Multidrug resistant

Cancer Res, 2003 Jul 15, 63(14), 4048 - 54
Transport of methotrexate, methotrexate polyglutamates, and 17beta-estradiol 17-(beta-D-glucuronide) by ABCG2: effects of acquired mutations at R482 on methotrexate transport; Chen ZS et al.; ABCG2 is a plasma membrane efflux pump that is able to confer resistance to several anticancer agents, including mitoxantrone, camptothecins, anthracyclines, and flavopiridol . The antimetabolite methotrexate (MTX) was inferred recently to be an additional substrate of the pump based on the analysis of ABCG2-overexpressing cell lines . However, the transport characteristics of the pump with regard to this agent have not been determined . In addition, physiological substrates of ABCG2 have not been identified . Here we examine the in vitro transport properties of the pump using membrane vesicles prepared from HEK293 cells transfected with ABCG2 expression vector . In so doing it is shown that MTX is a high capacity low affinity substrate of the pump, with K(m) and V(max) values of 1.34 +/- 0.18 mM and 687 +/- 87 pmol/mg/min, respectively . Unlike previously characterized multidrug resistance protein family members, ABCG2 is also able to transport MTX diglutamate and MTX triglutamate . However, addition of even one more glutamyl residue is sufficient to completely abrogate ABCG2-mediated transport . By contrast with the wild-type protein (ABCG2-R482), two ABCG2 variants that have been identified in drug selected cell lines, R482T and R482G, were unable to transport MTX to any extent . Similarly, folic acid was subject to efflux by the wild-type protein but not by the two mutants . However, transport of the reduced folate leucovorin was not detected for either the wild-type or the mutant proteins . Finally, it is shown that ABCG2 is capable of transporting E(2)17betaG with K(m) and V(max) values of 44.2 +/- 4.3 micro M and 103 +/- 17 pmol/mg/min, respectively . These results indicate that ABCG2 is a component of the energy-dependent efflux system for certain folates and antifolates, but that its transport characteristics with respect to polyglutamates and reduced folates are not identical to those of multidrug resistance protein family members . In addition, it is demonstrated that R482 mutations observed in drug-resistant cell lines have profound effects on the in vitro transport properties of the pump.

Cancer Res, 2003 Jul 15, 63(14), 3860 - 5
Glucosylceramide synthase and its functional interaction with RTN-1C regulate chemotherapeutic-induced apoptosis in neuroepithelioma cells; Di Sano F et al.; Glucosylceramide synthase (GCS), the key enzyme in the biosynthesis of glycosphingolipids, has been implicated in many biological phenomena, including multidrug resistance . GCS inhibition, by both antisense and the specific inhibitor (D-threo)-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), results in a drastic decrease of apoptosis induced by the p53-independent chemotherapeutic agent N-(4-hydroxyphenyl)retinamide in neuroepithelioma cells . By using the yeast two-hybrid system, we have identified a member of the reticulon (RTN) family (RTN-1C) as the major GCS-protein partner . Interestingly, RTN-1C not only interacts with GCS at Golgi/ER interface but also modulates its catalytic activity in situ . In fact, overexpression of RTN-1C sensitizes CHP-100 cells to fenretinide-induced apoptosis . These findings demonstrate a novel p53-independent pathway of apoptosis regulated by Golgi/endoplasmic reticulum protein interactions, which is relevant for cancer combined therapy.

Br J Pharmacol, 2003 Jul, 139(6), 1111 - 8
Antidepressant fluoxetine enhances glucocorticoid receptor function in vitro by modulating membrane steroid transporters; Pariante CM et al.; 1 . Incubation of LMCAT fibroblast cells with antidepressants potentiates glucocorticoid receptor (GR)-mediated gene transcription in the presence of dexamethasone and cortisol, but not of corticosterone . We have shown that antidepressants do so by inhibiting the LMCAT cell membrane steroid transporter (which is virtually identical to the multidrug resistance P-glycoprotein) and thus by increasing dexamethasone or cortisol intracellular concentrations . However, previous experiments with the antidepressant fluoxetine in the presence of dexamethasone have produced negative results (Pariante et al . (2001) . Br . J . Pharmacol., 134, 1335-1343) . 2 . We have since re-examined the effects of fluoxetine on GR-mediated gene transcription in the presence of dexamethasone . Moreover, we have examined the effects of fluoxetine on GR-mediated gene transcription in the presence of cortisol and corticosterone, and on the intracellular accumulation of radioactive cortisol and corticosterone . Finally, we have examined the effects of fluoxetine on inhibition of P-glycoprotein activity in Caco-2 cells . 3 . We now find that fluoxetine (1-10 micro M) enhances GR-mediated gene transcription in the presence of dexamethasone and cortisol (+140-170%), but not of corticosterone, and increases the intracellular accumulation of (3)H-cortisol (+5-15%), but not of (3)H-corticosterone . Moreover, fluoxetine (10 micro M) induces approximately 30% inhibition of PGP activity in Caco-2 cells . 4 . Our results show that fluoxetine, like other antidepressants, inhibits membrane steroid transporters.

Curr Drug Metab, 2003 Aug, 4(4), 313 - 8
An update on the extraneuronal monoamine transporter (EMT): characteristics, distribution and regulation; Martel F et al.; Biological membranes prevent transmembrane diffusion in the majority of organic molecules that bear net charges at physiological pH . Consequently, these compounds must use more or less specific membrane-bound transport systems to be imported into or exported from cells or organisms . The extraneuronal monoamine transporter (EMT) is a transmembranar transport system involved in the transfer of monoamine compounds across cell membranes . It was identified more than 30 years ago {1}, its functional characteristics being thereafter described {review by 2} . The recent cloning of this transporter in man and rat reopened investigation and interest in this entity . EMT is a Na(+) and Cl(-)-independent, potential-dependent carrier, known to have a broad tissue distribution (eg . myocardium, vascular and non-vascular smooth muscle cells, glandular cells, placenta and CNS glial cells) . According to its transport function and primary structure, EMT is included in the amphiphilic solute facilitator (ASF) family of transporters . Physiological substrates for EMT include the monoamines serotonin, dopamine, noradrenaline, adrenaline and histamine . Moreover, several xenobiotics including the neurotoxin 1-methyl-4-phenylpyridinium, clonidine, cimetidine and the K(+)-channel blocker tetraethylammonium interact with this transporter . The aim of this work is to review knowledge concerning EMT, making an update on its functional characteristics, physiological importance and regulation . A special emphasis will be given to very recent investigations concerning regulation of EMT by intracellular second messenger systems and the interaction of modulators of P-glycoprotein, the product of the multidrug resistance gene MDR1, with EMT.

Biochem Cell Biol, 2003 Apr, 81(2), 61 - 70
Examination of EmrE conformational differences in various membrane mimetic environments; Federkeil SL et al.; Ethidium multidrug resistance protein (EmrE) is a member of the small multidrug resistance family of proteins and is responsible for resistance in Escherichia coli to a diverse group of lipophilic cations . Research is beginning to elucidate structural information as well as substrate binding and extrusion mechanisms for this protein . However, the choice of membrane mimetic environment to perform structural studies needs to be made . In this study EmrE was solubilized in different membrane mimetic environments to investigate the influence of environment on the structure and dynamics of the protein by comparing the fluorescence properties of emission maxima, peak shifts, relative intensities, acrylamide quenching constants, and polarization . Taken together, the different fluorescence observations on EmrE in the various membrane mimetic systems tested suggest that the tryptophan residues in EmrE are present in the most flexible and exposed state when solubilized in methanol, followed by sodium dodecyl sulfate and urea . The two detergents N-dodecyl-beta-D-maltoside (DM) and polyoxyethylene(8)dodecyl ether, for the most part, only display subtle differences between the spectral properties with DM best representing the lipid environment . The conformation of EmrE is clearly more open and dynamic in detergent relative to being reconstituted in small unilamellar vesicles . The fluorescence observations of EmrE solubilized in trifluoroethanol shows an environment that is similar to that of EmrE solubilized in detergents . Additionally, secondary structure was monitored by circular dichroism (CD) . The CD spectra were similar among the different solubilizing conditions, suggesting little difference in alpha-helical content . This work establishes groundwork for the choice of solubilizing conditions for future structural, folding, and ligand binding studies.

Int J Tuberc Lung Dis, 2003 Jul, 7(7), 660 - 4
Chronic cases of tuberculosis in Casablanca, Morocco; El Baghdadi J et al.; OBJECTIVE: To evaluate the prevalence and patterns of drug resistance of Mycobacterium tuberculosis isolates collected from patients with chronic tuberculosis in Casablanca, Morocco . METHODS: Between February 1996 and September 2001, 122 isolates were recovered from 112 different patients . The male to female ratio was 2.4 . RESULTS: From February 1996 to May 1997, 77.5% of isolates were multidrug-resistant (MDR-TB), compared to 69.4% from February 1999 to May 2000 and 78.7% from June 2000 to September 2001 . The prevalence of MDR-TB is similar from the initial to the last period of this study . Analysis of the 69 bp hypervariable region of the rpoB gene by DNA sequencing on 42 M . tuberculosis isolates (37 resistant, 5 sensitive) showed nine different types of mutations on codons rpoB 513, rpoB 516, rpoB 522, rpoB 523 and rpoB 526 . A new point mutation was observed on codon rpoB 523 on one isolate . No mutation was detected on this rpoB region for four resistant isolates . CONCLUSION: The high rate of MDR-TB illustrates a serious problem . The public health authorities have introduced a new regimen protocol consisting of 3 months of kanamycin, ofloxacin, pyrazinamide and ethionamide, followed by 18 months of ofloxacin, pyrazinamide and ethionamide (3KOZEA/18OZEA) for this category of patients, and it is hoped that the additional use of ofloxacin during the intensive phase of treatment will reduce the rate of resistance.

Int J Tuberc Lung Dis, 2003 Jul, 7(7), 652 - 9
Comparison of DNA sequencing, PCR-SSCP and PhaB assays with indirect sensitivity testing for detection of rifampicin resistance in Mycobacterium tuberculosis; Mani C et al.; SETTING: Tuberculosis Research Centre, Chennai, India . OBJECTIVE: To rapidly identify multidrug-resistant Mycobacterium tuberculosis using phenotypic and genotypic methods . DESIGN: Two genotypic assays, DNA sequencing and polymerase chain reaction single strand conformation polymorphism (PCR-SSCP), and one phenotypic assay, phage amplified biological assay (PhaB) were standardised in-house and performed on coded 101 rifampicin-resistant and 100 rifampicin-sensitive M . tuberculosis clinical isolates for the identification of rifampicin resistance . RESULTS AND CONCLUSION: The results obtained using the three assays were compared with those from the conventional indirect sensitivity test . The sensitivities and specificities of DNA sequencing, PCR-SSCP and PhaB were 97% and 100%, 76% and 100%, and 97% and 84%, respectively . DNA sequencing was found to be more sensitive and specific than the other tests.

Int J Tuberc Lung Dis, 2003 Jul, 7(7), 637 - 44
Clinical and programmatic considerations in the treatment of MDR-TB in children: a series of 16 patients from Lima, Peru; Mukherjee JS et al.; SETTING: Since 2000, the directly observed treatment, short-course (DOTS) strategy has been expanded in several countries to include treatment of multidrug-resistant tuberculosis (MDR-TB) . This strategy is known as DOTS-Plus . Tuberculosis is a common cause of morbidity and mortality for children throughout the developing world . Children may also be infected with MDR-TB, yet most developing countries do not specifically address pediatric MDR-TB . OBJECTIVE: To present the intermediate outcomes of the first 16 children enrolled in the Peruvian DOTS-Plus program and to demonstrate the tolerability of second-line anti-tuberculosis drugs . RESULTS: Three children completed therapy and are cured, one child had bacteriologic and clinical failure after 12 months of therapy and died of respiratory insufficiency, and 12 have intermediate outcomes demonstrating favorable clinical, bacteriologic, and radiographic evidence of improvement after 9-19 months of therapy . CONCLUSIONS: Of the 16 pediatric DOTS-Plus patients, 15 have tolerated therapy well and have had favorable clinical evolution . However, the diagnosis of pediatric MDR-TB is often extremely delayed due to reliance on the adult case definition and should be changed to prevent progressive, chronic illness in such children . Programmatic changes could facilitate earlier diagnosis and treatment of pediatric MDR-TB in Peru and in other DOTS-Plus programs.

Int J Tuberc Lung Dis, 2003 Jul, 7(7), 631 - 6
Drug resistance among failure and relapse cases of tuberculosis: is the standard re-treatment regimen adequate?
Quy HT, Lan NT, Borgdorff MW, Grosset J, Linh PD, Tung LB, van Soolingen D, Raviglione M, Co NV, Broekmans J.
OBJECTIVE: To determine acquired drug resistance among failure and relapse cases after treatment of new smear-positive tuberculosis . METHODS: A cohort of 2901 patients with new smear-positive tuberculosis was enrolled in Vietnam . Sputum samples were stored at enrolment . Upon failure or relapse, another sputum sample was collected . Both were cultured and underwent drug susceptibility testing and restriction fragment length polymorphism (RFLP) typing . RESULTS: Of 40 failure cases, 17 had multidrug resistance (MDR) at enrolment . At failure, 15 of the 23 (65%) patients without primary MDR had acquired MDR . Of 39 relapse cases and 143 controls, none had primary MDR . CONCLUSION: Primary drug resistance was a strong risk factor for failure and relapse and for acquiring further resistance . As 80% of failure cases had MDR, the standard re-treatment regimen appears inadequate for failure cases in this control programme with a very high cure rate among new cases.

J Infect Dis, 2003 Aug 1, 188(3), 356 - 63 Epub 2003 Jul 18.
Persistence of a highly resistant strain of tuberculosis in New York City during 1990-1999; Munsiff SS et al.; One multidrug-resistant Mycobacterium tuberculosis (MDRTB) strain, strain W, caused several nosocomial outbreaks in New York City (NYC) during 1 January 1990-31 July 1993 . We reviewed all MDRTB cases verified during 1 August 1993-31 December 1999 that had isolates with either this DNA pattern or a variant of this strain, and we compared them to the outbreak cases . Of 427 DNA-confirmed cases from 1990-1999, 161 (37%) were from 1 August 1993-31 December 1999; these 161 cases, from 56 hospitals and 2 correctional sites, constituted 28% of all MDRTB cases in NYC during this period . Compared with those from 1 January 1990-31 July 1993, patients from 1 August 1993-31 December 1999 were less likely to be infected with human immunodeficiency virus, to have been born in the United States, to be homeless, to have been incarcerated, and to have epidemiological links; 16% of patients had nosocomial- and 9% had community-exposure links . This strain was disseminated widely in the community during the outbreaks; postoutbreak cases likely represent reactivated disease among individuals infected during the outbreak periods in the community.

Mol Pharmacol, 2003 Aug, 64(2), 382 - 94
Molecular modes of action of artesunate in tumor cell lines; Efferth T et al.; A profound cytotoxic action of the antimalarial, artesunate (ART), was identified against 55 cancer cell lines of the U.S . National Cancer Institute (NCI) . The 50% inhibition concentrations (IC50 values) for ART correlated significantly to the cell doubling times (P = 0.00132) and the portion of cells in the G0/G1 (P = 0.02244) or S cell cycle phases (P = 0.03567) . We selected mRNA expression data of 465 genes obtained by microarray hybridization from the NCI data base . These genes belong to different biological categories (drug resistance genes, DNA damage response and repair genes, oncogenes and tumor suppressor genes, apoptosis-regulating genes, proliferation-associated genes, and cytokines and cytokine-associated genes) . The constitutive expression of 54 of 465 (=12%) genes correlated significantly to the IC50 values for ART . Hierarchical cluster analysis of these 12 genes allowed the differentiation of clusters with ART-sensitive or ART-resistant cell lines (P = 0.00017) . For exemplary validation, cell lines transduced with 3 of the 12 genes were used to prove a causative relationship . The cDNAs for a deletion-mutated epidermal growth factor receptor (EGFR) and for gamma-glutamylcysteine synthetase increased resistance to ART . The conditional expression of the CDC25A gene using a tetracycline repressor expression vector increased sensitivity toward ART . Multidrug-resistant cells differentially expressing the MDR1, MRP1, or BCRP genes were not cross-resistant to ART . ART acts via p53-dependent and- independent pathways in isogenic p53+/+ p21WAF1/CIP1+/+, p53-/- p21WAF1/CIP1+/+, and p53+/+ p21WAF1/CIP1-/- colon carcinoma cells.

J Chemother, 2003 Jun, 15(3), 260 - 5
Reversal of cancer multidrug resistance by tea polyphenol in KB cells; Mei Y et al.; Tea polyphenols, (-)-epigallocatechin gallate in particular, were examined for their modulating effects on the drug resistance KB-A-1 cells and drug sensitive KB-3-1 cells . Both KB-3-1 and KB-A-1 cells were equally sensitive to tea polyphenol and (-)-epigallocatechin gallate . When 10 microgram/ml (-)-epigallocatechin gallate or 40 microgram/ml tea polyphenol were present simultaneously with doxorubicin, the IC50 of doxorubicin on KB-A-1 cells decreased from 10.3 +/- 0.9 microgram/ml to 4.2 +/- 0.2 or 2.0 +/- 0.1 microgram/ml . Tea polyphenol and (-)-epigallocatechin gallate enhanced the cytotoxicity of doxorubicin on KB-A-1 cells by 5.2 and 2.5 times, respectively, but did not show a modulating effect on KB-3-1 cells . Both tea polyphenol and (-)-epigallocatechin gallate showed reversal effects on the multidrug resistance phenotype.

Drug Metab Dispos, 2003 Aug, 31(8), 1016 - 26
Molecular cloning and pharmacological characterization of rat multidrug resistance protein 1 (mrp1); Nunoya K et al.; Multidrug resistance protein 1 (MRP1) transports a wide range of structurally diverse conjugated and nonconjugated organic anions and some peptides, including oxidized and reduced glutathione (GSH) . The protein confers resistance to certain heavy metal oxyanions and a variety of natural product-type chemotherapeutic agents . Elevated levels of MRP1 have been detected in many human tumors, and the protein is a candidate therapeutic target for drug resistance reversing agents . Previously, we have shown that human MRP1 (hMRP1) and murine MRP1 (mMRP1) differ in their substrate specificity despite a high degree of structural conservation . Since rat models are widely used in the drug discovery and development stage, we have cloned and functionally characterized rat MRP1 (rMRP1) . Like mMRP1 and in contrast to hMRP1, rMRP1 confers no, or very low, resistance to anthracyclines and transports the two estrogen conjugates, 17beta-estradiol-17-(beta-d-glucuronide) (E217betaG) and estrone 3-sulfate, relatively poorly . Mutational studies combined with vesicle transport assays identified several amino acids conserved between rat and mouse, but not hMRP1, that make major contributions to these differences in substrate specificity . Despite the fact that the rodent proteins transport E217betaG poorly and the GSH-stimulated transport of estrone 3-sulfate is low compared with hMRP1, site-directed mutagenesis studies indicate that different nonconserved amino acids are involved in the low efficiency with which each of the two estrogen conjugates is transported . Our studies also suggest that although rMRP1 and mMRP1 are 95% identical in primary structure, their substrate specificities may be influenced by amino acids that are not conserved between the two rodent proteins.

Curr Drug Targets, 2003 Aug, 4(6), 469 - 76
P-glycoprotein--a novel therapeutic target for immunomodulation in clinical transplantation and autoimmunity?
Pendse S, Sayegh MH, Frank MH.
P-glycoprotein, the human MDR1 gene product and cancer multidrug resistance-associated ATP-binding cassette transporter, is physiologically expressed on peripheral blood mononuclear cells, but its role in cellular immunity is only beginning to be elucidated . A role of P-glycoprotein in the secretion of several T cell- and antigen presenting cell-derived cytokines has been described, and additional functions of the molecule have been identified in lymphocyte survival and antigen presenting cell differentiation . Taken together, these findings provide compelling evidence that P-glycoprotein serves several distinct functions in the initiation of primary immune responses, and a critical role of the molecule in functional immune responses is now established . Here, we will review the current understanding of P-glycoprotein function in T cell activation and antigen presenting cell function, which are relevant to the fields of clinical transplantation and autoimmunity, and summarize the evidence for in vitro and in vivo immunomodulatory actions of several known P-glycoprotein-inhibiting agents currently in clinical use for other indications . We suggest that it is the P-glycoprotein-inhibitory function of many of these agents that underly their immunoregulatory capacities . Thus, the established immunoregulatory function of P-glycoprotein and the availability of P-glycoprotein-inhibitory drugs raise the possibility that P-glycoprotein may represent a promising novel therapeutic target for immune modulation in acute and chronic allograft rejection, and cell-mediated autoimmune disorders.

Indian J Chest Dis Allied Sci, 2003 Jul-Sep, 45(3), 215 - 9
Multi-drug resistant tuberculosis in context of RNTCP; Arora VK et al.; DOTS has been successful in improving cure rates in tuberculosis worldwide, but has remained an inefficient strategy in respect of multidrug-resistant tuberculosis (MDR TB) . The present article discusses its management in context of RNTCP and focuses specially on DOTS-plus, a strategy arising out of the constitution of Green Light Committee to effectively tackle the cases of MDR TB globally.

J Antimicrob Chemother, 2003 Aug, 52(2), 180 - 7 Epub 2003 Jul 15.
Modulation of the multidrug resistance (MDR) system in the nematode Haemonchus contortus by changing cholesterol content: effects on resistance to anthelmintics; Riou M et al.; OBJECTIVES: The efficiency of the anthelmintics used to treat small domestic ruminants infected with nematodes is compromised by the emergence of resistant parasites . Both specific and non-specific mechanisms of resistance exist . The non-specific mechanisms involve multiple resistance phenomena and are dependent on the multidrug resistance (MDR) system, which is also responsible for the development of chemotherapy-resistant tumour cells . We showed previously that the system also exists in nematodes . Membrane 'pumps', known as P-glycoproteins (Pgp), are activated in the MDR system . The nature of the membrane, in particular the lipids, appears to condition the activity of the pumps . Thus, we studied the effects of cholesterol on drug transport activity in the nematode Haemonchus contortus . MATERIALS AND METHODS: We used methyl-beta-cyclodextrin to carry out cholesterol depletion and cholesterol loading experiments . The resulting changes in resistance were estimated by measuring changes in drug transport (a) by means of in vitro egg hatch assays in the presence of a benzimidazole anthelmintic, thiabendazole and (b) by measuring the transport of rhodamine 123 (R123), a specific substrate of Pgp . We used biochemical assays to estimate the cholesterol concentration in the parasites . RESULTS: Changes in the cholesterol content induced changes in anthelmintic resistance; cholesterol depletion gave increased resistance and cholesterol loading gave decreased resistance . These changes also altered the transport of R123 . CONCLUSION: Cholesterol depletion or cholesterol loading allow modulation of xenobiotic resistance in nematode eggs as they do in tumour cells . The effect appears to be correlated with changes in the function of membrane P-glycoproteins . The lipid environment thus influences the nematode Pgp activity.

Mol Microbiol, 2003 Aug, 49(3), 671 - 83
Mapping of the Plasmodium falciparum multidrug resistance gene 5'-upstream region, and evidence of induction of transcript levels by antimalarial drugs in chloroquine sensitive parasites; Myrick A et al.; The Plasmodium falciparum multidrug resistance gene, pfmdr1, has been shown to be involved in the mediation of the parasite's response to various antimalarial drugs . Previous studies of pfmdr1 expression have shown that transcript levels are increased in drug-resistant isolates . However, a detailed examination of the transcriptional regulation of this gene has not been completed . The aim of this study was to map the 5' UTR of pfmdr1, and to examine the transcriptional profile of the gene in sensitive parasites treated with four different antimalarial drugs . RT-PCR and 5'-RACE mapping showed that the 5' UTR has a length of 1.94 kb . A putative promoter has been identified via transient transfection . Northern analysis revealed a 2.1- to 2.7-fold increase in pfmdr1 expression in 3D7 parasites treated with 50 nM chloroquine for 6 h, confirming results from Serial Analysis of Gene Expression . 3D7 parasites were subsequently treated with experimentally derived IC50 concentrations of mefloquine, quinine and pyrimethamine . pfmdr1 transcript levels specifically increased 2.5-fold at 6 h in mefloquine-treated parasites and threefold in parasites treated with quinine for 30 min . There was no evidence of transcript induction in pyrimethamine-treated parasites . This is the first evidence of induction of pfmdr1 expression in sensitive cells; and suggests a novel method of transcriptional control for this gene.

