the role of NF-H in drug resistance . RESULTS: Comparing with the expression profiles, we found upregulation of 5 transcripts and downregulation of 7 transcripts in response to MDR of K562/A02 cells . The overexpression of NF-H, one of the 5 upregulated genes, was confirmed . After being treated with antisense oligodeoxynucleotides of NF-H and mdr1, the cellular adriamycin concentration increased significantly, but antisense NF-H alone did not have significant effect on drug resistance phenotype . CONCLUSION: The development of MDR in K562/A02 cells is multifactorial . NF-H may be involved in the drug resistance of K562/A02, which may provide a new marker of diagnosis and a new target of therapy.

J Clin Periodontol, 2004 Sep, 31(9), 758 - 63
P-glycoprotein drug transporter MDR1 gene polymorphism in renal transplant patients with and without gingival overgrowth; Drozdzik M et al.; OBJECTIVE: To determine whether there is association between genotypes of drug transporter multidrug resistant (MDR)1 gene coding drug transporter P-glycoprotein and gingival overgrowth in kidney transplant patients . METHODS: Fifty-four unrelated kidney transplant patients suffering from gingival overgrowth as well 120 control transplant patients without overgrowth were enrolled into the study . Gingival overgrowth was assessed by two independent periodontal specialists at 6 months after transplantation . During the post-transplant period all patients were given medication, which included cyclosporine A, diltiazem or verapamil, prednisone, azathioprine . MDR1 C3435T polymorphism was determined using the polymerase chain reaction-restriction fragment length polymorphism assay . RESULTS: In kidney transplant patients suffering from gingival overgrowth mean score of gingival overgrowth was 1.43 +/- 0.63, whereas in control subjects was 0.0 . Patients with gingival overgrowth induced by immunosuppressive medication were characterized by similar distribution of MDR1 genotypes . There were no significant differences of 3435CC, 20.4% and 22.5%, 3435CT, 61.1% and 54.2% and 3435TT, 18.5% and 23.3% genotypes (frequencies) between patients with and without gingival overgrowth . The risk of gingival overgrowth was the highest among patients carrying 3435CT genotype (OD 1.33), but did not differ markedly from the other genotypes, i.e . 3435CC (OD 0.88) and 3435TT (OD 0.75) . Likewise to genotypes, distribution of alleles was similar in patients with gingival overgrowth and healthy gingiva . The wild-type allele 3435C was found in 50.9% and 49.6% of subjects whereas the mutated allele 3435T was revealed in 49.1% and 50.4% of patients with and without gingival overgrowth, respectively . The evaluated risk of gingival overgrowth in patients with 3435C allele was 1.06 versus 0.95 in those with healthy gingiva . The medication regimen administered in both groups of the study was comparable . Immunohistochemical studies revealed expression of P-glycoprotein in ducts of the salivary gland . CONCLUSION: No association between the MDR1 gene polymorphism and gingival overgrowth was revealed in kidney transplant patients administered cyclosporine A as a principal immunosuppressive agent . Further studies are needed to elucidate the role of P-glycoprotein in drug transport in salivary glands.

Mol Pharmacol, 2004 Nov, 66(5), 1332 - 9 Epub 2004 Aug 12.
Modulation of cellular cholesterol alters P-glycoprotein activity in multidrug-resistant cells; Troost J et al.; The drug transporter P-glycoprotein (ABCB1) plays an important role in drug distribution and elimination, and when overexpressed it may confer multidrug resistance (MDR) . P-glycoprotein is localized in the plasma membrane, especially within rafts and caveolae, characterized as detergent-resistant membranes (DRMs) . This study investigated the effect of cholesterol depletion and repletion as well as saturation on subcellular localization and function of P-glycoprotein to determine the effect of DRM localization on P-glycoprotein-mediated drug efflux . In L-MDR1 overexpressing human P-glycoprotein, cholesterol depletion removed P-glycoprotein from the raft membranes into non-DRM fractions, whereas repletion fully reconstituted raft localization . P-glycoprotein function was assessed by realtime monitoring with confocal laser scanning microscopy using BODIPY-verapamil as substrate . Cholesterol depletion reduced P-glycoprotein function in L-MDR1 cells resulting in intracellular substrate accumulation (159% +/- 43, p < 0.001; control = 100%) . Cholesterol repletion reduced intracellular substrate fluorescence (120% +/- 36, p < 0.001) and restored the transporter activity . Addition of surplus cholesterol (saturation) even enhanced drug efflux in L-MDR1 cells, leading to reduced intracellular accumulation of BODIPY-verapamil (69% +/- 10, p < 0.001) . Transport of BODIPY-verapamil in cells not expressing human P-glycoprotein (LLC-PK1) was not susceptible to cholesterol alterations . These results demonstrate that cholesterol alterations influence P-glycoprotein localization and function, which might contribute to the large interindividual variability of P-glycoprotein activity known from in vivo studies.

Zhonghua Liu Xing Bing Xue Za Zhi, 2004 Jul, 25(7), 582 - 5
{Study on the epidemiology and determinants of drug-resistant tuberculosis in northern rural area of Jiangsu province}; Yang BF et al.; OBJECTIVE: To understand the determinants and epidemiology of drug-resistant tuberculosis (TB) in rural area . METHODS: All the diagnosed TB patients in a county with directly observed treatment (DOTS) short-course program in 2002 and a sample of patients in another county without DOTS program located in northern Jiangsu province were surveyed with questionnaires . Drug susceptibility testing (DST) for positive cultures were performed by standardized proportion method . Univariable analysis and multivariate nonconditional logistic regression modeling were applied for data analysis . RESULTS: Among the 152 patients with DST results, 32.9% of the cases showed resistance to at least one of the first-line anti-tuberculosis drugs with 26.3% to isoniazid, 18.4% to rifampin and 17.1% to both isoniazid and rifampin respectively . Previous treatments for TB and residence in the county without DOTS program were independent risk factors for isoniazid and rifampin resistance . TB patients showing indifferent to their health and delayed health seeking for more than 1 month were more likely to have rifampin resistance . Independent predictors of multidrug-resistant TB would include delayed health seeking for more than 1 month (OR = 4.66, 95% CI: 1.26 - 17.24), residing in the county without a DOTS program (OR = 3.01, 95% CI: 1.10 - 8.22), indifference to their health condition (OR = 5.13, 95% CI: 1.06 - 24.90) and suffering from chronic diseases (OR = 0.22, 95% CI: 0.05 - 0.87) . CONCLUSION: Drug-resistant TB was quite serious in this rural areas, mainly associated with man-made factors but partly due to the availability of the transmission.

Trans R Soc Trop Med Hyg, 2003 Sep-Oct, 97(5), 592 - 4
Artesunate-atovaquone-proguanil rescue treatment of multidrug-resistant Plasmodium falciparum malaria in pregnancy: a preliminary report; McGready R et al.; Pregnant women are particularly vulnerable to malaria infections . Multidrug resistance in Plasmodium falciparum seriously compromises treatment in some endemic areas . Between April 1999 and October 2001, we treated and prospectively followed 27 Karen pregnant women with multiple recrudescent P . falciparum infections who were resistant to all other antimalarials with a triple combination of artesunate-atovaquone-proguanil . The treatment was well tolerated and we found no evidence of toxicity for the mothers and the fetus . All but 1 woman were cured (cure rate 96%, 95% CI 89-100) . The triple combination of artesunate (4 mg/kg/d), atovaquone (20 mg/kg/d), and proguanil (8 mg/kg/d) may provide a much needed, albeit expensive, 3-d rescue treatment for pregnant women exposed to multidrug- resistant P . falciparum malaria.

Int J Tuberc Lung Dis, 2004 Aug, 8(8), 1012 - 6
Costs of patients hospitalized for multidrug-resistant tuberculosis; Rajbhandary SS et al.; SETTING: From 1993 through 1998, 1846 cases of multidrug-resistant tuberculosis (MDR-TB) were reported in the United States . Costs associated with MDR-TB are likely to be much higher than for drug-susceptible tuberculosis due to longer hospitalization, longer treatment with more expensive and toxic medications, greater productivity losses, and higher mortality . OBJECTIVE: To measure the societal costs of patients hospitalized for MDR-TB . DESIGN: We detailed in-patient costs for 13 multidrug-resistant patients enrolled in a national study . We estimated costs for physician care, out-patient treatment, and productivity losses for survivors and for deceased patients . RESULTS: In-patient costs averaged US$25,853 per person and $1036 per person-day of hospitalization . Outpatient costs per person ranged from $5744 to $41,821 (average $19028, or $44 a day) . Direct medical costs averaged $44,881; indirect costs for those who survived averaged $32,964, and indirect costs for those who died averaged $686,381 per person . Total costs per person ranged from $28,217 to $181492 (average $89,594) for those who survived, and from $509490 to $1278066 (average $717555) for those who died . CONCLUSION: The societal costs of MDR-TB varied, mostly because of length of therapy (including in-patient), and deaths during treatment.

J Gastroenterol Hepatol, 2004 Sep, 19(9), 1016 - 22
Effects of colchicine on the maximum biliary excretion of cholephilic compounds in rats; Tachizawa H et al.; BACKGROUND AND AIM: Colchicine, an inhibitor of intracellular vesicular transport, has been reported to inhibit the biliary excretion of bile acids and organic anions, but the previous findings are controversial . In order to systematically evaluate the effect of colchicine on the biliary excretion of cholephilic compounds, we studied the effect of colchicine on the biliary excretion of substrates of various canalicular transporters, which were administered at or above the excretory maximum in rats . METHODS: Substrates of various canalicular adenosine triphosphate-binding-cassette transporters were infused at or above the rate of maximum excretion into rats, and the effect of colchicine (0.2 mg/100 g), which was intraperitoneally injected 3 h before, on the biliary excretion was studied . Furthermore, the effect of tauroursodeoxycholate (TUDC) co-infusion on the biliary excretion of taurocholate (TC) after colchicine treatment was also studied . RESULTS: The biliary excretion of TC and cholate administered at the rate of 1 micro mol/min/100 g was markedly inhibited by colchicine, whereas that of TUDC was not inhibited even with the infusion rate of 2 micro mol/min/100 g . TUDC co-infusion at the rate of 1 micro mol/min/100 g increased the biliary excretion of TC (1 micro mol/min/100 g), which was decreased by the colchicine pretreatment . The biliary excretory maximum of taurolithocholate-sulfate and sulfobromophthalein, substrates of the multidrug resistance protein 2, of erythromycin, a substrate of the P-glycoprotein, and of indocyanine green were not affected by colchicine . CONCLUSIONS: The different excretory maximums of TC and TUDC and the different effect of colchicine on the excretion of these bile acids are considered to be a result of different regulatory mechanisms of vesicular targeting of the bile salt export pump to the canalicular membrane by these bile acid conjugates . The vesicular targeting of the multidrug resistance protein 2 and the P-glycoprotein to the canalicular membrane is considered to be colchicine insensitive in the absence of bile acid coadministration.

J Pharmacol Exp Ther, 2004 Dec, 311(3), 1203 - 10 Epub 2004 Dec.
Hepatobiliary disposition of the metabolically stable opioid peptide {D-Pen2, D-Pen5}-enkephalin (DPDPE): pharmacokinetic consequences of the interplay between multiple transport systems; Hoffmaster KA et al.; {D-Pen2,D-Pen5}-Enkephalin (DPDPE) is excreted extensively into the bile . Although DPDPE is transported by P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (Mrp2) has been identified as an important mechanism for DPDPE transport across the canalicular membrane of the hepatocyte . The present studies determined the relative impact of Mrp2 and P-gp on the hepatobiliary disposition of {3H}DPDPE in isolated perfused rat livers (IPLs) . Perfusate clearance of {3H}DPDPE was not different between livers from control and Mrp2-deficient (TR-) rats . Biliary excretion of {3H}DPDPE in IPLs from Wistar control rats was rapid and extensive . However, when {3H}DPDPE was administered to livers from TR- rats, the rate and extent of excretion decreased significantly . Surprisingly, in the presence of the P-gp inhibitor GF120918 {N-(4-{2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl}-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide}, biliary excretion of {3H}DPDPE was not inhibited in control livers . In contrast, administration of GF120918 to TR- livers further reduced the maximal excretion rate and decreased net biliary excretion of {3H}DPDPE by 87% . GF120918 administration caused an unexpected increase in perfusate clearance in both control and TR- rat livers . At distribution equilibrium, {3H}DPDPE liver/perfusate partitioning was higher in GF120918-treated livers . Results of pharmacokinetic modeling were consistent with the hypothesis that GF120918 inhibited a {3H}DPDPE basolateral excretion mechanism . Mrp2 is the primary mechanism for {3H}DPDPE biliary excretion, and P-gp facilitates excretion of {3H}DPDPE only in the absence of functional Mrp2 . {3H}DPDPE is a substrate for a basolateral efflux mechanism that is sensitive to inhibition by GF120918 . These data emphasize the importance of using appropriate model systems and comprehensive pharmacokinetic modeling in elucidating the complex interplay between multiple transport systems.

Eukaryot Cell, 2004 Aug, 3(4), 880 - 92
Differential regulation of ceramide synthase components LAC1 and LAG1 in Saccharomyces cerevisiae; Kolaczkowski M et al.; In Saccharomyces cerevisiae, the essential ceramide synthase reaction requires the presence of one of a homologous pair of genes, LAG1 and LAC1 . Mutants that lack both of these genes cannot produce ceramide and exhibit a striking synthetic growth defect . While the regulation of ceramide production is critical for the control of proliferation and for stress tolerance, little is known of the mechanisms that ensure proper control of this process . The data presented here demonstrate that the pleiotropic drug resistance (Pdr) regulatory pathway regulates the transcription of multiple genes encoding steps in sphingolipid biosynthesis, including LAC1 . The zinc cluster transcriptional activators Pdr1p and Pdr3p bind to Pdr1p/Pdr3p-responsive elements (PDREs) in the promoters of Pdr pathway target genes . LAC1 contains a single PDRE in its promoter, but notably, LAG1 does not . Reporter gene, Northern blot, and Western blot assays indicated that the expression level of Lac1p is approximately three times that of Lag1p . Detailed analyses of the LAC1 promoter demonstrated that transcription of this gene is inhibited by the presence of the transcription factor Cbf1p and the anaerobic repressor Rox1p . LAG1 transcription was also elevated in cbf1Delta cells, indicating at least one common regulatory input . Although a hyperactive Pdr pathway altered the profile of sphingolipids produced, the loss of either LAC1 or LAG1 alone failed to produce further changes . Two other genes involved in sphingolipid biosynthesis (LCB2 and SUR2) were found to contain PDREs in their promoters and to be induced by the Pdr pathway . These data demonstrate extensive coordinate control of sphingolipid biosynthesis and multidrug resistance in yeast.

Ai Zheng, 2004 Aug, 23(8), 963 - 7
{Predictive value of drug resistance-related genes expression in neoadjuvant chemotherapy in patients with non-small cell lung cancer of stage III}; Peng ZM et al.; BACKGROUND & OBJECTIVE: P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and lung resistant protein (LRP) play important roles in multidrug resistance (MDR) . This study was to determine P-gp, MRP, and LRP expression in patients with non-small cell lung cancer (NSCLC) of stage III, and evaluate their predictive value in neoadjuvant chemotherapy . METHODS: Immunohistochemical analyses were performed on 31 patients with NSCLC of stage III before, and after neoadjuvant chemotherapy . RESULTS: The frequency of P-gp, MRP, and LRP expression were 29.0% (9/31), 45.2% (14/31), and 38.7% (12/31) before chemotherapy, and were 61.3% (19/31), 51.6% (16/31), and 41.9% (13/31) after chemotherapy . Of 31 patients, 10 (10/31, 32.3%) expressed both MRP and LRP before chemotherapy, which indicated significant positive correlation between MRP and LRP expression (r=0.061, P< 0.001) . In patients with P-gp, MRP, or LRP expression before chemotherapy, the response rates towards chemotherapy were 44.4% (4/9), 28.6% (4/14), and 16.7% (2/12) . Of 10 patients with both MRP and LRP expression, only 1 (1/10, 10.0%) responded to chemotherapy . The median survival time of patients who responded to chemotherapy was 31 months, while that of patients who did not responded to chemotherapy was 15 months, that of patients who didn't receive neoadjuvant chemotherapy before surgery was 18 months . CONCLUSION: Patients with both MRP and LRP expression are probably resistant to chemotherapy, the value of neoadjuvant chemotherapy is limit in such patients.

Ai Zheng, 2004 Aug, 23(8), 905 - 9
{Establishment of in vivo adriamycin-induced multidrug resistance models of subcutaneous and hepatic transplanted human liver cancer in nude mice}; Zhai BJ et al.; BACKGROUND & OBJECTIVE: Multidrug resistance (MDR) phenotype is the obstacle of chemotherapy in tumors and inspired research interesting . This study was to establish multidrug resistance (MDR) models induced with adriamycin in vivo of subcutaneous or in situ hepatic transplanted human liver cancer in nude mice (BALB/C nu/nu), and explore the biological characteristics and mechanism of multidrug resistance, which can provide an ideal animal model for the basic,and clinical study of MDR . METHODS: After successful performing either subcutaneous or hepatic transplantation in nude mice with liver cancer cell line HepG2, adriamycin (ADM) was injected into abdominal cavity to induce multidrug resistance . The chemosensitivity of HepG2 and ADM-cultured HepG2 cells was determined by MTT assay . P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), and cell pump-efflux to Rhodamine 123 (R123) were determined by flow cytometry . The mRNA expression of P-gp, MRP, and LRP was assessed with reverse transcriptase polymerase chain reaction(RT-PCR) . RESULTS: Most biological characteristics of either subcutaneous or hepatic transplanted HepG2 liver cancer in nude mice like the parent HepG2 liver cancer . Both of them showed significant multidrug resistance . Among them, the resistance index of ADM was 26.4 times in hepatic transplanted group, and 24.6 times in subcutaneous transplanted group higher than that of parent cell line HepG2 . In subcutaneous,and hepatic transplanted ADM-cultured groups, P-gp {(77.3+/-2.1)%, and (78.1+/-1.9)%}, MRP {(72.1+/-4.3)%, and (72.7+/-5.0)%}, and LRP {(31.1+/-1.0)%, and (32.2+/-1.4)%} positive cells increased significantly, their mRNA positive expression increased also, and the cell pump-efflux to R123 strengthened . However, no significant difference of above data was observed between subcutaneous,and hepatic transplanted tumor group (P >0.05) . CONCLUSION: Both MDR subcutaneous and hepatic HepG2 transplanted models induced by in vivo injection of adriamycin in nude mice have the same biological characteristics as parent HepG2 liver cancer . These 2 animal models could be used as in vivo MDR models to explore the mechanism of MDR and reversing therapeutic methods.

Ai Zheng, 2004 Aug, 23(8), 900 - 4
{Methylated oligonucleotide inhibiting expression of human MRP2 and reversing multidrug resistance in hepatocellular carcinoma Cell HepG2}; Li WH et al.; BACKGROUND & OBJECTIVE: Accumulating evidences showed that the overexpression of multidrug resistance-associated protein 2 (MRP2) was the main cause of multidrug resistance in hepatocellular carcinoma (HCC); and the methylated cytosine at cytosine-guanine (CpG) dinucleotides might silence the gene expression . This study was to evaluate the inhibiting effect of methylated oligonucleotide (MON) on MRP2 gene transcription . METHODS: MON complementary to human MRP2 promoter region was designed . The non- methylated oligonucleotide (NON) carried same nucleotide acid sequence with MON, but the cytosines were not methylated . Cells from human HCC cell line HepG2 were divided into 3 groups: control group, NON group, and MON group . HepG2 cells were transfected with liposome-encapsulated oligonucleotide, cytotoxicity was determined by MTT assay . Reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry (FCM) were used to analyze the expression of MRP2 . RESULTS: MON specifically inhibited MRP2 gene transcription and down-regulated the expression of MRP2 in a concentration- dependent manner in vitro, whereas NON had no effect . The positive rates of MRP2 protein were 18.0%, 9.15%, 5.73%, 3.73%, and 2.56% respectively after transfected with 1, 2, 4, 8, and 16 micromol/L MON . It was 24.5% in NON group . The 50% inhibitory concentration (IC50) of epirubicin, hydroxyl- camptothecin, and carboplatin were reduced in MON group . CONCLUSIONS: MON can inhibit MRP2 gene transcription, enhance chemosensitivity, and reverse multidrug resistance of HCC cells,it may become a new gene therapeutic agent for HCC.

Oncol Res, 2004, 14(7-8), 355 - 62
Screening novel, potent multidrug-resistant modulators from imidazole derivatives; Chen LM et al.; The overexpression of P-glycoprotein (P-gp) by tumor cells results in multidrug resistance (MDR) to structurally unrelated anticancer drugs . Combined therapy with MDR-related cytotoxins and MDR modulators is a promising strategy to overcome clinical MDR . This study was designed to screen potent MDR modulators from imidazole derivatives . Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay . The intracellular accumulation of doxorubicin (Dox) was detected by fluorescence spectrophotometry . The function of P-gp was examined by Rhodamine 123 accumulation detected with flow cytometry (FCM) . Among imidazole derivatives, FG020326, FG020327, and FG020318 were found to possess three- to fourfold stronger reversal MDR activity than verapamil, a well-known positive MDR modulator . Imidazole derivatives significantly increased the Dox accumulation and inhibited P-gp function exhibited by the increase of Rhodamine accumulation in MDR cells . The fold reversal of MDR was relative with the increase of Rhodamine accumulation . FG020326, FG020327, and FG020318 showed potent MDR reversal activity in vitro . Their mechanism of MDR reversal is associated with the inhibition of P-gp function and the increase of anticancer accumulation . These results suggest FG020326, FG020327, and FG020318 are promising to further study and develop.

Oncol Res, 2004, 14(7-8), 331 - 43
Age-related differences in vincristine toxicity and biodistribution in wild-type and transporter-deficient mice; Muramatsu T et al.; The impact of mouse multidrug resistance genes mdrla/b and mrpl on age-related differences in the toxicity and biodistribution of vincristine (VCR) was evaluated in wild-type, mrpl(-/-), mdrla/b(-/-), and combined mdrla/b(-/-), mrpl(-/-) weanling and adult mice given a single IP dose of VCR ranging from 0.0625 to 6 mg/kg . Weanling mice of all four genotypes were more sensitive than adult animals as determined by survival rate, average time of death, and pathologic findings . Wild-type animals were the least sensitive and combined mdrla/b(-/-), mrpl(-/-) mice the most sensitive to VCR toxicity . Mdrla/b(-/-) and mrpl(-/-) genotypes exhibited intermediate sensitivities, with mdrla/b(-/-) mice being more sensitive than mrpl(-/-) animals to the vinca alkaloid . Administration of {3H}VCR to wild-type and mdrla/b(-/-), mrpl(-/-) animals revealed relatively greater accumulation of radioactive VCR equivalents in weanlings over adults in several tissues, with weanling mdrla/b(-/-), mrpl(-/-) lung and heart exhibiting the greatest enhanced accumulation of 26- and 15-fold over adults, respectively . A similar cardiopulmonary differential accumulation of VCR was not observed in wild-type weanlings to adults . Semiquantitative RT-PCR expression analyses of ABC transporter genes in weanling and adult tissues of wild-type and combined mdrla/b(-/-), mrpl(-/-) mice did not reveal major age-related differences in these ABC transporters that would explain the relatively greater toxicity observed in weanling mice . However, the greater cardiopulmonary accumulation of VCR equivalents seen in the combined mdrla/b(-/-), mrpl(-/-) weanlings over that of adults underscores the potential for unique organ and age-related toxicities of this agent in the setting of transporter deficiency.

Cancer Sci, 2004 Aug, 95(8), 679 - 84
Docetaxel enhances the cytotoxicity of cisplatin to gastric cancer cells by modification of intracellular platinum metabolism; Maeda S et al.; We have examined the combined anticancer effects of docetaxel (DOC) and cisplatin (CDDP) in vitro using the gastric cancer cell lines MKN-45, MKN-74, and TMK-1 . Treatment of the cell lines with 30 microg/ml of DOC for 24 h followed by incubation with 3 or 10 microg/ml of CDDP for 24 h showed a clear synergistic effect . Sequence dependency of the agents was observed in these cell lines: DOC followed by CDDP (DC) showed a stronger antitumor effect than CDDP followed by DOC (CD) in all cell lines . To clarify the mechanism of action of the DC combination, total intracellular platinum (Pt) levels were evaluated after treatment with CDDP alone or combined with DC . For the MKN-45 and -74 cell lines, cells treated with DOC (10 microg/ml for 12 h) and then CDDP showed significantly increased intracellular Pt accumulation compared to cells treated with CDDP alone . We also investigated alterations in intracellular glutathione (GSH) concentration in response to DOC and CDDP . MKN-45 and -74 cells pretreated with DOC (10 microg/ml for 12 h) showed significantly increased intracellular GSH levels compared to cells administered CDDP only . To explain these findings, messenger RNA (mRNA) levels for multidrug resistance-associated protein-1 (MRP-1), the ATP-dependent pump for Pt-GSH complexes, were quantified in CDDP-treated MKN-45 cells with and without DOC pretreatment . While CDDP administration increased MRP-1 mRNA expression in MKN-45 cells, MRP-1 was not up-regulated after CDDP administration in DOC pretreated MKN-45 cells . Our results suggested that the enhanced CDDP toxicity due to DOC pretreatment may be related to the accumulation of intracellular Pt-GSH complexes, because DOC appears to suppress the MRP-1 up-regulation induced by CDDP exposure in gastric cancer cells.

Blood, 2004 Dec 1, 104(12), 3603 - 10 Epub 2004 Dec 1.
The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage; Jedlitschky G et al.; Platelet aggregation is initiated by the release of mediators as adenosine diphosphate (ADP) stored in platelet granules . Possible candidates for transport proteins mediating accumulation of these mediators in granules include multidrug resistance protein 4 (MRP4, ABCC4), a transport pump for cyclic nucleotides and nucleotide analogs . We investigated the expression of MRP4 in human platelets by immunoblotting, detecting a strong signal at 170 kDa . Immunofluorescence microscopy using 2 MRP4-specific antibodies revealed staining mainly in intracellular structures, which largely colocalized with the accumulation of mepacrine as marker for delta-granules and to a lower extent at the plasma membrane . Furthermore, an altered distribution of MRP4 was observed in platelets from a patient with Hermansky-Pudlak syndrome with defective delta-granules . Adenosine triphosphate (ATP)-dependent cyclic guanosine monophosphate (cGMP) transport codistributed with MRP4 detection in subcellular fractions, with highest activities in the dense granule and plasma membrane fractions . This transport was inhibited by dipyramidole, indomethacin, and MK571 with median inhibitory concentration (IC(50)) values of 12, 22, and 43 microM, and by ibuprofen . Transport studies with {(3)H}ADP indicated the presence of an orthovanadate-sensitive ADP transporting system, inhibited by dipyramidole, MK571, and cyclic nucleotides . The results indicate a function of MRP4 in platelet mediator storage and inhibition of MRP4 may represent a novel mechanism for inhibition of platelet function by some anti-inflammatory drugs.

Acta Biochim Biophys Sin (Shanghai), 2004 Aug, 36(8), 519 - 28
Identification and characterization of hmr19 gene encoding a multidrug resistance efflux protein from Streptomyces hygroscopicus subsp . yingchengensis strain 10-22; Qin L et al.; The hmr19 gene was cloned from Streptomyces hygroscopicus subsp . yingchengensis strain 10-22, a bacterium strain producing agricultural antibiotics . Sequence similarity comparison indicates that hmr19 gene may encode a predicted protein with 14 putative transmembrane alpha-helical spanners, belonging to the drug:H(+) antiporter-2 family of the major facilitator superfamily . The expression of hmr19 in the mycelium of strain 10-22 was detected by Western blotting analysis . Gene replacement technology was employed to construct an hmr19 disruption mutant . The growth inhibition test against different antibiotics indicated that the mutant strain was 5-20 fold more susceptible to tetracycline, vancomycin and mitomycin C than the parental wild type strain . The mutant took up tetracycline much faster and accumulated more antibiotics than the wild type strain 10-22 . While with the addition of an energy uncoupler, carbonyl cyanide m-chlorophenylhydrazone, the characteristics of the accumulation of {(3)H}tetracycline in these two strains were almost the same . It was thus concluded that hmr19 encoded a multidrug resistance efflux protein.

Biochem Pharmacol, 2004 Sep 1, 68(5), 843 - 55
Reversal of P-glycoprotein-mediated multidrug resistance by Alisol B 23-acetate; Wang C et al.; Herbal drugs were screened for their activity in reversing multidrug resistance (MDR) in P-glycoprotein (P-gp) over-expressing cancer cells . Through bio-assay guided fractionation an active compound was isolated from Rhizoma Alismatis, the underground part of Alisma orientale and the chemical structure of the isolate compound was confirmed by HPLC, LC-MS and NMR as Alisol B 23-acetate (ABA) . ABA restored the sensitivity of MDR cell lines HepG2-DR and K562-DR to anti-tumor agents that have different modes of action but are all P-gp substrates . It restored the activity of vinblastine, a P-gp substrate, in causing G2/M arrest in MDR cells . In a dose-dependent manner, ABA increased doxorubicin accumulation and slowed down the efflux of rhodamin-123 from MDR cells . ABA inhibited the photoaffinity labeling of P-gp by {125I}iodoarylazidoprazosin and stimulated the ATPase activity of P-gp in a concentration-dependent manner, suggesting that it could be a transporter substrate for P-gp . In addition, ABA was also a partial non-competitive inhibitor of P-gp when verapamil was used as a substrate . Our results suggest that ABA may be a potential MDR reversal agent and could serve as a lead compound in the development of novel drugs.

Mol Cell Probes, 2004 Oct, 18(5), 299 - 306
Rapid detection of ethambutol-resistant Mycobacterium tuberculosis strains by PCR-RFLP targeting embB codons 306 and 497 and iniA codon 501 mutations; Ahmad S et al.; Mutations at embB gene codons 306 and 497 and iniA gene codon 501 occur frequently in ethambutol (EMB)-resistant Mycobacterium tuberculosis strains worldwide . The identification of these mutations in resistant strains has been achieved by labor-intensive DNA sequencing or by tedious amplification protocols followed by restriction endonuclease digestion . In this report, we describe PCR-restriction fragment length polymorphism (RFLP)-based methods for determining substitutions at embB codons 306 and 497 and iniA codon 501 directly in BACTEC cultures of M . tuberculosis isolates . The wild-type and mutant alleles are revealed by easily interpretable and different RFLP patterns . The methods optimized initially on reference strains were tested directly on BACTEC cultures of 25 randomly selected clinical M . tuberculosis isolates, seven of which were determined to contain EMB-resistant strains by phenotypic drug susceptibility testing . The PCR-RFLP methods identified mutations in four of seven EMB-resistant strains with three isolates containing mutated embB codon 306 and one isolate containing mutated embB codon 497 . The results of PCR-RFLP were confirmed by DNA sequencing . The worldwide prevalence figures for mutations at embB codons 306 and 497 and iniA codon 501 suggest that nearly half of EMB-resistant M . tuberculosis strains could be identified within one working day even in developing countries equipped with simple PCR technology instead of weeks required for phenotypic drug susceptibility testing . Further, since EMB resistance is also associated with multiple-drug resistance from some geographical locations, detection of EMB resistance may also lead to rapid identification of multidrug-resistant strains of M . tuberculosis.

Br J Cancer, 2004 Sep 13, 91(6), 1205 - 12
No topoisomerase I alteration in a neuroblastoma model with in vivo acquired resistance to irinotecan; Calvet L et al.; CPT-11 (irinotecan) is a DNA-topoisomerase I inhibitor with preclinical activity against neuroblastoma (NB) xenografts . The aim was to establish in vivo an NB xenograft resistant to CPT-11 in order to study the resistance mechanisms acquired in a therapeutic setting . IGR-NB8 is an immature NB xenograft with MYCN amplification and 1p deletion, which is sensitive to CPT-11 . Athymic mice bearing advanced-stage subcutaneous tumours were treated with CPT-11 (27 mg kg(-1) day(-1) x 5) every 21 days (1 cycle) for a maximum of four cycles . After tumour regrowth, a new in vivo passage was performed and the CPT-11 treatment was repeated . After the third passage, a resistant xenograft was obtained (IGRNB8-R) . The tumour growth delay (TGD) was reduced from 115 at passage 1 to 40 at passage 4 and no complete or partial regression was observed . After further exposure to the drug, up to 28 passages, the resistant xenograft was definitively established with a TGD from 17 at passage 28 . Resistant tumours reverted to sensitive tumours after 15 passages without treatment . IGR-NB8-R remained sensitive to cyclophosphamide and cisplatin and cross-resistance was observed with the topoisomerase I inhibitor topotecan . No quantitative or qualitative topoisomerase I modifications were observed . The level of expression of multidrug resistance 1 (MDR1), MDR-associated protein 1 (MRP1) and, breast cancer resistance protein, three members of the ATP-binding cassette transporter family was not modified over passages . Our results suggest a novel resistance mechanism, probably not involving the mechanisms usually observed in vitro.

Pharm Res, 2004 Jul, 21(7), 1294 - 302
P-glycoprotein expression, localization, and function in sandwich-cultured primary rat and human hepatocytes: relevance to the hepatobiliary disposition of a model opioid peptide; Hoffmaster KA et al.; PURPOSE: The isolation of hepatocytes from intact liver involves collagenase digestion of the tissue, resulting in loss of cell polarization and functional vectorial excretion . These studies examined repolarization, localization of P-glycoprotein (P-gp) to the canalicular domain of the hepatocyte, and re-establishment of vectorial transport in sandwich-cultured (SC) rat and human primary hepatocytes . METHODS: Protein localization and expression were determined in SC hepatocytes by confocal microscopy and Western blotting, respectively . Transporter function was evaluated by measuring {D-penicillamine2,5}enkephalin (3H-DPDPE) and 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF) biliary excretion in SC hepatocytes . RESULTS: P-gp and the canalicular marker protein dipeptidyl peptidase IV (DPPIV) co-localized by Day 3 and Day 6 in SC rat hepatocytes and SC human hepatocytes, respectively, consistent with canalicular network formation visualized by light microscopy . Co-localization of multidrug resistance associated protein 2 (MRP2) and P-gp in SC human hepatocytes was observed on Day 6 in culture . Expression levels of P-gp increased slightly in both species over days in culture; similar expression was observed for MRP2 in SC human hepatocytes . Oatp1a1 expression in SC rat hepatocytes was maintained over days in culture, whereas Oatp1a4 expression decreased . OATP1B1 expression decreased slightly on Day 3 in SC human hepatocytes . OATP1B3 expression was constant in SC human hepatocytes . In vitro biliary excretion of the opioid peptide 3H-DPDPE correlated with the proper localization of canalicular proteins in both species . Excretion of CDF in SC human hepatocytes confirmed network formation and MRP2 function . CONCLUSIONS: These studies indicate that SC hepatocytes repolarize and traffic functional canalicular transport proteins to the appropriate cellular domain.

Pharm Res, 2004 Jul, 21(7), 1263 - 73
Combined effects of multiple flavonoids on breast cancer resistance protein (ABCG2)-mediated transport; Zhang S et al.; PURPOSE: The purpose of this study was to determine the dynamic parameter (EC50) of flavonoids apigenin, biochanin A, chrysin, genistein, kaempferol, hesperetin, naringenin, and silymarin for breast cancer resistance protein (BCRP) inhibition when used alone, and to evaluate their potential interactions (additive, synergistic, or antagonistic) with regards to BCRP inhibition when used in multiple-flavonoid combinations . METHODS: The effects of flavonoids on BCRP-mediated transport were examined by evaluating their effects on mitoxantrone accumulation and cytotoxicity in MCF-7 MX100 cells overexpressing BCRP . The EC50 values of these flavonoids for increasing mitoxantrone accumulation were estimated using a Hill equation . The potential interactions among multiple flavonoids with regard to BCRP inhibition were assessed by isobologram and Berenbaum's interaction index methods . RESULTS: The EC50 values of these flavonoids for increasing mitoxantrone accumulation ranged from 0.39+/-0.13 microM to 33.7+/-2.78 microM . Quantitative analysis of the combined effects of multiple flavonoids on mitoxantrone accumulation indicated that these flavonoids act additively in inhibiting BCRP when given as 2-, 3-, 5-, or 8-flavonoid combinations with equimolar concentrations of all constituents . The results of the mitoxantrone cytotoxicity studies were consistent with these findings . CONCLUSIONS: The additive effects of multiple flavonoids for BCRP inhibition suggests that prediction of BCRP-mediated food (herbal product)-drug interactions should also take into consideration the presence of multiple flavonoids and provides a rationale for using "flavonoid cocktails" as a potential approach for multidrug resistance reversal in cancer treatment.

Cancer Chemother Pharmacol . 2004 Jul 28; {Epub ahead of print}
A phase I and pharmacologic study of the MDR converter GF120918 in combination with doxorubicin in patients with advanced solid tumors; Planting AS et al.; BACKGROUND . Resistance to chemotherapy can partly be explained by the activity of membrane bound P-glycoprotein . Competitive inhibition of P-glycoprotein, by multidrug resistance (MDR) converters, may overcome this MDR . Previously studied MDR converters either have serious intrinsic side effects or considerably influence the pharmacokinetics of cytotoxic agents at concentrations theoretically required to convert MDR . GF120918 is a third-generation MDR converter with high affinity for P-glycoprotein and can be given orally . We performed a phase 1 study with escalating doses of GF120918 in combination with doxorubicin . PATIENTS AND METHODS . The study group comprised 46 patients with advanced solid tumors . Doxorubicin was administered on day 1 (cycle 1), GF120918 on days 22-24 (cycle 2), and on days 29-33 with doxorubicin administered on day 31 (cycle 3) . Pharmacokinetics of both GF120918 and doxorubicin were studied . The starting daily dose of GF120918 was 50 mg and was to be increased in subsequent cohorts until a steady state plasma level of 100 ng/ml was reached . The starting dose of doxorubicin was 50 mg/m(2) and was to be increased after reaching the target dose level of GF120918 . RESULTS . In 37 of the 46 patients, full pharmacokinetic data from the three scheduled cycles were obtained . Pharmacokinetics of GF120918 showed a less than linear increase in C (max) with increasing doses, with considerable interpatient variation . The target steady-state plasma level for GF120918 was exceeded in 12 out of 19 patients who received 400 mg GF120918 alone twice daily and in 12 of 17 patients who received 400 mg GF120918 twice daily in combination with doxorubicin . GF120918 pharmacokinetics were not influenced by coadministration of doxorubicin . The doxorubicin AUC was only marginally influenced by GF120918 and only at the highest dose levels . In these patients there was a significant increase in the AUC of doxorubicinol in cycle 3 as compared to cycle 1 . Hematologic toxicity mainly consisted of neutropenia and was more severe in cycle 3 than in cycle 1 (13 vs 5 patients with grade 4 neutropenia, P=0.003) . Neutropenic fever was the dose-limiting toxicity at a doxorubicin dose of 75 mg/m(2) with 400 mg GF120918 twice daily . The toxicity of GF120918 was limited to somnolence in eight patients and occasional gastrointestinal complaints . CONCLUSION . GF120918 is an MDR converter with only minimal side effects at a dose level yielding concentrations able to convert the action of P-glycoprotein in vitro . A doxorubicin dose of 60 mg/m(2) on day 3 in combination with 400 mg GF120918 twice daily on days 1-5 is an acceptable regimen for further clinical trials.

Proc Natl Acad Sci U S A, 2004 Aug 10, 101(32), 11725 - 30 Epub 2004 Aug 02.
Breed distribution and history of canine mdr1-1Delta, a pharmacogenetic mutation that marks the emergence of breeds from the collie lineage; Neff MW et al.; A mutation in the canine multidrug resistance gene, MDR1, has previously been associated with drug sensitivities in two breeds from the collie lineage . We exploited breed phylogeny and reports of drug sensitivity to survey other purebred populations that might be genetically at risk . We found that the same allele, mdr1-1Delta, segregated in seven additional breeds, including two sighthounds that were not expected to share collie ancestry . A mutant haplotype that was conserved among affected breeds indicated that the allele was identical by descent . Based on breed histories and the extent of linkage disequilibrium, we conclude that all dogs carrying mdr1-1Delta are descendants of a dog that lived in Great Britain before the genetic isolation of breeds by registry (ca . 1873) . The breed distribution and frequency of mdr1-1Delta have applications in veterinary medicine and selective breeding, whereas the allele's history recounts the emergence of formally recognized breeds from an admixed population of working sheepdogs.

Cancer Res, 2004 Aug 1, 64(15), 5063 - 7
Peloruside A does not bind to the taxoid site on beta-tubulin and retains its activity in multidrug-resistant cell lines; Gaitanos TN et al.; Peloruside A (peloruside), a microtubule-stabilizing agent from a marine sponge, is less susceptible than paclitaxel to multidrug resistance arising from overexpression of the P-glycoprotein efflux pump and is not affected by mutations that affect the taxoid binding site of beta-tubulin . In vitro studies with purified tubulin indicate that peloruside directly induces tubulin polymerization in the absence of microtubule-associated proteins . Competition for binding between peloruside, paclitaxel, and laulimalide revealed that peloruside binds to a different site on tubulin to paclitaxel . Moreover, laulimalide was able to displace peloruside, indicating that peloruside and laulimalide may compete for the same or overlapping binding sites . It was concluded that peloruside and laulimalide have binding properties that are distinct from other microtubule-stabilizing compounds currently under investigation.

Toxicol Appl Pharmacol, 2004 Aug 15, 199(1), 44 - 51
Disruption of mitochondrial membrane potential during apoptosis induced by PSC 833 and CsA in multidrug-resistant lymphoid leukemia; Bustamante J et al.; Previous findings from our laboratory demonstrated that when used at low concentration (0.1 microg ml(-1)), CsA as well as its analog PSC 833 were able to revert the MDR phenotype, while at high concentration (1 microg ml(-1)) were able to induce apoptosis . CsA induced apoptosis in leukemia cell lines sensitive (LBR-) and resistant to vincristine (LBR-V160), and doxorubicin (LBR-D160), while PSC 833 only induced apoptosis in vincristine-resistant cell line (LBR-V160) . In this work, we investigated mitochondrial-associated mechanisms during CsA- and PSC 833-induced apoptosis . Mitochondrial function was evaluated by recording changes in its transmembrane potential, cytochrome c release, and caspase activation cascade . Results showed that CsA- and PSC 833-induced apoptosis was associated with mitochondrial depolarization, through potentiometric measurements with JC-1 and DiOC(6) probes . Collapse of mitochondrial potential in these cell lines after CsA treatment was followed by cytochrome c release to the cytosol, reaching an increase of 2.61-fold in LBR-, 1.98-fold in LBR-V160, and 3.01-fold in the case of LBR-D160 . However, in the case of PSC 833 treatment, induction of apoptosis in LBR-V160 was associated with mitochondrial depolarization followed by a lower cytochrome c release of 1.15-fold as compared with untreated cells . Caspase 3 activation was clearly observed in LBR-, LBR-V160, and LBR-D160 after CsA treatment, while in LBR-V160, PSC 833 was less effective inducing activation of this caspase . Neither caspase 6 nor 8 activity was observed in these three cell lines . Our results suggest that during CsA- and PSC 833-induced apoptosis, mitochondrial dysfunction occurs . This is mediated through mitochondrial events, associated with an evident decrease in DeltaPsi(m), cytochrome c release and caspase 3 activation.

Lancet, 2004 Jul 31, 364(9432), 438 - 47
Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number; Price RN et al.; BACKGROUND: The borders of Thailand harbour the world's most multidrug resistant Plasmodium falciparum parasites . In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years . A combination of artesunate with mefloquine now cures more than 95% of acute infections . For both treatment regimens, the underlying mechanisms of resistance are not known . METHODS: The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years . pfmdr1 copy number was assessed by a robust real-time PCR assay . Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism . FINDINGS: Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro . In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6.3 (95% CI 2.9-13.8, p<0.001) after mefloquine monotherapy and 5.4 (2.0-14.6, p=0.001) after artesunate-mefloquine therapy . Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo . INTERPRETATION: Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria . RELEVANCE TO PRACTICE: Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat . Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance . In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with mefloquine . Increase in pfmdr1 copy number predicts failure even after chemotherapy with the highly effective combination of mefloquine and 3 days' artesunate . Monitoring of pfmdr1 copy number will be useful in epidemiological surveys of drug resistance in P falciparum, and potentially for predicting treatment failure in individual patients.

J Hepatol, 2004 Aug, 41(2), 201 - 8
Differential expression of bile salt and organic anion transporters in developing rat liver; Gao B et al.; BACKGROUND/AIMS: Differentiated hepatocytes express distinct transport systems at their basolateral and canalicular membrane domains . Here, we investigated the ontogenesis of the polar expression of hepatocellular organic anion and bile salt transport systems in rat liver . METHODS: mRNA levels (real time PCR) and protein expression (immunofluorescence microscopy) were investigated for the Na(+)-taurocholate cotransport protein (Ntcp), the organic anion transporting polypeptides (Oatp1a1, Oatp1a4, Oatp1b2), the multidrug resistance associated proteins (Mrp2, Mrp6) and the bile salt export pump (Bsep) . RESULTS: Expression of mRNA and protein was detected first for Oatp1b2, Mrp2 and Mrp6 at embryonic day 16 (E16), followed by Ntcp, Oatp1a1 and Bsep at E20 and by Oatp1a4 at postnatal day 5 (P5) . Intracellular localization of Oatps (e.g . Oatp1b2) preceded expression at the plasma membrane . Approximate adult phenotypes of polarized expression were achieved for Ntcp by P5, for Bsep, Mrp2 and Mrp6 by P12 and for Oatp1a1, Oatp1a4 and Oatp1b2 by P29 . CONCLUSIONS: The data demonstrate that full maturation of polarized transporter expression in rat liver requires several weeks . The findings provide a molecular explanation for the previously observed chronology of the functional maturation of bile salt-independent and dependent bile formation and of hepatic detoxification functions in developing rat liver.

J Pharm Pharmacol, 2004 Aug, 56(8), 1061 - 6
Reversal of P-gp mediated multidrug resistance in-vitro and in-vivo by FG020318; Chen LM et al.; Overexpression of P-glycoprotein (P-gp) by tumours results in multidrug resistance (MDR) to structurally and functionally unrelated chemotherapeutic drugs . Combined therapy with MDR-related cytotoxins and MDR modulators is a promising strategy to overcome clinical MDR . This study was performed to explore the MDR reversal activity of a novel compound 2-{4-(2-pyridin-2-yl-vinyl) phenyl}-4,5-bis-(4-N,N-diethylaminophenyl)-1(H)-imidazole (FG020318) in-vitro and in-vivo . Tetrazolium (MTT) assay was used to evaluate the ability of FG020318 to reverse drug resistance in two P-gp-expressing tumour cell lines, KBv200 and MCF-7/adr . Intracellular doxorubicin accumulation was determined by fluorescence spectrophotometry in MCF-7/adr cell line . The effect of FG020318 on P-gp function was demonstrated by rhodamine 123 (Rh123) accumulation in KBv200 cells . KBv200 cell xenograft models were established to study the in-vivo effect of FG020318 on reversing MDR . FG020318 was not cytotoxic by itself against P-gp expressing KBv200 cells and MCF-7/adr cells and their parental drug-sensitive KB cells and MCF-7 cells . FG020318 could significantly increase the sensitivity of MDR cells to antitumour drugs including doxorubicin and vincristine in MCF-7/adr cells and KBv200 cells, respectively . It was much stronger than the positive control verapamil in reversal of MDR . FG020318 also increased the intracellular accumulation of doxorubicin in a concentration-dependent manner in MCF-7/adr cells, but did not affect the accumulation of doxorubicin in drug-sensitive MCF-7 cells . The Rh123 accumulation in resistant KBv200 cells was also increased by the addition of FG020318, but Rh123 accumulation was not affected by FG020318 in drug-sensitive KB cells . FG020318 potentiated the antitumour activity of vincristine to KBv200 xenografts and was an efficacious modulator in-vivo . Our results suggested that FG020318 was a highly potent, efficacious MDR modulator not only in-vitro but also in-vivo . The reversal of drug resistance by FG020318 was probably related to the increased anticancer drug accumulation and its inhibition of P-gp function of MDR tumour cells.