Leuk Res, 2003 Oct, 27(10), 903 - 8
Expression of functional markers in acute lymphoblastic leukemia; Oh EJ et al.; We analyzed surface antigens, multidrug resistance (MDR) parameters (PGP, MRP, LRP), tissue infiltration parameters (CD18, CD44, VCAM, MMP2), receptors for colony stimulating factors (G-CSFr, GM-CSFr) and cell cycle parameters (Ki-67, topoisomerase IIalpha) in 86 patients with acute lymphoblastic leukemia (ALL) . LRP, PGP and CD18 were associated with poor clinical outcome, and LRP expression was related with CD18, CD44 and G-CSFr . Of the cell cycle parameters, Ki-67 (+) fraction was increased in ALL with hepato-splenomegaly and extramedullary involvement . In conclusion, analysis of LRP, PGP, CD18 and Ki-67 could be helpful to predict the clinical behavior of ALL.

Leuk Res, 2003 Oct, 27(10), 893 - 7
Gemtuzumab ozogamicin, fludarabine, cytarabine and cyclosporine combination regimen in patients with CD33+ primary resistant or relapsed acute myeloid leukemia; Tsimberidou A et al.; Clinical resistance to gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukemia (AML) is associated with blast multidrug resistance (MDR) phenotype . A Phase II study of Mylotarg, fludarabine, ara-C and the MDR-modifier, cyclosporine (CSA) (MFAC) was conducted in 32 patients with primary resistant (11, 34%) or relapsed (21, 66%) AML . Nine (28%) patients obtained complete remission (CR), two (6%) CR with incomplete platelet recovery . Overall median survival was 5.3 months, 12-month survival rate 19% . Fourteen patients (44%) developed grade 3/4 hyperbilirubinemia; six (18%) grade 3/4 hepatic transaminitis; three (9%) hepatic veno-occlusive disease (VOD) . CSA inclusion in gemtuzumab ozogamicin-based regimens is feasible . MFAC is an effective regimen for refractory AML.

Clin Cancer Res, 2003 Jul, 9(7), 2817 - 25
Marked activity of irofulven toward human carcinoma cells: comparison with cisplatin and ecteinascidin; Poindessous V et al.; PURPOSE: To characterize the activities of irofulven, a novelanticancer agent derived from the mushroom natural productilludin S toward human cancer cells . Experimental Design: We have determined the activity spectrum of irofulven toward a human tumor cell panel comprised of 10 different tumor types in comparison with cisplatin and ET-743 . We have also evaluated the influence of major resistance mechanisms, such as expression of multidrug resistance-associated drug efflux pumps, cisplatin resistance, loss of p53 function, and absence of mismatch repair on the cytotoxic activity of irofulven . RESULTS: The activity spectrum of irofulven is clearly different from that of ET-743 and cisplatin . Irofulven shows excellent cytotoxicity toward the majority of human carcinoma cell lines tested, but lesser activity toward sarcoma and leukemia cell lines . The cytotoxic activity of irofulven was particularly pronounced toward head and neck, non-small cell lung, colon, and ovary carcinoma cells, as well as toward malignant glioma cell lines . In addition, irofulven displayed good activity toward poorly differentiated, androgen-independent prostate cancer cells and cell lines expressing high levels of the detoxifying enzymes glutathione S-transferase and gamma-glutamyl cysteine synthetase . The cytotoxicity of irofulven was not affected by loss of p53 or mismatch repair function, and the drug was not a substrate for multidrug transporters, such as the P-glycoprotein and multidrug resistance protein 1 . CONCLUSIONS: Irofulven has an unusual activity spectrum with strong activity toward tumor cells of epithelial origin . Furthermore, irofulven is not or only marginally affected by resistance mechanisms limiting the efficacy of other alkylating agents.

World J Gastroenterol, 2003 Jul, 9(7), 1444 - 9
Overcoming multi-drug resistance by anti-MDR1 ribozyme; Wang H et al.; AIM: To reverse multidrug resistance (MDR) of HepG2 by anti-MDR1 hammerhead ribozyme . METHODS: We developed an anti-MDR1 hammerhead ribozyme and delivered it to P-gp-overproducing human hepatocarcinoma cell line HepG2 by a retroviral vector containing RNA polymerase III promoter . We detected the expression of mdr1/Pgp and Rz in HepG2, HepG2 multidrug-resistant cell line and HepG2 Rz-transfected cells by real-time RT-PCR, semi-quantitative RT-PCR and Western blot methods . Moreover, MTT assay was tested to detect sensitivity of these ribozyme-transfected cells, and Rhodamine123 (Rh123) applied to test the function of Pgp . RESULTS: The Rz- transfected HepG2 cells became doxorubicin-sensitive, concomitant with the decreases in MDR1 expression, P-gp amounts and efflux pump function . CONCLUSION: The approaches using either retrovirus or liposome-mediated transfer of anti-MDR1 ribozyme may be selectively applicable to the treatment of MDR cells.

J Med Chem, 2003 Jul 17, 46(15), 3382 - 94
Novel benzylidene-9(10H)-anthracenones as highly active antimicrotubule agents . Synthesis, antiproliferative activity, and inhibition of tubulin polymerization; Prinz H et al.; A novel series of 10-benzylidene-9(10H)-anthracenones and 10-(phenylmethyl)-9(10H)-anthracenones were synthesized and evaluated for antiproliferative activity in an assay based on K562 leukemia cells . The 3-hydroxy-4-methoxybenzylidene analogue 9h was found to be the most active compound (IC(50) K562: 20 nM) . Structure-activity relationships are also considered . The highly active compound 9h and the 2,4-dimethoxy-3-hydroxybenzylidene analogue 9l were tested against five tumor cell lines using the XTT assay, including multidrug resistant phenotypes . Induction of cell death in a variety of tumor cell lines was determined in a monolayer assay using propidium iodide . Noteworthy, all compounds within the series induced elongations in K562 cells similar to vinblastine-treated cells . The effect of the lead compound 9h on K562 cell growth was associated with cell cycle arrest in G2/M . Concentrations for 50% KB/HeLa cells arrested in G2/M after treatment with 9h and 9l were determined and found to be in the range of 0.2 microM . Additionally, we monitored the dose dependent caspase-3-like protease activity in K562 cells and MCF-7/Casp-3 cells treated with 9h, indicating induction of apoptosis . Western blotting analysis demonstrated that 9h caused a shift in tubulin concentration from the polymerized state found in the cell pellet to the unpolymerized state found in the cell supernatant . Seven compounds strongly inhibited tubulin polymerization with activities higher or comparable to those of the reference compounds such as colchicine, podophyllotoxin, and nocodazole . In general, the antiproliferative activity correlated with inhibition of tubulin polymerization . The most active compounds strongly displaced {(3)H}colchicine from its binding site in the tubulin, yielding IC(50) values 3- to 4-fold lower than that of colchicine . The novel benzylidene-9(10H)-anthracenones described in the present study constitute an interesting group of highly active and easily accessible antimitotic agents that inhibit tubulin polymerization.

Vitam Horm, 2003, 66, 403 - 56
Membrane transport of folates; Matherly LH et al.; The chapter reviews the current understanding of the transport mechanisms for folates in mammalian cells--their molecular identities and organization, tissue expression, regulation, structures, and their kinetic and thermodynamic properties . This encompasses a variety of diverse processes . Best characterized is the reduced folate carrier, a member of the SLC19 family of facilitative carriers . But other facilitative organic anion carriers (SLC21), largely expressed in epithelial tissues, transport folates as well . In addition to these bi-directional carrier systems are the membrane-localized folate receptors alpha and beta, that mediate folate uptake unidirectionally into cells via an endocytotic process . There are also several transporters, typified by the family of multidrug resistance-associated proteins, that unidirectionally export folates from cells . There are transport activities for folates, that function optimally at low pH, related in part to the reduced folate carrier, with at least one activity that is independent of this carrier . The reduced folate carrier-associated low-pH route mediates intestinal folate transport . This review considers how these different transport processes contribute to the generation of transmembrane folate gradients and to vectorial flows of folates across epithelia . The role of folate transporters in mouse development, as assessed by homologous deletion of folate receptors and the reduced folate carrier, is described . Much of the focus is on antifolate cancer chemotherapeutic agents that are often model surrogates for natural folates in transport studies . In particular, antifolate transport mediated by the reduced folate carrier is a major determinant of the activity of, and resistance to, these agents . Finally, many of the key in vitro findings on the properties of antifolate transporters are now beginning to be extended to patient specimens, thus setting the stage for understanding response to these drugs in the clinical setting at the molecular level.

Int J Oncol, 2003 Aug, 23(2), 509 - 17
Analysis of single nucleotide polymorphism C3435T of the multidrug resistance gene MDR1 in acute lymphoblastic leukemia; Efferth T et al.; Multidrug resistance is an important mechanism responsible for refractoriness of leukemia and worse outcome of patients . Overexpression of the multidrug resistance gene, MDR1, is of prognostic relevance in acute myeloid leukemia, while its role in acute lymphoblastic leukemia (ALL) is still under debate . Single nucleotide polymorphisms (SNP) have been detected in the MDR1 gene . The C3435T polymorphism in this gene seems to have functional and clinical consequences . In the present investigation, we have analyzed the role of the C3435T SNP for drug resistance and prognosis of human ALL . The C3435T SNP was analyzed in 20 T-ALL cell lines and in blood samples from 53 ALL patients and 7 healthy donors . The cell line panel consisted of cell lines not prior exposed in vitro to cytostatic drugs as well as of drug-resistant lines which were selected in vitro by exposure to doxorubicin, vincristine, methotrexate, or hydroxyurea . We have developed a highly sensitive matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based genotyping approach to survey the C3435T SNP . Furthermore, mRNA expression was determined by real time reverse-transcribed polymerase chain reaction and doxorubicin sensitivity by a growth inhibition assay . Surprisingly, we did not find a significant correlation between C3435T homo- or heterozygote genotypes and MDR1 mRNA expression of cell lines or blood samples from patients and healthy donors . Furthermore, there was no relationship between the response of the cell lines to doxorubicin and the C3435T genotypes . Homo- or heterozygosity did not correlate to survival of patients in the Kaplan-Meier analysis . In conclusion, we do not have reason to assume that the C3435T SNP contributes to drug resistance of ALL and prognosis of ALL patients.

Surv Ophthalmol, 2003 Jul-Aug, 48(4), 424 - 38
Pseudoxanthoma elasticum: a clinical, histopathological, and molecular update; Hu X et al.; Pseudoxanthoma elasticum is an autosomally inherited disorder that is associated with the accumulation of mineralized and fragmented elastic fibers in the skin, Bruch's membrane in the retina, and vessel walls . The ophthalmic and dermatologic expression of pseudoxanthoma elasticum and its vascular complications are heterogeneous, with considerable variation in phenotype, progression, and mode of inheritance . Using linkage analysis and mutation detection techniques, mutations in the ABCC6 gene were recently implicated in the etiology of pseudoxanthoma elasticum . ABCC6 encodes the sixth member of the ATP-binding cassette transporter and multidrug resistance protein family (MRP6) . In humans, this transmembrane protein is highly expressed in the liver and kidney . Lower expression was found in tissues affected by pseudoxanthoma elasticum, including skin, retina, and vessel walls . So far, the substrates transported by the ABCC6 protein and its physiological role in the etiology of pseudoxanthoma elasticum are not known . A functional transport study of rat MRP6 suggests that small peptides such as the endothelin receptor antagonist BQ123 are transported by MRP6 . Similar molecules transported by ABCC6 in humans may be essential for extracellular matrix deposition or turnover of connective tissue at specific sites in the body . One of these sites is Bruch's membrane . This review is an update on etiology of pseudoxanthoma elasticum, including its clinical and genetic features, pathogenesis, and biomolecular basis.

Biol Pharm Bull, 2003 Jul, 26(7), 964 - 8
Diethyldithiocarbamate-induced cytotoxicity and apoptosis in leukemia cell lines; Kanno S et al.; Diethyldithiocarbamate (DDTC) has been shown to induce cytotoxicity in several different systems . We examined whether the DDTC-induced cytotoxicity was via apoptosis, or in relation to intracellular glutathione (GSH) in various murine and human leukemia cell lines . The cells most sensitive to DDTC-induced cytotoxicity were P388 lymphoid neoplasma cells and NALM-6, a B cell line of acute lymphocytic leukemia (ALL) . The next level of susceptible cells included J774.1, having a macrophage function, HL-60 premyelocytic leukemia cells, MOLT-4, an acute lymphoblastic leukemia cell, and Jurkat, a T-cell leukemia . U937 (expressing many monocyte-like characteristics), K562 erythroleukemia and K562/DXR (a multidrug-resistant clone derived from K562) were almost unaffected by DDTC . P388 was also highly susceptible to H(2)O(2), a most useful exogenous reactive oxygen species generator, and was lower in intracellular total GSH content than other leukemia cells . DDTC-induced cytotoxicity was closely related to intracellular GSH, but the level of cellular GSH did not always correlate with H(2)O(2)-induced cytotoxicity in this experiment . K562 had a higher intracellular total GSH content and showed lower susceptibility to DDTC and H(2)O(2), but with the combination of DDTC and DL-buthionine-(S,R)-sulfoximine (BSO), cytotoxicity increased significantly . The ratio of GSH/GSSG in P388 was reduced by DDTC or H(2)O(2) . H(2)O(2)-induced cytotoxicity was completely blocked by catalase (CAT), while it was enhanced by superoxide dismutase (SOD) . CAT or SOD did not affect DDTC-induced cytotoxicity . N-Acetylcysteine (NAC: 1 mM), a vanguard substance of GSH, and aurintricarboxylic acid (ATA: 100 microM), an endonuclease inhibitor, ameliorated DDTC-induced cytotoxicity and apoptosis . In conclusion, we suggest that DDTC-induced cytotoxicity was via an oxidative shift in the intracellular redox state, and accompanied the activation of endonuclease through apoptosis in leukemia cell lines.

Cas Lek Cesk, 2003, 142(4), 226 - 8
{Results of treatment of multiresistant tuberculosis}; Bartu V et al.; BACKGROUND: Multidrug resistant tuberculosis (MDR TB) is an infectious disease with limited therapeutic possibilities . Beside its epidemiological importance, MDR TB represents a problem also for long-term therapy with antituberculous drug combination and for its economical costs . METHODS AND RESULTS: We evaluated retrospectively the effect of treatment of MDR TB according to the clinical, radiological and bacteriological findings of 11 patients hospitalised in our department in years 1996 to 2000 . Individual chemotherapy regimens were chosen according to the susceptibility test results for the first and second-line antituberculosis drugs . Despite the fully controlled and long-term treatment, seven patients died during the regimen; in three of them the cause of death was different from MDR TB . CONCLUSIONS: Prevalence of MDR TB cases in the Czech republic has remained in the same level for several previous years and it represents about 20-30 persons . To prevent acquired resistance with its negative consequences, it would be optimal to implement the recommended standardized treatment regimens.

J Tongji Med Univ, 2000, 20(4), 311 - 4
Correlative expression of glutathione S-transferase-pi and multidrug resistance associated protein in bladder transitional cell carcinoma; Yang W et al.; In order to elucidate the mechanisms of multidrug resistance (MDR) in bladder cancer, the expression of glutathione S-transferase-pi (GST-pi) and multidrug resistance associated protein (MRP) in tissue samples resected from 44 patients and 6 normal bladder mucosa as control was detected by using immunohistochemical method, and the results were analyzed by computer-assisted image analyzing system (IAS) to achieve semi-quantitative data . In addition, correlation between the expression of both factors was studied . The results showed that the positive expression rate of GST-pi and MRP in bladder cancer was 72.7% (32/44) and 68.2% (30/44) respectively, significantly higher than those in normal bladder mucosa, being 16.7% and 33.3% respectively . The rate of GST-pi positive staining was increased correspondingly with tumor grade and stage elevated, being higher in recurrent tumors treated by chemotherapy, but not significantly (P > 0.05) . There was no significant differences between the expression of MRP and tumors' behaviors and clinical characters . However, the results demonstrated that the correlation between the expression of both resistant factors was very evident (r = 0.695, P < 0.0025) . It was suggested that the activation of GST-pi and MRP might occur during malignant transformation of normal mucosa, but tumors' differentiation and progression could not be the unique factors that influenced both overexpression . Chemotherapy might be another important reason . The correlation of both indicated that there was a common mechanism regulating their expression probably, which made them play a pivotal role in chemotherapy drug resistance of bladder cancers.

Hua Xi Kou Qiang Yi Xue Za Zhi, 2003 Apr 20, 21(2), 127 - 9
{Reversal effect of hyperthemia on multidrug resistant phenomena}; Zhang P et al.; OBJECTIVE: The purpose of this study was investigate the effect of hyperthemia on multidrug resistance in K562/ADM cell . METHODS: The MDR1 (mulitdrug resistance gene) and MRP (multidrug resistant associated gene) gene expressions in Tca8113 and K562/ADM cell lines were analyzed by RT-PCR after treated with different cytotoxic drugs and different temperature (37 degrees C and 41 degrees C) . The function and expression of Pgp and MRP were detected by fluorescence photometeric assays . RESULTS: Inhibition rate of both cells was significantly enhanced by exposure to chemotherapeutic drugs and 41 degrees C temperature; Exposing to 41 degrees C hyperthemia reduced MDR1 and MRP expression and enhanced intracellular drug concentration as well in K562/ADM . CONCLUSION: 41 degrees C hyperthemia could effectively enhance the inhibition rate of chemotherapeutic drugs and partially reverse the multidrug resistance . It is suggested that hyperthemia could be used as a method to overcome multidrug resistance.

Mikrobiyol Bul, 2003 Jan, 37(1), 13 - 8
{Determination of rifampin resistance in Mycobacterium tuberculosis isolates by the RNA/RNA mismatch method}; Cavusoglu C et al.; Rifampin is one of the most potent antituberculosis drugs and therefore, rifampin resistance leads to high clinical relapse rates . Detection of rifampin resistance could be an indication of multidrug resistance . In the recent years several molecular methods have been developed to evaluate the mutations in rpoB gene for the detection of rifampin resistance . The aim of the present study was to evaluate the performance of the RNA/RNA Mismatch Assay for detection of the mutations in the rpoB gene in 20 M . tuberculosis isolates which were determined as resistant to rifampin by agar proportion method . While RNA/RNA Mismatch Assay detected the mutations in the rpoB region in 16 of 20 (80%) M . tuberculosis isolates, the remaining four isolates yielded no band pattern indicating resistance . However, there may be situations where interpretation of the results is difficult in RNA/RNA Mismatch Assay which is already cheaper than DNA sequencing and other molecular methods . In conclusion, if the RNA/RNA Mismatch Assay can be optimized, it can be used for the rapid detection of rifampin resistance.

Pharmacology, 2003 Aug, 68(4), 177 - 82
Biliary excretion of phenolphthalein sulfate in rats; Tanaka H et al.; Glucuronide and glutathione conjugates have been reported to be substrates of multidrug resistance protein 2 (Mrp2), whereas sulfates of nonbile acid organic anions have never been reported as substrates of Mrp2 . To further examine the substrate specificity of Mrp2, we examined the effects of bile acid sulfates on the biliary excretion of phenolphthalein sulfate in rats . The biliary excretion of phenolphthalein sulfate was markedly delayed in Eisai hyperbilirubinemic rats, an Mrp2-deficient strain, and was markedly inhibited by taurolithocholate-3-sulfate . The biliary excretion of leukotriene C(4) metabolites and sulfobromophthalein was inhibited by phenolphthalein sulfate infusion to some extent . These findings suggest that phenolphthalein sulfate is a unique sulfated nonbile acid organic anion which is a substrate of Mrp2 .

J Antimicrob Chemother, 2003 Aug, 52(2), 269 - 75 Epub 2003 Jul 01.
Apoptosis and proliferation kinetics of T cells in patients having experienced antiretroviral treatment interruptions; Roger PM et al.; Multiple failures of antiretroviral treatments, as a result of multidrug-resistant virus, have led to a proposal for structured therapeutic interruptions (STI) . However, a significant decrease in CD4+ T cells may occur . The aim of our study was to determine the kinetics of T cell subpopulation changes, T cell apoptosis and peripheral blood mononuclear cell proliferation after STI . The impact of resistance mutation disappearance on T cell apoptosis was also studied . Ten patients were enrolled prospectively, and blood sampling was performed at weeks 0, 2, 4, 6, 8 and 12 . The mean increase in viral load was 1.3 log(10) copies/ml, ranging from 0.1 to 3.2 . CD4+ T cell count decreased to a mean of 80 cells/mm(3) from baseline to week 12 . In the same period, CD8+ T cells decreased to a mean of 139 cells/mm(3) . A significant increase in both T cell apoptosis and proliferation of mononuclear cells was observed . However, proliferation was an early and brief event . The increase in CD4+ T cell apoptosis was obvious in patients exhibiting complete reversion of resistance mutations to antiviral drugs . Our results suggest that during STI, apoptosis is an overwhelming phenomenon compared with proliferation, and may explain the limited immunological impact of this therapeutic option.

Proc Natl Acad Sci U S A, 2003 Aug 5, 100(16), 9244 - 9 Epub 2003 Jun 30.
The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs; Reid G et al.; Prostaglandins are involved in a wide variety of physiological and pathophysiological processes, but the mechanism of prostaglandin release from cells is not completely understood . Although poorly membrane permeable, prostaglandins are believed to exit cells by passive diffusion . We have investigated the interaction between prostaglandins and members of the ATP-binding cassette (ABC) transporter ABCC {multidrug resistance protein (MRP)} family of membrane export pumps . In inside-out membrane vesicles derived from insect cells or HEK293 cells, MRP4 catalyzed the time- and ATP-dependent uptake of prostaglandin E1 (PGE1) and PGE2 . In contrast, MRP1, MRP2, MRP3, and MRP5 did not transport PGE1 or PGE2 . The MRP4-mediated transport of PGE1 and PGE2 displayed saturation kinetics, with Km values of 2.1 and 3.4 microM, respectively . Further studies showed that PGF1alpha, PGF2alpha, PGA1, and thromboxane B2 were high-affinity inhibitors (and therefore presumably substrates) of MRP4 . Furthermore, several nonsteroidal antiinflammatory drugs were potent inhibitors of MRP4 at concentrations that did not inhibit MRP1 . In cells expressing the prostaglandin transporter PGT, the steady-state accumulation of PGE1 and PGE2 was reduced proportional to MRP4 expression . Inhibition of MRP4 by an MRP4-specific RNA interference construct or by indomethacin reversed this accumulation deficit . Together, these data suggest that MRP4 can release prostaglandins from cells, and that, in addition to inhibiting prostaglandin synthesis, some nonsteroidal antiinflammatory drugs might also act by inhibiting this release.

J Biochem Biophys Methods, 2003 Jul 31, 57(1), 1 - 16
Rhodamine B as a mitochondrial probe for measurement and monitoring of mitochondrial membrane potential in drug-sensitive and -resistant cells; Reungpatthanaphong P et al.; In order to get more insight into the energetic state of multidrug-resistance (MDR) cell compared with its corresponding sensitive cell, a noninvasive fluorescence method for determining and monitoring the mitochondrial membrane potential (DeltaPsi(m)), using rhodamine B and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was established . Rhodamine B distributes across biological membranes in response to the electrical transmembrane potential . P-glycoprotein- and MRP1-protein-mediated efflux do not create a concentration gradient, leading the cell-rhodamine B system to reach a steady state, where the ratio of cytosolic to extracellular rhodamine B was equal to 1 . The mitochondrial matrix rhodamine B concentration was precisely determined as a decrease of rhodamine B fluorescence in the presence of formazan, a rhodamine B fluorescence quencher, which locally accumulates in the matrix of mitochondria . The kinetics of decrease in rhodamine B fluorescence (V(i)) can be used to estimate DeltaPsi(m) using the Nernst equation: DeltaPsi(m)=-61.54 log V(i)-258.46 . The DeltaPsi(m) values determined were -160+/-4 mV for K562 cell, -146+/-6 mV for K562/adr cell, -161+/-10 mV for GLC4 cell and -168+/-2 mV for GLC4/adr cell . An increase or a decrease in DeltaPsi(m) consequently followed an increase or a decrease in the cellular ATP contents . An increase ATP content in the two MDR cell lines can protect cells from cytotoxicity induced by pirarubicin.

Cancer, 2003 Jul 1, 98(1), 61 - 5
A phase II study of irinotecan in patients with advanced renal cell carcinoma; Fizazi K et al.; BACKGROUND: Patients with disseminated renal cell carcinoma (RCC) have a poor outcome, and the disease is considered highly resistant to chemotherapy . Irinotecan is an active drug in the treatment of a number of neoplastic diseases and is not concerned with the multidrug-resistance phenotype of tumor cells, a common mechanism of drug inactivation and resistance in patients with RCC . Therefore, the authors tested the antitumor activity of irinotecan in patients with RCC . METHODS: Patients with disseminated RCC received irinotecan (350 mg/m(2)) every 3 weeks . The primary objective of the study was to determine the overall response rate . Two groups of patients were defined: previously treated patients (Group A) and nonpretreated patients (Group B) . RESULTS: Forty-two eligible patients were recruited: Twenty-six patients (Group A) had received previous chemotherapy or immunotherapy, and 16 patients had received no previous systemic therapy (Group B) . The median number of cycles received per patient was 3 cycles (range, 1-6 cycles) . A dose reduction was required in only 8% of cycles . Two patients, one in each group, had minor responses . Eleven patients (42%) in Group A and 1 patient (12%) in Group B had disease stabilization . Overall, therapy was tolerated well . Grade 4 neutropenic fever occurred in 17% of patients . The 1-year overall survival rate was 61% (95% confidence interval, 42-80%) in Group A and 19% (95% confidence interval, 0-49%) in Group B . CONCLUSIONS: Irinotecan was tolerated well and had limited activity in patients with disseminated RCC at the dose and schedule used in the current study . A high percentage of disease stabilization was observed in cytokine-pretreated patients .

Nucl Med Biol, 2003 Jul, 30(5), 471 - 5
Usefulness of technetium-99m tetrofosmin liver imaging to detect hepatocellular carcinoma and related to expression of P-glycoprotein or multidrug resistance associated protein-a preliminary report; Ding HJ et al.; Technetium-99m Tetrofsomin (Tc-TF) has been shown to be useful in identifying several types of tumors, such as breast, lung, and thyroid cancers . There was no report in the literature for Tc-TF uptake in hepatocellular carcinoma (HCC) . The aim of this study was to evaluate the usefulness of Tc-TF liver imaging to detect HCC and investigate the relationship between Tc-TF liver imaging findings and P-glycoprotein (Pgp) and multidrug resistance associated protein (MRP) expression . Before any therapy, 22 patients with HCC were enrolled in this study . Tc-TF liver images were performed l0 minutes after intravenous injection of 20mCi Tc-TF . All patients had liver biopsy or surgery within l week after Tc-TF liver imaging . Immunohistochemical study of the biopsy or resected HCC specimens was performed using anti-human Pgp and MRP antibodies . Twenty of the 22 (90.9%) patients showed negative Tc-TF liver imaging results without significant Tc-TF uptake in HCC, whereas only the remaining 2 (9.1%) patients showed positive Tc-TF liver imaging results with significant Tc-TF uptake in HCC . Positive Pgp expression was observed in 13 of 20 patients with negative Tc-TF liver imaging results, whereas positive MRP expression was observed in 6 of the remaining 7 patients with negative both Tc-TF liver imaging results and Pgp expression . However, negative Pgp expression but positive MRP expression was observed in all of the remaining 2 patients with positive Tc-TF liver imaging results . The correlation between Tc-TF liver imaging findings and Pgp expression was significant and better than between Tc-TF liver imaging findings and MRP expression . Pgp or MRP expression in HCC may induce no significant Tc-TF uptake in HCC resulting in negative Tc-TF liver imaging findings . Therefore, Tc-TF liver imaging is potential to be a non-invasive method to predict Pgp or MRP expression in HCC . However, further studies with a larger series of patients and longer follow-up time are necessary to confirm our findings.

J Pharm Pharmacol, 2003 May, 55(5), 675 - 81
Effects of continuous exposure to digoxin on MDR1 function and expression in Caco-2 cells; Takara K et al.; The Caco-2 cell line has been used widely for studying intestinal permeability and several transport functions, and express the multidrug resistance transporter MDR1/P-glycoprotein . Previously, the transient exposure to digoxin for 24 h was found to induce MDR1 mRNA in Caco-2 cells . Here, a digoxin-tolerant Caco-2 subline (Caco/DX) was newly established by the continuous exposure of Caco-2 cells to digoxin, and the effects of continuous exposure to digoxin on MDR1 were examined . The 50% growth inhibitory concentration (IC(50)) values for digoxin in Caco-2 and Caco/DX cells were 17.2 and 81.4 nM, respectively . The IC(50) values for paclitaxel, an MDR1 substrate, were 1.0 and 547 nM, respectively, whereas the cytotoxicity of 5-fluorouracil was comparable in both cells . The uptake and efflux of Rhodamine123, an MDR1 substrate, in Caco/DX cells were significantly less and greater, respectively, than those in Caco-2 cells, and these transports were affected by the addition of ciclosporin . The expression of MDR1 mRNA in Caco/DX cells was approximately 2- and 1.7-fold compared with Caco-2 cells and Caco-2 cells treated with 100 nM digoxin for 24 h, respectively . On the other hand, MRP1 mRNA in Caco/DX cells was unchanged . These observations confirmed that the continuous exposure to digoxin, as well as the transient exposure, induced MDR1 in Caco-2 cells.

Eur J Nucl Med Mol Imaging, 2003 Aug, 30(8), 1147 - 54 Epub 2003 Jun 27.
In vivo and in vitro multitracer analyses of P-glycoprotein expression-related multidrug resistance; Marian T et al.; P-glycoprotein (Pgp) is an ABC (ATP binding cassette) transporter that is often overexpressed in tumours, contributing significantly to their multidrug resistance . In this study, we explored whether the radiotracers used in tumour diagnostics can be used for in vivo visualisation of Pgp-related multidrug resistance . We also examined the effects of different Pgp modulators on the accumulation of these radioligands in tumours with or without Pgp expression . In a SCID BC-17 mouse model, cells of the drug-sensitive KB-3-1 (MDR(-)) and the KB-V1 Pgp-expressing (MDR(+)) human epidermoid carcinoma cell lines were inoculated to yield tumours in opposite flanks . For in vivo scintigraphic (biodistribution) and positron emission tomography (PET) examinations, the mice were injected with technetium-99m hexakis-2-methoxybutylisonitrile ((99m)Tc-MIBI), carbon-11 labelled methionine and fluorine-18 fluoro-2-deoxy- d-glucose ((18)FDG) . For validation, in vitro cell studies with (99m)Tc-MIBI,( 99m)Tc-tetrofosmin, {(11)C}methionine and (18)FDG were carried out using a gamma counter . The expression and function of the MDR product were proved by immunohistochemistry and spectrofluorimetry . (99m)Tc-MIBI uptake was significantly lower in KB-V1 cells as compared with KB-3-1-derived tumours in vivo (Pgp(+)/Pgp(-) =0.61+/-0.13; P<0.01) and cells in vitro (Pgp(+)/Pgp(-) =0.08+/-0.01; P<0.001).()Cyclosporin A reversed (99m)Tc-MIBI uptake in the Pgp+ cells, while verapamil failed to modify it . (18)FDG uptake was significantly higher in KB-V1 tumours (Pgp(+)/Pgp(-) =1.36+/-0.05; P<0.01) and cells (Pgp(+)/Pgp(- )=1.52+/-0.12; P<0.001) . Whereas cyclosporin A eliminated the difference between FDG uptake in MDR(+) and MDR(-) cell lines, verapamil significantly increased it . When the animals were treated with verapamil, the ratio of (99m)Tc-MIBI uptake in the MDR(+) tumours to that in the MDR(-) tumours decreased to 0.38+/-0.05 ( P<0.01), while the ratio of (18)FDG uptake increased to 2.1+/-0.3 ( P<0.001) . There were no significant differences in the {(11)C}methionine uptake in the MDR(+) and MDR(-) tumours and cell lines, nor was {(11)C}methionine accumulation modified by cyclosporin A . Parallel administration of (18)FDG and (99m)Tc-MIBI combined with verapamil treatment seems to be a good candidate as a non-invasive marker for the diagnosis of MDR-related Pgp expression in tumours.

J Virol, 2003 Jul, 77(14), 7736 - 45
Infectivity and replication capacity of drug-resistant human immunodeficiency virus type 1 variants isolated during primary infection; Simon V et al.; It is believed that replication capacity is an important determinant of human immunodeficiency virus type 1 (HIV-1) pathogenicity and transmissibility . To explore this, we conducted a comprehensive analysis of the replication properties of nine drug-resistant and nine drug-susceptible viral isolates derived from patients with primary HIV-1 infection . Viral isolates were tested for single-cycle infectivity in the GHOST cell line . The infectivity of isolates carrying resistance-associated mutations was significantly higher than that of drug-susceptible isolates . Additionally, the growth kinetics of these isolates were determined in CD4+ T lymphocytes . Drug-resistant isolates replicated as well as drug-susceptible viruses . Insertion of the resistance-conferring regions into an NL4-3-based molecular background resulted in chimeras that displayed a modest but significant reduction in replication capacity compared to the drug-susceptible chimeric viruses . Of note, two multidrug-resistant isolates and one protease inhibitor-resistant isolate displayed higher rates of infectivity and growth kinetics than the other drug-resistant or drug-susceptible isolates . These distinct replicative features, however, were not seen in the corresponding chimeras, indicating that changes within the C-terminal region of Gag as well as within the protease and reverse transcriptase genes contribute to but are not sufficient for the level of compensatory adaptation observed . These findings suggest that some drug-resistant viruses isolated during primary infection possess unique adaptive changes that allow for both high viral replication capacity and resistance to one or more classes of antiretroviral drugs . Further studies are needed to elucidate the precise regions that are essential for these characteristics.

Exp Hematol, 2003 Jun, 31(6), 483 - 7
Multidrug resistance-1 (MDR-1) in autoimmune disorders III: increased P-glycoprotein activity in lymphocytes from immune thrombocytopenic purpura patients; Ruiz-Soto R et al.; OBJECTIVE: P-glycoprotein (P-gp) expression has been widely observed in normal and neoplastic cells . The physiologic role of P-gp involves hormone and metabolite secretion, bacterial product detoxification, and transport of several drugs to the extracellular space . Multidrug resistance-1 is characterized by drug extrusion through P-gp, reducing the intracellular levels of drugs and diminishing their pharmacological effects . Treatment of immune thrombocytopenic purpura (ITP) includes agents that are substrates of P-gp; hence, the objective of this study was to analyze the functional activity of P-gp in lymphocytes from patients with ITP . PATIENTS AND METHODS: 30 ITP patients (9 refractory, 5 dependent, 14 responders to treatment, and 2 with stable disease) and 25 healthy controls were studied . Peripheral blood mononuclear cells were isolated by gradient centrifugation and incubated with daunorubicin (a fluorescent drug extruded by P-gp) . Functional activity of P-gp was analyzed by flow cytometry . Results were expressed as the percentage of lymphocytes able to extrude daunorubicin . RESULTS: ITP patients showed an increased number of lymphocytes with P-gp activity (mean=12.3%+/-16%) when compared to controls (mean=0.87%+/-0.72%) (p<0.05) . P-gp function was higher in the refractory group (median=9.4%) than in the treatment-dependent (median=5.4%), responder (median=6.4%), and stable disease (median=5.2%) groups, although no statistical differences were found among them . CONCLUSION: Enhanced P-gp activity in ITP may be related to an unfavorable clinical outcome and poor response to treatment . Furthermore, P-gp function might affect therapeutic requirements for disease control.

Nature, 2003 Jun 26, 423(6943), 999 - 1002
Enhanced gravi- and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1; Noh B et al.; Many aspects of plant growth and development are dependent on the flow of the hormone auxin down the plant from the growing shoot tip where it is synthesized . The direction of auxin transport in stems is believed to result from the basal localization within cells of the PIN1 membrane protein, which controls the efflux of the auxin anion . Mutations in two genes homologous to those encoding the P-glycoprotein ABC transporters that are especially abundant in multidrug-resistant tumour cells in animals were recently shown to block polar auxin transport in the hypocotyls of Arabidopsis seedlings . Here we show that the mdr mutants display faster and greater gravitropism and enhanced phototropism instead of the impaired curvature development expected in mutants lacking polar auxin transport . We find that these phenotypes result from a disruption of the normal accumulation of PIN1 protein along the basal end of hypocotyl cells associated with basipetal auxin flow . Lateral auxin conductance becomes relatively larger as a result, enhancing the growth differentials responsible for tropic responses.

Cancer Sci, 2003 May, 94(5), 459 - 66
DJ-927, a novel oral taxane, overcomes P-glycoprotein-mediated multidrug resistance in vitro and in vivo; Shionoya M et al.; DJ-927 is a novel taxane, which was selected for high solubility, non-neurotoxicity, oral bioavailability, and potent antitumor activity . In this study, we compared the in vitro and in vivo efficacy of DJ-927 with those of paclitaxel and docetaxel . DJ-927 exhibited stronger cytotoxicity than paclitaxel and docetaxel in various tumor cell lines, especially against P-glycoprotein (P-gp)-expressing cells . The cytotoxicity of DJ-927, unlike those of other taxanes, was not affected by the P-gp expression level in tumor cells, or by the co-presence of a P-gp modulator . When intracellular accumulation of the three compounds was compared, intracellular amounts of DJ-927 were much higher than those of paclitaxel or docetaxel, particularly in P-gp-positive cells . In vivo, DJ-927 showed potent antitumor effects against two human solid tumors in male BALB/c-nu/nu mice, and yielded significant life-prolongation in a murine liver metastasis model with male C57BL/6 mice, in which neither paclitaxel nor docetaxel was effective . The results demonstrate the superior efficacy of orally administered DJ-927 over intravenously administered paclitaxel or docetaxel against P-gp-expressing tumors, probably due to higher intracellular accumulation . A phase I clinical trials of DJ-927 is currently ongoing in the US.

Cancer Sci, 2003 Jun, 94(6), 557 - 63
Reversal of the resistance to STI571 in human chronic myelogenous leukemia K562 cells; Mukai M et al.; STI571, an Abl-specific tyrosine kinase inhibitor, selectively kills Bcr-Abl-containing cells in vitro and in vivo . However, some chronic myelogenous leukemia (CML) cell lines are resistant to STI571 . We evaluated whether STI571 interacts with P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1), and examined the effect of agents that reverse multidrug resistance (MDR) on the resistance to SI571 in MDR cells . STI571 inhibited the {(125)I}azidoagosterol A-photolabeling of P-gp, but not that of MRP1 . K562/MDR cells that overexpress P-gp were 3.67 times more resistant to STI571 than the parental Philadelphia-chromosome-positive (Ph +) CML K562 cells, and this resistance was most effectively reversed by cepharanthine among the tested reversing agents . The concentration of STI571 required to completely inhibit tyrosine phosphorylation in K562/MDR cells was about 3 times higher than that in K562 cells, and cepharanthine abolished the difference . In KB-G2 cells that overexpress P-gp, but not Bcr-Abl, 2.5 micro M STI571 partly reversed the resistance to vincristine (VCR), paclitaxel, etoposide (VP-16) and actinomycin D (ACD) but not to Adriamycin (ADM) or colchicine . STI571 increased the accumulation of VCR, but not that of ADM in KB-G2 cells . STI571 did not reverse resistance to any agent in KB/MRP cells that overexpress MRP1 . These findings suggest that STI571 is a substrate for P-gp, but is less efficiently transported by P-gp than VCR, and STI571 is not a substrate for MRP1 . Among the tested reversing agents that interact with P-gp, cepharanthine was the most effective agent for the reversal of the resistance to STI571 in K562/MDR cells . Furthermore, STI571 itself was a potent reversing agent for MDR in P-gp-expressing KB-G2 cells.

J Biomed Sci, 2003 Jul-Aug, 10(4), 361 - 6
A light in multidrug resistance: photodynamic treatment of multidrug-resistant tumors; Capella MA et al.; The major drawback of cancer chemotherapy is the development of multidrug-resistant (MDR) tumor cells, which are cross-resistant to a broad range of structurally and functionally unrelated agents, making it difficult to treat these tumors . In the last decade, a number of authors have studied the effects of photodynamic therapy (PDT), a combination of visible light with photosensitizing agents, on MDR cells . The results, although still inconclusive, have raised the possibility of treating MDR tumors by PDT . This review examines the growing literature concerning the responses of MDR cells to PDT, while stressing the need for the development of new photosensitizers that possess the necessary characteristics for the photodynamic treatment of this class of tumor .

J Exp Ther Oncol, 2003 Mar-Apr, 3(2), 72 - 82
Combined treatment of Bcl-2 antisense oligodeoxynucleotides (G3139), p-glycoprotein inhibitor (PSC833), and sterically stabilized liposomal doxorubicin suppresses growth of drug-resistant growth of drug-resistant breast cancer in severely combined immunodeficient mice; Lopes de Menezes DE et al.; We studied the possibility of increasing sensitization of drug-resistant MDA435/LCC6 multidrug-resistant (MDR) human breast cancer cells to doxorubicin (DOX) by increasing cellular drug retention with P-glycoprotein (P-gp) inhibitor PSC833 in combination with induction of cell death through down-regulation of Bcl-2 protein using Bcl-2 antisense (G3139) . In in vitro cytotoxicity assays, the combination of G3139 with DOX exhibited 40% increased cytotoxicity in both wild-type (WT) and MDR cells . PSC833 increased the cytotoxicity of DOX and Taxol with complete and partial reversal of the resistance of MDR cells to DOX and Taxol, respectively . The presence of G3139 did not increase the cytotoxicity of PSC833 combined with DOX or Taxol in both cell lines . In vivo studies with WT and MDR cell lines transplanted into severely combined immunodeficient mice demonstrated that G3139 (5 mg/kg) was able to suppress the growth of both WT and MDR tumors to an equivalent extent . PSC833 (100 mg/kg) partially restored the sensitivity of resistant tumors to DOX, and the combination of G3139 and PSC833 with liposomal DOX showed maximum growth suppression of MDR tumors compared with individual treatments . The improved efficacy of this treatment was attributed to Bcl-2 antisense-induced apoptosis, combined with cellular retention of DOX in tumor cells via P-gp blockade.

Curr Opin Infect Dis, 2003 Jun, 16(3), 193 - 8
Host innate defenses in the lung: the role of cytokines; Strieter RM et al.; PURPOSE OF REVIEW: The lung has a unique relationship with the environment . Through evolution the lung has developed strategies to defend itself from microbial invasion . As we encounter increasing multidrug-resistant microorganisms, we need to further our knowledge of innate defense systems in order to design novel strategies to deal with these microbes without inducing over-exuberant inflammation and lung injury . RECENT FINDINGS: The development of lung innate immunity requires microbial molecular pattern recognition by the recently described Toll like receptors, the release of early response cytokines that further activate the 'master switch', nuclear factor-kappaB, leading to amplified host defense to invading microbes . A balance of Type 1 and Type 2 cytokines modulates the intensity of innate immunity . Cytokines/chemokines orchestrate the polarization and transition of innate to adaptive immunity . SUMMARY: The elucidation of the pathways involved in innate immunity and factors controlling the transition to adaptive immunity will improve our understanding of the host response to infection and improve our ability to design new therapies for the treatment of infectious disease.

Antimicrob Agents Chemother, 2003 Jul, 47(7), 2231 - 5
Allele-specific rpoB PCR assays for detection of rifampin-resistant Mycobacterium tuberculosis in sputum smears; Mokrousov I et al.; We describe an allele-specific PCR assay to detect mutations in three codons of the rpoB gene (516, 526, and 531) in Mycobacterium tuberculosis strains; mutations in these codons are reported to account for majority of M . tuberculosis clinical isolates resistant to rifampin (RIF), a marker of multidrug-resistant tuberculosis (MDR-TB) . Three different allele-specific PCRs are carried out either directly with purified DNA (single-step multiplex allele-specific PCR), or with preamplified rpoB fragment (nested allele-specific PCR {NAS-PCR}) . The method was optimized and validated following analysis of 36 strains with known rpoB sequence . A retrospective analysis of the 287 DNA preparations from epidemiologically unlinked RIF-resistant clinical strains from Russia, collected from 1996 to 2002, revealed that 247 (86.1%) of them harbored a mutation in one of the targeted rpoB codons . A prospective study of microscopy-positive consecutive sputum samples from new and chronic TB patients validated the method for direct analysis of DNA extracted from sputum smears . The potential of the NAS-PCR to control for false-negative results due to lack of amplification was proven especially useful in the study of these samples . The developed rpoB-PCR assay can be used in clinical laboratories to detect RIF-resistant and hence MDR M . tuberculosis in the regions with high burdens of the MDR-TB.

Antimicrob Agents Chemother, 2003 Jul, 47(7), 2169 - 78
High-level beta-lactam resistance associated with acquired multidrug resistance in Helicobacter pylori; Kwon DH et al.; Four clinical Helicobacter pylori isolates with high-level resistance to beta-lactams exhibited low- to moderate-level resistance to the structurally and functionally unrelated antibiotics ciprofloxacin, chloramphenicol, metronidazole, rifampin, and tetracycline . This pattern of multidrug resistance was transferable to susceptible H . pylori by natural transformation using naked genomic DNA from a clinical multidrug-resistant isolate . Acquisition of the multidrug resistance was also associated with a change in the genotype of the transformed multidrug-resistant H . pylori . DNA sequence analyses of the gene encoding penicillin binding protein 1A (PBP 1A) showed 36 nucleotide substitutions resulting in 10 amino acid changes in the C-terminal portion (the putative penicillin binding domain) . Acquisition of beta-lactam resistance was consistently associated with transfer of a mosaic block containing the C-terminal portion of PBP 1A . No changes of genes gyrA, rpoB, rrn16S, rdxA, and frxA, and nine other genes (ftsI, hcpA, llm, lytB, mreB, mreC, pbp2, pbp4, and rodA1) encoding putative PBPs or involved in cell wall synthesis were found among the transformed resistant H . pylori . Antibiotic accumulations of chloramphenicol, penicillin, and tetracycline were all significantly decreased in the natural and transformed resistant H . pylori compared to what was seen with susceptible H . pylori . Natural transformation also resulted in the outer membrane protein profiles of the transformed resistant H . pylori becoming similar to that of the clinical resistant H . pylori isolates . Overall, these results demonstrate that high-level beta-lactam resistance associated with acquired multidrug resistance in clinical H . pylori is mediated by combination strategies including alterations of PBP 1A and decreased membrane permeability.

Antimicrob Agents Chemother, 2003 Jul, 47(7), 2065 - 71
Biosynthetic origin of hygromycin A; Habib el-SE et al.; Hygromycin A, an antibiotic produced by Streptomyces hygroscopicus, is an inhibitor of bacterial ribosomal peptidyl transferase . The antibiotic binds to the ribosome in a distinct but overlapping manner with other antibiotics and offers a different template for generation of new agents effective against multidrug-resistant pathogens . Reported herein are the results from a series of stable-isotope-incorporation studies demonstrating the biosynthetic origins of the three distinct structural moieties which comprise hygromycin A . Incorporation of {1-(13)C}mannose and intact incorporation of D-{1,2-(13)C(2)}glucose into the 6-deoxy-5-keto-D-arabino-hexofuranose moiety are consistent with a pathway in which mannose is converted to an activated L-fucose, via a 4-keto-6-deoxy-D-mannose intermediate, with a subsequent unusual mutation of the pyranose to the corresponding furanose . The aminocyclitol moiety was labeled by D-{1,2-(13)C(2)}glucose in a manner consistent with formation of myo-inositol and a subsequent unprecedented oxidation and transamination of the C-2 hydroxyl group to generate neo-inosamine-2 . Incorporation of {carboxy-(13)C}-4-hydroxybenzoic acid and intact incorporation of {2,3-(13)C(2)}propionate are consistent with a polyketide synthase-type decarboxylation condensation to generate the 3,4-dihydroxy-alpha-methylcinnamic acid moiety of hygromycin A . No labeling of hygromycin A was observed when {3-(13)C}tyrosine, {3-(13)C}phenylalanine, or {carboxy-(13)C}benzoic acid was used, suggesting that the 4-hydroxybenzoic acid is derived directly from chorismic acid . Consistent with this hypothesis was the observation that hygromycin A titers could be reduced by addition of N-(phosphonomethyl)-glycine (an inhibitor of chorismic acid biosynthesis) and restored by coaddition of 4-hydroxybenzoic acid . The convergent biosynthetic pathway established for hygromycin A offers significant versatility for applying the techniques of combinatorial and directed biosynthesis to production of new antibiotics which target the ribosomal peptidyl transferase activity.