J Pharm Pharmacol, 2004 Aug, 56(8), 1001 - 5
Inhibition of P-glycoprotein function by tea catechins in KB-C2 cells; Kitagawa S et al.; We studied the effects of tea catechins, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) on the P-glycoprotein (P-gp) function in multidrug-resistant P-gp over-expressing KB-C2 cells . EC did not have any effects on cellular accumulation of P-gp substrates, rhodamine-123 and daunorubicin, but the other catechins increased the accumulation in the order of EGC < ECG < EGCG . The effects of EGCG were larger than those of verapamil and quercetin . Since these catechins inhibited the efflux of P-gp substrates, the elevation of substrate accumulation seemed to be induced by the inhibition of the efflux transporter . The results showed that the inhibitory effects of the catechins did not depend on their total hydrophobicity, but significantly depended on their chemical structure . The presence of the galloyl moiety on the C-ring markedly increased the n-octanol/PBS partition coefficients of the catechins and their activity on P-gp . On the other hand, the presence of the trihydric pyrogallol group as the B-ring decreased the partition coefficients but increased the activity on P-gp, compared with the action of the corresponding catechins with a dihydric catechol B-ring.

Immunol Cell Biol, 2004 Aug, 82(4), 377 - 82
Mycobacterium tuberculosis induces high production of nitric oxide in coordination with production of tumour necrosis factor-alpha in patients with fresh active tuberculosis but not in MDR tuberculosis; Sharma S et al.; Mycobacterium tuberculosis is an intracellular pathogen that readily survives and replicates in human macrophages . Host cells have developed various mycobactericidal and immunoregulatory mechanisms, such as the production of nitric oxide and inflammatory cytokines to control intracellular replication of M . tuberculosis . Inducible nitric oxide synthase (iNOS) is transcriptionally under the control of IFN-gamma and TNF-alpha . IL-12 provides a crucial link between activated mononuclear phagocytes and T cells by regulating the production of IFN-gamma . In this study, we investigated the production of nitric oxide (NO), TNF-alpha and IL-12 by the peripheral blood monocytes (PB Mn) of patients suffering from multidrug-resistant tuberculosis (MDR-TB) . The cells were infected with M . tuberculosis and stimulated with IFN-gamma or activated with mycobacterial subcellular components . The results were compared with those from cases of newly diagnosed TB and healthy controls . Nitric oxide production was significantly depressed in PB Mn from MDR-TB patients . Infected monocytes from newly diagnosed TB patients produced significantly higher levels of NO as compared to those from MDR-TB patients or normal controls . The subcellular fraction of M . tuberculosis-like whole cell lysate (WCL), culture filtrate protein (CFP) and lipoarabinomannan (LAM) induced higher concentrations of NO release in PB Mn from newly diagnosed TB patients as compared to those from MDR-TB patients . Cell culture supernatant from PB Mn assayed at 48 h after infection or stimulation demonstrated significantly depressed release of TNF-alpha and IL-12 from MDR-TB cases as compared to the fresh cases . We observed a definite correlation between nitric oxide release and TNF-alpha production, irrespective of low or high production in MDR-TB or fresh cases, respectively . The present data suggest that peripheral blood monocytes of MDR-TB patients typically show signs of immunosuppression . Whether such immunodepression is the cause or the effect of MDR-TB merits further investigation.

J Reprod Med, 2004 Jun, 49(6), 438 - 42
Salvage chemotherapy for high-risk gestational trophoblastic tumor; Matsui H et al.; OBJECTIVE: To evaluate the efficacy and safety of etoposide/methotrexate/actinomycin D (MEA regimen) as initial chemotherapy and 5-fluorouracil/actinomycin D (FA regimen) as salvage chemotherapy for high-risk gestational trophoblastic tumor (GTT) . STUDY DESIGN: From 1985 to 2001, 36 patients with World Health Organization (WHO)--defined high-risk GTT were treated with MEA or FA at Chiba University Hospital . Thirty-three patients were initially treated with MEA . FA was administered to 11 patients; 1 had had no previous chemotherapy, 7 had developed drug resistance to MEA, 1 had relapsed following MEA, and 2 had relapsed following etoposide/methotrexate/actinomycin D/ cyclophosphamide/vincristine (EMA/CO) combination chemotherapy . RESULTS: The primary remission rate with MEA was 69.7% (23 of 33) . With FA the survival rate was 81.8% (9 of 11) for a mean follow-up period of 11.5 years . Two patients died due to multidrug resistance, and 2 patients relapsed subsequently . The 2 relapse cases were successfully salvaged again with MEA . The toxicity of FA was evaluated in 89 cycles . Myelosuppression seemed to be the dose-limiting toxicity, and the incidence of WHO grade 4 leukocytopenia and thrombocytopenia were 5.6% and 3.4%, respectively . CONCLUSION: Although etoposide-containing chemotherapy is currently the most effective and well tolerated regimen for high-risk GTT, 20-30% of patients develop drug resistance to these regimens . Salvage combination chemotherapy with FA is effective for refractory patients, and the toxicity is predictable and manageable.

J Antimicrob Chemother, 2004 Sep, 54(3), 593 - 602 Epub 2004 Jul 28.
Which agents should we use for the treatment of multidrug-resistant Mycobacterium tuberculosis?
Di Perri G, Bonora S.
The inappropriate treatment of drug-susceptible tuberculosis can lead to the selection and transmission of multidrug-resistant tuberculosis (MDR-TB), indicating resistance to at least isoniazid and rifampicin . In the treatment of MDR-TB, residual first-line drugs, such as ethambutol, pyrazinamide and streptomycin must be appropriately combined with additional second-line drugs, guided by individual susceptibility patterns . The clinical pharmacology of these second-line antituberculous drugs is reviewed . Fluoroquinolones represent the only substantial therapeutic advance in the last 20 years . Many factors potentially affect the outcome of MDR-TB . Treatment adherence, prior exposure to antituberculous drugs, the number of drugs to which the infection is still susceptible and the time since the first diagnosis of tuberculosis are the most relevant . The management of MDR-TB requires considerable expertise . When initiating or revising therapy for MDR-TB, the process of selecting drugs should rely on prior treatment history, results of susceptibility testing and an evaluation of the patient's adherence . In making drug selection, we propose to follow a hierarchy based on the intrinsic activity against Mycobacterium tuberculosis and the clinical evidence of efficacy of the available active compounds.

J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Sep 25, 809(1), 87 - 97
Determination of the investigational anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid and its acyl glucuronide in Caco-2 monolayers by liquid chromatography with fluorescence detection: application to transport studies; Zhou S et al.; 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a potent cytokine inducer, with a bioavailability of >70% in the mouse . The aim of this study was to develop and validate HPLC methods for the determination of DMXAA and DMXAA acyl glucuronide (DMXAA-G) in the human intestinal cell line Caco-2 monolayers . The developed HPLC methods were sensitive and reliable, with acceptable accuracy (85-115% of true values) and precision (intra- and inter-assay CV < 15%) . The total running time was within 6.8 min, with acceptable separation of the compounds of interest . The limit of quantitation (LOQ) values for DMXAA and DMXAA-G were 14.2 and 24 ng/ml, respectively . The validated HPLC methods were applied to examine the epithelial transport of DMXAA and DMXAA-G by Caco-2 monolayers . The permeability coefficient (Papp) values (overall mean +/- S.D., n = 3-9) of DMXAA over 10-500 microM were independent of concentration for both apical (AP) to basolateral (BL) (4.0 +/- 0.4 x 10(-5)cm/s) and BL-AP (4.3 +/- 0.5 x 10(-5)cm/s) transport, and of similar magnitude in either direction, with net efflux ratio (Rnet) values of 1-1.3 . However, the Papp values for the BL to AP transport of DMXAA-G were significantly greater than those for the AP to BL transport, with Rnet values of 17.6, 6.7 and 4.5 at 50, 100 and 200 microM, respectively . Further studies showed that the transport of DMXAA-G was Na+- and energy-dependent, and inhibited by MK-571 {a multidrug resistance associated protein (MRP) 1/2 inhibitor}, but not by verapamil and probenecid . These data indicate that the HPLC methods for the determination of DMXAA and DMXAA-G in the transport buffer were simple and reliable, and the methods have been applied to the transport study of both compounds by Caco-2 monolayers . DMXAA across Caco-2 monolayers was through a passive transcellular process, whereas the transport of DMXAA-G was mediated by MRP1/2.

Curr Med Chem Anti-Canc Agents, 2004 Jul, 4(4), 363 - 78
Lamellarins, from A to Z: a family of anticancer marine pyrrole alkaloids; Bailly C; The lamellarins form a group of more than 30 polyaromatic pyrrole alkaloids, isolated from diverse marine organisms, mainly but not exclusively ascidians and sponges . These molecules fall in three structural groups, with the central pyrrole ring fused or unfused (lamellarins O-R) to adjacent aromatic rings and with the quinoline moiety containing a 5, 6-single--as in lamellarins I-L--or a double bond, as it is the case for lamellarins D and M which are both potent cytotoxic agents . The family also includes sulphated members, such as the integrase inhibitor lamellarin alpha 20-sulfate . This review presents the origin and structure of the lamellarins and summarizes the various chemical pathways which have been proposed to synthesize all lamellarins and different structurally related marine pyrrole alkaloids, including ningalins, storniamides and lukianols . The mechanisms of actions of these marine products are also discussed . Inhibition of HIV-1 integrase by lamellarin alpha 20-sulfate and human topoisomerase I by lamellarin D and Molluscum contagiosum virus topoisomerase by lamellarin H, along with other effects on nuclear proteins, provide an experimental basis indicating that DNA manipulating enzymes are important targets for the lamellarins . Some of these marine compounds exhibit cytotoxic activities against tumor cells in vitro and are insensitive to Pgp-mediated drug efflux . The structure-activity relationships are discussed . Other compounds in the series, without being strongly cytotoxic, can reverse the multidrug resistance phenotype and thus may be useful to promote the therapeutic activity of conventional cytotoxic drugs toward chemoresistant tumors . A complete description of the chemistry and pharmacological profiles of the lamellarins is presented here to shed light on this undervalued family of marine alkaloids.

J Environ Pathol Toxicol Oncol, 1999, 18(2), 103 - 8
Adaptive resistance of Saccharomyces cerevisiae to chronic treatment with genotoxic and nongenotoxic carcinogens; Fahrig R et al.; The exposure of mammalian cells or tumors for weeks or months to low nonlethal doses of cytostatic drugs may induce multidrug resistance, which can be enhanced by a variety of DNA-damaging agents . Multidrug resistance to a variety of drugs has been observed . But in yeast, DNA-damaging agents have not yet been tested . As the appearance of resistance is the result of longterm exposure, we decided to extend the application of test substances to a period of up to 400 days . In such long-term experiments S . cerevisiae MP1 adapted to treatment with low doses of mutagens . Consistent results were obtained for both genotoxic and nongenotoxic carcinogenic substances, which implies that there may be a single pathway for carcinogens with different modes of action.

AIDS, 2004 Aug 20, 18(12), 1653 - 60
Persistence of multidrug-resistant HIV-1 in primary infection leading to superinfection; Brenner B et al.; OBJECTIVE: The authors previous studies documented persistence of multidrug resistance (MDR) acquired in five primary HIV-1 infection (PHI) cases for 1-2 years in the absence of antiretroviral treatment . This study characterizes the evolution of transmitted wild-type (WT) (n = 15), resistant (n = 10), and MDR (n = 6) infections . Long-term persistence of MDR infections (2-7 years), leading to one observed MDR superinfection is documented . METHODS: Genotypic changes in circulating viral quasi-species were evaluated over 1.5-7 years in patients (n = 31) enrolled in the PHI study . Sequencing of reverse transcriptase and protease regions identified nucleotide substitutions in the viral quasi-species and mutations at sites implicated in resistance to antiretroviral drugs . Phylogenetic and clonal analysis were performed to confirm one observed superinfection . RESULTS: Patients acquiring WT, drug-resistant and MDR infections showed little quasi-species evolution (> 99.6% homology) for more than 1.5 years, regardless of route of transmission . Transmitted resistance mutations (other than 184V) persisted for 2-7 years . MDR persistence in two PHI cases contrasted with the corresponding rapid reversion of MDR infections to WT in their partners following treatment interruption . One MDR transmission eliciting low-level viremia resulted in clearance of the original MDR infection followed by re-infection with a second heterologous MDR strain from a different partner . Phylogenetic and clonal analysis of source and index partner confirmed the superinfection . Both MDR species showed approximately 13-fold reductions in replication capacity relative to the homologous WT strain isolated from the source partner . CONCLUSIONS: Genotypic analysis in PHI may identify superinfection and MDR infections that represent important determinants of virological and treatment outcome.

J Virol, 2004 Aug, 78(16), 8654 - 62
Spirodiketopiperazine-based CCR5 inhibitor which preserves CC-chemokine/CCR5 interactions and exerts potent activity against R5 human immunodeficiency virus type 1 in vitro; Maeda K et al.; We identified a novel spirodiketopiperazine (SDP) derivative, AK602/ONO4128/GW873140, which specifically blocked the binding of macrophage inflammatory protein 1alpha (MIP-1alpha) to CCR5 with a high affinity (K(d) of approximately 3 nM), potently blocked human immunodeficiency virus type 1 (HIV-1) gp120/CCR5 binding and exerted potent activity against a wide spectrum of laboratory and primary R5 HIV-1 isolates, including multidrug-resistant HIV-1 (HIV-1(MDR)) (50% inhibitory concentration values of 0.1 to 0.6 nM) in vitro . AK602 competitively blocked the binding to CCR5 expressed on Chinese hamster ovary cells of two monoclonal antibodies, 45523, directed against multidomain epitopes of CCR5, and 45531, specific against the C-terminal half of the second extracellular loop (ECL2B) of CCR5 . AK602, despite its much greater anti-HIV-1 activity than other previously published CCR5 inhibitors, including TAK-779 and SCH-C, preserved RANTES (regulated on activation normal T-cell expressed and secreted) and MIP-1beta binding to CCR5(+) cells and their functions, including CC-chemokine-induced chemotaxis and CCR5 internalization, while TAK-779 and SCH-C fully blocked the CC-chemokine/CCR5 interactions . Pharmacokinetic studies revealed favorable oral bioavailability in rodents . These data warrant further development of AK602 as a potential therapeutic for HIV-1 infection.

J Pharmacol Exp Ther, 2004 Dec, 311(3), 1179 - 87 Epub 2004 Dec.
Haplotype-oriented genetic analysis and functional assessment of promoter variants in the MDR1 (ABCB1) gene; Takane H et al.; Recently, a number of nucleotide variants have been described in the multidrug resistance 1 (MDR1/ABCB1) gene; however, most studies have focused on the coding region . In the present study, we identified promoter variants of the MDR1 gene and evaluated their phenotypic consequences using a reporter gene assay and the real-time polymerase chain reaction method . Ten allelic variants were detected in the promoter region (approximately 2 kilobases), seven of which were newly identified . Certain mutations occurred simultaneously, and a total of 10 haplotypes were observed . These promoter polymorphisms were found more frequently in Japanese than Caucasians . Some haplotypes were associated with changes in luciferase activity and placental and hepatic mRNA levels . We also determined DNA methylation status in the proximal promoter region of the MDR1 gene . The promoter region around potential binding sites for transcription factors was found to be hypomethylated and thus likely to be independent of the gene expression . Nucleotide and/or haplotype variants not only in the coding region but also in the promoter region of the MDR1 gene may be important for interindividual differences of P-glycoprotein expression.

J Cell Sci, 2004 Aug 15, 117(Pt 18), 4077 - 87 Epub 2004 Jul 27.
Small hepatocytes in culture develop polarized transporter expression and differentiation; Sidler Pfandler MA et al.; Rat small hepatocytes have been shown to proliferate in culture and to form organoids with differentiated hepatocytes in vitro . To evaluate the degree of polarized transporter differentiation of rat small hepatocytes during 9 weeks of culturing, we studied the time-dependent expression and subcellular localization of the major bile salt and organic anion transport systems of hepatocytes {i.e . the basolateral sodium-taurocholate co-transporting protein (Ntcp), organic-anion-transporting polypeptide 1b2 (Oatp1b2), the canalicular bile-salt export pump (Bsep) and multidrug-resistance-associated protein 2 (Mrp2)} . Small hepatocytes proliferated and differentiated in culture and formed sharply demarcated colonies as assessed by morphology, alpha-fetoprotein, albumin and Mrp1 expression . Polarized surface transporter expression was evident after 5 weeks of culturing for Ntcp, Oatp1b2 and Mrp2, and after 7 weeks for Bsep . After 9 weeks in culture, the vast majority of matured hepatocytes expressed Ntcp/Oatp1b2 at the basolateral and Bsep/Mrp2 at the canalicular plasma-membrane domains . This polarized transporter expression was accompanied by canalicular secretion of fluorescein-diacetate and cholylglycyl-fluorescein . Furthermore, an anastomizing three-dimensional network of bile canaliculi developed within piling-up colonies . These data demonstrate that cultured rat small hepatocytes acquire a fully differentiated transporter expression phenotype during their development into hepatic 'organoid-like' clusters of mature hepatocytes . Thereby, the time-dependent sequence of transporter expression mirrored the ontogenesis of transporter expression in developing rat liver, supporting the concept that small hepatocytes correspond to the hepatocyte lineage derived from embryonic hepatoblasts and/or from a different pool of 'committed hepatocyte progenitor cells'.

Drug Metab Dispos, 2004 Nov, 32(11), 1272 - 8 Epub 2004 Jul 27.
Hepatic transport of PKI166, an epidermal growth factor receptor kinase inhibitor of the pyrrolo-pyrimidine class, and its main metabolite, ACU154; Takada T et al.; PKI166, a specific inhibitor of the tyrosine kinase activity of two epidermal growth factor receptors, was under development for the treatment of cancer . In preclinical studies PKI166 was mainly cleared by metabolism, and its metabolites were eliminated by biliary excretion, emphasizing the role of liver transport processes for its disposition . Here the transport properties of {14C}PKI166 and its main metabolite {14C}ACU154, an O-glucuronide, were analyzed using 1) Madin-Darby canine kidney II (MDCKII) cells stably transfected with human multidrug resistance-associated protein 2 (MRP2) and/or human organic anion-transporting peptide 2 (OATP2) and 2) liver canalicular membrane vesicles (CMVs) prepared from Wistar and mrp2-deficient TR- rats . Analysis of transport through MDCKII cells revealed that {14C}ACU154 was a substrate of MRP2 and OATP2 . Rat mrp2 was shown to transport {14C}ACU154 with a Km of approximately 1 microM . {14C}PKI166 efficiently crossed MDCKII cells, particularly toward the apical side, but expression of MRP2 and/or OATP2 did not increase the flux . The effect of PKI166 and ACU154 on transport of {3H}estradiol-17beta-d-glucuronide (EG; via mrp2/MRP2 and OATP2) or {3H}taurocholic acid (TCA; via bile salt export pump (bsep) was analyzed . PKI166 inhibited the transport of {3H}EG by OATP2 . ACU154 did strongly inhibit {3H}TCA uptake into CMVs from Wistar but not from TR- rats, demonstrating a dependence of bsep inhibition on mrp2 activity . ATP-dependent uptake of {3H}EG into CMVs from Wistar rats was inhibited by ACU154 but up to 4-fold increased by PKI166 . In conclusion, OATP2 and MRP2/mrp2 were identified as transporters involved in ACU154 transport into bile . Both PKI166 and its O-glucuronide ACU154 affected mrp2/MRP2-, OATP2-, and/or bsep-mediated transport processes .

Drug Metab Dispos, 2004 Oct, 32(10), 1139 - 45 Epub 2004 Jul 27.
Estradiol 3-glucuronide is transported by the multidrug resistance-associated protein 2 but does not activate the allosteric site bound by estradiol 17-glucuronide; Gerk PM et al.; beta-estradiol 17-(beta-D-glucuronide) (E217G) is a well known cholestatic agent and substrate of multidrug resistance-associated protein 2 (Mrp2), whereas beta-estradiol 3-(beta-D-glucuronide) (E23G) is a noncholestatic regioisomer of E217G with unknown transport properties . The purpose of this study was to compare and contrast the Mrp2-mediated transport of E217G and E23G . The full coding region of rat Mrp2 was cloned into the baculovirus genome, the recombinant baculovirus used to infect Sf9 cells, and ATP-dependent transport of 3H-E23G and 3H-E217G in Sf9 cell membranes was characterized . Mrp2 transported E23G into an osmotically sensitive space, requiring ATP, with S50=55.7 microM, Vmax=326 pmol.mg(-1).min(-1), and a Hill coefficient of 0.88 . ATP-dependent Mrp2-mediated E217G transport was markedly stimulated at high E217G concentrations, consistent with positive cooperativity (Hill coefficient 1.5) . E217G (5-125 microM) increased S50 but not Vmax for E23G transport, consistent with competitive inhibition . E23G (0.4-400 microM) completely, potently (IC50=14.2 microM), and competitively inhibited E217G transport, but E217G (0.01-250 microM) inhibited only 53% of E23G transport (IC50=33.4 microM) . Estriol 16alpha-(beta-D-glucuronide) potently and completely inhibited transport of E23G (IC50=2.23 microM), as did beta-estradiol 3-sulfate 17-(beta-D-glucuronide) (5-50 microM) . In summary, E217G binds not only to an Mrp2 transport site, but also to an allosteric site that activates Mrp2 with positive cooperativity, thus activating its own transport and potentially that of other Mrp2 substrates, such as E23G . The noncholestatic E23G is an Mrp2 substrate and competes with E217G for transport, but does not activate the allosteric site.

Curr Med Chem, 2004 Aug, 11(15), 2093 - 113
Multidrug resistance and anticonvulsants: new studies with some enaminones; Salama NN et al.; The multidrug resistance (MDR), often conferred by the active extrusion of drugs from the cell, is a phenomenon often seen in cancer cells that may become resistant to a wide spectrum of drugs with varying chemical structures or cellular targets . This event has recently been reported for anticonvulsants . Studies in our laboratories on this occurrence with some enaminones have shown that the enaminones display high efflux ratios and are recognized by P-glycoprotein (P-gp) and/or the multidrug resistance protein (MRP), which have been reported as the main efflux transporters responsible for the development of MDR . Recent studies have uncovered interesting structural analogues that can modulate the functional activity of P-gp, suggesting a possible increase in the bioavailabillity of P-gp substrate drugs when administered concurrently .

J Formos Med Assoc, 2004 Jun, 103(6), 411 - 5
Antituberculosis drug resistance among retreatment tuberculosis patients in a referral center in Taipei; Chiang CY et al.; BACKGROUND AND PURPOSE: To determine the prevalence of antituberculosis drug resistance among retreatment tuberculosis patients in a referral center in Taipei . METHODS: We reviewed the register of susceptibility testing of the mycobacteriology laboratory of the Chronic Disease Control Bureau to identify patients with positive culture for Mycobacterium tuberculosis in the year 2000-2001 . Medical charts were reviewed to determine patients' tuberculosis treatment histories . Patients who had multidrug-resistant (MDR) tuberculosis, defined as documentation of isolates resistant to at least isoniazid and rifampin, were identified . Retreatment tuberculosis patients without prior evidence of MDR tuberculosis were classified into 3 categories, i.e., relapse, treatment after default and treatment after failure, and the frequency and patterns of antituberculosis drug resistance were determined . RESULTS: A total of 317 patients who had received antituberculosis treatment for more than 1 month were identified . Among them, 183 were retreatment cases without prior evidence of MDR tuberculosis, including 93 with relapse, 57 with treatment after default, and 33 with treatment after failure . Among the 183 patients, the prevalence of resistance to any drug was 42.6%; 14.2% were resistant to 1 drug, 13.7% to 2 drugs, 7.1% to 3 drugs, 7.7% to 4 drugs or more, and 24.6% had MDR tuberculosis . The prevalence of any drug resistance among patients with relapse, treatment after default and treatment after failure was 33.3%, 42.1%, and 69.7%, respectively, while the prevalence of MDR tuberculosis in these groups was 12.9%, 19.3% and 66.7%, respectively . CONCLUSIONS: If susceptibility results are unavailable, the World Health Organization-recommended retreatment regimen may be used in retreatment tuberculosis patients . However, the high proportion of MDR tuberculosis among patients with treatment after failure poses a challenge to the efficacy of the retreatment regimen.

Med Sci Monit, 2004 Aug, 10(8), BR300 - 5 Epub 2004 Jul 23.
Lack of relationship between metallothionein (MT) expression and proliferation exponents in cells of primary ductal breast cancer of G2 grade of differentiation; Surowiak P et al.; BACKGROUND: The metallothioneins (MTs) are a group of proteins which, due to their unique structure, fulfil numerous functions in the cell . They participate in growth, differentiation, and reparative processes, protect cells against free radicals, and are responsible for heavy metal homeostasis . Their involvement has been reported in the multidrug resistance to cytostatic drugs . Numerous reports document MT presence in cells of various tumors, including breast cancer . Augmented expression of MTs has been reported in less differentiated tumors . MT expression used to be linked to higher proliferative activity of tumor cells, shorter survival of the patients, and tamoxifen-resistance . The present study aimed at examining the relation between MT expression and the manifestation of proliferation exponents (Ki67, nucleolar organizers--AgNORs) in cells of ductal breast cancer of G2 grade of malignancy . MATERIAL/METHODS: Reactions were performed to detect MTs (clone E9), Ki67 (clone MIB-1) (immunocytochemistry), and AgNORs (silver impregnation) in paraffin sections of breast cancers in G2 grade originating from 60 females . Results of the reactions were subjected to statistical analysis using Statistica 98 PL software . RESULTS: Statistical analysis (Spearman's rank correlation) demonstrated no relationships between the studied markers (p>0.05) . CONCLUSIONS: There is no correlation between metallothionein expression and proliferation and between Ki67 and AgNORs in ductal breast cancers of G2 grade of differentiation.

Ann Oncol, 2004 Aug, 15(8), 1194 - 203
Single nucleotide polymorphisms and outcome in docetaxel-cisplatin-treated advanced non-small-cell lung cancer; Isla D et al.; BACKGROUND: Platinum-based doublets are the standard chemotherapy for advanced non-small-cell lung cancer (NSCLC) . Excision-repair cross-complementing 1 (ERCC1), xeroderma pigmentosum group D (XPD) and ribonucleotide reductase subunit M1 (RRM1) are essential to the repair of cisplatin DNA adducts . Multidrug resistance 1 (MDR1) has been related to antimicrotubule resistance . We assessed whether single nucleotide polymorphisms (SNPs) in ERCC1, XPD, RRM1 and MDR1, and ERCC1 mRNA expression, predicted survival in docetaxel-cisplatin-treated stage IV NSCLC patients . PATIENTS AND METHODS: Using the TaqMan 5' nuclease assay, we examined ERCC1 118, XPD 751 and 312, RRM1 -37C/A, and MDR1 C3435T SNPs in peripheral blood lymphocytes (PBLs) obtained from 62 docetaxel-cisplatin-treated advanced NSCLC patients . ERCC1 expression was measured in RNA isolated from PBLs using real-time reverse transcriptase PCR . RESULTS: Overall median survival was 10.26 months . Median survival was 9.67 months for 34 patients with ERCC1 118 C/T, 9.74 months for 17 patients with T/T, and not reached for 11 patients with C/C (P=0.04) . Similar significant differences in time to progression were observed according to ERCC1 118 genotype (P=0.03) . No other significant differences were observed . CONCLUSIONS: Patients homozygous for the ERCC1 118 C allele demonstrated a significantly better survival . ERCC1 SNP assessment could be an important component of tailored chemotherapy trials .

Res Vet Sci, 2004 Dec, 77(3), 223 - 9
Quantification of MDR-1 gene expression in canine tissues by real-time reverse transcription quantitative polymerase chain reaction; Culmsee K et al.; MDR-1 gene product mediated multidrug resistance is thought to play a major role in the outcome of chemotherapy in some canine tumors, especially malignant lymphoma . In the present study, MDR-1 RNA expression in normal lymph node and liver tissue as well as in tumor biopsies from 23 dogs with lymphomas and two dogs with liver tumors was measured by real-time RT-quantitative PCR . MDR-1 gene expression was detected in all samples analyzed . Comparably high MDR-1 RNA levels were measured in all normal liver tissues, one of the lymphomas and a cholangiocarcinoma . MDR-1 expression levels in canine lymphomas were found to vary over a wide range with most tumors expressing relative low levels . Interestingly, gastrointestinal lymphomas expressed higher MDR-1 RNA levels than multicentric lymphomas (p = 0.03) . In conclusion, real-time RT-quantitative PCR appears to be a suitable method for sensitive and quantitative determination of MDR-1 gene expression in canine normal and neoplastic tissues.

J Am Soc Mass Spectrom, 2004 Aug, 15(8), 1181 - 90
Characterization of noncovalent complexes of antimalarial agents of the artemisinin-type and FE(III)-heme by electrospray mass spectrometry and collisional activation tandem mass spectrometry; Pashynska VA et al.; In this study, we demonstrate, using electrospray ionization mass spectrometry (ESI-MS) and collision-induced dissociation tandem mass spectrometry (ESI-MS/CID/MS), that stable noncovalent complexes can be formed between Fe(III)-heme and antimalarial agents, i.e., quinine, artemisinin, and the artemisinin derivatives, dihydroartemisinin, alpha- and beta-artemether, and beta-arteether . Differences in the binding behavior of the examined drugs with Fe(III)-heme and the stability of the drug-heme complexes are demonstrated . The results show that all tested antimalarial agents form a drug-heme complex with a 1:1 stoichiometry but that quinine also results in a second complex with the heme dimer . ESI-MS performed on mixtures of pairs of various antimalarial agents with heme indicate that quinine binds preferentially to Fe(III)-heme, while ESI-MS/CID/MS shows that the quinine-heme complex is nearly two times more stable than the complexes formed between heme and artemisinin or its derivatives . Moreover, it is found that dihydroartemisinin, the active metabolite of the artemisinin-type drugs in vivo, results in a Na(+)-containing heme-drug complex, which is as stable as the heme-quinine complex . The efficiency of drug-heme binding of artemisinin derivatives is generally lower and the decomposition under CID higher compared with quinine, but these parameters are within the same order of magnitude . These results suggest that the efficiency of antimalarial agents of the artemisinin-type to form noncovalent complexes with Fe(III)-heme is comparable with that of the traditional antimalarial agent, quinine . Our study illustrates that electrospray ionization mass spectrometry and collision-induced dissociation tandem mass spectrometry are suitable tools to probe noncovalent interactions between heme and antimalarial agents . The results obtained provide insights into the underlying molecular modes of action of the traditional antimalarial agent quinine and of the antimalarials of the artemisinin-type which are currently used to treat severe or multidrug-resistant malaria.

Biochem Pharmacol, 2004 Aug 15, 68(4), 791 - 8
Effect of acetaminophen on expression and activity of rat liver multidrug resistance-associated protein 2 and P-glycoprotein; Ghanem CI et al.; We evaluated the effect of acetaminophen (APAP), given as a single, 1g/kg body weight dose, on expression and activity of rat liver multidrug resistance-associated protein 2 (Mrp2) and P-glycoprotein (P-gp), two major canalicular drug transporters . The studies were performed 24h after administration of the drug . APAP induced an increase in plasma membrane content of Mrp2 detected by western blotting, consistent with increased detection of the protein at the canalicular level by immunoflourescence microscopy . In vivo biliary excretion of dinitrophenyl-S-glutathione, a well known Mrp2 substrate, was slightly but significantly increased by APAP, agreeing well with upregulation of the transporter . Basal biliary excretion of oxidized glutathione, an endogenous Mrp2 substrate, was also increased by APAP, likely indicating increased hepatic synthesis as a result of APAP-induced oxidative stress followed by accelerated canalicular secretion mediated by Mrp2 . APAP also increased the expression of P-gp detected by western blotting and immunofluorescence microscopy as well as the in vivo biliary secretory rate of digoxin, a model P-gp substrate . Because specific APAP-conjugated metabolites are Mrp2 substrates, we postulate that induction of Mrp2 by APAP may represent an adaptive mechanism to accelerate liver disposition of the drug . In addition, increased Mrp2-mediated elimination of oxidized glutathione may be essential in maintaining the redox equilibrium in the hepatocyte under conditions of APAP-induced oxidative stress.

Parasitol Today, 1996 Apr, 12(4), 135 - 40
The ATP-binding cassette (ABC) gene family of Plasmodium falciparum; Rubio JP et al.; Multidrug resistance (MDR) in mammalian tumour cells is mediated by P-glycoproteins . The apparent similarities between MDR and the chloroquine-resistance phenotype (CQR) in Plasmodium falciparum suggests that homologous proteins may be involvea . In mammals, P-glycoproteins are encoded by mdr genes that are a subset of a super-family characterized by ATP-binding cassettes (ABC) . Three genes, pfmdr1, pfmdr2 and pfef3-rl, have been identified in P . falciparum that have homology to the ABC transporter gene family . Each protein encoded by these genes has a distinct structure, suggesting functional differences between the three . Justin Rubio and Alan Cowman here discuss the structure and possible function of the ABC proteins from P . falciparum and evidence that the protein encoded by the pfmdr1 gene can influence quinoline-containing antimalarial drug-resistance phenotypes.

Anticancer Res, 2004 May-Jun, 24(3a), 1631 - 6
Synthesis and multidrug resistance reversal activity of 1,2-disubstituted tetrahydroisoquinoline derivatives; Mihalyi A et al.; BACKGROUND: Cancer treatment often fails due to multidrug resistance (MDR) of the tumor cells . One of the major causes is overexpression of P-glycoprotein (P-gp) . MATERIALS AND METHODS: By N-substitution reactions of diamine, amino acid and amino alcohol derivatives with 1-substituted tetrahydroisoquinoline skeleton, structurally diverse 1,2-disubstituted 1,2,3,4-tetrahydroisoquinolines were synthesized . The compounds were assayed as P-gp inhibitors using a standard functional assay with rhodamine (6G) on MCF-7/Adr cells . Cytotoxicity was investigated on HeLa cells using an antiproliferative assay . RESULTS: Five of the 24 compounds showed greater P-gp inhibition than the control compound verapamil with AC50 values (concentration of the compound eliciting 50% of the maximal rhodamine 6G accumulation) significantly lower than that of verapamil . CONCLUSION: Novel compounds were synthesized that showed MDR-reversal effect . One of them, (1'R*,2R*)-2-{2'-{2''-hydroxy-3''-(alpha-naphthyloxy)propyl}-6',7'-dimethoxy-1',2',3',4'-tetrahydro-1'-isoquinolyl}propan-1-ol hydrochloride, showed two times higher efficacy than verapamil at 10 times lower concentrations . The outcome makes this molecule an attractive subject for further investigation and development.

Anticancer Res, 2004 May-Jun, 24(3a), 1529 - 44
Metals and metal compounds in cancer treatment; Desoize B; Metals and metal compounds have been used in medicine for several thousands of years . In this review we summarized the anti-cancer activities of the ten most active metals: arsenic, antimony, bismuth, gold, vanadium, iron, rhodium, titanium, gallium and platinum . The first reviewed metal, arsenic, presents the anomaly of displaying anti-cancer and oncogenic properties simultaneously . Some antimony derivatives, such as Sb2O3, salt (tartrate) and organic compounds, show interesting results . Bismuth directly affects Helicobacter pylori and gastric lymphoma; the effects of bismuth complexes of 6-mercaptopurine are promising . Gold(I) and (III) compounds show anti-tumour activities, although toxicity remains high . Research into the potential use of gold derivatives is still ongoing . Several derivatives of vanadium show anti-proliferative activity, but their toxicity must be overcome . Several pieces of evidence indicate that iron deprivation could be an excellent therapeutic approach; furthermore, it is synergistic with classic anti-cancer drugs . Rhodium belongs to the same group as platinum and it also presents interesting activity, but with the same nephrotoxicity . Several rhodium compounds have entered phase I clinical trials . In contrast to the platinum complexes, titanium derivatives showed no evidence of nephrotoxicity or myelotoxicity; titanocene dichloride is undergoing clinical trial . The anti-proliferative effect of gallium could be related to its competition with the iron atom; in addition a derivative appears to reverse the multidrug resistance . The last metal reviewed, platinum, has given some of the very best anti-cancer drugs . Four derivatives are used today in the clinic; their mechanism of action and of resistance are described.

J Pharmacol Exp Ther, 2004 Dec, 311(3), 1062 - 70 Epub 2004 Dec.
Relationship between antiapoptotic molecules and metastatic potency and the involvement of DNA-dependent protein kinase in the chemosensitization of metastatic human cancer cells by epidermal growth factor receptor blockade; Um JH et al.; The failure to treat metastatic cancer with multidrug resistance is a major problem for successful cancer therapy, and the molecular basis for the association of metastatic phenotype with resistance to therapy is still unclear . In this study, we revealed that various metastatic cancer cells showed consistently higher levels of antiapoptotic proteins, including Bcl-2, nuclear factor-kappaB, MDM2, DNA-dependent protein kinase (DNA-PK), and epidermal growth factor receptor (EGFR), and lower levels of proapoptotic proteins, including Bax and p53 than low metastatic parental cells . This was followed by chemo- and radioresistance in metastatic cancer cells compared with their parental cells . EGFR and DNA-PK activity, which are known to be associated with chemo- and radioresistance, were demonstrated to be mutually regulated by each other . Treatment with PKI166, an EGFR inhibitor, suppressed etoposide-induced activation of DNA-PK in A375SM metastatic melanoma cells . In addition, PKI166 enhanced markedly the chemosensitivities of metastatic cancer cell sublines to various anticancer drugs in comparison with those of low metastatic cancer cells . These results suggest that the activities of DNA-PK and EGFR, which is positively correlated with each other, may contribute to metastatic phenotype as well as therapy resistance, and the EGFR inhibitor enhances the effect of anticancer drugs against therapy-resistant metastatic cancer cells via suppression of stress responses, including activation of DNA-PK.

Antimicrob Agents Chemother, 2004 Aug, 48(8), 3133 - 5
Lack of activity of orally administered clofazimine against intracellular Mycobacterium tuberculosis in whole-blood culture; Janulionis E et al.; The activity of oral clofazimine against intracellular Mycobacterium tuberculosis was compared to that of ofloxacin in healthy volunteers by the use of whole-blood cultures . Clofazimine was inactive whether it was tested alone or combined with other drugs that are used to treat multidrug-resistant tuberculosis, despite a total dose of 2 g . Kanamycin was the most active drug tested.

J Chem Inf Comput Sci, 2004 Jul-Aug, 44(4), 1497 - 505
Prediction of P-glycoprotein substrates by a support vector machine approach; Xue Y et al.; P-glycoproteins (P-gp) actively transport a wide variety of chemicals out of cells and function as drug efflux pumps that mediate multidrug resistance and limit the efficacy of many drugs . Methods for facilitating early elimination of potential P-gp substrates are useful for facilitating new drug discovery . A computational ensemble pharmacophore model has recently been used for the prediction of P-gp substrates with a promising accuracy of 63% . It is desirable to extend the prediction range beyond compounds covered by the known pharmacophore models . For such a purpose, a machine learning method, support vector machine (SVM), was explored for the prediction of P-gp substrates . A set of 201 chemical compounds, including 116 substrates and 85 nonsubstrates of P-gp, was used to train and test a SVM classification system . This SVM system gave a prediction accuracy of at least 81.2% for P-gp substrates based on two different evaluation methods, which is substantially improved against that obtained from the multiple-pharmacophore model . The prediction accuracy for nonsubstrates of P-gp is 79.2% using 5-fold cross-validation . These accuracies are slightly better than those obtained from other statistical classification methods, including k-nearest neighbor (k-NN), probabilistic neural networks (PNN), and C4.5 decision tree, that use the same sets of data and molecular descriptors . Our study indicates the potential of SVM in facilitating the prediction of P-gp substrates.

Chemotherapy, 2004 Jun, 50(3), 119 - 26
Hypoxia-inducible factor 1alpha- mediated resistance to phenolic anticancer; Hyun JY et al.; BACKGROUND: Phenolic compounds EGCG {(-)-epigallocatechin-3-gallate}, resveratrol (3,4',5-trihydroxy-trans-stilbene) and capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) are worth investigating for clinical application in cancer prevention and chemotherapy . Hypoxia-induced drug resistance is a major obstacle in the development of effective cancer chemotherapy . Therefore, we examined whether drug resistance to these phenolic compounds is acquired by hypoxia . METHODS: Hep3B hepatoma, Caki-1 renal carcinoma, SK-N-MC neuroblastoma, and HEK293 cell lines were cultured under normoxic or hypoxic conditions . Drug sensitivities to the phenolic compounds and expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and the multidrug resistance genes were examined in these cell lines . RESULTS: Drug resistance was acquired 24 h after hypoxia and subsided 8 h after reoxygenation . Protein synthesis inhibitors abolished this drug resistance . A transfection study demonstrated that HIF-1alpha enhanced this hypoxia-induced resistance and that its dominant-negative isoform suppressed resistance acquisition . However, MDR1 and MRP1, which provide multidrug resistance to conventional anticancer agents, were not induced by hypoxia . CONCLUSIONS: These results suggest that HIF-1alpha-dependent gene expression participates in the cellular process of the hypoxia-induced resistance to phenolic compounds.

Nucleic Acids Res, 2004 Jul 22, 32(13), 3864 - 76 Print 2004.
New invMED1 element cis-activates human multidrug-related MDR1 and MVP genes, involving the LRP130 protein; Labialle S et al.; The MDR1 gene is a key component of the cytotoxic defense network and its overexpression results in the multidrug resistance (MDR) phenotype . However, the molecular mechanisms that regulate the MDR1 gene and coordinate multiple MDR-related genes expression are poorly understood . In a previous study, we identified a new 12 bp cis-activating region in the 5'-flanking region of the human MDR1 gene, which we called inverted MED1 . In the present study, we characterized the precise binding element, which we named invMED1, and revealed the presence of the LRP130 protein as the nuclear factor . Its binding intensity increases with the endogenous MDR1 geneexpression and with the MDR level of CEM leukemia cells . Interestingly, the LRP130 level did not vary with the chemoresistance level . We observed the involvement of LRP130 in the transcriptional activity of the MDR1 gene promoter, and moreover, in that of the MDR-related, invMED1-containing, MVP gene promoter . We used siRNAs and transcriptional decoys in two unrelated human cancer cell lines to show the role of the invMED1/LRP130 couple in both MDR1 and MVP endogenous genes activities . We showed that invMED1 was localized in the -105/-100 and -148/-143 regions of the MDR1 and MVP gene promoters, respectively . In addition, since the invMED1 sequence is primarily located in the -160/-100 bp region of mammalian MDR-related genes, our results present the invMED1/LRP130 couple as a potential central regulator of the transcription of these genes.

J Biol Chem, 2004 Sep 17, 279(38), 39620 - 7 Epub 2004 Jul 21.
The DeltaF508 mutation disrupts packing of the transmembrane segments of the cystic fibrosis transmembrane conductance regulator; Chen EY et al.; The most common mutation in cystic fibrosis (deletion of Phe-508 in the first nucleotide binding domain (DeltaF508)) in the cystic fibrosis transmembrane conductance regulator (CFTR) causes retention of the mutant protein in the endoplasmic reticulum . We previously showed that the DeltaF508 mutation causes the CFTR protein to be retained in the endoplasmic reticulum in an inactive and structurally altered state . Proper packing of the transmembrane (TM) segments is critical for function because the TM segments form the chloride channel . Here we tested whether the DeltaF508 mutation altered packing of the TM segments by disulfide cross-linking analysis between TM6 and TM12 in wild-type and DeltaF508 CFTRs . These TM segments were selected because TM6 appears to line the chloride channel, and cross-linking between these TM segments has been observed in the CFTR sister protein, the multidrug resistance P-glycoprotein . We first mapped potential contact points in wild-type CFTR by cysteine mutagenesis and thiol cross-linking analysis . Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wild-type background . The disulfide cross-linking occurs intramolecularly and was reducible by dithiothreitol . Introduction of the DeltaF508 mutation into these cysteine mutants, however, abolished cross-linking . The results suggest that the DeltaF508 mutation alters interactions between the TM domains . Therefore, a potential target to correct folding defects in the DeltaF508 mutant of CFTR is to identify compounds that promote correct folding of the TM domains.

Am J Physiol Heart Circ Physiol, 2004 Dec, 287(6), H2501 - 9 Epub 2004 Dec.
cAMP modulates cGMP-mediated cerebral arteriolar relaxation in vivo; Xu HL et al.; No studies have specifically addressed whether cAMP can influence nitric oxide (NO)/cGMP-induced cerebral vasodilation . In this study, we examined whether cAMP can enhance or reduce NO-induced cerebral vasodilation in vivo via interfering with cGMP efflux or through potentiating phosphodiesterase 5 (PDE5)-mediated cGMP breakdown, respectively, in cerebral vascular smooth muscle cells (CVSMCs) . To that end, we evaluated, in male rats, the effects of knockdown {via antisense oligodeoxynucleotide (ODN) applications} of the cGMP efflux protein multidrug resistance protein 5 (MRP5) and PDE5 inhibition on pial arteriolar NO donor {S-nitroso-N-acetyl penicillamine (SNAP)}-induced dilations in the absence and presence of cAMP elevations via forskolin . Pial arteriolar diameter changes were measured using well-established protocols in anesthetized rats . In control (missense ODN treated) rats, forskolin elicited a leftward shift in the SNAP dose-response curves (approximately 50% reduction in SNAP EC50) . However, in MRP5 knockdown rats, cAMP increases were associated with a substantial reduction in SNAP-induced vasodilations (reflected as a significant 35-50% lower maximal response) . In the presence of the PDE5 inhibitor MY-5445, the repression of the NO donor response accompanying forskolin was prevented . These findings suggest that cAMP has opposing effects on NO-stimulated cGMP increases . On the one hand, cAMP limits CVSMC cGMP loss by restricting cGMP efflux . On the other, cAMP appears to enhance PDE5-mediated cGMP breakdown . However, because increased endogenous cAMP seems to potentiate NO/cGMP-induced arteriolar relaxation when MRP5 expression is normal, the effect of cAMP to reduce cGMP efflux appears to predominate over cAMP stimulation of cGMP hydrolysis.