J Pharm Sci, 2003 Jul, 92(7), 1343 - 55
Amphiphilic block copolymers for drug delivery; Adams ML et al.; Amphiphilic block copolymers (ABCs) have been used extensively in pharmaceutical applications ranging from sustained-release technologies to gene delivery . The utility of ABCs for delivery of therapeutic agents results from their unique chemical composition, which is characterized by a hydrophilic block that is chemically tethered to a hydrophobic block . In aqueous solution, polymeric micelles are formed via the association of ABCs into nanoscopic core/shell structures at or above the critical micelle concentration . Upon micellization, the hydrophobic core regions serve as reservoirs for hydrophobic drugs, which may be loaded by chemical, physical, or electrostatic means, depending on the specific functionalities of the core-forming block and the solubilizate . Although the Pluronics, composed of poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide), are the most widely studied ABC system, copolymers containing poly(L-amino acid) and poly(ester) hydrophobic blocks have also shown great promise in delivery applications . Because each ABC has unique advantages with respect to drug delivery, it may be possible to choose appropriate block copolymers for specific purposes, such as prolonging circulation time, introduction of targeting moieties, and modification of the drug-release profile . ABCs have been used for numerous pharmaceutical applications including drug solubilization/stabilization, alteration of the pharmacokinetic profile of encapsulated substances, and suppression of multidrug resistance . The purpose of this minireview is to provide a concise, yet detailed, introduction to the use of ABCs and polymeric micelles as delivery agents as well as to highlight current and past work in this area .

J Am Soc Nephrol, 2003 Jul, 14(7), 1889 - 96
Association of the multidrug resistance-1 gene single-nucleotide polymorphisms with the tacrolimus dose requirements in renal transplant recipients; Anglicheau D et al.; The immunosuppressive drug tacrolimus, whose pharmacokinetic characteristics display large interindividual variations, is a substrate for P-glycoprotein (P-gp), the product of the multidrug resistance-1 (MDR1) gene . Some of the single nucleotide polymorphisms (SNP) of MDR1 reported correlated with the in vivo activity of P-gp . Because P-gp is known to control tacrolimus intestinal absorption, it was postulated that these polymorphisms are associated with tacrolimus pharmacokinetic variations in renal transplant recipients . The objective of this study was to evaluate in a retrospective study of 81 renal transplant recipients the effect on tacrolimus dosages and concentration/dose ratio of four frequent MDR1 SNP possibly associated with P-gp function (T-129C in exon 1b, 1236C>T in exon 12, 2677G>T,A in exon 21, and 3435C>T in exon 26) . As in the general population, the SNP in exons 12, 21, and 26 were frequent (16, 17.3, and 22.2% for the variant homozygous genotype, respectively) and exhibited incomplete linkage disequilibrium . One month after tacrolimus introduction, exon 21 SNP correlated significantly with the daily tacrolimus dose (P < or = 0.05) and the concentration/dose ratio (P < or = 0.02) . Tacrolimus dose requirements were 40% higher in homozygous than wild-type patients for this SNP . The concentration/dose ratio was 36% lower in the wild-type patients, suggesting that, for a given dose, their tacrolimus blood concentration is lower . Haplotype analysis substantiated these results and suggested that exons 26 and 21 SNP may be associated with tacrolimus dose requirements . Genotype monitoring of the MDR1 gene reliably predicts the optimal dose of tacrolimus in renal transplant recipients and may predict the initial daily dose needed by individual patients to obtain adequate immunosuppression.

Biochem Pharmacol, 2003 Jul 1, 66(1), 163 - 70
Decreased expression of P-glycoprotein during differentiation in the human intestinal cell line Caco-2; Goto M et al.; The expression profile of the multidrug resistance (MDR) 1 gene product P-glycoprotein (Pgp) was examined during culture using Caco-2 cells as an in vitro model . Levels of MDR1 and cyclooxygenase 2 mRNA expression in Caco-2 cells were the highest on day 3 and decreased with days in culture, but the level of cyclooxygenase 1 was stable throughout the culture period . The stability of MDR1 mRNA was 7-fold higher on day 3 than on day 9, and the run-on assay suggested the transcription rate of the MDR1 gene on day 3 tended to be higher than on day 9 . In addition, the expression of Pgp was comparable with that of MDR1 mRNA, but was inversely correlated with villin expression . The Pgp-mediated tacrolimus transport was the highest on day 1 and the lowest on day 11 . These results suggested that the changeable mRNA stability rather than transcription rate of MDR1 contributed to its up-regulation during cell proliferation and down-regulation after post-confluent differentiation in Caco-2 cells . Therefore, the temporal induction and subsequent down-regulation of the enterocyte Pgp could affect bioavailability of several drugs during the regeneration of the intestinal wall.

Life Sci, 2003 Jul 11, 73(8), 981 - 91
Reversal of P-glycoprotein expressed in Escherichia coli leaky mutant by ascorbic acid; El-Masry EM et al.; It has been reported that functional expression of the multidrug resistance protein P-glycoprotein (P-gp) in E . coli is useful for screening P-gp substrates and inhibitors . In the present study, we have constructed by nitrosoguanidine and UV mutagenesis 28 leaky mutants of E . coli UT5600 . These mutants are significantly susceptible to the toxic effect of known P-gp substrates and lipophilic cancer drugs . Mouse mdr1 was functionally expressed in the most permeable E . coli mutant (UTP17) . Expression of P-gp in this mutant confers cross-resistance to mitomycin C, tegafur, daunorubicin, rhodamine 6G, tetraphenylphosphonium bromide and ciprofloxacin . To examine the reversal of P-gp expressed in this heterologous system, UTP17 cells expressing mouse mdr1 or lac permease as negative control were treated with various concentrations of mitomycin C with or without ascorbic acid . We found that ascorbic acid abrogated P-gp mediated multidrug resistance, suggesting that ascorbic acid might be used in combination with anticancer drugs to reduce emergence of multidrug resistance . We also demonstrated that tomato lectin antagonized the inhibitory action of ascorbic acid . This study provide a heterologous system for mdr1 expression in E . coli leaky mutant that can be used as a system for the screening of P-gp inducers and inhibitors, since it is quick and simple.

Blood, 2003 Dec 15, 102(13), 4493 - 8 Epub 2003 Jun 19.
The multidrug resistance-associated protein 3 (MRP3) is associated with a poor outcome in childhood ALL and may account for the worse prognosis in male patients and T-cell immunophenotype; Steinbach D et al.; The family of multidrug resistance-associated proteins (MRPs) belongs to the superfamily of adenosine triphosphate-binding-cassette (ABC) transporters, which have the ability to function as outward pumps for chemotherapeutic drugs and therefore might be involved in drug resistance . In this study the expression of the MRP2, MRP3, MRP4, MRP5, and SMRP genes was measured using TaqMan real-time polymerase chain reaction (PCR) in 103 children with previously untreated acute lymphoblastic leukemia (ALL) (precursor B-cell ALL {B-ALL}, n = 71; T-cell ALL {T-ALL}, n = 32) . All 5 genes were expressed with a great variability . Only MRP3 expression was associated with a significantly worse prognosis (P =.008) . The median expression of MRP3 was 10-fold higher in T-ALL than in precursor B-ALL (P <.001) and 4-fold higher in male patients than in female patients (P <.001) . The prognostic impact of MRP3 was independent of immunophenotype or sex . Higher levels of MRP3 were found in patients with a poor in vivo response to prednisone, but this could not be confirmed in an independent case-control study (40 patients) for prednisone response . In healthy donors, the median expression of MRP4 was 4-fold higher in bone marrow and 8-fold higher in CD34+ stem cells compared with peripheral blood (P =.002) . Our results suggest that MRP3 is involved in drug resistance in childhood ALL . It therefore represents an interesting target to overcome multidrug resistance . High levels of MRP3 could possibly be the reason for the poorer prognosis of male patients or patients who have T-ALL . Similar to other members of the family of ABC transporters, MRP4 seems to be a marker for immature stem cells.

Zhonghua Nei Ke Za Zhi, 2003 Mar, 42(3), 169 - 72
{The relationship between cyclin B1 and multidrug resistance in adult patients with acute leukemia}; Ma WD et al.; OBJECTIVE: To investigate the relation between the expression of cyclin B1 and multidrug resistance in adult patients with acute leukemia . METHODS: The proteins expression of cyclin B1, p170 was measured with flow cytometric analysis in 85 adult de novo acute leukemia patients (AL) and 17 normal control (NC) . The expression of cyclin B1, multidrug resistance gene (mdr-1), topoisomerase IIalpha, beta (TOPOIIalpha, beta) and bcl-2 mRNA in these patients was measured with semi-quantify reverse transcription polymerase chain reaction (RT-PCR) . RESULTS: (1) The expression of cyclin B1 protein (M = 12.3%) and mRNA (M = 0.217) in the treatment resistance group was significantly lower than the sensitive group cyclin B1 protein (M = 22.7%) and mRNA (M = 0.563) (P < 0.05), so was the mRNA of TOPOIIalpha (M = 0.236), TOPOIIbeta (M = 0.328) than the sensitive group TOPOIIalpha (M = 0.514), TOPOIIbeta (M = 0.635) (P < 0.01) . Cyclin B1 protein expression was lower than 5% and there were no expression of cyclin B1, TOPOIIalpha, mdr-1 mRNA in the NC group under the same condition . (2) The p170 protein (M = 14.3%) and mdr-1 (M = 1.071), bcl-2 (M = 0.941) mRNA expression in the resistant group was significant higher than the sensitive group p170 protein (M = 3.6%) and mdr-1 (M = 0.094), bcl-2 (M = 0.153) (P < 0.01) . (3) The expression of cyclin B1 protein and TOPOIIalpha, TOPOIIbeta mRNA was positive correlated (r(TOPOIIalpha) = 0.472, P < 0.01; r(TOPOIIbeta) = 0.683, P < 0.01), so was the cyclin B1 mRNA (r(TOPOIIalpha) = 0.319, P < 0.05; r(TOPOIIbeta) = 0.527, P < 0.05) . (4) There was no correlation between cyclin B1 and p170, mdr-1, bcl-2 . (5) By Binary logistic forward conditional analysis we concluded that cyclin B1 correlated with atypital mutidrug resistance . CONCLUSIONS: Low expression of cyclin B1 might be a unfavorable prognostic factor for patients with AL and measurement of both cyclin B1 and TOPOIIalpha, TOPOIIbeta gene expression would predict drug resistance in adult acute leukemia patients.

Zhonghua Jie He He Hu Xi Za Zhi, 2003 Mar, 26(3), 165 - 8
{Multidrug resistance and its relationship with neuroendocrine differentiation in non-small cell lung carcinoma}; Yu SP et al.; OBJECTIVE: To study the multidrug resistance (MDR) of non-small lung cancer (NSCLC) and its relationship with neuroendocrine (NE) differentiation . METHODS: NSCLC samples from 113 untreated patients were analyzed immunohistochemically with antibodies to glutathion-s-transferase-pi (GST-pi), multidrug resistance associated protein (MRP), lung resistance associated protein (LRP), neuro-specific enolase (NSE), synaptophysin (SYN) and chromogranin (CgA) . RESULTS: (1) The expression of the three proteins was significantly associated with the type of lung carcinoma (P < 0.05), but not with the differentiation and lymph node metastasis . The expression of GST-pi was significantly related with MRP, MRP and LRP (P < 0.05) . (2) The positive rates of the NE markers were: NSE, 53.1%; SYN, 26.6%; CgA, 6.2%; and 21.2% for at least two markers . The expression of at least 2 markers was associated with the degree of differentiation (P < 0.05), but not with the type of lung cancer and lymph node metastasis . (3) The expression of the three multidrug resistance related proteins in the positive group for at least 2 markers was significantly lower than that in the negative group (P < 0.05) . CONCLUSIONS: The over-expressions of GST-pi, MRP and LRP are important causes of primary multidrug resistance in NSCLC . The differentiation of NE may be one of the factors involved in multidrug resistance.

Mol Pharmacol, 2003 Jul, 64(1), 154 - 9
Phenobarbital alters hepatic Mrp2 function by direct and indirect interactions; Patel NJ et al.; Phenobarbital (PB) treatment impairs the biliary excretion of some organic anions . One mechanism may involve direct competition for biliary excretion by PB and/or a PB metabolite . Alternatively, PB may alter the expression and/or function of hepatic organic anion transport proteins . The role of multidrug resistance-associated protein 2 (Mrp2) in the biliary excretion of PB and metabolites was studied using isolated perfused livers (IPLs) from Wistar and Mrp2-deficient TR- rats . In normal livers, 4.19 +/- 0.53% of the PB dose was recovered in bile as PB metabolites {2.21 +/- 0.69% as 5-ethyl-5-(4-OH phenyl) barbituric acid (PBOH)-glucuronide; 1.98 +/- 0.09% as PBOH-sulfate} . In TR- livers, only PBOH-sulfate was recovered in bile (0.35 +/- 0.16% of dose) during the 2-h perfusion . Mrp2 message was increased (2.3-fold) by PB pretreatment (80 mg/kg i.p . x 4 days) but decreased to control values after a 48-h washout . Mrp2 protein was increased slightly in PB-treated livers and remained slightly elevated after a 24-h washout, but it was decreased significantly to 62 +/-7% of control values after a 48-h washout . The 120-min cumulative biliary excretion of the Mrp2 substrate 5-(and-6)-carboxy-2', 7'-dichlorofluorescein in IPLs from PB-treated rats after a 48-h washout was significantly lower than in vehicle-treated livers (66.3 +/- 9.2% versus 83.4 +/- 2.4% of the dose, respectively) . These data support two mechanisms for impaired biliary excretion of some organic anions by PB treatment: 1) PBOH-glucuronide is a substrate for Mrp2 and may compete with other organic anions for biliary excretion and 2) Mrp2 protein expression and functional capacity is decreased 48 h after PB treatment.

Monaldi Arch Chest Dis, 2002 Oct-Dec, 57(5-6), 285 - 90
Problems to control TB in eastern Europe and consequences in low incidence countries; Migliori GB et al.; The regular decline in tuberculosis (TB) notification rates observed in the majority of industrialised countries over the past decades has levelled off or reversed in recent years in both the USA and in Europe, where relevant differences between Central/Eastern and Western countries have become more and more apparent . In the countries of the Central and Eastern European sub-region, notification rates have ceased to decline or markedly increased since the 1990s, particularly in the Baltic States, in Romania, Russian Federation and other countries belonging to the previous USSR . The evaluation of age-specific notification rates shows that the majority of the countries in the Central and Eastern European sub-region still have a pattern of middle-income countries, where TB is affecting significantly the younger cohorts, and the infection in the community is still rampant . The aim of the present paper is to discuss the main determinants of the deteriorated TB control in Eastern Europe, the main consequences of this situation for Western European Countries, and the possible solutions . The major constraints described are: socio-economic crisis, health system weaknesses, HIV pandemic, multidrug-resistant TB (MDRTB) and failure to control TB in prisons and in other risk groups . The main consequences of the sub-optimal TB control achieved in Eastern Europe, are the exportation of TB trough immigration and, as part of this phenomenon, the exportation of drug resistance and MDRTB . Proper TB control is possible in the sub-region, as testified by the successful results achieved in the Czech republic, Slovakia and Slovenia . A global reform of health systems is necessary, particularly in the previous USSR countries, based on cost-effective interventions . MDRTB should be managed promptly as an international emergency, based on a cooperative approach of donor and assisted countries . As opportunities for improved funding of TB control in the region exist, there is the potential to reverse to TB pandemic before the explosion of the HIV epidemic expected in Eastern Europe.

Cell Mol Biol Lett, 2003, 8(2), 311 - 5
There is no evidence for the existence of complex formation between doxorubicin and glutathione; Marszalek M et al.; Doxorubicin is co-transported with glutathione by several multidrug resistance proteins (MRPs) . In order to check whether weak non-covalent aggregates between doxorubicin and glutathione can be formed, which might be substrates for the transporter, the effect of glutathione on the partition coefficient of doxorubicin was studied . No evidence of an effect of glutathione (at levels up to 20 microM) on the partition coefficient of doxorubicin was found in the pH range of 4.0-7.4 . These results indicate that non-covalent doxorubicin-glutathione complexes do not form.

Oncogene, 2003 Jun 19, 22(25), 3888 - 900
Peroxisome proliferator-activated receptor-gamma upregulates caveolin-1 and caveolin-2 expression in human carcinoma cells; Burgermeister E et al.; Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor for eicosanoids that promotes differentiation of human epithelial and mesenchymal cells in vitro and in vivo . PPARgamma was proposed as a target for drug-induced differentiation therapy of cancer . Caveolin-1 is a constituent of plasma membrane caveolae in epithelial cells that is often downregulated upon oncogenic transformation . Caveolin-1 has growth-inhibitory activities and its disruption is sufficient to induce transformation in fibroblasts . Herein we have tested the hypothesis that caveolins are transcriptional target genes for PPARgamma . In human HT-29 colon carcinoma cells, thiazolidinedione PPARgamma ligands increased the levels of caveolin-1 and caveolin-2 proteins two to fivefold in a concentration-dependent manner within 24 h . In human MCF-7 breast adenocarcinoma cells, nonthiazolidinedione PPARgamma ligands elevated caveolin-2 protein three to fourfold, while the thiazoli-dinediones were less effective . Caveolin-1 mRNA levels were found to be upregulated by PPARgamma ligands already after 3 h in both the cell lines . Ectopic expression of a dominant-negative PPARgamma construct attenuated ligand-induced upregulation of caveolins in both HT-29 and HEK-293T cells, indicating that ligand action is mediated by PPARgamma . Ligand-treated MCF-7 cells exhibited a differentiated phenotype, as evinced by analysis of cell-specific differentiation markers: protein levels of maspin were elevated and perinuclear lipid droplets accumulated . In contrast, in HT-29 cells, caveolin expression was not correlated with differentiation . Interestingly, PPARgamma partially cofractionated in lipid rafts and could be coimmunoprecipitated from cell lysates with caveolin-1, indicating that PPARgamma and caveolin-1 may coexist in a complex . Our data indicate that PPARgamma participates in the regulation of caveolin gene expression in human carcinoma cells and suggest that caveolin-1 may mediate some of the phenotypic changes induced by this nuclear receptor in cancer cells . These findings may have potentially important functional implications in the context of cancer differentiation therapy and multidrug resistance.

Zhonghua Yi Xue Za Zhi, 2003 Feb 25, 83(4), 328 - 32
{The overexpression of prion protein in drug resistant gastric cancer cell line SGC7901/ADR and its significance}; Du JP et al.; OBJECTIVE: To investigate the overexpression of prion protein (PrP) in drug-resistant gastric cancer cell line SGC7901/ADR and its role in multidrug resistance in gastric cancer . METHODS: (1) The expression of PrP in SGC7901/ADR, SGC790/VCR and their parental cell line SGC7901 was detected with Northern and Western blot at the mRNA and protein level . (2) Eukaryotic sense and antisense expression vector were constructed based on DNA recombination technology and (3) introduced into SGC7901 and SGC7901/ADR cell lines through electroporation . (4) The accumulation and retention of ADR in transiently transfected cells were detected by flow cytometry . RESULTS: (1) Northern and western blot suggested significantly higher expression of PrP in SGC7901/ADR and SGC7901/VCR than that in SGC7901 . (2) 48 hours after the vectors transfection, the average fluorescence intensity of Adr in transfected cells were detected . The accumulation intensity were 8.9 +/- 0.7 in BS, 6.6 +/- 0.3 in PS and 7.5 +/- 0.6 in PA . The retention intensity were 9.3 +/- 0.6 in SGC7901, 5.9 +/- 0.5 in PS and 7.1 +/- 0.5 in PA . There were significant difference between PS and BS with P < 0.01, as well as RA and BA with P < 0.01 . These data suggested that PrP gene could affect the drug accumulation in gastric cancer cells after its transfected into cells . CONCLUSION: PrP was highly expressed in gastric cancer cell lines SGC7901/ADR and SGC7901/VCR . Overexpression of PrP had certain effect on drug accumulation in gastric cancer cells.

Cancer Res, 2003 Jun 15, 63(12), 3228 - 33
Differential effects of the breast cancer resistance protein on the cellular accumulation and cytotoxicity of 9-aminocamptothecin and 9-nitrocamptothecin; Rajendra R et al.; Breast cancer resistance protein (BCRP)/MXR/ABCG2 is a new member of the family of ATP-dependent drug efflux proteins . Whereas overexpression of another member of this family, P-glycoprotein, minimally affects the cytotoxicity of camptothecins (CPTs), overexpression of wild-type as well as certain mutant BCRPs confers resistance to CPT analogues that are used clinically, including topotecan and irinotecan . Relatively little is known regarding the effects of BCRP on other CPT analogues . We now report studies of 9-aminocamptothecin (9-AC) and 9-nitrocamptothecin (9-NC) using mammalian cells stably transfected with constructs expressing a variety of efflux proteins, including wild-type BCRP and a mutant BCRP that contains a threonine rather than an arginine at position 482 (R482T) . The results indicate that overexpression of either P-glycoprotein, multidrug resistance protein type 1, or multidrug resistance protein type 2 has little effect on the cytotoxicity of 9-NC or 9-AC . By contrast, overexpression of either wild-type or R482T BCRP confers resistance to 9-AC, but not to 9-NC . Furthermore, overexpression of wild-type or mutant BCRP is associated with reduced intracellular accumulation of 9-AC, but not 9-NC . In addition, immunoblotting studies indicate that whereas increased BCRP expression is evident in cells selected for resistance to irinotecan, BCRP expression is not detectable in two different cell lines selected for resistance to 9-NC . Taken together, these findings suggest that wild-type as well as R482T BCRP mediates cellular efflux of 9-AC but not 9-NC . Furthermore, the results suggest that polar groups at the 9 or 10 position of the CPT A ring facilitate interaction with BCRP and have implications for the clinical development of new CPT analogues.

Cancer Res, 2003 Jun 15, 63(12), 3211 - 20
Taccalonolides E and A: Plant-derived steroids with microtubule-stabilizing activity; Tinley TL et al.; During the course of a mechanism-based screening program designed to identify new microtubule-disrupting agents from natural products, we identified a crude extract from Tacca chantrieri that initiated Taxol-like microtubule bundling . Bioassay-directed purification of the extract yielded the highly oxygenated steroids taccalonolides E and A . The taccalonolides caused an increased density of cellular microtubules in interphase cells and the formation of thick bundles of microtubules similar to the effects of Taxol . Mitotic cells exhibited abnormal mitotic spindles containing three or more spindle poles . The taccalonolides were evaluated for antiproliferative effects in drug-sensitive and multidrug-resistant cell lines . The data indicate that taccalonolide E is slightly more potent than taccalonolide A in drug-sensitive cell lines and that both taccalonolides are effective inhibitors of cell proliferation . Both taccalonolides are poorer substrates for transport by P-glycoprotein than Taxol . The ability of the taccalonolides to circumvent mutations in the Taxol-binding region of beta-tubulin was examined using the PTX 10, PTX 22, and 1A9/A8 cell lines . The data suggest little cross-resistance of taccalonolide A as compared with Taxol, however, the data from the PTX 22 cell line indicate a 12-fold resistance to taccalonolide E, suggesting a potential overlap of binding sites . Characteristic of agents that disrupt microtubules, the taccalonolides caused G(2)-M accumulation, Bcl-2 phosphorylation, and initiation of apoptosis . The taccalonolides represent a novel class of plant-derived microtubule-stabilizers that differ structurally and biologically from other classes of microtubule-stabilizers.

Cancer Res, 2003 Jun 15, 63(12), 3084 - 91
P-glycoprotein, expressed in multidrug resistant cells, is not responsible for alterations in membrane fluidity or membrane potential; Aleman C et al.; Expression of P-glycoprotein (P-gp), the multidrug resistance (MDR) 1 gene product, can lead to MDR in tumors . However, the physiological role of P-gp in normal tissues is not well understood . Previous studies on multidrug-resistant cells have suggested changes in membrane fluidity and membrane potential associated with P-gp expression, but interpretation of these studies is difficult, because most experimental cells have been selected for long periods in the presence of cytotoxic drugs and may have other host alterations . Therefore, we created two cell lines in which a transfected human MDR1 cDNA is repressed by tetracycline and induced in the absence of tetracycline . One cell line was derived from a mouse embryonic fibroblast cultured from a double (mdr1a/1b) knockout mouse, and the other was from a human HeLa cell line . Analysis of the kinetics of expression of P-gp showed that the mRNA had a half-life of approximately 4 h, and the protein had a half-life of approximately 16 h . P-gp cell surface expression (measured with monoclonal antibody MRK-16) and P-gp function (measured with a fluorescent substrate, rhodamine 123) was characterized by using fluorescence-activated cell sorting . No differences in membrane potential using the fluorescent probe oxonol or in membrane "fluidity" using fluorescent anisotropy probe or electron spin resonance probe were observed in the tet-repressible P-gp-expressing cells . In contrast, several drug-selected cells that express P-gp showed an increase in membrane fluidity and membrane potential . These results suggest that expression of P-gp per se has little effect on membrane fluidity or membrane potential, and it does not have H(+) pump activity . The changes in these parameters observed in drug-selected cells must reflect other host adaptations to drug selection.