J Biol Chem, 2004 Sep 24, 279(39), 40980 - 6 Epub 2004 Jul 21.
A novel fold revealed by Mycobacterium tuberculosis NAD kinase, a key allosteric enzyme in NADP biosynthesis; Garavaglia S et al.; NAD kinase catalyzes the magnesium-dependent phosphorylation of NAD, representing the sole source of freshly synthesized NADP in all organisms . The enzyme is essential for the growth of the deadly multidrug-resistant pathogen Mycobacterium tuberculosis and is an attractive target for novel antitubercular agents . The crystal structure of NAD kinase has been solved by multiwavelength anomalous dispersion at a resolution of 2.3 A in its T state . Two crystal forms have been obtained revealing either a dimer or a tetramer . The enzyme architecture discloses a novel molecular arrangement, with each subunit consisting of an alpha/beta N-terminal domain and a C-terminal 12-stranded beta sandwich domain, connected by swapped beta strands . The C-terminal domain shows a striking internal approximate 222 symmetry and an unprecedented topology, revealing a novel fold within the family of all beta structures . The catalytic site is located in the long crevice that defines the interface between the domains . The conserved GGDG structural fingerprint of the catalytic site is reminiscent of the related region in 6-phosphofructokinase, supporting the hypothesis that NAD kinase belongs to a newly reported superfamily of kinases .

Drug Metab Dispos, 2004 Oct, 32(10), 1075 - 82 Epub 2004 Jul 21.
Tocotrienols activate the steroid and xenobiotic receptor, SXR, and selectively regulate expression of its target genes; Zhou C et al.; Vitamin E is an essential nutrient with antioxidant activity . Vitamin E is comprised of eight members, alpha-, beta-, gamma-, and delta-tocopherols and alpha-, beta-, gamma-, and delta-tocotrienols . All forms of vitamin E are initially metabolized by omega-oxidation, which is catalyzed by cytochrome P450 enzymes . The steroid and xenobiotic receptor (SXR) is a nuclear receptor that regulates drug clearance in the liver and intestine via induction of genes involved in drug and xenobiotic metabolism . We show here that all four tocotrienols specifically bind to and activate SXR, whereas tocopherols neither bind nor activate . Surprisingly, tocotrienols show tissue-specific induction of SXR target genes, particularly CYP3A4 . Tocotrienols up-regulate expression of CYP3A4 but not UDP-glucuronosyltransferase 1A1 (UGT1A1) or multidrug resistance protein-1 (MDR1) in primary hepatocytes . In contrast, tocotrienols induce MDR1 and UGT1A1 but not CYP3A4 expression in intestinal LS180 cells . We found that nuclear receptor corepressor (NCoR) is expressed at relatively high levels in intestinal LS180 cells compared with primary hepatocytes . The unliganded SXR interacts with NCoR, and this interaction is only partially disrupted by tocotrienols . Expression of a dominant-negative NCoR enhanced the ability of tocotrienols to induce CYP3A4 in LS180 cells, suggesting that NCoR plays an important role in tissue-specific gene regulation by SXR . Our findings provide a molecular mechanism explaining how vitamin supplements affect the absorption and effectiveness of drugs . Knowledge of drug-nutrient interactions may help reduce the incidence of decreased drug efficacy.

Clin Cancer Res, 2004 Jul 15, 10(14), 4652 - 60
Alternative splicing of the multidrug resistance protein 1/ATP binding cassette transporter subfamily gene in ovarian cancer creates functional splice variants and is associated with increased expression of the splicing factors PTB and SRp20; He X et al.; PURPOSE: Overexpression of multidrug resistance protein 1 (MRP1) confers resistance to a range of chemotherapeutic agents in cell lines and could be involved in clinical drug resistance of some tumor types also . We examined MRP1 expression in a small series of untreated human ovarian tumors and matched normal tissues . EXPERIMENTAL DESIGN: We analyzed ten pairs of snap-frozen ovarian tumor and matched normal total ovarian tissues from the same patients for expression of MRP1 by reverse transcription-PCR . Amplified PCR products were sequenced to reveal splicing events of MRP1 . MRP1 splice variants were expressed as enhanced green fluorescent fusion proteins in HEK293T cells to demonstrate their localization in the cell and their activity in conferring resistance to doxorubicin . The expression of splicing factors PTB and SRp20 was examined by Western blot . RESULTS: MRP1 was expressed in all 10 of the pairs of specimens . Multiple MRP1 cDNA fragments of various sizes were amplified between exons 10 and 19 . Of interest, more MRP1 cDNA fragments were detected in ovarian tumors than in matched normal tissues in 9 of 10 pairs . We identified 10 splicing forms between exons 10 and 19 of the MRP1 gene with exon skipping ranging from 1 to 7 . Amplification of the entire coding region of MRP1 from 1 ovarian tumor revealed >20 splice variants . We found whole and partial exon skipping and partial intron inclusion in these splice variants . We expressed 3 of these MRP1 splice variants in HEK293T cells and found that they appeared to localize to the plasma membrane and were functional in conferring resistance to doxorubicin . In addition, we identified a few nucleotide variations in this gene . To understand the basis for increased splice variants in the tumors, we examined splicing factor expression in these tissues . Western blot analysis revealed that two splicing factors, PTB and SRp20, were overexpressed in most ovarian tumors compared with their matched normal ovarian tissues . Importantly, overexpression of both of these splicing factors was associated with the increased number of MRP1 splicing forms in the ovarian tissues . CONCLUSION: The MRP1 gene undergoes alternative splicing at a higher frequency in ovarian tumors than in matched normal tissues . Some of these splice variants confer resistance to doxorubicin . Expression of splicing factors PTB and SRp20 is strongly associated with the alternative splicing of the MRP1 gene.

Am J Ther, 2004 Jul-Aug, 11(4), 262 - 77
In vitro interaction of the HIV protease inhibitor ritonavir with herbal constituents: changes in P-gp and CYP3A4 activity; Patel J et al.; The purpose of this study was to evaluate in vitro interactions of commercially obtained pure herbal constituents with p-glycoprotein P-gp and cytochrome P-450 3A4 (CYP3A4) activities, which can further modulate the transcellular transport and metabolism kinetics of orally administered drugs . Caco-2 cells grown in the presence of 0.25 micromol/L 1alpha,25-dihydroxy vitamin D3 and multidrug-resistant 1 (MDR1) transfected MDCK cells were used as models to evaluate the effect of purified herbal constituents (quercetin, hypericin, hyperforin from St . John's wort, kaempferol from ginseng, silibinin from milk thistle, and allicin from garlic) on P-gp-mediated efflux of the human immunodeficiency virus (HIV) protease inhibitor ritonavir . In addition, the inhibitory effect of these constituents on CYP3A4-mediated metabolism was determined by using cortisol as a model compound . Silibinin and hyperforin did not significantly alter cellular uptake of H-ritonavir in Caco-2 cells . A similar result was also observed for silibinin when tested in MDR1-MDCK cells . Quercetin, hypericin, and kaempferol exhibited a remarkable inhibition of P-gp-mediated efflux of ritonavir by increasing its cellular uptake in these models . These values were also comparable with the inhibitory effect of quinidine in Caco-2 cells, a well-known inhibitor of P-gp, on ritonavir efflux from Caco-2 cells . Allicin exhibited a concentration-dependent inhibition of ritonavir efflux when tested on MDR1-MDCK cells . There was a significant decrease in the Apical to Basal/Basal to Apical (AP-BL/BL-AP) transport ratio of ritonavir in presence of hypericin, kaempferol, and quercetin . These herbal constituents inhibited the CYP3A4 activity when tested with the Vivid CYP3A4 assay kit, whereas silibinin did not alter cortisol metabolism . Hypericin showed a significant inhibition in reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent metabolism of cortisol with 64.6% of intact drug at the end of a 1-hour study . Similarly, kaempferol and quercetin also caused substantial inhibition of cortisol metabolism with 89.7% and 90.1% of intact cortisol, respectively, compared with 45.9% in the control . Prolonged exposure of quercetin resulted in significant increase of mRNA expression of both MDR1 and CYP3A4 levels in Caco-2 cells . However, hyperforin caused upregulation of CYP3A4 and downregulation of MDR1, whereas the effect of silibinin and kaempferol remained inconclusive on these gene expressions . Hypericin, kaempferol, quercetin, and allicin inhibit the efflux and CYP3A4-mediated metabolism of xenobiotics in vitro . Hence, this study warns against the use of herbal constituents along with prescribed HIV protease inhibitors that are substrates for P-gp and/or CYP3A4.

Nucl Med Commun, 2004 Aug, 25(8), 777 - 85
99mTc tetrofosmin scintigraphy in acute leukaemia: the relationship between marrow uptake of tetrofosmin and P-glycoprotein and chemotherapy response; Sukan A et al.; BACKGROUND: The non-invasive detection of P-glycoprotein (Pgp) and multidrug resistance related proteins in vivo, will represent the greatest challenge in overcoming multidrug resistance . Although 99mTc tetrofosmin has been used previously as a myocardial perfusion agent, it is now also being used in the imaging of various tumours . In the current study, Tc tetrofosmin was used in the investigation of acute leukaemia . AIM: To show the uptake pattern of 99mTc tetrofosmin in the bone marrow of patients with acute leukaemia, and to ascertain the relationship between 99mTc tetrofosmin uptake and the level of Pgp expression and their relation to the response to chemotherapy . In addition, CD95, which is an indicator of apoptosis (programmed cell death), has also been assessed . MATERIALS AND METHODS: Pgp and CD95 were detected by using flow cytometry . Of the 27 acute leukaemia patients assessed, nine had previously received chemotherapy, and 18 had had an initial diagnosis . All patients had undergone 99mTc tetrofosmin scintigraphy, and their Pgp and CD95 levels had been determined . The same parameters were studied again for 14 patients . The responses to chemotherapy were assessed by patients' clinicians . A control group of 37 patients without bone marrow pathology was also studied in order to provide comparisons for the scintigraphy results . The control images were assessed only qualitatively . RESULTS: In leukaemia patients the uptake of 99mTc tetrofosmin into bone marrow was found to be considerably higher than in control patients (P=0.000) . An analysis of the relationship between Pgp, CD95, and the qualitative and quantitative tetrofosmin uptake ratios (URs) showed that there was an inverse correlation only between Pgp and the quantitative uptake ratio (P=0.016, r=-0.461) . When the patients were grouped as 'good' and 'poor', as related to the chemotherapy response, there were no meaningful differences between these two groups regarding Pgp, CD95 and tetrofosmin URs (P>0.05) . By evaluating the scintigraphic findings of the 'repeated' 14 patients, we showed that if the 99mTc tetrofosmin UR in the second imaging test was reduced by >0.08, the response to chemotherapy tended to be good . This method, based on follow-up scanning with tetrofosmin, showed a sensitivity of 83% and a specificity of 62% in the prediction of a 'good' response, if a decrease of 0.08 was taken into consideration . CONCLUSION: In this study, patients with acute leukaemia showed significant uptake of tetrofosmin into the bone marrow . The addition of basal and repeated 99mTc tetrofosmin scintigraphy to the management protocol for leukaemia could lead to the preferential determination of responses to chemotherapy, by evaluating whole bone marrow non-invasively . This method seems promising, but it needs further support from various similar investigations comprising more patients in order to confirm our results.

Mol Pharmacol, 2004 Aug, 66(2), 268 - 75
Strategies for inhibition of MDR1 gene expression; Xu D et al.; Several distinct strategies have been used to modulate the expression of cancer-associated genes, including antisense oligonucleotides, small interfering RNAs (siRNAs), and artificial transcriptional factors . One major cause for chemotherapeutic treatment failure in cancer is the overexpression of P-glycoprotein, the product of the multidrug resistance gene MDR1 . In this study, we tested the ability of siRNAs to inhibit MDR1 gene expression . We evaluated the efficiency of chemically synthesized dsRNAs as well as vector-based hairpin siRNAs and investigated the behavior of clones of multidrug-resistant NCI/ADR-RES breast carcinoma cells stably transfected with hairpin siRNA vectors . The effects of siRNA on the MDR phenotype were compared with those elicited by antisense oligonucleotides or by designed transcription factors targeting the MDR1 promoter . These studies suggest that there are several comparably effective strategies for inhibiting MDR1 expression.

Curr Opin Mol Ther, 2004 Jun, 6(3), 308 - 17
The role of phenotyping and replication capacity in anti-HIV therapeutics; Baliga CS et al.; The management of antiretroviral-experienced and -naive HIV-infected patients is becoming increasingly complex with the emergence of multidrug-resistant viruses and with the inter-relationships between numerous virological, immunological and therapeutic factors . In addition to standard surrogate markers of peripheral CD4+ T-cell counts and plasma viral loads, viral resistance phenotype, replication capacity and genotype assays serve as objective measures of viral properties, which presumably reflect, to a certain degree, what is occurring in vivo . This review explores the principles behind, and the basic science and clinical evidence supporting the application of viral resistance phenotypic assays and replication capacity assays in the therapeutic management of HIV disease.

Rev Port Pneumol, 2003 Nov, 9(5 Suppl), 14 - 5
{Gene expression profiling by real-time system PCR analysis in hiv negative patients with multidrug-resistant tuberculosis}; Santos P et al.; In response to chronic stimulation of M . tuberculosis, multidrug-resistant tuberculosis (MDR-TB) patients up-regulate and down-regulate the expression of several genes . Thus, relative quantification of mRNA from genes involved with the immune response could be helpful to elucidate the immunopathology of MDR-TB giving clues to immune modulation . The purpose of this study was to evaluate immunologic gene expression profile associated with MDR-TB . Peripheral blood drawn from 18 MDR-TB patients and 17 healthy control individuals was analysed . Real-time PCR analysis with Taqman(R) probes was used for relative quantification of mRNAs from genes with important involvement in immune response (cytokines, chemokines, activatory and inhibitory receptors, etc) . A panel of primers and probes specific for the following 20 genes was used: IL-1 a, IL-1 b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN- g, TGF- b, TNF- a, TNF- b, RANTES, MCP1, MIP1 b, IP-10, CTLA-4, CD28, CD80 and CD86 . In the MDR-TB group, the following cytokine genes were found significantly up-regulated: IFN- g (1.8 logs; p<0.001), IL-10 (2.3 logs; p<0.05), IL-2 (3.0 logs; p<0.001), IL-4 (3.4 logs; p<0.00001) and TNF- b (1.1 logs; p<0.05) . A trend for IL-6 up-regulation (1.0 logs) was also observed (p=0.054) . A slight down-regulation of IL-1 b was noticed although lacking statistic significance . Additional genes with important immunologic functions showed altered levels of mRNA (e.g . CD28, RANTES, CD86) . Taken together these results corroborate the concept of chronic immune activation ongoing in MDR-TB patients . However, the gene expression of inhibitory Th1-response cytokines (v.g., IL-10 and IL-4) suggest the maintenance of an ineffective immune response in MDR tuberculosis.

J Biol Chem, 2004 Sep 24, 279(39), 40412 - 8 Epub 2004 Jul 19.
Inhibition of glucosylceramide synthase does not reverse drug resistance in cancer cells; Norris-Cervetto E et al.; The multidrug-resistant cancer cell lines NCI/AdR(RES) and MES-SA/DX-5 have higher glycolipid levels and higher P-glycoprotein expression than the chemosensitive cell lines MCF7-wt and MES-SA . Inhibiting glycolipid biosynthesis by blocking glucosylceramide synthase has been proposed to reverse drug resistance in MDR cells by causing an increased accumulation of proapoptotic ceramide during treatment of cells with cytotoxic drugs . We treated both multidrug-resistant cell lines with the glucosylceramide synthase inhibitors PDMP (d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol), C9DGJ (N-nonyl-deoxygalactonojirimycin) or C4DGJ (N-butyl-deoxygalactonojirimycin) . PDMP achieved a significant reversal of drug resistance in agreement with previous reports . However, the N-alkylated iminosugars C9DGJ and C4DGJ, which are more selective glucosylceramide synthase inhibitors than PDMP, failed to cause any reversal of drug resistance despite depleting glycolipids to the same extent as PDMP . Our results suggest that (a) inhibition of glucosylceramide synthase does not reverse multidrug resistance and (b) the chemosensitization achieved by PDMP cannot be caused by inhibition of glucosylceramide synthase alone .

Virology, 2004 Aug 15, 326(1), 103 - 12
Relative replication fitness of multi-nucleoside analogue-resistant HIV-1 strains bearing a dipeptide insertion in the fingers subdomain of the reverse transcriptase and mutations at codons 67 and 215; Prado JG et al.; A two-serine insertion at position 69 (i69SS) of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) appears to be critical to enhance multi-nucleoside RT inhibitor resistance (MNR) in the sequence context of multiple zidovudine (AZT) resistance mutations (i.e., M41L, L210W, T215Y) . In this study, we measured the replication capacity relative to the wild-type (WT) HIV-1 of a series of recombinant viruses carrying the i69SS in the background of a clinical isolate with MNR in which we introduced mutations D67N, Y215T, Y215S, or Y215N . In vitro measurements included replication kinetics and growth competition assays at different multiplicities of infection (MOI) . While the addition of D67N had a minor effect on replication capacity, the reversion of Tyr-215 to Thr, Ser, or Asn was sufficient to increase the virus ability to replicate in a drug-free environment . The same genotypic changes at position 215 rendered the MNR virus susceptible to AZT and stavudine . Interestingly, the presence of the insertion together with mutation T215Y in an otherwise WT sequence background was not sufficient to confer high-level resistance to AZT, although its replication capacity was clearly impaired . Therefore, the RT residue 215 plays a critical role in both replication capacity and drug resistance of multidrug-resistant viruses containing the i69SS.

Brain Res, 2004 Aug 20, 1018(1), 1 - 9
Contribution of glial cells and pericytes to the mRNA profiles of P-glycoprotein and multidrug resistance-associated proteins in an in vitro model of the blood-brain barrier; Berezowski V et al.; P-glycoprotein (P-gp) and the multidrug resistance-associated proteins (MRP), whose expression is associated with multidrug resistance, have been recently located in the brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB), without taking into account a possible influence or contribution of glial cells and pericytes . Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), the present study analysed the transcriptional expression of P-gp and the seven homologues of MRP transporters in BCECs in solo culture or in an in vitro model of the BBB consisting of a co-culture of BCECs and glial cells . Pericytes, glial cells, isolated brain capillaries and bovine grey matter extracts were also tested . P-gp mRNA, absent in glial cells, was found in brain capillaries and in co-cultured BCECs with an increased signal compared to the in solo culture . No amplification was observed in pericytes or grey matter . While MRP2, MRP3 and MRP7 remained undetected, MRP1, absent in capillaries or grey matter, was amplified in BCECs, glial cells and pericytes . MRP4 gave a low signal in most cultures . MRP5 was ubiquitously expressed, displaying a potent signal in all conditions . In spite of its presence in cultured glial cells, MRP6 mRNA expression appeared to be restricted to BCECs, with the same upregulation in the co-cultured condition as observed with P-gp . Moreover, MRP6 was the only transporter whose endothelial mRNA expression was influenced by the presence of pericytes . The tissue distribution of the expression of these transporters and the contribution of the different cell populations are discussed.

Gynecol Oncol, 2004 Jul, 94(1), 67 - 73
The role of topotecan for extending the platinum-free interval in recurrent ovarian cancer: an in vitro model; Horowitz NS et al.; OBJECTIVE: Topotecan, a novel topoisomerase-I inhibitor, is an active agent of second-line chemotherapy for extending the platinum-free interval (PFI) and improving the chances of a response to platinum in recurrent ovarian cancer patients . The aim of this study was to understand the molecular mechanism of topotecan-based second-line chemotherapy through an in vitro cell culture model and to gain clinical insight into sequencing issues for second-line treatment with novel agents versus retreatment with platinum . STUDY DESIGN: The human ovarian cancer cell line A2780 and the cisplatin resistance cell line A2780-CR were separately seeded in 6-well cell culture plates and then exposed to multiple concentrations of cisplatin plus paclitaxel or topotecan for 7 days . Surviving cells were recovered and cultured in drug-free media for 3 weeks and then replated in a 96-well microtiter plate . The LD(50) for these cells was determined by a cytotoxic MTT assay after exposure to multiple clinically relevant concentrations of cisplatin or topotecan . Surviving cells were cultured in drug-free media for an additional 4 weeks at which time the LD(50) was reassessed for each cell population by a second MTT assay . Using RT-PCR and Northern blot hybridization to measure mRNA expression, the molecular profile of these cells in terms of resistance was evaluated for the multidrug-resistant gene (MDR-1), multidrug-resistant protein (MRP), Topoisomerase-I, and beta-Actin . RESULTS: The LD(50) to cisplatin was unchanged in A2780-CR cells treated by topotecan . Those A2780-CR cells originally exposed to higher concentrations of cisplatin became more resistant to cisplatin in the MTT assays, while those A2780-CR cell lines treated with a combination of lower cisplatin concentrations and paclitaxel became more sensitive to cisplatin in the MTT assay (P < 0.01) . The second MTT assay demonstrated that the LD(50) for cisplatin in every cell line decreased significantly after a 4-week drug-free interval (P < 0.01) . There was no difference in the mRNA expression for MRP or topoisomerase-I regardless of cell line, or type or concentration of chemotherapeutic exposure . The mRNA for MDR-1 was uniquely overexpressed in the cisplatin-resistant cell line A2780-CR9 initially treated with low doses of cisplatin and paclitaxel, but was not amplified in A2780 (P < 0.01) . CONCLUSIONS: The acquired resistance to cisplatin in A2780 is potentially due to P-glycoprotein-mediated multidrug resistance . This acquired resistance to cisplatin is an unstable phenotype in that some cell populations become sensitive after a drug-free interval and topotecan treatment . This reversal of resistance, however, does not appear to be simply due to loss of MDR-1 expression . While in vivo confirmation is required, agents with novel mechanisms of action offer a strategy to extend the platinum-free interval and thereby improve survival in patients with recurrent ovarian cancer.

Lancet, 2004 Jul 17, 364(9430), 285 - 94
Antimalarial combinations; Kremsner PG et al.; Multidrug resistance has rendered monotherapy for malaria useless in most parts of the world, and has also compromised the usefulness of many of the available combination chemotherapies . New antimalarial regimens are, therefore, urgently needed . We review the various antimalarial combinations that can be used to treat otherwise drug-resistant disease, and discuss what defines an ideal antimalarial combination regimen.

Biochemistry, 2004 Jul 27, 43(29), 9448 - 56
Mutational analysis of ABCG2: role of the GXXXG motif; Polgar O et al.; ABCG2 (BCRP/MXR/ABCP) is a half-transporter associated with multidrug resistance that presumably homodimerizes for function . It has a conserved GXXXG motif in its first transmembrane segment, a motif that has been linked with dimerization in other proteins, e.g., glycophorin A . We substituted either or both glycines of this GXXXG motif with leucines to evaluate the impact on drug transport, ATP hydrolysis, cross-linking, and susceptibility to degradation . All mutants also carried the R482G gain-of-function mutation, and all migrated to the cell surface . The mutations resulted in lost transport for rhodamine 123 and impaired mitoxantrone, pheophorbide a, and BODIPY-prazosin transport, particularly in the double leucine mutant (G406L/G410L) . Basal ATPase activity of the G406L/G410L mutant was comparable to the empty vector transfected cells with no substrate induction . Despite impaired function, the mutants retained susceptibility to cross-linking using either disuccinimidyl suberate (DSS) or the reducible dithiobis(succinimidyl propionate) (DSP) and demonstrated a high molecular weight complex under nonreducing conditions . Mutations to alanine at the same positions yielded fully functional transporters . Finally, we exposed cells to mitoxantrone to promote folding and processing of the mutant proteins, which in the leucine mutants resulted in increased amounts detected on immunoblot and by immunofluorescence . These studies support a hypothesis that the GXXXG motif promotes proper packing of the transmembrane segments in the functional ABCG2 homodimer, although it does not solely arbitrate dimerization.

Biochemistry, 2004 Jul 27, 43(29), 9413 - 25
Transmembrane helix 11 of multidrug resistance protein 1 (MRP1/ABCC1): identification of polar amino acids important for substrate specificity and binding of ATP at nucleotide binding domain 1; Zhang DW et al.; Human multidrug resistance protein 1 (MRP1) is an ATP binding cassette (ABC) transporter that confers resistance to many natural product chemotherapeutic agents and can transport structurally diverse conjugated organic anions . MRP1 has three polytopic transmembrane domains (TMDs) and a total of 17 TM helices . Photolabeling and mutagenesis studies of MRP1 indicate that TM11, the last helix in the second TMD, may form part of the protein's substrate binding pocket . We have demonstrated that certain polar residues within a number of TM helices, including Arg(593) in TM11, are determinants of MRP1 substrate specificity or overall activity . We have now extended these analyses to assess the functional consequences of mutating the remaining seven polar residues within and near TM11 . Mutations Q580A, T581A, and S585A in the predicted outer leaflet region of the helix had no detectable effect on function, while mutation of three residues close to the membrane/cytoplasm interface altered substrate specificity . Two of these mutations affected only drug resistance . N597A increased and decreased resistance to vincristine and VP-16, respectively, while S605A decreased resistance to vincristine, VP-16 and doxorubicin . The third, S604A, selectively increased 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport . In contrast, elimination of the polar character of the residue at position 590 (Asn in the wild-type protein) uniformly impaired the ability of MRP1 to transport potential physiological substrates and to confer resistance to three different classes of natural product drugs . Kinetic and photolabeling studies revealed that mutation N590A not only decreased the affinity of MRP1 for cysteinyl leukotriene 4 (LTC(4)) but also substantially reduced the binding of ATP to nucleotide binding domain 1 (NBD1) . Thus, polar interactions involving residues in TM11 influence not only the substrate specificity of MRP1 but also an early step in the proposed catalytic cycle of the protein.

Int J Tuberc Lung Dis, 2004 Jul, 8(7), 905 - 9
Successful treatment of multidrug-resistant tuberculosis following drug-induced hepatic necrosis requiring liver transplant; Marra F et al.; A 28-year-old female developed multidrug-resistant (MDR) tuberculous lymphadenitis following a trip to India . She was initially treated with a four-drug regimen of first-line anti-tuberculosis medications, but when sensitivities indicated resistance to isoniazid and rifampin, her regimen was altered to ciprofloxacin (CFX), pyrazinamide (PZA) and ethambutol . She subsequently developed a rash, flu-like symptoms and fever, which progressed to acute hepatic necrosis despite discontinuation of medication . The clinical presentation and subsequent investigations suggested a hypersensitivity reaction, possibly related to the quinolone . The patient subsequently had an orthoptic liver transplant; second-line anti-tuberculosis medications were restarted to which she responded clinically and radiologically . Our findings raise the possibility that the CFX and PZA combination was responsible for the hepatic necrosis . The patient also illustrates that active, even MDR tuberculosis is not a contraindication to hepatic transplant.

Int J Tuberc Lung Dis, 2004 Jul, 8(7), 837 - 41
Isolation of multidrug-resistant tuberculosis strains in patients from private and public health care facilities in Nairobi, Kenya; Githui WA et al.; SETTING: Health care facilities in Nairobi, Kenya . OBJECTIVE: To document the presence of multidrug-resistant tuberculosis (MDR-TB) strains in patients from Nairobi between September 1999 and October 2001 . DESIGN: Descriptive study . RESULTS: Of the 983 referred patients who submitted sputum for culture and drug susceptibility testing (DST), 59% were males . Two hundred and nine (21.3%) patients had a positive culture, of whom 15.2% had a request for DST against isoniazid, rifampicin, streptomycin and ethambutol . Of these, 65 (43.6%) had an isolate resistant to one or more drugs, while 17 (11.4%) had MDR-TB . Ten (59.0%) cases were referred from public health care facilities while seven (41%) were from the private sector . Sixteen isolates were resistant to all four drugs . All MDR-TB cases but one were from Nairobi . CONCLUSION: The emergence of MDR-TB in Nairobi is a cause for concern . An outbreak would be catastrophic, creating not only increased morbidity and mortality but also a tremendous strain on already limited health care resources . Lack of policies for the treatment and management of MDR-TB and the unavailability of appropriate diagnostic facilities may increase its spread . Efforts to prevent outbreaks of MDR-TB should be emphasised.

Antivir Ther, 2004 Jun, 9(3), 415 - 21
Persistence of multidrug-resistant HIV-1 without antiretroviral treatment 2 years after sexual transmission; Delaugerre C et al.; OBJECTIVES: To understand the virological mechanisms of 2-year persistence of multidrug-resistant virus without selective antiretroviral pressure in HIV-1-infected patients . PATIENTS AND METHODS: Two patients were contaminated recently by their HIV-1-infected partners, who had received, before the transmission, all available antiretroviral drugs and who exhibited a severe therapeutic failure . The resistance mutations analysis was performed by clonal sequencing of 1.2 kb of pol gene in plasma of index and sources patients . Sequencing of HIV-1 DNA was performed in PBMCs of index patients . RESULTS: Genotypic testing performed in index patients at time of seroconversion showed resistance mutations to three classes of drugs . All mutations were linked on the same viral genome and all quasispecies carried all mutations . No wild-type virus was detected . The same results were found in source patients and showed that all mutations were transmitted . In the index patients, all mutations persisted over 2 years without antiretroviral treatment . Moreover, the resistance mutations were all archived in the cellular reservoir . Viral load and CD4 count of index patients remained unchanged during 2 years of follow-up . DISCUSSION: Only multidrug-resistant viruses were detected in the source patients and could be transmitted in index patients . In the latter, an expansion of predominant multidrug-resistant quasispecies and the 'archival' of all mutations were observed . These results explain the persistence of mutations and suggest that it is highly difficult to return to a wild-type viral population, sensitive to an antiretroviral treatment . The treatment of index patients is limited and the major risk is the transmission of these multidrug-resistant viruses . This work was presented in part in the XII International HIV Drug Resistance Workshop, Los Cabos, Mexico, June 2003; and in the 2nd IAS Conference on HIV Pathogenesis & Treatment, Paris, France, July 2003.

Antivir Ther, 2004 Jun, 9(3), 375 - 84
Infrequent transmission of HIV-1 drug-resistant variants; Yerly S et al.; Transmission of drug-resistant variants is influenced by several factors, including the prevalence of drug resistance in the population of HIV-1-infected patients, HIV-1 RNA levels and transmission by recently infected patients . In order to evaluate the impact of these factors on the transmission of drug-resistant variants, we have defined the population of potential transmitters and compared their resistance profiles to those of newly infected patients . Sequencing of pol gene was performed in 220 recently infected patients and in 373 chronically infected patients with HIV-1 RNA >1000 copies/ml . Minimal and maximal drug-resistance profiles of potential transmitters were estimated by weighting resistance profiles of chronically infected patients with estimates of the Swiss HIV-1-infected population, the prevalence of exposure to antiviral drugs and the proportion of infections attributed to primary HIV infections . The drug-resistance prevalence in recently infected patients was 10.5% (one class drug resistance: 9.1%; two classes: 1.4%; three classes: 0%) . Phylogenetic analysis revealed significant clustering for 30% of recent infections . The drug-resistance prevalence in chronically infected patients was 72.4% (one class: 29%; two classes: 27.6%; three classes: 15.8%) . After adjustment, the risk of transmission relative to wild-type was reduced both for one class drug resistance (minimal and maximal estimates: odds ratio: 0.39, P<0.001; and odds ratio: 0.55, P=0.011, respectively), and for two to three class drug resistance (odds ratios: 0.05 and 0.07, respectively, P<0.001) . Neither sexual behaviour nor HIV-1 RNA levels explained the low transmission of drug-resistant variants . These data suggest that drug-resistant variants and in particular multidrug-resistant variants have a substantially reduced transmission capacity.

Drug Metab Dispos, 2004 Aug, 32(8), 834 - 9
Effect of dexamethasone treatment on the expression and function of transport proteins in sandwich-cultured rat hepatocytes; Turncliff RZ et al.; Dexamethasone (DEX) is a well established inducer of CYP3A . These studies examined the influence of DEX treatment on transport protein expression and function in sandwich-cultured (SC) rat hepatocytes . Freshly isolated hepatocytes were cultured between two layers of gelled collagen and maintained in Dulbecco's modified Eagle's medium supplemented with DEX (0.1 microM, 0-48 h and 0.1-100 microM, 48-96 h) . The expression of sinusoidal {(organic anion transporting polypeptide 1a1 (Oatp1a1), Oatp1a4, multidrug resistance-associated protein 3 (Mrp3), and Na(+)-dependent taurocholate cotransporting polypeptide (Ntcp)} and canalicular {bile salt export pump (Bsep), multidrug resistance protein 1a/b (Mdr1a/b), and Mrp2} transport proteins was determined by Western blot analysis . The accumulation and biliary excretion index (BEI; percentage of accumulated substrate in canalicular networks) of the probe substrates taurocholate (TC; 1 microM, 10 min), rhodamine 123 (Rh123; 10 microM, 30 min), and carboxy-2',7'-dichlorofluorescein (CDF; 10 microM, 10 min) were employed as measures of canalicular transport protein function in SC rat hepatocytes . DEX treatment increased CYP3A1/2, Oatp1a4, and Mrp2 expression, decreased the expression of Ntcp, and did not seem to alter the expression of Oatp1a1, Mrp3, Mdr1a/b, or Bsep . The BEI of CDF, an Mrp2 substrate, increased from 18 to 37% after DEX treatment (100 microM) . The accumulation of TC, an Ntcp substrate, was reduced (<50% of control), whereas the BEI of TC, also a Bsep substrate, was unchanged . Treatment of SC rat hepatocytes with DEX resulted in alterations in the expression of CYP3A1/2 and some hepatic transport proteins . Modest alterations in hepatic transport protein function were consistent with changes in protein expression.

Haematologica, 2004 Jul, 89(7), 782 - 90
Clinical effects and P-glycoprotein inhibition in patients with acute myeloid leukemia treated with zosuquidar trihydrochloride, daunorubicin and cytarabine; Gerrard G et al.; BACKGROUND AND OBJECTIVES: P-glycoprotein (P-gp) is a major cause of multidrug resistance (MDR) in acute myelogenous leukemia (AML) and is thought to contribute to the failure of chemotherapy . Zosuquidar trihydochloride (Z.3HCL) is a potent and selective inhibitor of P-gp which rapidly and effectively inhibits drug efflux . DESIGN AND METHODS: The aim of this study was to evaluate the clinical effects of Z.3HCL and determine its influence on P-gp activity . Sixteen AML patients were entered into a phase 1 dose ranging clinical trial of Z.3HCL, co-administered intravenously with daunorubicin and cytosine arabinoside (ARA-C) . Clinical outcomes, toxicity abd adverse events were assessed . P-gp function was analyzed by flow cytometry . In vitro cytotoxicity was studied using the MTT assay . RESULTS: Eleven patients achieved a complete remission and one a partial remission with a median survival of 559 (range 38-906) days . Non-hematologic grade 3 and 4 toxicities were seen in 4 patients . Z.3HCL infusion was associated with rapid inhibition of Rh123 efflux in CD56+ cells in 16/16 patients and in CD33+ cells from 6/10 patients . The median inhibition was 95% for CD56+ cells and 85.25% for CD33+ cells was significantly elevated in 6/16 patients . The median IC50, using a MTT assay for daunorubicin, decreased significantly between Z.3HCL modulated and unmodulated cells (n=11,153 and 247 ng/mL respectively, p=0.01) . INTERPRETATION AND CONCLUSIONS: The modulator Z.3HCL is a specific inhibitor of P-gp efflux and can be given safely to patients with AML in combination with induction doses of conventional cytotoxic drugs.

Di Yi Jun Yi Da Xue Xue Bao, 2004 Jul, 24(7), 779 - 81
{Expression of glucosylceramide synthase mRNA in vincristine-resistant KBV200 cell line in association with multidrug resistance}; Yang Q et al.; OBJECTIVE: To investigate the relationship between the expression of glucosylceramide synthase (GCS) mRNA in vincristine-resistant KBV(200) human cancer cell line and multidrug resistance (MDR) of the cancer cells . METHODS: Reverse transcriptional polymerase chain reaction (RT-PCR) was employed to analyze the differential expression of GCS mRNA between KBV(200) and KB cell lines and the changes in the mRNA expressions of GCS and mdr1 gene in KBV(200) cells after reversion of MDR . The effects of de-phenyl-z-palmaitoylamino-3-morpholine-1-propanol (DL-PPMP) and verapamil in reversing MDR of the cells were evaluated by MTT assay . RESULTS: KBV(200) cells exhibited significantly increased expressions of GCS and mdr1 gene, whereas mdr1 gene failed to be detected in the parental KB cells . DL-PPMP within the concentrations ranging from 5 to 25 micromol/L could inhibit the expression of GCS gene, with the maximum inhibition achieved at 25 micromol/L . Verapamil at the concentration of 10 micromol/L was already sufficient to induce inhibition of GCS expression in KVB(200) cells, which was more manifest with the concentration of 15 micromol/L . DL-PPMP and verapamil were found to inhibit mdr1 gene expression in KBV(200) cells at the mRNA level, and complete inhibition occurred after a 48-hour DL-PPMP treatment at 25 micromol/L . CONCLUSION: The inhibition of GCS and mdr1 gene expressions is positively correlated with the concentrations of DL-PPMP and verapamil, which can reverse MDR by inhibiting synthesis of GCS and mdr1 gene, indicating the positive correlation between the expression of GCS gene and MDR in KBV(200) cells . GCS gene might play an important role in MDR during tumor progression.

Cancer Res, 2004 Jul 15, 64(14), 4950 - 6
Verapamil and its derivative trigger apoptosis through glutathione extrusion by multidrug resistance protein MRP1; Trompier D et al.; This study demonstrates that verapamil and a newly synthesized verapamil derivative, NMeOHI(2), behave as apoptogens in multidrug resistance protein 1 (MRP1)-expressing cells . When treated with either verapamil or NMeOHI(2), surprisingly, baby hamster kidney-21 (BHK) cells transfected with human MRP1 were killed . Because parental BHK cells were not, as well as cells expressing an inactive (K1333L) MRP1 mutant, this indicated that cell death involved functional MRP1 transporter . Cell death was identified as apoptosis by using annexin V-fluorescein labeling and was no longer observed in the presence of the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH(2)F (Z-VAD-FMK) . In vitro, both verapamil and its derivative inhibited leukotriene C4 transport by MRP1-enriched membrane vesicles in a competitive manner, with a K(i) of 48.6 microm for verapamil and 5.5 microm for NMeOHI(2,) and stimulated reduced glutathione (GSH) transport 3-fold and 9-fold, respectively . Treatment of MRP1-expressing cells with either verapamil or the derivative quickly depleted intracellular GSH content with a strong decrease occurring in the first hour of treatment, which preceded cell death beginning at 8-16 h . Furthermore, addition of GSH to the media efficiently prevented cell death . Therefore, verapamil and its derivative trigger apoptosis through stimulation of GSH extrusion mediated by MRP1 . This new information on the mechanism of induced apoptosis of MDR cells may represent a novel approach in the selective treatment of MRP1-positive tumors.

Cancer Res, 2004 Jul 15, 64(14), 4927 - 30
Analysis of the drug resistance profile of multidrug resistance protein 7 (ABCC10): resistance to docetaxel; Hopper-Borge E et al.; The multidrug resistance protein (MRP) family consists of nine members that can be categorized according to whether or not a third (NH(2)-terminal) membrane-spanning domain is present . Three (MRP1, MRP2, and MRP3) of the four members that have this structural feature are able to confer resistance to natural product anticancer agents . We previously established that MRP7, the remaining family member that has three membrane-spanning domains, possesses the cardinal biochemical activity of MRPs in that it is able to transport amphipathic anions such as 17beta-estradiol 17-(beta-d-glucuronide) . However, the drug resistance profile of the pump has not been determined . In this study, the drug resistance capabilities of MRP7 are evaluated by analyzing the resistance profiles of two clones of HEK293 cells in which the pump was ectopically expressed . MRP7-transfected HEK293 cells exhibited the highest levels of resistance toward docetaxel (9-13-fold) . In addition, lower levels of resistance were observed for paclitaxel (3-fold), vincristine (3-fold), and vinblastine (3-4-fold) . Consistent with the operation of an ATP-dependent efflux pump, MRP7-transfected cells exhibited reduced accumulation of radiolabeled paclitaxel compared with HEK293 cells transfected with parental plasmid . These results indicate that MRP7, unlike other MRPs, is a resistance factor for taxanes.

Cancer Res, 2004 Jul 15, 64(14), 4887 - 92
Efflux kinetics and intracellular distribution of daunorubicin are not affected by major vault protein/lung resistance-related protein (vault) expression; van Zon A et al.; Vaults may contribute to multidrug resistance by transporting drugs away from their subcellular targets . To study the involvement of vaults in the extrusion of anthracyclines from the nucleus, we investigated the handling of daunorubicin by drug-sensitive and drug-resistant non-small lung cancer cells, including a green fluorescent protein (GFP)-tagged major vault protein (MVP)-overexpressing transfectant (SW1573/MVP-GFP) . Cells were exposed to 1 microm daunorubicin for 60 min, after which the cells were allowed to efflux the accumulated drug . No significant differences in daunorubicin efflux kinetics were observed between the sensitive SW1573 and SW1573/MVP-GFP transfectant, whereas the drug-resistant SW1573/2R120 cells clearly demonstrated an increased efflux rate . It was noted that the redistribution of daunorubicin from the nucleus into distinct vesicular structures in the cytoplasm was not accompanied by changes in the intracellular localization of vaults . Similar experiments were performed using mouse embryonic fibroblasts derived from wild-type and MVP knockout mice, which were previously shown to be devoid of vault particles . Both cell lines showed comparable drug efflux rates, and the intracellular distribution of daunorubicin in time was identical . Reintroduction of a human MVP tagged with GFP in the MVP(-/-) cells results in the formation of vault particles but did not give rise an altered daunorubicin handling compared with MVP(-/-) cells expressing GFP . Our results indicate that vaults are not directly involved in the sequestration of anthracyclines in vesicles nor in their efflux from the nucleus.

Zhonghua Bing Li Xue Za Zhi, 2004 Jun, 33(3), 251 - 4
{Reversal of multidrug resistance of tumor cells by anti-mdr1 ribozyme}; Gao P et al.; OBJECTIVE: To stably reverse the multidrug resistance (MDR) of breast carcinoma cells in vitro . METHODS: Two anti-mdr-1 ribozyme plasmids, RZ196 and RZ179, were constructed with EGFP as reporter gene and transfected into drug-resistant breast carcinoma cells in vitro . The expression of EGFP was observed by laser confocal microscopy . Flow cytometry, RT-PCR and Rhodamine123 efflux assay were used to detect P-glyco protein (p-gp) and mdr-1 mRNA . RESULTS: After transfection with RZ196 and RZ179, the mdr-1 indices were reduced from 2.20 to 0.76 and 1.40, the expression rates of p-gp were reduced from 55.0% to 4.6% and 18.2%, the fluorescence intensity increased from 22.0% to 46.2% and 70.1%, TCL reduced from 75% to 28% and 43% respectively . In addition, the expression of ribozyme plasmid in tumor cells was stable under G418 selection . After two months, the mdr-1 indices remained at 0.81 and 1.47 in the cells transfected RZ196 and RZ179 respectively . The expression rates of p-gp were 5.2% and 19.5% and the Rh123 fluorescence intensity was 51.4% and 71.6% respectively . CONCLUSIONS: Both anti-mdr-1 ribozyme RZ196 and RZ179 can stably reverse MDR phenotype of breast carcinoma cells in vitro . RZ196 construct appears to be more effective.

Neoplasma, 2004, 51(3), 169 - 74
Expression of the multidrug resistance-associated protein 1 (MRP1) and the lung resistance-related protein (LRP) in human lung cancer; Rybarova S et al.; Fifty lung cancer samples (41 non-small cell lung cancer-NSCLC and 9 small cell lung cancer-SCLC) were immunohistochemically analyzed for lung resistance-related protein (LRP) and multidrug resistance-associated protein 1 (MRP1) expressions which were then correlated with histopathological subtype of the tumor . To detect these proteins, monoclonal antibodies LRP-56 and MRPm6 were used . NSCLC samples were divided into two groups, adenocarcinomas (17 samples) and squamous cell carcinomas (24 samples) . Four categories of LRP and MRP1 quantity were distinguished: +++ = high level--90--100% of positive cells, ++ = lower level--10--90% of positive cells, + = low level--up to 10% of positive cells, - = negative cells--0% of positive cells . Within the NSCLC group the most samples (36/41) had the similar level of LRP and MRP1 . Significantly higher expression of both proteins was observed in the adenocarcinomas in comparison with squamous cell carcinomas . The lowest positive staining for LRP and MRP1 proteins has been found in SCLC . It is suggested that our finding can confirm the overall empirical clinical knowledge about much higher chemosensitivity of untreated SCLC comparing to NSCLC.

Am J Med Sci, 2004 Jul, 328(1), 10 - 6
The antiretroviral-experienced patient; Clark RA et al.; There are many considerations for stopping and changing antiretroviral (ARV) therapy in the ARV-experienced individual . Given the potential for possible long-term toxicities and the shift to initiating ARV therapy later, it may be reasonable to stop ARV therapy among asymptomatic patients with high CD4 cell counts and low viral loads and carefully monitor them . Ongoing studies are currently evaluating this strategy . Treatment regimen failure may be due to problems with tolerability, adherence, pharmacokinetic issues, or emergence of resistance . Clinicians can utilize two types of resistance testing-genotype and phenotype assays . Generally, continuation of an optimized regimen in the patient with a multidrug resistant (MDR) virus is the best strategy . Structured treatment interruption among patients with an MDR virus is not recommended . New drugs, either recently licensed, such as enfuvirtide, or under investigation, may offer hope to patients with an MDR virus.

Kidney Int, 2004 Aug, 66(2), 532 - 41
Antiretroviral and immunosuppressive drug-drug interactions: an update; Izzedine H et al.; With the introduction of highly active antiretroviral therapy (HAART), human immunodeficiency virus (HIV) infection has become a chronic disease with more frequent end-stage organ failures . As a result, the question of transplantation in HIV patients is raised more often . However, some of the HAART regimen medications require elimination or metabolism via the P-glycoprotein (P-gp) and multidrug-resistant protein (MRP) transporters or via the cytochrome P450 enzyme system . Since these transporters and enzymes are also responsible for the clearance of immunosuppressive drugs, drug-drug interactions are likely to occur . Indeed, profound drug-drug interactions between protease inhibitors and immunosuppressive drugs have been observed and they required reductions in drug dosage . In contrast, HAART using nucleoside or nonnucleoside reverse transcriptase inhibitors without the use of protease inhibitors has been shown to produce less significant drug-drug interactions . It is thus crucial to take into account those potential pharmacokinetic and/or pharmacodynamic drug-drug interactions in order to avoid drug toxicity or a lack of efficacy . The aim of this work was to review and synthesize the international literature on this field in order to give practical recommendations on how to manage immunosuppressive drugs in HIV patients who get transplanted and on how to handle HAART therapy in transplant-recipient patients who get infected with HIV.