Biochem Biophys Res Commun, 2003 Jul 4, 306(3), 644 - 9
Expression of functional multidrug-resistance protein 1 in Saccharomyces cerevisiae: effects of N- and C-terminal affinity tags; Lee SH et al.; Studies of the multidrug-resistance protein 1 (MRP1) have been hampered by the lack of a simple expression system allowing for rapid generation of mutants and yielding milligram amounts of protein . Here, we describe a Saccharomyces cerevisiae expression system that meets those conditions . MRP1 was expressed under the control of the constitutive PMA1 (yeast proton pump) promoter . The best conditions for expression were determined, including the use of the chemical chaperone glycerol, which increased MRP1 expression . N-terminal poly-histidine or FLAG affinity tags reduce MRP1 expression, whereas the same tags fused to the C-terminus had no effect . All the fusion proteins were functional . We conclude that because of its low cost and simplicity, the S . cerevisiae-based MRP1-expression system will be useful for studies where a large number of mutants or milligram amounts of purified MRP1 are needed.

Ann Biol Clin (Paris), 2003 May-Jun, 61(3), 305 - 9
{A new, rapid and robust genotyping method for CYP2C9 and MDR1}; Verstuyft C et al.; Single nucleotide polymorphisms (SNPs) can significantly affect human phenotypes . Detection of allelic variant carriers has become a major goal for clinical pharmacologists in order to study phenotype-genotype relationships . However, there is a crucial need for rapid, and validated pharmacogenetic tests . The aim of the study was to validate a new fluorescence PCR strategy for cytochrome P450 2C9 (CYP2C9) and multidrug resistance gene (MDR1) genotyping . Results of CYP2C9 and MDR1 genotypes determined with reference techniques were compared to those obtained by allelic discrimination assays employing fluorescent TaqMan probes . Sixteen subjects carrying CYP2C9*2 and CYP2C9*3 allelic variants (heterozygous and homozygous) previously identified by sequencing and 55 subjects previously genotyped for MDR1 exon 26 (C3435T) SNP by conventional PCR-RFLP were genotyped with fluorescent PCR . Fluorescent PCR gave 100 % accuracy with the results obtained with reference genotyping strategies for each of the 3 SNPs . Genotyping results with fluorescent PCR repeated on three consecutive occasions remained constant over time for each of the 3 SNPs . Allelic discrimination assays based on fluorescent PCR gave entire satisfaction for CYP2C9 and MDR1 genotyping . This reliable genotyping strategy can be easily used in clinical practice and should be further developed for additional SNPs identification.

FEBS Lett, 2003 Jun 19, 545(2-3), 144 - 50
Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi); Nieth C et al.; For reversal of MDR1 gene-dependent multidrug resistance (MDR), two small interfering RNA (siRNA) constructs were designed to inhibit MDR1 expression by RNA interference . SiRNA duplexes were used to treat human pancreatic carcinoma (EPP85-181RDB) and gastric carcinoma (EPG85-257RDB) cells . In both cellular systems, siRNAs could specifically inhibit MDR1 expression up to 91% at the mRNA and protein levels . Resistance against daunorubicin was decreased to 89% (EPP85-181RDB) or 58% (EPG85-257RDB) . The data indicate that this approach may be applicable to cancer patients as a specific means to reverse tumors with a P-glycoprotein-dependent MDR phenotype back to a drug-sensitive one.

Leuk Lymphoma, 2003 May, 44(5), 783 - 9
In vitro chemosensitivity testing of selected myeloid cells in acute myeloid leukemia; Mollgard L et al.; In several studies different chemosensitivity assays have been examined in acute myeloid leukemia (AML) . Some have shown that in vitro chemosensitivity testing is an independent prognostic factor but so far no one has been able to show that the use of these methods can improve treatment outcome . In an attempt to improve in vitro chemosensitivity testing in AML we wanted to establish and evaluate a new flow cytometry chemosensitivity assay . After 4 days of incubation viable mononuclear myeloid cells were identified by the exclusion of propidium iodide in CD13 or CD33 positive cells . Sixty-eight samples from 64 AML patients were included . In this study, we showed that the flow cytometry method is feasible in AML and we also found some correlations to clinical data . The secondary AML at diagnosis showed an in vitro resistance to etoposide and amsacrine that was significantly higher compared to de novo AML at diagnosis (p = 0.04 and p = 0.02) . When AML patients at diagnosis were compared to resistant disease/relapse patients there was a significantly higher effect of ara-C in the diagnosis group (p = 0.03) . Responders and non-responders were compared in vitro but we found no significant differences . In vitro mitoxantrone was more effective in multidrug resistance (MDR) negative cells compared to MDR positive cells (p < 0.01) . This new method is feasible and makes it possible to selectively evaluate the effect of cytotoxic drugs in myeloid cells . Further studies with a larger group of patients are needed to evaluate the predictive value of the assay.

Clin Infect Dis, 2003 Jun 15, 36(12), e152 - 4 Epub 2003 Jun 03.
Incidence of multidrug-resistant tuberculosis in urban and rural India and implications for prevention; Almeida D et al.; We compared the incidence of multidrug resistance in 150 consecutive Mycobacterium tuberculosis isolates obtained from a rural center (in Sakawar, India) and an urban tertiary care center (in Mumbai, India) . The study highlights an alarmingly high percentage of multidrug-resistant M . tuberculosis isolates in Mumbai (51%) as compared with that at the rural center (2%).

Gut, 2003 Jul, 52(7), 1060 - 7
ATP binding cassette transporter gene expression in rat liver progenitor cells; Ros JE et al.; BACKGROUND AND AIM: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs) . Under these conditions they must be able to withstand the toxic milieu of the damaged liver . ATP binding cassette (ABC) transporters are cytoprotective efflux pumps that may contribute to the preservation of these cells . The aim of this study was to determine the ABC transporter phenotype of HPCs . METHODS: HPC activation was studied in rats treated with 2- acetylaminofluorene (2-AAF) followed by partial hepatectomy (PHx) . ABC transporter gene expression was determined by real time detection reverse transcription-polymerase chain reaction in isolated HPCs, hepatocytes, cholangiocytes, and cultured progenitor cell-like RLF phi 13 cells and by immunohistochemistry of total liver samples . ABC transporter efflux activity was studied in RLF phi 13 cells by flow cytometry . RESULTS: 2-AAF/PHx treated animals showed increased hepatic mRNA levels of the genes encoding multidrug resistance proteins Mdr1b, Mrp1, and Mrp3 . Immunohistochemistry demonstrated expression of Mrp1 and Mrp3 proteins in periportal progenitor cells and of the Mdr1b protein in periportal hepatocytes . Freshly isolated Thy-1 positive cells and cultured RLF phi 13 progenitor cells highly expressed Mrp1 and Mrp3 mRNA while the hepatocyte specific transporters Mdr2, Bsep, Mrp2, and Mrp6 were only minimally expressed . Blocking Mrp activity by MK-571 resulted in accumulation of the Mrp specific substrate carboxyfluorescein in RLF phi 13 cells . CONCLUSION: HPCs express high levels of active Mrp1 and Mrp3 . These may have a cytoprotective role in conditions of severe hepatotoxicity.

Eur J Haematol, 2003 Jul, 71(1), 1 - 8
Clinical characteristics, prognostic factors and multidrug-resistance related protein expression in 36 adult patients with acute promyelocytic leukemia; Candoni A et al.; We report on a single-center experience about the characteristics and outcome of 36 acute promyelocytic leukemia (APL) patients observed at our Department of Hematology between 1990 and 2002 . The expression, of multidrug-resistance (MDR) associated proteins (PGP, LRP, MRP1) was also analyzed . There were 12 males and 24 females (median age 37 yr), 89% (32 of 36) with classic morphology, and 11% (four of 36) with a microgranular variant . Risk class (according to GIMEMA/PETHEMA): 25% (nine of 36) high risk (HR), 53% (nineteen of 36) intermediate risk (IR), 22% (eight of 36) low risk (LR) . PGP, LRP, and MRP1 expression at onset and at first relapse was low . CD33 antigen expression was high in all cases . The patients were treated according to GIMEMA protocols (LAP0389 and AIDA) including ATRA in induction in 75% (27 of 36) of cases and 94% (34 of 36) achieved a complete remission (CR) after induction therapy while 6% (two of 36) died early (DDI) of hemorrhage . Outcome: 71% (24 of 34) of evaluable patients remain in CR at a median follow-up of 57 months (range 4-158 months) while 29% (10 of 34) relapsed at a median time of 12 months (range 8-43 months) and, of them, eight of 10 died early . The majority of patients that relapsed were in high-risk group . The overall survival (OS) of the whole population at 32 months was 66% and the DFS at 42 months was 62% . A statistically significant difference in terms of DFS was observed between HR and IR/LR patients (P = 0.04 by log-rank) . DFS was not affected by age, sex, Hb levels, karyotype, and BCR isoform . At conclusion, our data confirm that despite the high rate of success with ATRA plus chemotherapy as induction (more than 90% of CR), about 30% of APL patients have a relapse (without a long-lasting second remission) and underline the importance of patient stratification in distinct risk groups at diagnosis in order to better adapt the type and intensity of treatment (risk-adapted therapy) . Taking into account the high expression of CD33 and the low expression of MDR proteins in APL, new and investigational approaches like gemtuzumab-ozogamicin, with or without ATRA and other new drugs, should be strongly considered expecially in HR APL.

Br J Cancer, 2003 Jun 16, 88(12), 1963 - 70
Increased sensitivity to gemcitabine of P-glycoprotein and multidrug resistance-associated protein-overexpressing human cancer cell lines; Bergman AM et al.; Gemcitabine (2',2'-difluorodeoxycytidine) is a deoxycytidine analogue that is activated by deoxycytidine kinase (dCK) to its monophosphate and subsequently to its triphosphate dFdCTP, which is incorporated into both RNA and DNA, leading to DNA damage . Multidrug resistance (MDR) is characterised by an overexpression of the membrane efflux pumps P-glycoprotein (P-gP) or multidrug resistance-associated protein (MRP) . Gemcitabine was tested against human melanoma, non-small-cell lung cancer, small-cell lung cancer, epidermoid carcinoma and ovarian cancer cells with an MDR phenotype as a result of selection by drug exposure or by transfection with the mdr1 gene . These cell lines were nine- to 72-fold more sensitive to gemcitabine than their parental cell lines . The doxorubicin-resistant cells 2R120 (MRP1) and 2R160 (P-gP) were nine- and 28-fold more sensitive to gemcitabine than their parental SW1573 cells, respectively (P<0.01), which was completely reverted by 25 micro M verapamil . In 2R120 and 2R160 cells, dCK activities were seven- and four-fold higher than in SW1573, respectively, which was associated with an increased dCK mRNA and dCK protein . Inactivation by deoxycytidine deaminase was 2.9- and 2.2-fold decreased in 2R120 and 2R160, respectively . dFdCTP accumulation was similar in SW1573 and its MDR variants after 24 h exposure to 0.1 micro M gemcitabine, but dFdCTP was retained longer in 2R120 (P<0.001) and 2R160 (P<0.003) cells . 2R120 and 2R160 cells also incorporated four- and six-fold more {(3)H}gemcitabine into DNA (P<0.05), respectively . P-glycoprotein and MRP1 overexpression possibly caused a cellular stress resulting in increased gemcitabine metabolism and sensitivity, while reversal of collateral gemcitabine sensitivity by verapamil also suggests a direct relation between the presence of membrane efflux pumps and gemcitabine sensitivity.

Genome Res, 2003 Jun, 13(6A), 1180 - 9
Genome analysis of F . nucleatum sub spp vincentii and its comparison with the genome of F . nucleatum ATCC 25586; Kapatral V et al.; We present the draft genome sequence and its analysis for Fusobacterium nucleatum sub spp . vincentii (FNV), and compare that genome with F . nucleatum ATCC 25586 (FN) . A total of 441 FNV open reading frames (ORFs) with no orthologs in FN have been identified . Of these, 118 ORFs have no known function and are unique to FNV, whereas 323 ORFs have functional orthologs in other organisms . In addition to the excretion of butyrate, H2S and ammonia-like FN, FNV has the additional capability to excrete lactate and aminobutyrate . Unlike FN, FNV is likely to incorporate galactopyranose, galacturonate, and sialic acid into its O-antigen . It appears to transport ferrous iron by an anaerobic ferrous transporter . Genes for eukaryotic type serine/threonine kinase and phosphatase, transpeptidase E-transglycosylase Pbp1A are found in FNV but not in FN . Unique ABC transporters, cryptic phages, and three types of restriction-modification systems have been identified in FNV . ORFs for ethanolamine utilization, thermostable carboxypeptidase, gamma glutamyl-transpeptidase, and deblocking aminopeptidases are absent from FNV . FNV, like FN, lacks the classical catalase-peroxidase system, but thioredoxin/glutaredoxin enzymes might alleviate oxidative stress . Genes for resistance to antibiotics such as acriflavin, bacitracin, bleomycin, daunorubicin, florfenicol, and other general multidrug resistance are present . These capabilities allow Fusobacteria to survive in a mixed culture in the mouth.

Eukaryot Cell, 2003 Jun, 2(3), 588 - 98
A region within a lumenal loop of Saccharomyces cerevisiae Ycf1p directs proteolytic processing and substrate specificity; Mason DL et al.; Ycf1p, a member of the yeast multidrug resistance-associated protein (MRP) subfamily of ATP-binding cassette proteins, is a vacuolar membrane transporter that confers resistance to a variety of toxic substances such as cadmium and arsenite . Ycf1p undergoes a PEP4-dependent processing event to yield N- and C-terminal cleavage products that remain associated with one another . In the present study, we sought to determine whether proteolytic cleavage is required for Ycf1p activity . We have identified a unique region within lumenal loop 6 of Ycf1p, designated the loop 6 insertion (L6(ins)), which appears to be necessary and sufficient for proteolytic cleavage, since L6(ins) can promote processing when moved to new locations in Ycf1p or into a related transporter, Bpt1p . Surprisingly, mutational results indicate that proteolytic processing is not essential for Ycf1p transport activity . Instead, the L6(ins) appears to regulate substrate specificity of Ycf1p, since certain mutations in this region lower cellular cadmium resistance with a concomitant gain in arsenite resistance . Although some of these L6(ins) mutations block processing, there is no correlation between processing and substrate specificity . The activity profiles of the Ycf1p L6(ins) mutants are dramatically affected by the strain background in which they are expressed, raising the possibility that another cellular component may functionally impact Ycf1p activity . A candidate component may be a new full-length MRP-type transporter (NFT1), reported in the Saccharomyces Genome Database as two adjacent open reading frames, YKR103w and YKR104w, but which we show here is present in most Saccharomyces strains as a single open reading frame.

Zhonghua Zhong Liu Za Zhi, 2003 Mar, 25(2), 134 - 6
{Establishment of human multidrug-resistant lung carcinoma cell line (D6/MVP)}; Ma SL et al.; OBJECTIVE: To establish human multidrug-resistant lung carcinoma cell line (D6/MVP) with its characteristics studied . METHODS: Intermittent administration of high-dose MMC, VDS and DDP (MVP) was used to induce human lung carcinoma cell line (D6) to a multidrug-resistant variety (D6/MVP) . MTT assay was used to study the multidrug resistance of D6/MVP to multianticarcinogen . Flow cytometry was used to study the cell cycle distribution and the expression of P-gp, multidrug resistance-associated protein (MRP) and GSH/GST . RESULTS: 1 . D6/MVP was resistant to many anti-tumor agents, with the IC(50) 13.3 times higher and the drug resistance 2 - 6 times higher than D6, 2 . The multiplication time of D6/MVP was prolonged and the cell number of S-phase decreased while that of G1- and G(2)-phase increased and 3 . The expression of P-gp and MRP was enhanced significantly (96.2% vs 51.7%), but the expression of GSH/GST kept stable . CONCLUSION: D6/MVP is a multidrug-resistant cell line possessing the basic characteristics of drug-resistance.

J Gastroenterol Hepatol, 2003 Jul, 18(7), 815 - 21
Effects of lipopolysaccharide on the biliary excretion of bile acids and organic anions in rats; Hojo M et al.; BACKGROUND: Lipopolysaccharide is known to be a cause of cholestasis associated with sepsis . It has also recently been reported to down-regulate the basolateral and canalicular transporters . The aim of the present study was to examine simultaneously the effect of lipopolysaccharide on the biliary excretion of typical substrates of bile salt export pump and multidrug resistance protein 2 in vivo, and the effect of lipopolysaccharide on the amount of these transporters . METHODS: After intravenous administration of O127:B8-derived lipopolysaccharide (2.5 mg/kg), the biliary excretion of taurocholate and various organic anions was studied, and the protein levels of bile salt export pump and multidrug resistance protein 2 in the crude liver membrane was determined by western blot analysis . RESULTS: Lipopolysaccharide decreased the biliary excretion of tracer amounts of taurocholate, leukotriene C4, taurolithocholate-3-sulfate and temocapril without affecting bile flow . The biliary excretion of high doses of taurocholate and sulfobromophthalein was markedly inhibited by lipopolysaccharide . Lipopolysaccharide decreased bile salt export pump levels in the liver plasma membrane fraction to 48% of control rats, and markedly decreased multidrug resistance protein 2 levels to 17% of control rats . CONCLUSIONS: These findings support the hypothesis that down-regulation of the canalicular transporters by lipopolysaccharide causes the impairment of the biliary excretion of bile acids and organic anions in cholestasis of sepsis prior to the decrease of bile flow .

Invest New Drugs, 2003 Feb, 21(1), 75 - 84
A phase I and pharmacologic study of pyrazoloacridine and cisplatin in patients with advanced cancer; Dees EC et al.; Pyrazoloacridine (PZA, NSC366140, PD115934) is an acridine derivative currently undergoing clinical evaluation . In preclinical testing, PZA has shown selectivity for solid tumor cell lines, activity in hypoxic, noncycling, and multidrug-resistant cell lines, and synergy with cisplatin in a variety of lung cancer cell lines . In early phase I clinical studies PZA has shown modest activity in ovarian, cervical, and colon cancer . The purpose of the present study was threefold: to determine the maximally tolerated doses of the combination of PZA (3-h infusion) and cisplatin administered with and without Filgrastim (G-CSF) (Amgen, Thousand Oaks, CA) every 3 weeks in untreated or minimally pretreated patients, to describe and quantify the clinical toxicities of combination chemotherapy with PZA and cisplatin, and to evaluate the effects of drug sequencing on the toxicity profile and pharmacologic behavior of PZA . The starting doses in this dose-escalation trial were PZA 400 mg/m2 as a 3-h intravenous infusion and cisplatin 50 mg/m2 as a 1 mg/min intravenous infusion . The sequence of drugs was alternated with each successive course in each patient treated . Twenty-one patients with refractory solid tumors received 43 courses of therapy through four dose levels . Neutropenia was dose-limiting and defined the maximum tolerated dose of PZA 400 mg/m2 and cisplatin 50 mg/m2 without G-CSF support . With G-CSF support, nausea and vomiting were dose-limiting . The maximum tolerated and recommended doses for further study of this combination are PZA 600 mg/m2 over 3 h and cisplatin 50 mg/m2 followed by G-CSF support . Pharmacokinetic analysis showed that sequence does not impact on the pharmacokinetics of PZA when given in combination with cisplatin.

Invest New Drugs, 2003 Feb, 21(1), 33 - 45
Discovery and preclinical antitumor efficacy evaluations of LY32262 and LY33169; Corbett TH et al.; The discoveries of a new antitumor agent (LY32262) (N-{2,4-dichlorobenzoyl}phenylsulfonamide) and a close analog (LY33169) are described . For this discovery, a disk-diffusion-soft-agar-colony-formation-assay was used to screen a portion of the Eli Lilly inventory, with the evaluation of each agent against normal cells, leukemic cells and several solid tumors, including a multidrug-resistant solid tumor (with marked selective cytotoxicity for Colon-38 and Human-Colon-15/MDR compared to normal fibroblasts and L1210 leukemic cells characterizing the discovery) . In mice, LY32262 and/or LY33169 had curative activity against Colon Adenocarcinoma-38, Human Colon-116, Human Prostate LNCaP, and Human Breast WSU-Br-1 . In addition, many other tumors were highly sensitive: Panc-03 = 2.4 log kill (LK); Panc-02 = 2.9-4.1 LK; Squamous Lung LC-12 = 2.1 LK; Colon-26 = 2.2 LK; AML1498 = 2.7 LK; Human Sm Cell Lung DMS-273 = 6.3 LK; Human Squamous Lung 165 = 3.7 LK; Human Ovarian BG-1 = 3.7 LK; Human Colon CX-1 (H29) = 1.6 LK; Human Colon-15/MDR (a p-glycoprotein positive multidrug resistant tumor) = 2.3 LK; Human CNS-gliosarcoma-SF295 = 3.8 LK . Several tumors were only marginally responsive or totally unresponsive: Mammary Adenocarcinoma-16/C = 0.6 LK; Mammary Adenocarcinoma-17 = no kill; Colon Adenocarcinoma-11 = no kill; L1210 leukemia = 1.3 LK; Human Prostate PC-3 = 0.5 LK; Human Adenosquamous Lung H125 = no kill; and Human Breast Adenocarcinoma MX-1 = 0.9 LK . There was no absolute tissue of origin correlation with antitumor efficacy, although colon tumors were most responsive and mammary tumors least responsive . The cause of the "hit and miss" efficacy has not been determined.

Int J Cancer, 2003 Aug 10, 106(1), 108 - 15
Cytotoxicity, apoptosis induction and downregulation of MDR-1 expression by the anti-topoisomerase II agent, salvicine, in multidrug-resistant tumor cells; Miao ZH et al.; Salvicine, a novel topoisomerase II inhibitor and a diterpenoid quinone compound, exerts potent in vitro and in vivo antitumor effects . In our study, we show that salvicine effectively kills multidrug-resistant (MDR) sublines, such as K562/A02, KB/VCR and MCF-7/ADR, and parental K562, KB and MCF-7 cell lines to an equivalent degree . These cytotoxic activities of salvicine were much more potent than those of several classical anticancer drugs (average resistance factor: 1.42 for salvicine vs . 344.35, 233.19 and 71.22 for vincristine, doxorubicin and etoposide, respectively) . Flow cytometry and DNA agarose gel electrophoresis demonstrated that salvicine induced similar levels of apoptosis in MDR K562/A02 and parental cells . The compound activated caspase-1 and -3 (but not caspase-8) and increased the ratio of bax to bcl-2 mRNA via reduction of bcl-2 mRNA expression in the same cells . Furthermore, salvicine induced the downregulation of mdr-1 gene and P-gp expression but had no effect on MRP and LRP gene expression in MDR K562/A02 cells . These results suggest that the reduction of mdr-1 and bcl-2 expression by salvicine possibly contributes to its cytotoxicity and apoptotic induction in this system . The effectiveness, broad-spectrum activity and possibly novel mechanism of killing MDR tumor cells in vitro of salvicine signify promising in vivo and clinical activity . The novel chemical structure of this compound further implies a role for salvicine in future MDR tumor therapy .

In Vivo, 2003 Mar-Apr, 17(2), 145 - 9
Reversal of multidrug resistance in mouse lymphoma cells by phenothiazines; Molnar J et al.; Various compounds were tested with regard to their reversal of multidrug resistance (MDR) in mouse tumor cells transfected with the human MDR1 gene . Phenothiazines containing aromatic moieties were bound through stacking interaction involving the polarization of the aromatic aminoacid substituents at the target site of p-glycoprotein (Pgp) 170, as a consequence of their large dipoles (as in the binding of phenothiazine to calmodulin-like structures) . Acting as a calcium channel blocker, verapamil may induce conformational changes in the calcium channel-like structures of the transmembrane regions of Pgp . Most probably the tyrosine moieties of Pgp are involved in the action of verapamil and phenothiazines . Tomato lectin specifically binds to the polylactosamine moiety of Pgp170 at the first loop of Pgp . Other targets in the membrane may exist in close proximity to Pgp170, such as conA-reactive glycoproteins with terminal mannosyl residues . WGA-reactive N-acetyl glucosamine residues can also be modified resulting in conformational changes in trans-membrane regions of the ABC transporter . Our results demonstrate that MDR can be reversed by interaction of various compounds with Pgp or by modification of the membrane structure around the Pgp.