Glia, 2004 Aug 15, 47(3), 233 - 40
Defining pathways of loss and secretion of chemical messengers from astrocytes; Evanko DS et al.; It is becoming evident that glia, and astrocytes in particular, are intimately involved in neuronal signaling . Astrocytic modulation of signaling in neurons appears to be mediated by the release of neuroactive compounds such as the excitatory amino acid glutamate . Release of these transmitters appears to be driven by two different processes: (1) a volume regulatory response triggered by hypo-osmotic conditions that leads to the release of osmotically active solutes from the cytoplasm into the extracellular space, and (2) intracellular calcium-dependent vesicle-mediated excytotic release . The regulatory volume decrease may be mediated by any of several different pathways that increase membrane permeability, thus allowing osmolytes to travel down their concentration gradient into the extracellular space . Such pathways include anion channels, hemichannels, P2X receptor channels, and transporters or multidrug resistance proteins . The excytotic release process may use calcium triggered synaptic like vesicle fusion or alterations in constitutive vesicle trafficking to the membrane . Determining the contribution of any of these release mechanisms requires agents that can be used to specifically block pathways of interest . Currently, many of the pharmacological compounds being used exhibit a great deal of cross-reactivity between several of these pathways . For example, the popular anion channel inhibitor 5-nitro-2-(3-phenyl-propylamino)benzoic acid (NPPB) is an efficient blocker of both hemichannels and vesicle loading . This demonstrates the need to more fully characterize the activities of the agents currently available and to choose pathway blockers carefully when designing experiments .

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2004 Jun, 21(3), 424 - 7
{Construction of the recombinant adenovirus vector carrying antisense multidrug resistance (MDR1) gene}; Li B et al.; The fragment of MDR1 gene obtained from the plasmid pHaMDR1-1 carrying the whole human MDR1 cDNA, was cloned reversely into the shuttle plasmid pAdTrack-CMV . With the resultant plasmid and the backbone plasmid pAdEasy-1, the homologous recombination took place in the Escherichia coli BJ5183 and the recombinant adenoviral plasmid was generated . The adenoviruses were packaged in the 293 cells . The recombinant adenovirus MDR1 vector would introduce the antisense MDR1 gene into the human multidrug resistance hepatocellular cell line effectively, which would provide an experimental basis for studies on the multidurg resistance in human hepatocellular carcinoma.

Cancer Chemother Pharmacol, 2004 Dec, 54(6), 525 - 30 Epub 2004 Jul 10.
Inhibition of P-glycoprotein activity and reversal of cancer multidrug resistance by Momordica charantia extract; Limtrakul P et al.; PURPOSE: Multidrug resistance (MDR) is known as a problem limiting the success of therapy in patients treated long term with chemotherapeutic drugs . The drug resistance is mainly due to the overexpression of the 170 kDa P-glycoprotein (Pgp), which causes a reduction in drug accumulation in the cancer cells . In this study, novel chemical modulator(s) from bitter melon (Momordica charantia L.) extracts obtained from leaves, fruits and tendrils were tested for their abilities to modulate the function of Pgp and the MDR phenotype in the multidrug-resistant human cervical carcinoma KB-V1 cells (high Pgp expression) in comparison with wildtype drug-sensitive KB-3-1 cells (lacking Pgp) . METHODS: The KB-V1 and KB-3-1 cells were exposed to bitter melon extracts in the presence of various concentrations of vinblastine, and cytotoxicity was assessed by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay . Relative resistance was calculated as the ratio of the IC50 value of the KB-V1 cells to the IC50 value of the KB-3-1 cells . Accumulation and efflux of vinblastine in KB-V1 and KB-3-1 cells were measured using a {3H}-vinblastine incorporation assay . RESULTS: The leaf extracts increased the intracellular accumulation of {3H}-vinblastine in KB-V1 cells in a dose-dependent manner, but extracts from the fruits and tendrils had no effect . By modulating Pgp-mediated vinblastine efflux, the leaf extracts decreased the {3H}-vinblastine efflux in KB-V1 cells in a dose-dependent manner, but not in KB-3-1 cells . Treatment of drug-resistant KB-V1 cells with bitter melon leaf extracts increased their sensitivity to vinblastine, but similar treatment of KB-3-1 cells showed no modulating effect . The fruit and tendril extracts did not affect the MDR phenotype in either cell line . CONCLUSION: The leaf extracts from bitter melon were able to reverse the MDR phenotype, which is consistent with an increase in intracellular accumulation of the drug . The exact nature of the active components of bitter melon leaf extracts remains to be identified.

Pharmacogenetics, 2004 Jun, 14(6), 381 - 5
Modulation of multidrug resistance P-glycoprotein 1 (ABCB1) expression in human heart by hereditary polymorphisms; Meissner K et al.; OBJECTIVES: Variable expression of the ABC-type multidrug resistance membrane protein P-glycoprotein (P-gp, MDR1, ABCB1) in human heart is a potential modulator of drug effects or drug-induced cardiotoxicity . Expression of P-gp is known to be affected by single nucleotide polymorphisms in the MDR1 gene . Therefore, genotype-dependent expression of P-gp could be an important modulator of action of cardiac drugs . METHODS: Heart tissue (auriculum) from 51 patients undergoing coronary artery bypass graft surgery was screened for genotype-dependent P-gp expression . P-gp was identified by immunoblotting and localized using immunohistochemistry . MDR1 mRNA was quantified by real-time PCR and immunohistochemistry and related to the MDR1 genotypes G2677T/A (Ala893Ser/Thr) and C3435T . RESULTS: MDR1/18S rRNA mRNA copy numbers in heart auriculum were 3.48 +/- 2.25 x 10(-6) compared to 4.56 +/- 0.58 x 10(-6) in non-failing ventricular samples studied before . While the exon 26 C3435T genotype did not influence MDR1 mRNA expression, we found significantly elevated MDR1 mRNA expression in 10 patients carrying the exon 21 2677 AT or TT genotype as compared to 12 patients carrying the GG-variant with intermediate MDR1 mRNA expression in 29 heterozygous samples . P-gp was detected in the endothelial wall . Quantitative immunohistochemistry of protein expression, however, did not reveal significant influence of the studied SNPs . CONCLUSION: The present study based on auricular samples suggests that genetic factors play a rather limited role in modulating P-gp expression in human heart . Therefore, the substantial interindividual variability in cardiac P-gp expression is likely related to environmental or disease related factors.

J Biol Chem, 2004 Sep 10, 279(37), 38395 - 401 Epub 2004 Jul 09.
Processing mutations located throughout the human multidrug resistance P-glycoprotein disrupt interactions between the nucleotide binding domains; Loo TW et al.; The most common cause of cystic fibrosis is misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR) protein because of deletion of residue Phe-508 (DeltaF508) . P-glycoprotein (P-gp) is an ideal model protein for studying how mutations disrupt folding of ATP-binding cassette proteins such as CFTR because specific chemical chaperones can be used to correct folding defects . Interactions between the nucleotide binding domains (NBDs) are critical because ATP binds at the interface between the NBDs . Here, we used disulfide cross-linking between cysteines in the Walker A sites and the LSGGQ signature sequences to test whether processing mutations located throughout P-gp disrupted interactions between the NBDs . We found that mutations present in the cytoplasmic loops, transmembrane segments, and linker regions or deletion of Tyr-490 (equivalent to Phe-508 in CFTR) inhibited cross-linking between the NBDs . Deletion of Phe-508 in the P-gp/CFTR chimera also inhibited cross-linking between the NBDs . Cross-linking was restored, however, when the mutants were expressed in the presence of the chemical chaperone cyclosporin A . The "rescued" mutants exhibited drug-stimulated ATPase activity, and cross-linking between the NBDs was inhibited by vanadate trapping of nucleotide . These results together with our previous findings (Loo, T . W., Bartlett, M . C., and Clarke, D . M . (2002) J . Biol . Chem . 277, 27585-27588) indicate that processing mutations disrupt interactions among all four domains . It appears that cross-talk between the cytoplasmic and the transmembrane domains is required for establishment of proper domain-domain interactions that occur during folding of ATP-binding cassette protein transporters.

Soc Sci Med, 2004 Oct, 59(7), 1529 - 39
Community-based treatment of multidrug-resistant tuberculosis in Lima, Peru: 7 years of experience; Shin S et al.; Programs implementing community-based directly observed therapy (DOT) have demonstrated success in the treatment of patients with tuberculosis . However, given complexities in the management and treatment of patients infected with multidrug-resistant tuberculosis (MDR-TB), the utilization of community-based DOT to treat MDR-TB patients has only recently been successfully attempted . We describe the first such program and highlight the crucial components and most critical challenges to creating a successful community-based MDR-TB treatment program.

Biochem J, 2004 Oct 15, 383(Pt 2), 335 - 41
The human multidrug-resistance-associated protein MRP1 mediates ATP-dependent transport of unconjugated bilirubin; Rigato I et al.; Results of previous studies have suggested that UCB (unconjugated bilirubin) may be transported by MRP1/Mrp1 (multidrug-resistance-associated protein 1) . To test this hypothesis directly, {3H}UCB transport was assessed in plasma-membrane vesicles from MDCKII cells (Madin-Darby canine kidney II cells) stably transfected with human MRP1 or MRP2; wild-type MDCKII cells served as controls . As revealed by Western blotting, transfection achieved abundant expression of MRP1 and MRP2 . {3H}UCB uptake was measured in the presence of 60 microM human serum albumin at a free (unbound) concentration of UCB (B(F)) ranging from 5 to 72 nM and in the presence of 3 mM ATP or 3 mM AMP-PCP (adenosine 5'-{beta,gamma-methylene}triphosphate) . MRP1-transfected vesicles showed transport activity three and five times higher respectively compared with MRP2 or wild-type vesicles, whose transport did not differ significantly . {3H}UCB transport was stimulated 4-fold by 1.5 mM GSH, occurred into an osmotically sensitive space, was inhibited by 3 microM MK571 and followed saturative kinetics with K(m)=10+/-3 nM (B(F)) and V(max)=100+/-13 pmol x min(-1) x (mg of protein)(-1) . UCB significantly inhibited the transport of LTC4 (leukotriene C4), a leukotriene substrate known to have high affinity for MRP1 . Collectively, these results prove directly that MRP1 mediates ATP-dependent cellular export of UCB and supports its role in protecting cells from bilirubin toxicity.

J Clin Microbiol, 2004 Jul, 42(7), 3046 - 51
Molecular epidemiology of Mycobacterium tuberculosis in western Sweden; Brudey K et al.; The genetic diversity of Mycobacterium tuberculosis isolates among patients from Sweden was determined by a combination of two PCR-based techniques (spoligotyping and variable number of tandem repeats analysis) . It resulted in a clustering of 23.6% of the isolates and a rate of recent transmission of 14.1% . The clustered isolates mainly belonged to the Haarlem family (23.2%), followed by the Beijing (9.8%), Latin American and Mediterranean (LAM; 8%), and East African-Indian (EAI; 6.2%) families . A comparison of the spoligotypes with those in the international spoligotyping database showed that 62.5% of the clustered isolates and 36.6% of all isolates typed were grouped into six major shared types . A comparison of the spoligotypes with those in databases for Scandinavian countries showed that 33% of the isolates belonged to an ill-defined T family, followed by the EAI (22%), Haarlem (20%), LAM (11%), Central Asian (5%), X (5%), and Beijing (4%) families . Both the highest number of cases and the proportion of clustered cases were observed in patients ages 15 to 39 years . Nearly 10% of the isolates were resistant to one or more drugs (essentially limited to isoniazid monoresistance) . However, none of the strains were multidrug resistant . Data on the geographic origins of the patients showed that more than two-thirds of the clustered patients with tuberculosis were foreign-born individuals or refugees . These results are explained on the basis of both the historical links within specific countries and recently imported cases of tuberculosis into Sweden.

J Gastroenterol Hepatol, 2004 Aug, 19(8), 879 - 83
Effects of pravastatin and bezafibrate on biliary lipid excretion and hepatic expression of Abcg5 and Abcg8 in the rat; Kamisako T et al.; BACKGROUND AND AIM: Multidrug resistance associated gene product 2 (Mdr2) is believed to have a significant role in biliary cholesterol and phospholipid secretions . Both pravastatin and bezafibrate resulted in Mdr2 induction, but increased cholesterol secretion was observed only in pravastatin treatment . To explore the mechanism, the hepatic expression of genes that are responsible for the metabolism of the lipids was studied . METHODS: Rats were divided into three experimental groups: (i) the control group; (ii) the bezafibrate group, which was fed a diet containing 0.45% bezafibrate for 5 days; and (iii) the pravastatin group, which was fed a diet containing 0.1% pravastatin for 5 days . Serum, hepatic and biliary lipids were measured by colorimetric assays and hepatic mRNA related to lipid metabolism was studied by reverse transcription-polymerase chain reaction (RT-PCR) . RESULTS: In the bezafibrate group biliary phospholipid secretion was increased although cholesterol secretion was not increased . In the pravastatin group, biliary cholesterol and phospholipid secretions were significantly increased . The biliary cholesterol/phospholipid ratio was decreased in the bezafibrate group, but the ratio did not change in the pravastatin group . Hepatic Mdr2, Abcg5 and Abcg8 mRNA expression was remarkably increased in the pravastatin group in comparison with the control group (184%, 264% and 247% of control value, respectively) . In the bezafibrate group the hepatic gene expression of Mdr2 was increased (157% of control value), but there were no significant changes in hepatic Abcg5 and Abcg8 mRNA expression compared with the control group . CONCLUSIONS: Compared with Mdr2, Abcg5 and Abcg8 seem to be more essential transporters for biliary secretion of cholesterol . Pravastatin upregulated Abcg5/Abcg8 while bezafibrate did not, which appears to explain the different effects of these compounds on biliary lipid secretion .

Transplantation, 2004 May 27, 77(10), 1507 - 12
Metabolic interaction between cyclosporine and sirolimus; Bai S et al.; BACKGROUND: When the immunosuppressants cyclosporine (CsA) and sirolimus (SRL) are co-administered to transplant patients, lower doses are used than when either drug is given alone . Since both drugs share similar transport and metabolic pathways, there is the potential for an interaction leading to unpredictable effects . Furthermore, both drugs affect the activity of cytochrome P450 3A1/2 (CYP3A1/2), the rat parallel to human CYP3A4, and the multidrug transporter P-glycoprotein (Pgp) . METHODS: To clarify the role of metabolic enzymes and membrane transporters involved in the disposition of both drugs, we examined hepatic CYP3A1/2, Pgp, and multidrug resistance gene (mdr) mRNA during chronic therapy with CsA and SRL in salt-depleted rats . Specifically, rats were given intravenous doses of CsA 2.5 mg/kg and SRL 1 mg/kg, alone or in combination, for two weeks via constant rate intravenous infusion . RESULTS: CsA treatment inhibited hepatic CYP3A1/2 protein expression, catalytic activity, and mRNA levels . SRL dosing suppressed CYP3A1/2 protein expression and catalytic activity, without affecting mRNA . With combined dosing, however, there was a much greater reduction . Hepatic Pgp protein levels were elevated after treatment with either drug alone, as well as with combined dosing . Compared to controls, there were significant increases in mdr1a and mdr1b mRNA levels in all treatment groups, with the combined drugs causing the greatest increase . CONCLUSIONS: Both CYP3A1/2 and Pgp participate in the disposition of CsA and SRL in rats . Changes in the individual activities of CYP3A1/2 and Pgp may contribute to an interaction between CsA and SRL resulting in unanticipated effects during chronic therapy.

Int J Cancer, 2004 Sep 10, 111(4), 522 - 9
Overexpression of caveolin-1 induces alteration of multidrug resistance in Hs578T breast adenocarcinoma cells; Cai C et al.; Caveolin-1 is a major caveolae-coat protein involved in a variety of cell signaling processes . Some studies have suggested that the level of caveolin-1 expression positively correlates with multi-drug resistance in cancer cells . We demonstrated for the first time that Hs578T doxorubicin resistant cells (Hs578T/Doxo), which contain low levels of endogenous caveolin-1 and high levels of P-glycoprotein, are rendered drug-sensitive by overexpression of exogenous caveolin-1 . MTT assays showed that after overexpressing caveolin-1, the drug resistance of Hs578T/Doxo cells to doxorubicin and cisplatin was reduced from 25.4 +/- 1.5 and 65.3 +/- 2.5 microg/ml to 0.8 +/- 0.15 and 23.2 +/- 2.1 microg/ml, respectively (i.e . reduced by 97% and 64%, respectively) . Furthermore, using rhodamine-123 efflux assays, we observed a significant decrease in P-glycoprotein activity in caveolin-1 overexpressing cells, similar to that observed with 5 microM cyclosporine A or 10 microM verapamil, 2 inhibitors of P-glycoprotein activity . Using confocal microscopy, subcellular fractionation and co-immunoprecipitation assays, a possible physical interaction between caveolin-1 and P-glycoprotein in the caveolae membrane was observed in Hs578T/Doxo cells overexpressing caveolin-1 . These results suggest that overexpression of caveolin-1 changes the state of the cells from drug-resistant to drug-sensitive by inhibiting P-glycoprotein transport activity .

Am J Trop Med Hyg, 2004 Jul, 71(1), 40 - 42
SHORT REPORT: POLYMORPHISMS IN THE CHLOROQUINE RESISTANCE TRANSPORTER GENE IN PLASMODIUM FALCIPARUM ISOLATES FROM LOMBOK, INDONESIA; Huaman MC et al.; The polymorphisms in the Plasmodium falciparum multidrug resistance 1 (pfmdr1) and P . falciparum chloroquine resistance transporter (pfcrt) genes, which are associated with chloroquine resistance, were examined in 48 P . falciparum isolates from uncomplicated malaria patients from the West Lombok District in Indonesia . The point mutation N86Y in pfmdr1 was present in 35.4% of the isolates and mutation K76T in pfcrt was found in all but one of the samples studied . Identified pfcrt haplotypes were mainly identical to the Papua New Guinea type S(agt)VMNT (42 of 48, 87.5%), and a few isolates had the Southeast Asia type CVIET (5 of 48, 10.4%) . Moreover, one P . falciparum isolate harbored the K76N mutation, giving rise to the haplotype CVMNN, which was not previously reported in field isolates . Our findings suggest that chloroquine resistance in this area might have the same origin as in Papua New Guinea.

J Pharm Sci, 2004 Aug, 93(8), 2076 - 89
Nuclear magnetic resonance and molecular modeling analysis of the interaction of the antimalarial drugs artelinic acid and artesunic acid with beta-cyclodextrin; Hartell MG et al.; The artemisinin derivatives artelinic acid and artesunic acid are members of a class of compounds that have shown promise for the treatment of multidrug resistant strains of Plasmodium falciparum . Unfortunately, these compounds exhibit poor solubility and stability in aqueous solution . The research presented herein was conducted to determine whether complexation of artelinic acid or artesunic acid with beta-cyclodextrin would result in complexes with increased aqueous solubility while retaining the potent antimalarial activity of these compounds . Preliminary complexation studies with natural beta-cyclodextrins were conducted as a proof of concept, with a primary focus on understanding the electrostatic interactions that stabilize the resulting complexes . Complex formation was monitored using UV spectroscopy . The structures of the resulting complexes were determined using multidimensional nuclear magnetic resonance spectroscopy (NMR) and molecular modeling . NMR results are most consistent for artelinic acid and beta-cyclodextrin forming complexes in a ratio of 2:1; however, the presence of 1:1, 2:2, and 3:1 complexes in solution cannot be excluded based on the experimental data collected . The NMR data also indicate selective insertion of artelinic acid into the hydrophobic cavity of the beta-cyclodextrin via the primary face . NMR results indicate artesunic acid forms a similar complex with beta-cyclodextrin in a ratio of 1:1; again however, the presence of 1:1, 2:2, and 3:1 complexes in solution cannot be ruled out .

Gastroenterology, 2004 Jul, 127(1), 261 - 74
Regurgitation of bile acids from leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4) knockout mice; Fickert P et al.; BACKGROUND & AIMS: Because the mechanisms leading to bile duct damage in sclerosing cholangitis are unknown, we aimed to determine the pathogenesis of bile duct injury in multidrug resistance gene (Mdr2) (Abcb4) knockout mice (Mdr2(-/-)) as a novel model of the disease . METHODS: Mdr2(-/-) and wild-type controls (Mdr2(+/+)) were studied at 2, 4, and 8 weeks of age . Liver histology, ultrastructure, immunofluorescence microscopy (to study inflammatory cells, tight junction protein ZO-1, basement membrane protein laminin, fluorescence-labeled ursodeoxycholic acid), immunohistochemistry (for alpha-smooth muscle actin, nitrotyrosine), sirius red staining, bacterial cultures of intra-abdominal organs, and polymerase chain reaction (PCR) for Helicobacter bilis DNA were compared between both genotypes . Hepatic cytokine expression was determined by reverse-transcription PCR . RESULTS: Bile ducts of Mdr2(-/-) showed disrupted tight junctions and basement membranes, bile acid leakage into portal tracts, induction of a portal inflammatory (CD11b, CD4-positive) infiltrate, and activation of proinflammatory (tumor necrosis factor {TNF}-alpha, interleukin {IL}-1beta) and profibrogenic cytokines (transforming growth factor {TGF}-beta1) . This resulted in activation of periductal myofibroblasts, leading to periductal fibrosis, separating the peribiliary plexus from bile duct epithelial cells and, finally, causing atrophy and death of the bile duct epithelium . Bacterial translocation was not increased and H . bilis was not detectable in Mdr2(-/-) . CONCLUSIONS: Sclerosing cholangitis in Mdr2(-/-) mice is a multistep process with regurgitation of bile from leaky ducts into the portal tracts, leading to induction of periductal inflammation, followed by activation of periductal fibrogenesis, finally causing obliterative cholangitis owing to atrophy and death of bile duct epithelial cells.

Mol Cancer Res, 2004 Jun, 2(6), 339 - 47
TEL/AML1 overcomes drug resistance through transcriptional repression of multidrug resistance-1 gene expression; Asakura K et al.; The t(12;21)(p12;q22) chromosomal aberration, which is frequently observed in pediatric precursor B-cell acute lymphoblastic leukemia (ALL), generates the TEL/AML1 chimeric gene and protein . TEL/AML1-positive ALL has a favorable prognosis, and one possible reason is that this subtype of ALL rarely shows drug resistance . AML1/ETO, another AML1-containing chimeric protein, has been shown to transcriptionally repress the activity of the multidrug resistance-1 (MDR-1) gene promoter; thus, we examined whether TEL/AML1 also represses MDR-1 gene expression, possibly preventing the emergence of multidrug resistance . In this study, we show that the TEL/AML1 protein binds to the consensus AML1 binding site in the MDR-1 promoter and transcriptionally represses its activity . Following transient transfection of TEL/AML1 protein into Adriamycin-resistant K562/Adr cells, we also demonstrate that TEL/AML1 can down-regulate the expression of P-glycoprotein, a product of the MDR-1 gene, and restore the chemosensitivity to the cells . Furthermore, we report that MDR-1 mRNA levels in leukemic cells obtained from TEL/AML1-positive ALL patients are lower than those from TEL/AML1-negative ALL patients . Thus, TEL/AML1 protein acts as a transcriptional repressor of MDR-1 gene expression, and although TEL/AML1 has been implicated in leukemogenesis, its effects on the MDR-1 gene may contribute to the excellent prognosis of TEL/AML1-positive ALL with current therapy.

Leuk Res, 2004 Sep, 28(9), 973 - 7
Caveolin-1 gene is coordinately regulated with the multidrug resistance 1 gene in normal and leukemic bone marrow; Pang A et al.; Caveolin-1 is a structural protein that may function as a scaffold for plasma membrane proteins, one of which is P-glycoprotein (P-gp), product of the multidrug resistance-1 (MDR-1) gene . We tested the hypothesis that if P-gp and caveolin-1 interacted physically, caveolin-1 and MDR-1 genes might be coordinately regulated; by quantifiying their gene expression with quantitative-polymerase chain reaction . MDR-1 and caveolin-1 gene expressions were normalized to an internal control and related to a fixed calibrator by a comparative cycle-threshold (CT) method . In four different groups of marrow samples (20 normal, 56 acute myeloid leukemias (AML) at diagnosis, 48 AMLs at relapse, and 51 regenerating marrows), caveolin-1 and MDR-1 gene expressions were positively correlated . In 65 samples with MDR-1 over-expression, caveolin-1 and MDR-1 expressions were also correlated . The coordinate expression of caveolin-1 and MDR-1 suggests that they may either interact physically, or are involved in the same aberrant pathway(s) activated during MDR-1 up-regulation.