Int J Oncol, 2003 Jul, 23(1), 213 - 20
Evaluation of MDR1, LRP, MRP, and topoisomerase IIalpha gene mRNA transcripts before and after interferon-alpha, and correlation with the mRNA expression level of the telomerase subunits hTERT and TEP1 in five unselected human melanoma cell lines; Miracco C et al.; Intrinsic and acquired multidrug-resistance (MDR) and the activity of the enzyme telomerase have been demonstrated in human melanoma . A direct regulation of the MDR pathways and of telomerase by interpheron-alpha (IFN-alpha), which is currently used in the therapy of advanced cutaneous melanoma, has also been hypothesized . In this study, we used five melanoma cell lines not selected in vitro for drug resistance (Me665/2/21, Me665/2/60, HT-144, SK-MEL-28, and SK-MEL-5), which in a previous study, had shown different responses to IFN-alpha in terms of proliferation, apoptosis, telomerase activity and expression of mRNA for the human telomerase reverse transcriptase (hTERT) . We investigated the expression of the multidrug resistance (MDR1) gene, multidrug resistance protein (MRP), lung resistance protein (LRP), topoisomerase IIalpha (Topo IIalpha), hTERT, and telomerase-associated protein (TEP1), which is shared by telomerase and vault MDR proteins at the mRNA expression level, using the reverse transcription-PCR (RT-PCR) . All cell lines showed an intrinsic expression of hTERT, TEP1, and MDR gene transcripts (only MDR1 mRNA was under the detection level in SK-MEL-28 cells) . After IFN-alpha exposure, we observed either no effect, a trend towards a decrease of hTERT, MRP, and Topo IIalpha, or an increase of TEP1, MDR1, and LRP mRNA expression in some cell lines . Effects were usually temporary and not always significant . No correlation was found between hTERT and TEP1 mRNA expression, whereas significant positive correlations were found between TEP1 and MDR1 mRNA, and between TEP1 and LRP mRNA . IFN-alpha modulates differently MDR gene transcripts in human melanoma cell lines . Positive correlation between TEP1 and LRP also seems to identify them as common targets of IFN-alpha effects.

J Clin Microbiol, 2003 Jun, 41(6), 2647 - 9
Utility of an in-house mycobacteriophage-based assay for rapid detection of rifampin resistance in Mycobacterium tuberculosis clinical isolates; Gali N et al.; A rapid in-house mycobacteriophage-based assay to identify multidrug resistance by detecting the rifampin susceptibility of Mycobacterium tuberculosis in a microtiter plate format was evaluated . The sensitivity, specificity, and overall accuracy of the assay were 100% . This test is rapid to perform and suitable for widespread implementation.

Eur J Pharm Sci, 2003 Jun, 19(2-3), 133 - 40
Arbekacin is actively secreted in the rat intestine via a different efflux system from P-glycoprotein; Saitoh H et al.; This study was undertaken to examine the secretory transport of arbekacin, an aminoglycoside antibiotic, in the rat small intestine and to compare it with those in Caco-2 and LLC-PK1 cells . In vitro permeation of arbekacin was examined using an Ussing chamber technique . Serosal-to-mucosal (secretory)/mucosal-to-serosal (absorptive) permeation ratios of 0.5 mM arbekacin were 2.8 in the jejunum and 7.0 in the ileum, respectively, indicating that arbekacin permeation was highly secretory-oriented . In the ileum, the ratios became smaller with increase in arbekacin concentration applied . When D-glucose was replaced with 3-o-methyl-D-glucose in the experimental medium, the directionality of the arbekacin permeation disappeared almost completely . Absorptive permeation of arbekacin was not significantly influenced by verapamil, cyclosporin A, or probenecid . On the other hand, when gentamicin sulfate was added to the serosal medium, secretory transport of arbekacin was significantly inhibited . The results of this study strongly suggest that a specialized efflux system other than P-glycoprotein and multidrug resistance proteins was involved in the secretory transport of arbekacin in the rat intestine . There was no directionality in arbekacin permeation across Caco-2 cell monolayers, suggesting the absence or very slight expression of the secretory system for arbekacin in this cell line.

Oncogene, 2003 Jun 5, 22(23), 3518 - 29
Triggering of p73-dependent apoptosis in osteosarcoma is under the control of E2Fs-pRb2/p130 complexes; La Sala D et al.; Mechanisms underlying multidrug resistance (MDR), one of the major causes of cancer treatment failure, are still poorly understood . We selected the osteosarcoma MDR HosDXR150 cell line by culturing Hos cells in the presence of increasing doxorubicin doses and showed that it is crossresistant to vinblastine . Similarly to the Hos parental cell line, HosDXR150 cells present mutated p53, functionally inactivated pRb/p105 and wild-type pRb2/p130 . Owing to p53 mutation, MDR-1 gene, codifying for P-glycoprotein, is upregulated . Evasion of apoptosis in HosDXR150 cells is only partially explained by drug extrusion because of P-glycoprotein overexpression . Analysis of gene expression level profiles showed that parental cell line undergoes apoptosis through an E2F1/p73-dependent pathway while its resistant variant evades it . This result can be explained by the presence of distinct E2Fs-pRb2/p130 complexes on the p73 promoter . Namely, in Hos p73 transcription is activated by E2F1-Rb2/p130-p300 complexes, while in HosDXR150 it is kept repressed by E2F4-Rb2/p130-HDAC1 complexes.

Bioorg Med Chem, 2003 Jul 3, 11(13), 2889 - 99
QSAR and 3D-QSAR of phenothiazine type multidrug resistance modulators in P388/ADR cells; Tsakovska IM; A series of 25 phenothiazines and structurally related compounds was investigated by QSAR (quantitative structure activity relationship) and 3D-QSAR methods with respect to their MDR (multidrug resistance) reversing activity in P388/ADR- murine leukemia cell line resistant to ADR (adriamycin) . The objective was to outline structural properties important for the investigated activity . Different measures for MDR reversal were used and compared . Two 3D-QSAR approaches were applied-CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis) . Both, neutral and protonated forms of the compounds were investigated . Molecular models with good predictive power were derived using a hydrophobic field alone and a combination of steric, hydrophobic, and hydrogen bond acceptor fields of the compounds . In the combined models highest contribution of the hydrogen bond acceptor field was noticed . Thus, the dominant role of the hydrophobic and hydrogen bond acceptor fields for MDR reversing activity of the investigated compounds was demonstrated . The structural regions responsible for the differences in anti-MDR activity were analyzed in respect to their hydrophobic, hydrogen bond acceptor and steric nature . The results may direct design of new phenothiazines and related compounds as MDR modulators.

Biochem Biophys Res Commun, 2003 Jun 20, 306(1), 116 - 20
Digoxin up-regulates multidrug resistance transporter (MDR1) mRNA and simultaneously down-regulates steroid xenobiotic receptor mRNA; Takara K et al.; A steroid xenobiotic receptor (SXR) is involved in the induction of MDR1/P-glycoprotein . MDR1 up-regulation by digoxin was previously demonstrated in human colon adenocarcinoma Caco-2 cells, but the participation of SXR remains unclear . Herein, the participation of SXR in MDR1 up-regulation was examined using reverse transcription-polymerase chain reaction in Caco-2 cells, and digoxin-tolerant cells (Caco/DX) as well as human colon carcinoma LS180 cells, which expressed SXR . MDR1 mRNA expression in Caco-2 or LS180 cells was increased by exposure to 1 microM digoxin for 24h, in a concentration-dependent manner, but SXR mRNA decreased concentration-dependently and was undetectable or significantly lower at 1 microM digoxin, indicating antithetical changes in MDR1 and SXR mRNA expression . Moreover, the MDR1 mRNA level was higher in Caco/DX cells than Caco-2 cells, whereas the SXR mRNA level was lower in Caco/DX cells . Consequently, digoxin was demonstrated to up-regulate MDR1 mRNA and simultaneously down-regulate SXR mRNA expression.

J Biol Chem, 2003 Aug 15, 278(33), 30764 - 71 Epub 2003 Jun 03.
ATP binding to the first nucleotide binding domain of multidrug resistance-associated protein plays a regulatory role at low nucleotide concentration, whereas ATP hydrolysis at the second plays a dominant role in ATP-dependent leukotriene C4 transport; Yang R et al.; Multidrug resistance-associated protein (MRP1) transports solutes in an ATP dependent manner by utilizing its two nonequivalent nucleotide binding domains (NBDs) to bind and hydrolyze ATP . The two NBDs possess different properties (Gao, M., Cui, H . R., Loe, D . W., Grant, C . E., Almquist, K . C., Cole, S . P., and Deeley, R . G . (2000) J . Biol . Chem . 275, 13098-13108; Hou, Y., Cui, L., Riordan, J . R., and Chang, X . (2000) J . Biol . Chem . 275, 20280-20287) and may play different roles during solute transport . We now report that NBD1 has moderately higher affinity for ATP than NBD2 . The consequence of this difference is that the overall Kd value for wild-type MRP1 is mainly determined by ATP binding at NBD1 . This conclusion is supported by the following: 1) mutation of the cysteine residue at 682 to alanine (C682A) in Walker A motif in NBD1 decreases the Kd value, indicating increased affinity for ATP; 2) mutation of the alanine residue at 1331 to cysteine (A1331C) in the Walker A motif of NBD2 does not have an effect on the Kd value; and 3) photolabeling of the protein with a cysteine residue in the Walker A motif of NBD1 is much more sensitive to N-ethylmaleimide modification than the protein with a cysteine residue in the Walker A motif of NBD2 . In contrast, the Km for ATP in support of LTC4 transport is mainly determined by ATP hydrolysis at NBD2 . This conclusion is supported by the following: 1) although mutation of A1331C does not have an effect on the Kd value, the Km values measured from LTC4 transport by proteins with this mutation in NBD2 are much higher than the proteins with wild-type NBD2, implying that the A1331C mutation affects ATP binding/hydrolysis at NBD2; and 2) ATP-dependent LTC4 transport by the protein with a cysteine residue in the Walker A motif of NBD2 is much more sensitive to N-ethylmaleimide modification than the protein with a cysteine residue in the Walker A motif of NBD1 . Our previous results indicated that ATP binding at NBD1 at low concentration enhanced ATP binding/hydrolysis at NBD2 . All of these results support the notion that ATP binding at NBD1 at low concentration plays a more important regulatory role than the binding at high ATP concentration and that ATP hydrolysis at NBD2 plays a dominant role in the ATP-dependent LTC4 transport.

Zhonghua Yan Ke Za Zhi, 2003 Feb, 39(2), 73 - 6
{Effect of bcl-2 antisense oligonucleotides on multidrug resistance of cultured uveal melanoma cells}; Guo LJ et al.; OBJECTIVE: To evaluate the chemotherapeutic sensitivity of uveal melanoma in vitro and reverse its drug resistance by the delivery of bcl-2 antisense oligodeoxynucleotide (AS-ODN) . METHODS: The drug sensitivity tests of primary cultured uveal melanoma cells toward 5-flurouracil (5-FU), thiophosphamide (TSPA), cisplatin (DDP), adriamycin (ADM), vinblastine (VLB), dacarbazine (DTIC) were performed with 3, -4, 5 dimethyliazol-2,5 diphenyl tetrazolium bromide (MTT) . AS-ODN bcl-2 were delivered with cationic lipid to down-regulate bcl-2 expression . Immunohistochemistry and Western-blot methods were used to detect bcl-2 expression . According to the principle of multi-drug mutual action, the influence of anti-apoptosis gene bcl-2 on tumor cell drug sensitivity was measured . RESULTS: Uveal melanoma was resistant to different chemotherapeutic drugs in different degrees . AS-ODN bcl-2 could down-regulate bcl-2 expression, was synergetic with all tested drugs and partially reversed the multi-drug resistance . CONCLUSIONS: Clinically, with ordinary dosages the above 6 drugs have little cytotoxicity to uveal melanoma cells, bcl-2 over-expression is associated with multi-drug resistance and AS-ODN bcl-2 can partially reverse the multi-drug resistance.

Eur J Clin Microbiol Infect Dis, 2003 Jun, 22(6), 342 - 8 Epub 2003 Jun 03.
PCR-based methodology for detecting multidrug-resistant strains of Mycobacterium tuberculosis Beijing family circulating in Russia; Mokrousov I et al.; The Beijing genotype of Mycobacterium tuberculosis has been identified in 40-50% of the clinical isolates studied in Russia during the last decade . This genotype has been reported to be associated with multiple drug resistance and possesses some significant pathogenic properties . Therefore, early identification of such strains is of extreme importance in the timely detection of drug resistance . The present study was performed on 354 strains isolated in Russia from 1996 to 2002 and previously characterised by IS 6110-restriction fragment length polymorphism (RFLP) typing and spoligotyping . These strains included 198 Beijing family strains and 156 strains of other genotypes (IS 6110-RFLP profiles) . A subsequent polymerase chain reaction (PCR) analysis with IS 6110-derived outwardly oriented primers (IS 6110-PCR) easily discriminated the Beijing strains from non-Beijing strains . The multiplex allele-specific (MAS)-PCR assays were further used to detect mutations in katG315 and rpoB531, associated with resistance to isoniazid and rifampin, respectively . The katG315 and rpoB531 mutations were found to be more prevalent among Beijing (96.8% and 77.3%) than among non-Beijing strains (85.7% and 28%) . Consequently, we propose a two-step methodology based on routine PCR and simple agarose gel electrophoresis in order to detect (i) a Beijing family strain using IS 6110-PCR, and, (ii) its possible resistance to the major anti-tuberculosis drugs using specific MAS-PCR assays.

Postgrad Med J, 2003 May, 79(931), 272 - 8
Paediatric tuberculosis; Hoskyns W; Children are important in the epidemiology of tuberculosis as a marker of recent disease transmission and a reservoir for the future . Once infected they have a higher risk of progressing to tuberculous disease . Chest radiography and tuberculin testing with or without tissue for culture are still the standard tools for confirming the diagnosis once this is considered . Well researched treatment protocols are available but multidrug resistant tuberculosis and coexistent HIV are a challenge . Ensuring compliance with treatment is a major concern . Controversy still surrounds the place of BCG . Advances in the molecular genetics of tuberculosis hold out the possibility of better vaccines.

Biochem Pharmacol, 2003 Jun 1, 65(11), 1843 - 52
Human MDR1 polymorphism: G2677T/A and C3435T have no effect on MDR1 transport activities; Morita N et al.; The two most frequently observed single nucleotide polymorphisms (SNPs) of the human multidrug resistance 1 (MDR1) gene are 2677G/T/A (893Ala/Ser/Thr) and 3435C/T (no amino acid substitution) . In this study, six forms of MDR1 cDNAs with the SNPs were expressed in LLC-PK1 cells and their transport activities were determined . Nearly identical amounts of the recombinant MDR1 proteins were expressed in the established cell lines using the Flp recombinase, which integrates a gene of interest at a specific genomic location . Four structurally diverse compounds: verapamil, digoxin, vinblastine and cyclosporin A, were examined for transcellular transport activities and intracellular accumulation . No significant differences were observed between cells expressing five polymorphic types of the MDR1 cDNAs (2677G/3435T, 2677A/3435C, 2677A/3435T, 2677T/3435C, 2677T/3435T) and cells expressing the wild-type (2677G/3435C) . These results suggested that the two frequently observed MDR1 SNPs had no effect on the transport activities of MDR1 proteins expressed in LLC-PK1 cells in vitro, and other genetic or environmental factors might control the expression of MDR1 and the in vivo activity of MDR1.

Clin Exp Immunol, 2003 Jun, 132(3), 443 - 9
The production of tumour necrosis factor-alpha is decreased in peripheral blood mononuclear cells from multidrug-resistant tuberculosis patients following stimulation with the 30-kDa antigen of Mycobacterium tuberculosis; Lee JS et al.; The clearance of intracellular bacteria requires the appropriate induction of proinflammatory cytokines and chemokines to recruit macrophages and T cells to the site of infection . In this study, we investigated the production of tumour necrosis factor (TNF)-alpha, interleukin (IL)-8 and interferon (IFN)-gamma by the peripheral blood mononuclear cells (PBMC) of patients with multidrug-resistant tuberculosis (MDR-TB) in response to in vitro stimulation with the 30-kDa antigen of Mycobacterium tuberculosis . The results were compared with those from cases of newly diagnosed TB (N-TB) and TB with treatment failure (TF-TB), and healthy tuberculin reactors (HTR) . The most significantly depressed TNF-alpha levels were found in MDR-TB patients . IFN-gamma production was depressed significantly in all groups of TB patients compared with the HTR group . TNF-alpha secretion in response to the 30-kDa antigen was unchanged by coculturing with recombinant human interferon (rhIFN)-gamma, and was increased dramatically following IL-10 neutralization with an anti-human IL-10 antibody . The IL-8 levels were depressed significantly in MDR-TB patients compared with N-TB patients, but were similar to the IL-8 levels in TF-TB patients . Furthermore, rhTNF-alpha directly increased IL-8 secretion, and neutralizing antibody to TNF-alpha inhibited IL-8 production by the PBMC of MDR-TB patients that were stimulated with the 30-kDa antigen . Taken together, these data suggest that the PBMC of MDR-TB patients typically show TNF-alpha depression in response to the 30-kDa antigen, and this effect is modulated by IL-10 . In addition, we highlight the role of TNF-alpha in IL-8 secretion in MDR-TB patients.

Gene Ther, 2003 Jun, 10(12), 1061 - 5
Impact of splice-site mutations of the human MDR1 cDNA on its stability and expression following retroviral gene transfer; Cmejlova J et al.; The multidrug resistance 1 (MDR1) gene transfer to hematopoietic cells for protection against cytotoxic drugs has received considerable attention in gene therapy . However, ectopic expression of MDR1 from retroviral vectors has been hampered by its genetic instability resulting from cryptic splice sites within the cDNA . We have evaluated the efficiency of retroviral MDR1 vectors with introduced mutations of the MDR1 cryptic splice donor (cSD) located at nucleotide +339 and of the cryptic splice acceptor (cSA) at nucleotide +2319 of the cDNA . Sequence alterations of the cSD reduced the expression of MDR1 P-glycoprotein (P-gp), even when generated as silent mutations . A silent mutation of the cSA reduced the splicing activity shifting the splice acceptor site one base downstream; however, it significantly improved the expression of P-gp . The incidence of wild-type MDR1 pregenome splicing was markedly reduced when vectors were produced in human 293 packaging cells as opposed to murine PG13 and GP+envAm12 . We conclude that complete splice correction of MDR1 in retroviral vectors may only be achieved with extensive alterations of the cDNA or neighboring vector sequences and that the splicing is significantly influenced by the choice of the packaging cells.

Anal Cell Pathol, 2003, 25(3), 115 - 22
Cytogenetic evolution of human ovarian cell lines associated with chemoresistance and loss of tumorigenicity; Struski S et al.; In order to identify genomic changes associated with a resistant phenotype acquisition, we used comparative genomic hybridization (CGH) to compare a human ovarian cell line, Igrov1, and four derived subcell lines, resistant to vincristine and presenting a reversion of malignant properties . Multicolor FISH (Multiplex-FISH and Spectral Karyotype) and conventional FISH are also used to elucidate the karyotype of parental cell line . The drug-resistant subcell lines displayed many chromosomal abnormalities suggesting the implication of different pathways leading to a multidrug resistance phenotype . However, these cell lines shared two common rearrangements: an unbalanced translocation der(8)t(8;13)(p22;q?) and a deletion of the 11p . These chromosomal imbalances could reflected the acquisition of the chemoresistance (der(8)) or the loss of tumorigenicity properties (del(11p)).

Zhongguo Zhong Yao Za Zhi, 2002 Jan, 27(1), 46 - 50
{Reversal of anti-apoptotic action by tetrandrine in human breast carcinoma multidrug-resistant MCF-7 cells}; Wang JH et al.; OBJECTIVE: To study whether the anti-apoptotic action is reversed by tetrandrine in a combination with vincristine in human breast carcinoma MCF-7 multidrug-resistant cells . METHOD: Chromatin condensation was observed by co-staining of fluorescent dyes Hoechst 33342 and propidium iodide; and G1 sub-peak was detected by flow cytometry . Apoptotic cells were detected with TUNEL method . Cellular free ca2+ was determined with Fluo-3 staining method . RESULT: Two types of chromatin condensation were observed after the sensitive and drug-resistant MCF-7 cells were treated with an antitumor drug vincristine 5 mumol.L-1 for 24 h . The number of cell with chromatin condensation was obviously reduced in the drug-resistant cells treated with the same concentration of vincristine, as compared with the sensitive MCF-7 cells . The number of the apoptotic cells was increased by a combination of non-cytotoxic tetrandrine 20 mumol.L-1 and vincristine in both the sensitive and drug-resistant cells, which was confirmed with fluorescent indication and TUNEL method . The increment of introcellular free Ca2+ level in the cells treated with tetrandrine in a combination of vincristine was detected with Fluo-3 staining method . CONCLUSION: The anti-apoptotic action of human breast carcinoma MCF-7 cells can be effectively reversed by tetrandrine.

Onkologie, 2003 Apr, 26(2), 175 - 81
Multidrug resistance in prostate cancer; van Brussel JP et al.; Advanced hormone-refractory prostate cancer remains a therapeutic challenge, because all available pharmaceutical concepts have been ineffective in improving cancer-specific survival . Failure of chemotherapy may be caused by multidrug resistance (MDR) mechanisms protecting cancer cells against cytotoxic drugs, and the question arises whether prostate cancer is also using MDR principles resulting in resistance against chemotherapeutic agents . In consequence, an array of diverse pathways known to lead to MDR such as MDR1, MRPs, glutathione, and apoptosis have been examined and partially established at varying degrees in hormone-refractory prostate cancer . Thus, evidence keeps accumulating for the involvement of some MDR mechanisms in the chemoresistance of prostate cancer in vitro and in vivo . For some of them, e.g . MRP1, functional expression appears to be probable . This lends credit to the idea that reversal, circumvention, or overcoming of MDR pathways in advanced prostate cancer may be feasible and will lead to new avenues with improved treatment efficacy in otherwise intractable disease .

Zhonghua Er Bi Yan Hou Ke Za Zhi, 2000 Dec, 35(6), 454 - 6
{Study on the relationship between the abnormal expression of c-myc and multidrug resistance in KB cell lines}; He Y et al.; OBJECTIVE: To investigate the relationship between expression of c-myc and the regulation of mdr1, and find out the best way for gene therapy of the proliferative diseases with c-myc overexpression and reverse the multidrug resistance phenotype . METHODS: The changes of c-Myc protein and P-gp in the KB cell lines were detected by using immunohistochemistry and flow cytometry methods before and after treatment with anti-mdr1- ribozyme . RESULTS: All KB cell lines could express c-Myc protein . The expression of c-myc in multidrug resistant cell KBv displayed higher than that in sensitive cell KB . However, after reversing the multidrug resistance phenotype by anti-mdr1-ribozyme, the level of c-Myc protein expression was lower in KBv/5mR3 and KB/5mR3 . The level of P-gp expression was lower in KBv/5mR3 than that in KBv but no difference in KB/5mR3 and KB . CONCLUSION: There are a close relationship between Myc protein and MDR phenotype . The c-myc involves in regulating expression of mdr1 . Myc protein can be used as a new assistant implement to monitor the formation of MDR.

J Control Release, 2003 Jun 5, 90(1), 37 - 48
Relationships between the hydrophilic-lipophilic balance values of pharmaceutical excipients and their multidrug resistance modulating effect in Caco-2 cells and rat intestines; Lo YL; The effects of a series of pharmaceutical excipients, including Span 80, Brij 30, Tween 20, Tween 80, Myrj 52, and sodium lauryl sulfate (with increasing hydrophilic-lipophilic balance (HLB) values) on the intracellular accumulation, transport kinetics, and intestinal absorption of epirubicin were investigated in both the human colon adenocarcinoma (Caco-2) cell line and the everted gut sacs of rat jejunum and ileum . The possible use of these excipients as multidrug resistance (MDR) reversing agents also was examined . Epirubicin uptake experiments using a flow cytometer showed that these selected excipients markedly enhanced the intracellular accumulation of epirubicin in Caco-2 cells in a dose-dependent manner . The optimal effect on the epirubicin uptake was characteristic of excipients with intermediate HLB values ranging from 10 to 17 . Moreover, the optimal net efficacy was observed for excipients with polyoxyethylene chains and intermediate chain length of fatty acid and fatty alcohol (monolaurate for Tween 20, monooleate for Tween 80, monostearate for Myrj 52, and lauryl alcohol for Brij 30) . These excipients significantly increased apical to basolateral absorption and substantially reduced basolateral to apical efflux of epirubicin across Caco-2 monolayers . Furthermore, the addition of Tween 20, Tween 80, Myrj 52, and Brij 30 markedly enhanced mucosal to serosal absorption of epirubicin in the rat jejunum and ileum . This study suggests that inhibition of intestinal P-glycoprotein (P-gp), multidrug resistance associated protein family (MRPs), or other transporter proteins by pharmaceutical excipients may improve oral absorption of drugs in MDR spectrum . The optimal HLB values of surfactant systems with suitable hydrocarbon chains and polar groups are an important factor in designing promising epirubicin formulations for reversing MDR . In conclusion, therapeutic efficacy of epirubicin may be enhanced by the use of such low toxicity excipients as absorption enhancers and MDR modulators in formulations . This provides a potential strategy for improving bioavailability in the optimization of formulations for drugs performing intestinal absorption and secretion.

Int J Cancer, 2003 Jul 20, 105(6), 784 - 9
Novel plant triterpenoid drug amooranin overcomes multidrug resistance in human leukemia and colon carcinoma cell lines; Ramachandran C et al.; Amooranin (AMR), a plant terpenoid, isolated from Amoora rohituka, was investigated for its ability to overcome multidrug resistance in human leukemia and colon carcinoma cell lines . AMR IC(50) values of multidrug-resistant leukemia (CEM/VLB) and colon carcinoma (SW620/Ad-300) cell lines were higher (1.9- and 6-fold) than parental sensitive cell lines (CEM and SW620) . AMR induced G(2)+M phase-arrest during cell cycle traverse in leukemia and colon carcinoma cell lines and the percentage of cells in G(2)+M phase increased in a dose-dependent manner . Coincubation of tumor cells with both DOX and AMR reversed DOX resistance in 104-fold DOX-resistant CEM/VLB and 111-fold DOX-resistant SW620/Ad-300 cell lines with a dose modification factor of 50.9 and 99.6, respectively . Flow cytometric assay showed that AMR causes enhanced cellular DOX accumulation in a dose-dependent manner . AMR inhibits photolabeling of P-glycoprotein (P-gp) with {(3)H}-azidopine and the blocking effect enhanced with increasing concentrations of AMR . Our results show that AMR competitively inhibits P-gp-mediated DOX efflux, suggestive of a mechanism underlying the enhanced DOX accumulation and reversal of multidrug resistance by AMR .

Toxicol Lett, 2003 Apr 30, 142(1-2), 61 - 70
The multidrug resistance-associated protein 1 transports methoxychlor and protects the seminiferous epithelium from injury; Tribull TE et al.; We examined the ability of the multidrug resistance-associated protein 1 (MRP1/ABCC1) to transport pesticides, as this transporter mediates the cellular efflux of a variety of xenobiotics, typically as glucuronide, sulfate, or glutathione conjugates . NIH3T3 cells stably expressing MRP1 were 3.37-fold more resistant to the toxicity of fenitrothion, 3.12-fold more resistant to chlorpropham, and 2.5-fold more resistant to methoxychlor, a pesticide with estrogenic and anti-androgenic metabolites . The cells expressing MRP1 also eliminated methoxychlor two times more rapidly than their mock-transfected counterparts . We then examined whether mrp1 expression could alter the toxicity of methoxychlor in vivo using male FVB/mrp1 knockout mice (FVB/mrp1-/-) . Both control and knockout mice were fed 25 mg/kg methoxychlor in honey for 39 days, and its effects on testicular morphology were examined . Methoxychlor treatment did not significantly affect testicular morphology in the FVB mice, but markedly reduced the number of developing spermatocytes in the FVB/mrp1-/- mice . These results suggest that MRPI may play a role in protecting the seminiferous tubules from methoxychlor-induced damage.

Zhonghua Er Bi Yan Hou Ke Za Zhi, 1999 Jun, 34(3), 173 - 6
{Expression and clinical implication of multidrug resistance gene and multidrug resistant-associated protein gene in patients with hypopharyngeal squamous cell carcinoma}; Cai X et al.; OBJECTIVE: To study the relationship between the expression of MDR-1 mRNA/P-glycoprotein (P-gp) . MRP mRNA and the clinical pathological characteristics of the patients with hypopharyngeal squamous cell carcinoma . METHODS: Reverse transcription polymerase chain reaction(RT-PCR) assay was used to detect the expression of MDR-1 mRNA and MRP mRNA in 25 specimens of the untreated advanced hypopharyngeal carcinoma . Meantime, immunohistochemistry SABC technique was used to test P-gp in 60 tumor specimens . RESULTS: The positive expression rates of MDR-1mRNA and P-gp in hypopharyngeal carcinoma were 48 percent (12/25) and 38.3 percent(23/60), respectively . There was a correlation between expression of MDR-1 mRNA and its product P-gp in 25 patients using RT-PCR assay(P < 0.05) . The positive expression rate of MRP mRNA was 52 percent (13/25), which was significantly related to metastases of cervical lymph node (P < 0.01) . CONCLUSION: Forty-eight percent of the untreated patients with hypopharyngeal carcinoma presented positive expression of MDR-1 mRNA, which was correlated with its gene product P-gp . The expression of MRP mRNA was associated with increased tumor dissemination and metastases.

Leukemia, 2003 Jun, 17(6), 1175 - 82
Bone marrow stromal-derived soluble factors and direct cell contact contribute to de novo drug resistance of myeloma cells by distinct mechanisms; Nefedova Y et al.; The tumor microenvironment plays a critical role in determining the fate of tumor cells . We have previously reported that adhesion of human myeloma and leukemia cell lines to the extracellular matrix protein, fibronectin, confers a multidrug-resistant phenotype . Mechanisms associated with this cell adhesion-mediated drug resistance are drug-type specific . In the present study, we examined the influence of bone marrow stromal cells (BMSCs) on myeloma cell response to the topoisomerase II inhibitor, mitoxantrone . Apoptosis was inhibited by more than 50% when cells were adhered to BMSCs as compared to myeloma cells maintained in suspension . To investigate the mechanisms contributing to the resistance of myeloma cells in contact with BMSCs, we examined the protective effects of BMSCs under four separate conditions: (1) direct cell contact; (2) BMSCs conditioned medium; (3) medium conditioned by coculturing myeloma cells in direct contact with BMSCs; and (4) medium conditioned by coculturing myeloma cells and BMSCs without direct physical contact . Conditioned medium from BMSCs alone was not sufficient to protect myeloma cells from drug-induced apoptosis; however, soluble factors produced during the myeloma-BMSCs interaction decreased the sensitivity of myeloma cells to mitoxantrone, suggesting a dynamic interaction between myeloma cells and BMSCs . We also found that myeloma cells in direct contact with BMSCs underwent growth arrest, whereas soluble factors produced by myeloma cells-BMSCs coincubation stimulated the proliferation of myeloma cells . These data show that both cell-cell adhesion of BMSCs with myeloma cells and soluble factors induced by this cell-cell interaction are involved in the protection of myeloma cells from mitoxantrone-induced apoptosis; however, the mechanisms contributing to the drug resistance are different.

J Biol Chem, 2003 Aug 8, 278(32), 29509 - 14 Epub 2003 May 22.
MRP8, ATP-binding cassette C11 (ABCC11), is a cyclic nucleotide efflux pump and a resistance factor for fluoropyrimidines 2',3'-dideoxycytidine and 9'-(2'-phosphonylmethoxyethyl)adenine; Guo Y et al.; MRP8 (ABCC11) is a recently identified cDNA that has been assigned to the multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporters, but its functional characteristics have not been determined . Here we examine the functional properties of the protein using transfected LLC-PK1 cells . It is shown that ectopic expression of MRP8 reduces basal intracellular levels of cAMP and cGMP and enhances cellular extrusion of cyclic nucleotides in the presence or absence of stimulation with forskolin or SIN-1A . Analysis of the sensitivity of MRP8-overexpressing cells revealed that they are resistant to a range of clinically relevant nucleotide analogs, including the anticancer fluoropyrimidines 5'-fluorouracil (approximately 3-fold), 5'-fluoro-2'-deoxyuridine (approximately 5-fold), and 5'-fluoro-5'-deoxyuridine (approximately 3-fold), the anti-human immunodeficiency virus agent 2',3'-dideoxycytidine (approximately 6-fold) and the anti-hepatitis B agent 9'-(2'-phosphonylmethoxynyl)adenine (PMEA) (approximately 5-fold) . By contrast, increased resistance was not observed for several natural product chemotherapeutic agents . In accord with the notion that MRP8 functions as a drug efflux pump for nucleotide analogs, MRP8-transfected cells exhibited reduced accumulation and increased efflux of radiolabeled PMEA . In addition, it is shown by the use of in vitro transport assays that MRP8 is able to confer resistance to fluoropyrimidines by mediating the MgATP-dependent transport of 5'-fluoro-2'-deoxyuridine monophosphate, the cytotoxic intracellular metabolite of this class of agents, but not of 5'-fluorouracil or 5'-fluoro-2'-deoxyuridine . We conclude that MRP8 is an amphipathic anion transporter that is able to efflux cAMP and cGMP and to function as a resistance factor for commonly employed purine and pyrimidine nucleotide analogs.

J Pharm Sci, 2003 Jun, 92(6), 1250 - 61
The role of surfactants in the reversal of active transport mediated by multidrug resistance proteins; Bogman K et al.; A variety of seven nonionic, one amphoteric and, one anionic surfactant that are applied or investigated as surfactants in drug formulation, were analyzed for their capacity to modulate carrier-mediated transport by efflux pumps . Two cell lines, murine monocytic leukemia cells overexpressing P-glycoprotein (P-gp) and Madin-Darby canine kidney cells stably overexpresssing human multidrug resistance-associated protein 2 (MRP2), were used as test systems . The modulation of P-gp and of MRP2 function was studied by the reversal of rhodamine 123 and of methylfluorescein-glutathione conjugate transport, respectively . Mechanisms that were not transporter related and could lead to misinterpretations were identified, such as probe quenching, probe encapsulation by micelles, and membrane damage . P-gp-mediated rhodamine 123 transport was inhibited by five nonionic surfactants in a concentration-dependent manner and in the order TPGS > Pluronic PE8100 > Cremophor EL > Pluronic PE6100 approximately Tween 80 . In contrast, none of the surfactants showed a significant inhibition of MRP2-mediated efflux in Madin-Darby canine kidney/MRP2 cells . In conclusion, the results indicate that surfactants demonstrate a transporter-specific interaction, rather than unspecific membrane permeabilization . The present analysis offers insight in the possible mechanisms of surfactant interactions with biological membranes and could help to identify specific drug formulations .

Oncogene, 2003 May 22, 22(21), 3205 - 12
HER2/PI-3K/Akt activation leads to a multidrug resistance in human breast adenocarcinoma cells; Knuefermann C et al.; Growth factor receptor-mediated signal transduction has been implicated in conferring resistance to conventional chemotherapy on cancer cells . In this study, we delineated a pathway that involves HER2/PI-3K/Akt in mediating multidrug resistance in human breast cancer cells . We found that the cell lines that express both HER2 and HER3 appear to have a higher phosphorylation level of Akt (activated Akt) . Transfection of HER2 in MCF7 breast cancer cells that express HER3 caused a phosphoinoside-3 kinase (PI-3K)-dependent activation of Akt, and was associated with an increased resistance of the cells to multiple chemotherapeutic agents (paclitaxel, doxorubicin, 5-fluorouracil, etoposide, and camptothecin) . Selective inhibition of PI-3K or Akt activity with their respective dominant-negative expression vectors sensitized the cells to the induction of apoptosis by the chemotherapeutic agents . We further demonstrated that MCF7 cells expressing a constitutively active Akt, in which the phospholipid-interactive PH domain of Akt was replaced by a farnesylation sequence for constitutive membrane anchorage (DeltaPH-Akt1-farn), showed a similar increased resistance to the chemotherapeutic agents . Our results suggest that activation of Akt1 by HER2/PI-3K plays an important role in conferring a broad-spectrum chemoresistance on breast cancer cells and that Akt may therefore be a novel molecular target for therapies that would improve the outcome of patients with breast cancer.

Ann Ist Super Sanita, 2002, 38(4), 387 - 92
Modulation of the multidrug resistance (MDR) phenotype in CEM MDR cells simultaneously exposed to anti HIV-1 protease inhibitors (PI's) and cytotoxic drugs; Dupuis ML et al.; Vinblastine, vincristine and doxorubicyn are currently used in chemotherapeutic treatments of several malignancies including HIV-1 associated tumours Kaposi's sarcoma (KS) and non-Hodgkin lymphoma (NHL) . Hence, AIDS patients also affected by KS and NHL may be simultaneously subjected to highly active antiretroviral therapy (HAART) and cytotoxic drugs to combat HIV-1 infection and cancer aggressiveness . In order to assess if the combination of these therapies may affect cell growth and survival of P-glycoprotein expressing MDR variants of the human CD4+ T-lymphoblastoid CEM cell line, the protease inhibitors (PI's) ritonavir, saquinavir and indinavir were tested in an in vitro assay for their ability to potentiate the vinblastine, vincristine and doxorubicyn cytotoxicity . The results we obtained demonstrated that at the concentration of 10 micrograms/ml, ritonavir and in a lesser extent saquinavir act as MDR reversing agents . By contrast, the PI indinavir at least in the CEM cell system, does not affect the patterns of drug resistance . The level of chemosensitization exerted by ritonavir and saquinavir suggests that these PI's may render P-glycoprotein expressing MDR cells de novo susceptible to the antineoplastic drugs vinblastine, vincristine and doxorubicyn.

Nephron Physiol, 2003, 93(4), p87 - 93
Nephrotoxicity and the proximal tubule . Insights from cadmium; Thevenod F; Cadmium (Cd(2+)) is a non-essential heavy metal, which is taken up from the environment into the body through pulmonary and enteral pathways . The S1 segment of the kidney proximal tubule (PT) is a major target of chronic Cd(2+) toxicity . Renal dysfunction develops in up to 7% of the general population and in its most severe form displays major features of Fanconi syndrome, such as a defective protein, amino acid, glucose, bicarbonate and phosphate reabsorption . The major pathway for Cd(2+) uptake by PT cells (PTCs) in vivo is apical endocytosis of Cd(2+) complexed to the high-affinity metal-binding protein metallothionein (MT), which may be receptor-mediated . MT is subsequently degraded in endo-lysosomes, and Cd(2+) is liberated for translocation into the cytosolic compartment, possibly using transporters for Fe(2+), Zn(2+) or Cu(2+), such as the divalent metal transporter DMT1 . Free Cd(2+) ions in the extracellular space are translocated across apical and/or basolateral PTC membranes into the cytosol via transporters, whose identity remains unknown . Cytosolic Cd(2+) generates reactive oxygen species (ROS), which deplete endogenous radical scavengers . ROS also damage a variety of transport proteins, including the Na(+)/K(+)-ATPase, which are subsequently degraded by the proteasome and endo-lysosomal proteases . Cd(2+) causes mitochondrial swelling and release of cytochrome C . If these ROS-mediated stress events are not balanced by repair processes, affected cells undergo apoptosis . But Cd(2+) also induces the upregulation of cytoprotective stress and metal-scavenging proteins, such as MT . In addition, Cd(2+) upregulates the detoxifying pump multidrug resistance P-glycoprotein, which appears to protect PTCs against Cd(2+)-induced apoptosis . Thus, Cd(2+) interferes with various cellular events ranging from mechanisms of induction of programmed cell death to activation of cell survival genes . A better understanding of the cellular mechanisms involved in Cd(2+) nephrotoxicity should provide insights into other heavy metal (e.g . Pb(2+), Hg(2+)) nephropathies and various forms of acquired Fanconi syndrome .

Oncology, 2003, 64(4), 399 - 406
Sensitivity of non-small-cell lung cancer cell lines established from patients treated with prolonged infusions of Paclitaxel; Fujishita T et al.; OBJECTIVE: Regimens with prolonged infusions of taxanes have been developed for patients with cancer to overcome drug resistance . Our objective of the present study was to examine the impact of prolonged exposure on the cytotoxicity of taxanes against non-small-cell lung carcinoma (NSCLC) cell lines and the clinical response and outcome of the patients . METHODS: Five cell lines (NCI-H2882, -H2887, -H2973, -H3122, -H3255) were derived from previously untreated patients with NSCLC who participated in clinical trials of continuous 96-hour infusions of paclitaxel followed by bolus cisplatin . Two additional cell lines (NCI-H838, -H1299) with previously published data were used as controls . Drug sensitivities were assessed by the MTS (Promega) assay . Multidrug resistance (MDR) phenotypes were assessed by quantitative real-time PCR and HER-2/NEU by both immunohistochemistry and ELISA . RESULTS: The median of mean IC(50) values of docetaxel at the exposure durations of 3, 24, 72 and 120 h were 0.52, 0.06, 0.03 and 0.06 microM, respectively . The median of mean IC(50) values of paclitaxel at the exposure duration of 3, 24, 72 and 120 h were 0.48, 0.13, 0.03 and 0.02 microM, respectively . In all cell lines studied, there was a less than 4-fold difference in the IC(50) values between docetaxel and paclitaxel at 3-, 72-, and 120-hour exposure times . The single cell line with moderate MDR1 expression (NCI-H2887) was the only cell line established from a patient with progressive disease when treated with paclitaxel . CONCLUSIONS: This study demonstrates prolonged exposure to both docetaxel and paclitaxel inhibits the growth of NSCLC cell lines in similar fashion .

Tuberculosis (Edinb), 2003, 83(1-3), 135 - 42
Shifting the focus of tuberculosis research in India; Narayanan PR et al.; India has a long and distinguished tradition of research in the field of tuberculosis (TB) . Pioneering studies from India demonstrated the efficacy and safety of domiciliary treatment, the necessity of direct observation of treatment, the feasibility of case detection through sputum smear microscopy in primary health care institutions, and the effectiveness of intermittent short-course chemotherapy . These findings laid the foundation of directly observed treatment, short course (DOTS), which has been adopted by nearly 150 countries worldwide . Today, India has the second-largest and the fastest-growing DOTS programme in the world . A strong component of programme evaluation and operational research is needed to sustain and expand DOTS in the context of a suboptimal primary health care system, a large and unregulated private health care system, and the dual threats of HIV and multidrug-resistant TB (MDR-TB) . Therefore, the focus of TB research in India has shifted to the following operational research areas: evaluating models to involve the private health sector; assessing the role of incentives in increasing treatment compliance; examining gender differentials in the access to TB services; assessing risk factors for delay in diagnosis; evaluating diagnosis, treatment and prevention of TB among HIV-infected persons; monitoring MDR-TB; estimating cost-effectiveness of the DOTS programme; monitoring the quality of smear microscopy services; and measuring the current burden of TB . Research for developing newer diagnostic tools, drugs and vaccines remains a long-term priority . Greater networking is needed among national researchers, programme managers and policy-makers to translate the findings of research into policies and programmes to make TB control in India more effective and efficient.

Tuberculosis (Edinb), 2003, 83(1-3), 59 - 65
From multidrug-resistant tuberculosis to DOTS expansion and beyond: making the most of a paradigm shift; Kim JY et al.; This review examines the paradigm shift around multidrug-resistant tuberculosis (MDR-TB) . This shift has centered largely on the activities of institutions participating in the World Health Organization's Working Group on DOTS-Plus for MDR-TB . We review the important milestones since 1995, namely, the emergence of new evidence, the construction of new mechanisms, and the building of consensus to support new policy guidelines . This paper offers a case study of the construction of a model of good global governance that--if the opportunity is taken--can improve access to effective TB care through improving and helping to expand the WHO-recommended DOTS strategy.

Tuberculosis (Edinb), 2003, 83(1-3), 52 - 8
DOTS-Plus for multidrug-resistant tuberculosis in the Philippines: global assistance urgently needed; Tupasi TE et al.; SETTING: The Philippines, a high burden country for tuberculosis (TB) . STUDY DESIGN: Health Operational Study . OBJECTIVE: To describe preliminary data from the Makati Medical Center (MMC)-DOTS Plus pilot project . METHODS: Patients were consecutively enrolled after confirmation of MDR-TB status . Individualized treatment regimens were based on drug susceptibility testing and history of previous intake for the other drugs that were not tested . Treatment outcome in those who had completed at least 18 months of therapy and interim outcome for those who received more than 12 months but less than 18 months were analyzed . RESULTS: One hundred forty-nine patients with MDR-TB were enrolled from April 1999 to 30 May 2002 at the MMC DOTS Clinic . Referrals were from private institutions and practicing physicians in 73.2% of cases . Approximately 30% of isolates tested were resistant to all five first-line drugs, 39.4% to four, 16.8% to three, 12.1% to two . Fluoroquinolone resistance was noted in 40.9% of all the isolates, including 54.5% of those resistant to five drugs and 34.6% of those resistant to four drugs . The outcome of 23 patients who completed therapy and 62 who have received more than 12 months therapy showed cure and likely cure in 73.4% of cases and failure in 3.8% and likely failure in 6.3% . Death occurred in 3.8% and default was observed in 11.4% . CONCLUSION: The MMC DOTS-Plus pilot project is a public-private collaboration in TB Control . Response to therapy was encouraging . Complete subsidy of medicines and laboratory and clinic services and DOT were essential in the successful implementation of the program . DOTS-Plus and DOTS should go hand in hand in TB control if MDR-TB is highly prevalent.

Tuberculosis (Edinb), 2003, 83(1-3), 44 - 51
The global situation of MDR-TB; Espinal MA; Drug-resistant tuberculosis has been reported since the early days of the introduction of chemotherapy . However, most of the evidence was limited to developed countries . In 1992, the Third World Congress on Tuberculosis concluded that there was little recent information on the global magnitude of multidrug-resistant tuberculosis (MDR-TB), defined as resistance to at least isoniazid and rifampicin . Through the WHO/IUATLD Global Project on Drug-Resistance Surveillance launched in 1994, a large number of reliable and accurate data have allowed us to understand the magnitude of the problem of MDR-TB . The data available suggest that globally MDR-TB is not a problem (median = 1% in 64 countries/geographical sites surveyed) of the same magnitude as that of drug-susceptible tuberculosis . However, MDR-TB is at critical levels in specific regions of the world . Hot spots for MDR-TB include Estonia, Latvia, the Oblasts of Ivanovo and Tomsk in Russia, and the provinces of Henan and Zhejiang Provinces in China . Trends confirm that MDR-TB is limited to local epidemics but the evidence is not yet irrefutable, as many countries have only provided short-term data . Two-thirds of the world's countries and, more importantly, half of the 22 tuberculosis high-burden countries, have not yet provided data . Mathematical modelling suggests that 3.2% (or 273,000) of the world's estimated new tuberculosis cases (95% confidence intervals: 185,000 and 414,000) were MDR-TB in 2000 . Adoption of DOTS to prevent the generation of resistant strains and careful introduction of second-line drugs to treat patients with MDR are the top priorities for proper control/containment of MDR-TB.

Bioorg Med Chem, 2003 Jun 12, 11(12), 2575 - 89
Synthesis and antiproliferative activity of basic thioanalogues of merbarone; Ranise A et al.; Three series of 5-substituted 1,3-diphenyl-6-(omega-dialkyl- and omega-cyclo-aminoalkyl)thio-2-thiobarbiturates (11-13) were synthesized as polysubstituted thioanalogues of merbarone, a topoisomerase II inhibitor acting on the catalytic site . To better understand pharmacophore requirements, a forth series of conformationally constrained analogues 14 was also prepared . Derivatives 11b,e, 14b,e,h,i,j were active in the low micromolar concentration range (IC(50): 3.3-4.3 microM), whereas compounds 11a,c,d,f,h,j and 13a,b,d,g,j and 14a,d,f showed IC(50) values between 10 and 15.5 microM . In contrast, compounds 12a-c,g-j, 13e,f,h and 14k were inactive . Cytotoxicity data provided from N.C.I . on selected compounds provided evidence that 11b,d, 13d,g and 14b,d,f,h,i,j were endowed with potent antiproliferative activity against leukemia and prostate cell lines (GI(50) up to 0.01 microM) . In general, bicyclic derivatives 14 were up to 10-fold more potent than monocyclic counterparts against solid tumor-derived cell lines . SAR studies indicated that, in general, a certain tolerability in length of the alkyl side chains and in shape of distal amines is allowed in the four series, but in the monocyclic derivatives (11-13) antiproliferative activity was strongly affected by the nature of the 5-substituents (COOC(2)H(5)>COCH(3)>>C(6)H(5)) . Compounds 11b and 14b were also evaluated against KB cell subclones expressing altered levels of topoisomerases or the multidrug resistance phenotype (MDR) . In both cases the above compounds showed a decrease in potency . In enzyme assays, 11b and 14b turned out to be inhibitors of topoisonerase II as merbaron.

Int J Tuberc Lung Dis, 2003 May, 7(5), 493 - 7
Para-aminosalicylic acid (PAS) desensitization review in a case of multidrug-resistant pulmonary tuberculosis; Wilson JW et al.; SETTING: Tertiary care hospital in the Upper Midwest, United States . OBJECTIVE: Rapid desensitization to para-aminosalicylic acid (PAS) in a patient with previous hypersensitivity reaction and a review of published PAS desensitization protocols . DESIGN: Composition and implementation of a short-course PAS desensitization protocol for a 34-year-old woman with multidrug-resistant (MDR) pulmonary tuberculosis, incorporating published experiences of PAS desensitization over the past 50 years . RESULTS: We composed a protocol and successfully desensitized our patient to PAS (Paser granules) . By starting with a low dose (50 mg), then doubling the PAS dose on each successive day, our patient was able to tolerate full dose in 1 week . No steroids were required and no adverse reactions were encountered . Previous published PAS desensitization protocols used starting doses of 10-500 mg, desensitization time ranges from 7 to 54 days and commonly used steroids or corticotropin . CONCLUSION: Rapid desensitization to PAS can be successfully conducted within 1 week without the use of steroids or corticotropin . Given the limited number of drugs available for many patients with MDR-TB, desensitization to PAS is a valid alternative to drug discontinuation for patients with hypersensitivity reactions.