J Pharm Pharmacol, 2004 Jul, 56(7), 899 - 907
Effects of monensin liposomes on the cytotoxicity, apoptosis and expression of multidrug resistance genes in doxorubicin-resistant human breast tumour (MCF-7/dox) cell-line; Shaik MS et al.; We have evaluated the effects of monensin liposomes on drug resistance reversal, induction of apoptosis and expression of multidrug resistance (MDR) genes in a doxorubicin-resistant human breast tumour (MCF-7/dox) cell line . Monensin liposomes were prepared by the pH-gradient method . MCF-7/dox cells were treated with various anticancer drugs (doxorubicin, paclitaxel and etoposide) alone and in combination with monensin liposomes . The cytotoxicity was assessed using the crystal violet dye uptake method . The induction of apoptosis in MCF-7/dox cells was assessed by established techniques such as TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labelling) staining and caspase-3 assay . The effect of monensin liposomes on doxorubicin accumulation in MCF-7/dox cells was monitored by fluorescent microscopy . Finally, the expression of MDR genes (MDR1 and MRP1) in MCF-7/dox cells following the exposure to doxorubicin alone and in combination with monensin liposomes was evaluated by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) . Our results indicated that monensin liposomes overcame drug resistance in MCF-7/dox cells to doxorubicin, etoposide and paclitaxel by 16.5-, 5.6- and 2.8-times, respectively . The combination of doxorubicin (2.5 microg mL(-1)) with monensin liposomes (20 x 10(-8)M) induced apoptosis in approximately 40% cells, whereas doxorubicin (2.5 microg mL(-1)) or monensin liposomes (20 x 10(-8)M) alone produced minimal apoptosis (<10%) in MCF-7/dox cells . Fluorescent microscopy revealed that monensin liposomes increased the accumulation of doxorubicin in MCF-7/dox cells . RT-PCR studies demonstrated that the expression of MDR1 and MRP1 was increased by 33 and 57%, respectively, in MCF-7/dox cells following treatment with doxorubicin (2.5 microg mL(-1)) for 72 h as compared with control MCF-7/dox cells . Furthermore, the levels of MDR1 and MRP1 in MCF-7/dox cells exposed to both doxorubicin and monensin liposomes showed a modest decrease as compared with MCF-7/dox cells treated with doxorubicin alone . In conclusion, the delivery of monensin via liposomes provided an opportunity to overcome drug resistance.

Cancer Res, 2004 Jul 1, 64(13), 4654 - 63
Antiangiogenic and antitumoral activity of phenyl-3-(2-chloroethyl)ureas: a class of soft alkylating agents disrupting microtubules that are unaffected by cell adhesion-mediated drug resistance; Petitclerc E et al.; The development of new anticancer agents with lower toxicity, higher therapeutic index, and weaker tendency to induce resistant phenotypes in tumor cells is a continuous challenge for the scientific community . Toward that end, we showed previously that a new class of soft alkylating agents designed as phenyl-3-(2-chloroethyl)ureas (CEUs) inhibits tumor cell growth in vitro and that their efficiency is not altered by clinically relevant mechanisms of resistance such as overexpression of multidrug resistance proteins, increase in intracellular concentration of glutathione and/or glutathione S-transferase activity, alteration of topoisomerase II, and increased DNA repair . Mechanistic studies have showed recently that the cytotoxic activity of several CEUs was mainly related to the disruption of microtubules . Here, we present results supporting our assumption that 4-tert-butyl-{3-(2-chloroethyl)ureido}phenyl (tBCEU) (and its bioisosteric derivative 4-iodo-{3-(2-chloroethyl)ureido}phenyl (ICEU) are potent antimicrotubule agents both in vitro and in vivo . They covalently bind to beta-tubulin, leading to a microtubule depolymerization phenotype, consequently disrupting the actin cytoskeleton and altering the nuclear morphology . Accordingly, tBCEU and ICEU also inhibited the migration and proliferation of endothelial and tumor cells in vitro in a dose-dependent manner . It is noteworthy that ICEU efficiently blocked angiogenesis and tumor growth in three distinct animal models: (a) the Matrigel plug angiogenesis assay; (b) the CT-26 tumor growth assay in mice; and (c) the chick chorioallantoic membrane tumor assay . In addition, we present evidence that CEU cytotoxicity is unaffected by additional resistance mechanisms impeding tumor response to DNA alkylating agents such as cisplatin, namely the cell adhesion mediated-drug resistance mechanism, which failed to influence the cytocidal activity of CEUs . On the basis of the apparent innocuousness of CEUs, on their ability to circumvent many classical and recently described tumor cell resistance mechanisms, and on their specific biodistribution to organs of the gastrointestinal tract, our results suggest that CEUs represent a promising new class of anticancer agents.

Cancer Res, 2004 Jul 1, 64(13), 4621 - 8
BPR0L075, a novel synthetic indole compound with antimitotic activity in human cancer cells, exerts effective antitumoral activity in vivo; Kuo CC et al.; BPR0L075 is a novel synthetic compound discovered through research to identify new microtubule inhibitors . BPR0L075 inhibits tubulin polymerization through binding to the colchicine-binding site of tubulin . Cytotoxic activity of BPR0L075 in a variety of human tumor cell lines has been ascertained, with IC(50) values in single-digit nanomolar ranges . As determined by flow cytometry, human cervical carcinoma KB cells are arrested in G(2)-M phases in a time-dependent manner before cell death occurs . Terminal deoxynucleotidyl transferase-mediated nick end labeling assay indicates that cell death proceeds through an apoptotic pathway . Additional studies indicate that the effect of BPR0L075 on cell cycle arrest is associated with an increase in cyclin B1 levels and a mobility shift of Cdc2 and Cdc25C . The changes in Cdc2 and Cdc25C coincide with the appearance of phosphoepitopes recognized by a marker of mitosis, MPM-2 . Furthermore, phosphorylated forms of Bcl-2, perturbed mitochondrial membrane potential, and activation of the caspase-3 cascade may be involved in BPR0L075-induced apoptosis . Notably, several KB-derived multidrug-resistant cell lines overexpressing P-gp170/MDR and MRP are resistant to vincristine, paclitaxel, and colchicine but not to BPR0L075 . Moreover, BPR0L075 shows potent activity against the growth of xenograft tumors of the gastric carcinoma MKN-45, human cervical carcinoma KB, and KB-derived P-gp170/MDR-overexpressing KB-VIN10 cells at i.v . doses of 50 mg/kg in nude mice . These findings indicate BPR0L075 is a promising anticancer compound with antimitotic activity that has potential for management of various malignancies, particularly for patients with drug resistance.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2004 Jun, 12(3), 321 - 3
{Reversal effect of tetrandrine in combination with droloxifene on cell line K562/A02 and its correlation with inducted apoptosis}; Zhou Y et al.; To study the correlation of the reversal effect of tetrandrine (Tet) in combination with droloxifene (DRL) on multidrug resistant cell line K562/A02 and induction apoptosis, the apoptosis of K562 cells and K562/A02 cells after the treatment of Tet and DRL alone or their combination was detected by flow cytometry (FCM) . The results showed 0.62 microg/ml Tet and 1.94 microg/ml DRL alone could induce apoptosis of K562 cells in time-dependent manner after culture for 48 hours . Neither Tet at 0.62 microg/ml nor DRL at 1.94 microg/ml alone could induce apoptosis of K562/A02 cells, but in the combination of them two induced apoptosis of few K562/A02 cells in culture for 72 hours . In conclusion, the mechanism of Tet and DRL reversing multidrug resistance has no correlation with the apoptosis of K562/A02 cells.

Pharmacogenetics, 2004 Jul, 14(7), 429 - 40
High plasma pravastatin concentrations are associated with single nucleotide polymorphisms and haplotypes of organic anion transporting polypeptide-C (OATP-C, SLCO1B1); Niemi M et al.; This study aimed to characterize possible relationships between polymorphisms in the drug transporter genes organic anion transporting polypeptide-C (OATP-C, SLCO1B1), OATP-B (SLCO2B1), multidrug resistance-associated protein 2 (MRP2, ABCC2) and multidrug resistance transporter (MDR1, ABCB1) and the pharmacokinetics of pravastatin . We studied 41 healthy Caucasian volunteers who had previously participated in pharmacokinetic studies with pravastatin . Six volunteers had a very high pravastatin AUC value and were defined as outliers according to statistical criteria . The OATP-C gene was sequenced completely in all subjects, and they were also genotyped for selected single nucleotide polymorphisms (SNP) in the OATP-B, MDR1 and MRP2 genes . Of the six outliers, five were heterozygous for the OATP-C 521T>C (Val174Ala) SNP (allele frequency 42%) and three were heterozygous for a new SNP in the promoter region of OATP-C (-11187G>A, allele frequency 25%) . Among the remaining 35 subjects, two were homozygous and six were heterozygous carriers of the 521T>C SNP (allele frequency 14%, P = 0.0384 versus outliers) and three were heterozygous carriers of the -11187G>A SNP (allele frequency 4%, P = 0.0380 versus outliers) . In subjects with the -11187GA or 521TC genotype, the mean pravastatin AUC0-12 was 98% (P = 0.0061) or 106% (P = 0.0034) higher, respectively, compared to subjects with the reference genotype . These results were substantiated by haplotype analysis . In heterozygous carriers of *15B (containing the 388A>G and 521T>C variants), the mean pravastatin AUC0-12 was 93% (P = 0.024) higher compared to non-carriers and, in heterozygous carriers of *17 (containing the -11187G>A, 388A>G and 521T>C variants), it was 130% (P = 0.0053) higher compared to non-carriers . No significant associations were found between OATP-B, MRP2 or MDR1 polymorphisms and the pharmacokinetics of pravastatin . These results suggest that haplotypes are more informative in predicting the OATP-C phenotype than single SNPs.

J Clin Oncol, 2004 Jul 1, 22(13), 2662 - 70
Liposome-encapsulated doxorubicin in combination with standard agents (cyclophosphamide, vincristine, prednisone) in patients with newly diagnosed AIDS-related non-Hodgkin's lymphoma: results of therapy and correlates of response; Levine AM et al.; PURPOSE: To evaluate the safety and efficacy of liposomal doxorubicin (Myocet; Medeus Pharma Ltd, Herts,UK) when substituted for doxorubicin in the CHOP regimen (cyclophosphamide, doxorubicin, vincristine, prednisone) in patients with newly diagnosed AIDS-related non-Hodgkin's lymphoma (AIDS-NHL) . Secondary objectives were to assess the impact of HIV viral control on response and survival, and to correlate MDR-1 expression with outcome . PATIENTS AND METHODS: Liposomal doxorubicin at doses of 40, 50, 60, and 80 mg/m(2) was given with fixed doses of cyclophosphamide, vincristine, and prednisone every 21 days . All patients received concurrent highly active antiretroviral therapy . NHL tissues were evaluated for multidrug resistance (MDR-1) expression . RESULTS: Twenty-four patients were accrued . 67% had high or high-intermediate International Prognostic Index scores; the median CD4 lymphocyte count was 112/mm(3) (range, 19/mm(3) to 791/mm(3)) . No dose-limiting toxicities were observed at any level, with myelosuppression being the most frequent toxicity . Overall response rate was 88%, with 75% complete responses (CRs), and 13% partial responses . The median duration of CR was 15.6+ months (range, 1.7 to 43.5+ months) . Effective HIV viral control during chemotherapy was associated with significantly improved survival (P =.027), but CRs were attained independent of HIV viral control . MDR-1 expression did not correlate with response, suggesting that the liposomal doxorubicin may evade this resistance mechanism . CONCLUSION: Liposomal doxorubicin in combination with cyclophosphamide, vincristine, and prednisone is active in AIDS-NHL, with complete remissions achieved in 75% independent of HIV viral control or tissue MDR-1 expression . HIV viral control is associated with a significant improvement in survival . Additional studies are warranted.

Mol Microbiol, 2004 Jul, 53(1), 283 - 95
Identification of mutator genes and mutational pathways in Escherichia coli using a multicopy cloning approach; Yang H et al.; We searched for genes that create mutator phenotypes when put on to a multicopy plasmid in Escherichia coli . In many cases, this will result in overexpression of the gene in question . We constructed a random shotgun library with E . coli genomic fragments between 3 and 5 kbp in length on a multicopy plasmid vector that was transformed into E . coli to screen for frameshift mutators . We identified a total of 115 independent genomic fragments that covered 17 regions on the E . coli chromosome . Further studies identified 12 genes not previously known as causing mutator phenotypes when overproduced . A striking finding is that overproduction of the multidrug resistance transcription regulator, EmrR, results in a large increase in frameshift and base substitution mutagenesis . This suggests a link between multidrug resistance and mutagenesis . Other identified genes include those encoding DNA helicases (UvrD, RecG, RecQ), truncated forms of the DNA mismatch repair protein (MutS) and a primosomal component (DnaT), a negative modulator of initiation of replication/GATC-binding protein (SeqA), a stationary phase regulator AppY, a transcriptional regulator PaaX and three putative open reading frames, ycgW, yfjY and yjiD, encoding hypothetical proteins . In addition, we found three genes encoding proteins that were previously known to cause mutator effects under overexpression conditions: error-prone polymerase IV (DinB), DNA methylase (Dam) and sigma S factor (RpoS) . This genomic strategy offers an approach to identify novel mutator effects resulting from the multicopy cloning (MCC) of specific genes and therefore complementing the conventional gene inactivation approach to finding mutators.

Mol Microbiol, 2004 Jul, 53(1), 275 - 82
The folate pathway is a target for resistance to the drug para-aminosalicylic acid (PAS) in mycobacteria; Rengarajan J et al.; The increasing rate of multidrug-resistant tuberculosis has led to more use of second-line antibiotics such as para-aminosalicylic acid (PAS) . The mode of action of PAS remains unclear, and mechanisms of resistance to this drug are undefined . We have isolated PAS-resistant transposon mutants of Mycobacterium bovis BCG with insertions in the thymidylate synthase (thyA) gene, a critical determinant of intracellular folate levels . BCG thyA mutants have reduced thymidylate synthase activity and are resistant to known inhibitors of the folate pathway . We also find that mutations in thyA are associated with clinical PAS resistance . We have identified PAS-resistant Mycobacterium tuberculosis isolates from infected patients, which harbour mutations in thyA and show reduced activity of the encoded enzyme . Thus, PAS acts in the folate pathway, and thyA mutations probably represent a mechanism of developing resistance not only to PAS but also to other drugs that target folate metabolism.

World J Gastroenterol, 2004 Jul 1, 10(13), 1979 - 83
Reversal of 5-flouroucial resistance by adenovirus-mediated transfer of wild-type p53 gene in multidrug-resistant human colon carcinoma LoVo/5-FU cells; Yu ZW et al.; AIM: To observe the reversal effects of wide-type p53 gene on multi-drug resistance to 5-FU (LOVO/5-FU) . METHODS: After treatment with Ad-p53, LOVO/5-FU sensitivity to 5-Fu was investigated using tetrazolium dye assay . Multidrug resistance gene-1 (MDR1) gene expression was assayed by semi-quantitative reverse transcription-polymerase chain reaction and the expression of p53 protein was examined by Western blotting . RESULTS: The reversal activity after treatment with wide-type p53 gene was increased up to 4.982 fold at 48 h . The expression of MDR1 gene decreased significantly after treatment with wide-type p53 gene, and the expression of p53 protein lasted for about 5 d, with a peak at 48 h, and began to decrease at 72 h . CONCLUSION: Wide-type p53 gene has a remarkable reversal activity for the high expression of MDR1 gene in colorectal cancers . The reversal effects seem to be in a time dependent manner . It might have good prospects in clinical application.

Amino Acids, 2004 Jun, 26(3), 273 - 82 Epub 2003 Dec 22.
Mitochondrial alterations induced by serum amine oxidase and spermine on human multidrug resistant tumor cells; Arancia G et al.; Multidrug resistance (MDR) has been studied extensively because it is one of major problems in cancer chemotherapy . The MDR phenotype is often due to overexpression of P-glycoprotein (P-gp), that acting as an energy-dependent drug efflux pump exports various anticancer drugs out of cells . The major goal of our investigation is to establish whether bovine serum amine oxidase (BSAO), which generates the products H(2)O(2) and aldehyde(s), from the polyamine spermine, is able to overcome MDR of human cancer cells . The cytotoxicity of the products was evaluated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells . A clonogenic cell survival assay demonstrated that LoVo DX cells were more sensitive than LoVo WT cells . Exogenous catalase protected cells against cytotoxicity mainly due to the formation of H(2)O(2) . However, spermine-derived aldehyde(s) still induced some cytotoxicity . The cytotoxic effect was totally inhibited in the presence of both enzymes, catalase and NAD-dependent aldehyde dehydrogenase (ALDH) . Transmission electron microscopy investigations showed that BSAO and spermine induced evident mitochondria alterations, more pronounced in MDR than in LoVo WT cells . The mitochondrial activity was checked by flow cytometry studies, labelling cells with the probe JC1, that displayed a basal hyperpolarized status of the mitochondria in multidrug-resistant cells . After treatment with amine oxidase in the presence of polyamine-spermine, the cells showed a marked increase in mitochondrial membrane depolarization higher in LoVo DX than in LoVo WT cells . Our findings suggest that toxic oxidation products formed from spermine and BSAO could be a powerful tool in the development of new anticancer treatments, mainly against MDR tumor cells.

J Pharmacol Exp Ther, 2004 Nov, 311(2), 449 - 55 Epub 2004 Jun 24.
Plasma membrane localization of multidrug resistance-associated protein homologs in brain capillary endothelial cells; Zhang Y et al.; Several multidrug resistance-associated protein (MRP) homologs are expressed in brain microvessel endothelial cells forming the blood-brain barrier (BBB) . The influence of these MRP transporters on BBB permeability will be dependent on their localization within the brain microvessel endothelial cells . Using two different and complementary approaches, the localization of various MPR homologs (MRP1, MRP4, and MRP5) was examined in primary cultured bovine brain microvessel endothelial cells (BBMECs) . The first approach involved centrifugal separation of apical and basolateral plasma membranes of cultured BBMECs . The membrane fractions were then subjected to Western blot analysis for MRPs . The second approach used confocal laser scanning microscopy to determine membrane localization of MRPs in BBMECs . Results show a predominantly apical plasma membrane distribution for MRP1 and MRP5, and an almost equal distribution of MRP4 on the apical and basolateral plasma membrane of BBMECs . These studies provide the first demonstration of the localization of MRP1, MRP4, and MRP5 homologs in brain microvessel endothelial cells . The present studies also indicate that the localization of MRPs in the endothelial cells forming the BBB is different from that observed in polarized epithelial cells and thus may contribute to the reduced entry and enhanced elimination of organic anions and nucleotides in the brain.

Blood, 2004 Oct 1, 104(7), 1940 - 51 Epub 2004 Jun 24.
Targeting the multidrug resistance-1 transporter in AML: molecular regulation and therapeutic strategies; Mahadevan D et al.; The multidrug resistance-1 (MDR1) gene product, P-glycoprotein (P-gp), and the multidrug resistance-related proteins (MRPs) are members of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter gene superfamily that regulates the trafficking of drugs, peptides, ions, and xenobiotics across cell membrane barriers . Three-dimensional modeling of human MDR1/P-gp indicates that these glycoproteins function as efficient, ATP-dependent gate-keepers, which scan the plasma membrane and its inner leaflet to flip lipophilic substrates to the outer membrane leaflet . Delineation of the adverse prognostic power of MDR1 in adult acute myeloid leukemia (AML) raised hopes that pharmacologic blockade of P-gp would improve the outcome of conventional cytotoxic therapy, perhaps more so than in any other human malignancy . Phase 3 clinical trials investigating first- and second-generation P-gp antagonists have yielded conflicting results, emphasizing the importance of applying preclinical principals to realistically appraise expectations for clinical benefit . Structure-based design strategies and the delineation of transcriptional regulators of survival gene cassettes promise to yield novel, more-effective strategies to overcome drug resistance . Lessons learned from investigations of these and other mechanisms of cellular defense hold promise for a renaissance in the development of targeted therapeutics in acute leukemia.

Am J Physiol Gastrointest Liver Physiol, 2004 Oct, 287(4), G749 - 56 Epub 2004 Jun 24.
ATP-dependent transport of organic anions into isolated basolateral membrane vesicles from rat intestine; Shoji T et al.; The mechanism for the cellular extrusion of organic anions across the intestinal basolateral membrane was examined using isolated membrane vesicles from rat jejunum, ileum, and colon . It was found that 17beta-estradiol 17beta-D-glucuronide (E217betaG) is taken up in an ATP-dependent manner into the basolateral membrane vesicles (BLMVs) but not into the brush-border or microsomal counterparts . The ATP-dependent uptake of E217betaG into BLMVs from jejunum and ileum was described by a single component with a Km value of 23.5 and 8.31 microM, respectively, whereas that into the BLMVs from colon was described by assuming the presence of high (Km=0.82 microM)- and low-affinity (Km=35.4 microM) components . Taurocholate, 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole glucuronide and taurolithocholate sulfate, but not leukotriene C4, were significantly taken up by the BLMVs . In addition to such substrate specificity, the inhibitor sensitivity of the ATP-dependent transport in BLMVs was similar to that of rat multidrug resistance-associated protein 3 (Mrp3), which is located on the basolateral membrane of enterocytes . Together with the fact that the rank order of the extent of the expression of Mrp3 (jejunum < ileum << colon) is in parallel with that of the extent of the transport of ligands, these results suggest that the ATP-dependent uptake of organic anions into isolated intestinal BLMVs is at least partly mediated by Mrp3.

J Infect Dis, 2004 Jul 15, 190(2), 285 - 92 Epub 2004 Jun 11.
Antiretroviral resistance among HIV-infected persons who have died in British Columbia, in the era of modern antiretroviral therapy; Recsky MA et al.; BACKGROUND: The prevalence of antiretroviral resistance among persons enrolled in the centralized HIV/AIDS Drug Treatment Program in British Columbia, Canada, who had died between July 1997 and December 2001, was investigated, to determine the degree to which antiretroviral resistance contributed to mortality . METHODS: During this period, 637 deaths had occurred . The last plasma sample obtained during therapy was genotyped retrospectively for treated individuals who had died of a nonaccidental cause . Samples with plasma human immunodeficiency virus (HIV) loads <500 copies/mL were not genotyped . Drug resistance among 1220 living HIV-infected persons who had experienced virologic therapy failure during the study period also was examined . RESULTS: Of 554 individuals who had died of nonaccidental causes, 58 (10.4%) were antiretroviral naive, and 99 (17.9%) had very brief exposure to antiretroviral therapy (median, 2 months) . The majority of isolates from the remaining 397 individuals harbored either no major resistance mutations or represented samples with plasma HIV suppression of <500 copies/mL . Resistance to >/=1, >/=2, or 3 drug classes was observed in 76%, 42%, and 11% of individuals, respectively, in the group of 1220 living individuals experiencing virologic therapy failure, compared with only 44%, 23%, and 5% of individuals, respectively, who had died (P<.001) . CONCLUSION: Only a relatively low prevalence of multidrug resistance was observed in this cohort, indicating that the exhaustion of treatment options because of drug resistance was not a significant contributor to mortality.

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