Int J Tuberc Lung Dis, 2003 May, 7(5), 472 - 7
Resistance to anti-tuberculosis drugs among smear-positive cases in Thai prisons 2 years after the implementation of the DOTS strategy; Pleumpanupat W et al.; SETTING: Three prisons in Bangkok and vicinity, Thailand . OBJECTIVE: To examine the prevalence of drug-resistant tuberculosis among smear-positive cases in Thai prisons 2 years after the implementation of the DOTS strategy, and to identify factors associated with resistance to anti-tuberculosis drugs . METHODS: In a cross-sectional study, 154 consecutive tuberculosis patients with at least one positive sputum smear and at least one positive sputum culture registered between 1 May and 31 October 2000 were enrolled . Drug susceptibility testing was performed by the Ministry of Public Health Tuberculosis Division . Patient characteristics were obtained by face-to-face interview . RESULTS: Resistance to at least one drug was found in 50.6% of the subjects, including multidrug-resistant tuberculosis (MDR-TB) in 19.5% . The proportion of resistance to any anti-tuberculosis drug in prisons A, B and C was respectively 52.7%, 37.8% and 61.5% . The only factor significantly associated with resistance to at least one drug (P = 0.011) and MDR-TB (P < 0.001) was a history of previous tuberculosis treatment . CONCLUSION: After 2 years of the DOTS strategy, resistance to anti-tuberculosis drugs, an indicator of the quality of tuberculosis treatment, was found to be high . The DOTS strategy currently used in Thai prisons should be reviewed, in order to reduce and prevent drug-resistant tuberculosis.

Int J Tuberc Lung Dis, 2003 May, 7(5), 451 - 7
Differential decline in tuberculosis incidence among US- and non-US-born persons in New York City; Li JH et al.; SETTING: A large urban tuberculosis control program . OBJECTIVES: To examine changes in tuberculosis incidence and characteristics of cases in New York City (NYC), and assess the epidemiology of tuberculosis among non-US-born persons . DESIGN: Tuberculosis surveillance data (1995-1999) for NYC were analyzed . RESULTS: Tuberculosis incidence decreased by 56.6% in US-born and 19.6% in non-US-born persons (age-adjusted) over the study period . The decline in tuberculosis incidence among US-born persons was more substantial in the first half of the study period (23-24%) than in the second half (13-15%) . The greatest decline in incidence was among US-born Hispanic or Black males aged 25-64 . However, although there was an overall decline in incidence among non-US-born persons, there was no significant change in any sex or racial/ethnic subgroup . The percent of multidrug-resistant (MDR) cases among non-US-born patients remained stable, but recent arrivals accounted for 79% of non-US-born MDR-TB patients in 1999, a significant increase from 16% in 1997 . CONCLUSIONS: Continuing current tuberculosis control efforts and treatment of immigrants with latent tuberculosis infection are of highest priority for reducing incident cases in NYC . Global collaboration towards earlier detection and treatment of active tuberculosis cases in high incidence countries is also essential.

Ai Zheng, 2003 May, 22(5), 496 - 9
{Expression and significance of MRP, GST-pi, Topo IIalpha, and LRP in gastric carcinoma}; Yu DQ et al.; BACKGROUND & OBJECTIVE: Multidrug resistance-associated protein (MRP), glutathione-S-transferase-pi(GST-pi), topoisomerase IIalpha(Topo IIalpha)and lung resistance protein (LRP) play important roles in the multidrug resistance(MDR)of tumor chemotherapy . There were few reports on combined determination of the expression of MRP, GST-pi, Topo IIalpha and LRP in gastric carcinoma . This study was designed to investigate the expression and significance of MRP, GST-pi, Topo IIalpha and LRP in gastric carcinomas . METHODS: Immunohistochemistry SP method was used to determine the expression of MRP, GST-pi, Topo IIalpha, and LRP in 90 tumor samples from the patients with gastric carcinoma . Chi-square test and Fisher exact test were used to analyze the significance of the expression . RESULTS: (1)The positive expression rates of MRP, GST-pi, Topo IIalpha and LRP in gastric carcinoma were 88.9%, 91.1%, 74.4%, and 87.7%, respectively . They were all significantly higher than those in normal stomach tissues (P< 0.05) . (2)The expression levels of MRP, GST-pi, and LRP in well-moderated differentiation adenocarcinoma were significantly higher than those in poor differentiation adenocarcinoma . The expression of Topo IIin well-moderated differentiation adenocarcinoma was significantly lower than that in poor differentiation adenocarcinoma . There was no difference between the expression levels of them in different degree of invasion or with lymph nodes metastasis and without lymph nodes metastasis (P > 0.05) . (3) There was no correlation in any two items among the expression levels of MRP,GST-pi, topo IIalpha,and LRP . CONCLUSION: MRP, GST-pi, topo IIalpha,and LRP play important roles in the primary MDR of gastric carcinoma . The expression of them are associated with the differentiation, but are not associated with the invasion degree and lymph node metastasis.

Biol Chem, 2003 Apr, 384(4), 505 - 16
Glutathione pathways in the brain; Dringen R et al.; The antioxidant glutathione (GSH) is essential for the cellular detoxification of reactive oxygen species in brain cells . A compromised GSH system in the brain has been connected with the oxidative stress occuring in neurological diseases . Recent data demonstrate that besides intracellular functions GSH has also important extracellular functions in brain . In this respect astrocytes appear to play a key role in the GSH metabolism of the brain, since astroglial GSH export is essential for providing GSH precursors to neurons . Of the different brain cell types studied in vitro only astrocytes release substantial amounts of GSH . In addition, during oxidative stress astrocytes efficiently export glutathione disulfide (GSSG) . The multidrug resistance protein 1 participates in both the export of GSH and GSSG from astrocytes . This review focuses on recent results on the export of GSH and GSSG from brain cells as well as on the functions of extracellular GSH in the brain . In addition, implications of disturbed GSH pathways in brain for neurodegenerative diseases will be discussed.

Cancer Res, 2003 May 15, 63(10), 2492 - 8
Effect of the multidrug resistance protein on the transport of the antiandrogen flutamide; Grzywacz MJ et al.; Prostate cancer is the most common noncutaneous malignancy of American men . Although it can be initially treated with androgen deprivation therapy, tumors that relapse become resistant to future hormonal manipulation . We previously found that the multidrug resistance protein (MRP), MRP1, is overexpressed in advanced stage and grade human prostate cancer and is negatively regulated by p53 . In this study, we sought to determine whether the cellular accumulation of the antiandrogen flutamide, a drug commonly used in the treatment of prostate cancer, is affected by MRP1 expression . There were significant differences between the wild-type and MRP1-overexpressing cells in efflux and accumulation of flutamide and hydroxyflutamide, its active metabolite . In contrast, transport of dihydrotestosterone was not affected by MRP1 . Treating the cells with leukotriene D4, a known MRP1 substrate, or VX-710, an MRP1 modulator, restored flutamide and hydroxyflutamide accumulation . Finally, intracellular glutathione depletion with buthionine sulfoximine or energy depletion using 2-deoxy-D-glucose/sodium azide restored flutamide accumulation to that of parental cells while incubating the cells at 4 degrees C abolished MRP1-mediated transport . In summary, these studies indicate that flutamide and hydroxyflutamide but not dihydrotestosterone are transported by MRP1 and that these findings may contribute to our understanding of resistance to hormone refractory prostate cancer.

Phytother Res, 2003 May, 17(5), 495 - 500
Biological activity of persimmon (Diospyros kaki) peel extracts; Kawase M et al.; Fractionated extracts of persimmon (Diospyros kaki) peels were studied for cytotoxic activity, multidrug resistance (MDR) reversal activity, anti-human immunodeficiency virus (HIV) activity and anti-Helicobacter pylori (H . pylori) activity . The potent cytotoxic activity against human oral squamous cell carcinoma cells (HSC-2) and human submandibular gland tumor (HSG) cells was found in the acetone fractions (A4 and A5) with IC(50) ranging from 21 to 59 micro g/mL . However, the cytotoxic activity was not correlated with the radical intensity of the fractions . Three 70% MeOH extract fractions (70M2-4) produced radical and efficiently scavenged the O(2)(-) produced by hypoxanthine and xanthine oxidase reaction . All of the fractions tested were not effective for anti-H . pylori and anti-HIV . Fractions H3 and H4 of hexane extract, and M2 and M3 of MeOH extract showed a remarkable MDR reversal activity comparable with that of (+/-)-verapamil (a positive control) . These results indicate the therapeutic value of persimmon peel extracts as potential antitumor and MDR-reversing agents .

J Med Chem, 2003 May 22, 46(11), 2125 - 31
Modulation of P-glycoprotein-mediated multidrug resistance by flavonoid derivatives and analogues; Hadjeri M et al.; Flavonoid derivatives were synthesized and tested for their ability to modulate P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) in vitro . These compounds belong to various flavonoid subclasses, namely: chromones, azaisoflavones, and aurones . Among the investigated compounds, three showed potent reversing activity . 2-(4-methylpiperazin-1-ylcarbonyl)-5-hydroxychromone (4a), 5,7-dimethoxy-3-phenyl-4-quinolone (5), and 4,6-dimethoxyaurone (6) potentiated daunorubicin cytotoxicity on resistant K562 cells . They were also able to increase the intracellular accumulation of rhodamine-123, a fluorescent molecule which acts as a probe of P-glycoprotein-mediated MDR . This suggests that these compounds act, at least in part, by inhibiting P-glycoprotein activity . The most active compound, 5-hydroxy-2-(4-methylpiperazin-1-ylcarbonyl)chromone (4a) was found to be a powerful reversal agent, more potent than cyclosporin A, used as the reference molecule . No effect was observed on MRP transport nor on cell proliferation . Little apoptosis was induced on K562S cells with 4a compared to K562R, probably due to the extrusion of the compound by Pgp.

Mol Membr Biol, 2003 Jan-Mar, 20(1), 53 - 60
Neither lipophilicity nor membrane-perturbing potency of phenothiazine maleates correlate with the ability to inhibit P-glycoprotein transport activity; Hendrich AB et al.; Although phenothiazines are known as multidrug resistance modifiers, the molecular mechanism of their activity remains unclear . Since phenothiazine molecules are amphiphilic, the interactions with membrane lipids may be related, at least partially, to their biological effects . Using the set of phenothiazine maleates differing in the type of phenothiazine ring substitution at position 2 and/or in the length of the alkyl bridge-connecting ring system and side chain group, we investigated if their ability to modulate the multidrug resistance of cancer cells correlated with model membrane perturbing potency . The influence exerted on lipid bilayers was determined by liposome/buffer partition coefficient measurements (using the absorption spectra second-derivative method), fluorescence spectroscopy and calorimetry . Biological effects were assessed by a flow cytometric functional test based on differential accumulation of fluorescent probe DiOC(2)(3) by parental and drug-resistant cells . We found that all phenothiazine maleates were incorporated into lipid bilayers and altered their biophysical properties . With only few exceptions, the extent of membrane perturbation induced by phenothiazine maleates correlated with their lipophilicity . Within the group of studied derivatives, the compounds substituted with CF(3)- at position 2 of phenothiazine ring were the most active membrane perturbants . No clear relation was found between effects exerted by phenothiazine maleates on model membranes and their ability to modulate P-glycoprotein transport activity.

Virchows Arch, 2003 Jun, 442(6), 529 - 37 Epub 2003 May 13.
Expression of drug resistance related proteins in sarcomas of the pulmonary artery and poorly differentiated leiomyosarcomas of other origin; Gaumann A et al.; Sarcomas are known to develop resistance to current chemotherapeutic strategies, displaying a multidrug-resistant phenotype . Mechanisms involved in drug resistance include reduced cellular drug accumulation, drug detoxification as well as alterations in drug target specificity . In seven sarcomas of the pulmonary artery (SPA) and ten leiomyosarcomas of other origin, we studied the immunohistochemical expression of P-glycoprotein (P-gp), multidrug-resistance protein (MRP), lung resistance protein (LRP), metallothionein (MT) and topoisomerase IIalpha . Upregulation was found in tumour cells for P-gp but not for MRP in SPA and other leiomyosarcomas . Topoisomerase IIalpha was expressed at high levels in tissue of primary tumours as well as recurrent tumours . Both P-gp and topoisomerase IIalpha were present in numerous tumour-associated vessels . LRP was expressed at high levels in SPA but to a lesser extent in the other leiomyosarcomas . MT was expressed at low levels but was markedly present at the border of necrosis . The overall survival and the relapse-free survival did not correlate with the expression of these factors . There was no significant relationship between treated and non-treated patients with respect to the expression of the examined molecules . P-gp, but not MRP, may play a role in the development of drug resistance . P-gp, LRP and topoisomerase IIalpha contribute to drug resistance through expression in tumour-associated vessels . Unique high levels of topisomerase IIalpha reflect the high proliferation rate of these tumours . MT seems to serve as a detoxifying agent of metabolites at the border of necrosis . Our findings underline the fact that multiple factors contribute to chemoresistance and that examination of a spectrum of relevant molecules is probably necessary to plan the best therapy.

Appl Microbiol Biotechnol, 2003 May, 61(4), 278 - 88 Epub 2003 Mar 01.
Aminoacyl-tRNA synthetases and their inhibitors as a novel family of antibiotics; Kim S et al.; The emergence of multidrug-resistant strains of pathogenic microorganisms and the slow progress in new antibiotic development has led in recent years to a resurgence of infectious diseases that threaten the well-being of humans . The result of many microorganisms becoming immune to major antibiotics means that fighting off infection by these pathogens is more difficult . The best strategy to get around drug resistance is to discover new drug targets, taking advantage of the abundant information that was recently obtained from genomic and proteomic research, and explore them for drug development . In this regard, aminoacyl-tRNA synthetases (ARSs) provide a promising platform to develop novel antibiotics that show no cross-resistance to other classical antibiotics . During the last few years there has been a comprehensive attempt to find the compounds that can specifically target ARSs and inhibit bacterial growth . In this review, the current status in the development of ARS inhibitors will be briefly summarized, based on their chemical structures and working mechanisms.

Biochem J, 2003 Aug 1, 373(Pt 3), 767 - 74
A functional study on polymorphism of the ATP-binding cassette transporter ABCG2: critical role of arginine-482 in methotrexate transport; Mitomo H et al.; Overexpression of the ATP-binding cassette transporter ABCG2 reportedly causes multidrug resistance, whereas altered drug-resistance profiles and substrate specificity are implicated for certain variant forms of ABCG2 . At least three variant forms of ABCG2 have been hitherto documented on the basis of their amino acid moieties (i.e., arginine, glycine and threonine) at position 482 . In the present study we have generated those ABCG2 variants by site-directed mutagenesis and expressed them in HEK-293 cells . Exogenous expression of the Arg(482), Gly(482), and Thr(482) variant forms of ABCG2 conferred HEK-293 cell resistance toward mitoxantrone 15-, 47- and 54-fold, respectively, as compared with mock-transfected HEK-293 cells . The transport activity of those variants was examined by using plasma-membrane vesicles prepared from ABCG2-overexpressing HEK-293 cells . {Arg(482)}ABCG2 transports {(3)H}methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly(482) and Thr(482)) . Transport of methotrexate by {Arg(482)}ABCG2 was significantly inhibited by mitoxantrone, doxorubicin and rhodamine 123, but not by S -octylglutathione . Furthermore, ABCG2 was found to exist in the plasma membrane as a homodimer bound via cysteinyl disulphide bond(s) . Treatment with mercaptoethanol decreased its apparent molecular mass from 140 to 70 kDa . Nevertheless, ATP-dependent transport of methotrexate by {Arg(482)}ABCG2 was little affected by such mercaptoethanol treatment . It is concluded that Arg(482) is a critical amino acid moiety in the substrate specificity and transport of ABCG2 for certain drugs, such as methotrexate.

Neoplasma, 2003, 50(2), 91 - 6
Modulation of HLA class I expression in multidrug-resistant human rhabdomyosarcoma cells; Melguizo C et al.; An abnormal HLA expression has been detected in some tumors including rhabdomyosarcoma (RMS) . Classical cytotoxic treatment of these tumors, the most common childhood soft tissue malignancy, may induce multidrug resistance (MDR) associated with the expression of a 170-kDa membrane-associated glycoprotein (P-glycoprotein) . In order to analyse the connection between modulation of HLA expression and the development of the MDR phenotype mediated by P-glycoprotein in RMS, we used three resistant RMS cell lines; two of these resistant cell lines (TE.32.7.DAC and RD-DAC) were established by in vitro exposure to actinomycin D, a drug of choice in the treatment of RMS; the resistant RMS- GR cell line was established from an embryonal RMS tumor after polychemotherapy . Our results showed that all the resistant cell lines showed a significant increase in the expression of HLA class I surface antigens in comparison to drug-sensitive cells . Blockade of P-glycoprotein with verapamil led to a decrease in HLA class I expression in RMS resistant cell lines . However, no modulation of HLA class II expression was observed in any of the three analyzed cell lines . These findings support the hypothesis that the development of resistance mediated by mdr 1/P-glycoprotein, directly influences the expression of HLA class I in RMS cells, inducing to upregulation . This effect may be relevant to the application in RMS of immunotherapy against tumor-associated antigens presented by HLA class I molecules.

J Biol Chem, 2003 Jul 11, 278(28), 25285 - 8 Epub 2003 May 08.
Regulation of multidrug resistance in cancer cells by hyaluronan; Misra S et al.; Multidrug resistance in cancer cells is often due to ATP-dependent efflux pumps, but is also linked to alterations in cell survival and apoptotic signaling pathways . We have found previously that perturbation of hyaluronan-tumor cell interaction by treatment with hyaluronan oligosaccharides suppresses the phosphoinositide 3-kinase/Akt cell survival signaling pathway in cancer cells and reduces tumor growth in vivo . Here we find that these oligomers suppress both the MAP kinase and phosphoinositide 3-kinase pathways in multidrug resistant tumor cells and sensitize these cells to a variety of chemotherapeutic drugs . On the other hand, increased hyaluronan production induces resistance in drug-sensitive tumor cells . Likewise, increased expression of emmprin, which is a glycoprotein that is present on the surface of most malignant cancer cells and that stimulates hyaluronan production, also induces increased resistance . Thus, perturbation of hyaluronan signaling may provide a dual therapeutic role, since it has intrinsic suppressive effects on tumor growth as well as sensitizing cancer cells to chemotherapeutic agents.

Cell Mol Life Sci, 2003 Mar, 60(3), 526 - 35
RU49953: a non-hormonal steroid derivative that potently inhibits P-glycoprotein and reverts cellular multidrug resistance; Perez-Victorias FJ et al.; Progesterone and the antiprogestin RU38486 have been reported as non-transported modulators of P-glycoprotein-mediated drug efflux . However, their hormonal properties limit their potential for clinical trials . The present work shows that some derivatives from either progesterone/RU38486 or estradiol, displaying differential interaction with hormone receptors, bind to P-glycoprotein and chemosensitize the growth of MDR1-transfected cells to vinblastine more strongly than does RU38486 . Structure comparison of the compounds indicates that the highly hydrophobic estradiol derivative RU49953, which does not interact with any hormone receptor, inhibits P-glycoprotein-mediated drug efflux very efficiently, as monitored by flow cytometry, and prevents drug site photoaffinity labeling by azidopine . It induces a much higher chemosensitization than the well-known P-glycoprotein modulator verapamil, which is itself more efficient than RU38486 . RU49953 therefore constitutes a promising new lead for steroid-type modulators of multidrug resistance.

New Microbiol, 2003 Apr, 26(2), 181 - 6
Evaluation of susceptibility of Mycobacterium bovis to antituberculous drugs by radiometric BACTEC 460TB system; Cavirani S et al.; Susceptibility of Mycobacterium bovis strains to antituberculous drugs (isoniazid and rifampin) was detected by radiometric BACTEC 460TB system . M.bovis strains were isolated from tissue samples showing tuberculous lesions collected at an abbattoir from cattle belonging to 47 tuberculosis outbreaks occurring in Northern Italy in 1995-1999 . Forty-six out of 61 strains (75.4%) resulted susceptible to both isoniazid and rifampin . Thirteen strains (21.3%) were resistant to isoniazid only . No strains showed resistance to rifampin only . Two strains (3.3%) resulted resistant to both drugs, showing antituberculous multidrug-resistance . Given the compulsory eradication program of bovine tuberculosis by elimination of infected animals and the ban on antituberculous drug treatments in animals, detection of resistant M . bovis strains appears of great interest.

Ann Thorac Surg, 2003 May, 75(5), 1613 - 7
Median sternotomy for pneumonectomy in patients with pulmonary complications of tuberculosis; Connery CP et al.; BACKGROUND: Traditionally, a thoractomy incision is used for pulmonary complications of tuberculosis . An attractive alternative is being presented by the use of midline sternotomy in such patients, which is the aim of this study . METHODS: Five patients (four male, one female) with pulmonary complications of tuberculosis requiring surgical therapy in 1993 and 1994 were treated through a median sternotomy . The median patient age at time of surgery was 40.2 years and the median patient follow-up was 4.0 years (range 1.0 to 5.0 years) in this retrospective review . RESULTS: All patients had uncomplicated operative courses and were discharged from the hospital . One patient's in-hospital postoperative course was complicated by prolonged ventilator dependency requiring temporary tracheostomy; he died 1 year postoperatively after hospital discharge due to recurrent multidrug-resistant tuberculosis . Sternal wound infections and bronchopleural fistulas were not observed in any patients . CONCLUSIONS: Surgical treatment of pulmonary complications of tuberculosis was traditionally performed through a thoracotomy approach . Many patients with tuberculous lungs have pulmonary adhesions or intrathoracic scarring from previous surgery, which would require extrapleural resection . Bleeding was a frequent complication of this procedure . Sternotomy offers excellent exposure of the intrapericardial vessels, and reduced postoperative disability compared to the standard thoracotomy, which may be an advantage given that the majority of patients in this population have poor pulmonary function . We recommend median sternotomy as an alternative operative approach in selected patients with pulmonary complications of tuberculosis.

J Clin Microbiol, 2003 May, 41(5), 2209 - 12
Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis isolates from China; Yue J et al.; Mutations in the 81-bp rifampin resistance determining region (RRDR) and mutation V176F locating at the beginning of the ropB gene were analyzed by DNA sequencing of 86 Mycobacterium tuberculosis clinical isolates (72 resistant and 14 sensitive) from different parts of China . Sixty-five mutations of 22 distinct kinds, 21 point mutations, and 1 insertion were found in 65 of 72 resistant isolates . The most common mutations were in codons 531 (41%), 526 (40%), and 516 (4%) . Mutations were not found in seven (10%) of the resistant isolates . Six new alleles within the RRDR, along with five novel mutations outside the RRDR, are reported . None of isolates contained the V176 mutation.

Biochem Pharmacol, 2003 May 1, 65(9), 1419 - 25
Multidrug resistance correlates with overexpression of Muc4 but inversely with P-glycoprotein and multidrug resistance related protein in transfected human melanoma cells; Hu YP et al.; Due to the size, glycosylation, and location in the plasma membrane of the sialomucin complex Muc4, which has been implicated in ErbB2 signaling, in the repression of apoptosis and cell adhesion, and in tumor metastasis, studies were initiated to determine whether its presence could influence cell sensitivity to anticancer drugs . Growth inhibition assays using melanoma cell lines that either express the glycoprotein (Muc4(+)) or do not (Muc4(-)) showed that Muc4 renders cells resistant to taxol, doxorubicin, vinblastine, rhodamine 123, and 2-deoxyglucose . When treated with various concentrations of doxorubicin, Muc4(+) cells were blocked less frequently in G(2) and underwent less DNA fragmentation (apoptosis and/or necrosis) than Muc4(-) cells . All of the drugs tested (except for 2-deoxyglucose) are well recognized by P-glycoprotein-mediated multidrug resistance 1 (MDR1) and to a lesser degree by multidrug resistance related protein 1 (MRP1) transporters . Therefore, transporter gene expression in these cells was assayed . Surprisingly, Muc4(+) cells expressed lower levels of both transporter genes than Muc4(-) cells . Moreover, rhodamine 123 was retained more highly in the Muc4(+) than in the Muc4(-) cells, demonstrating that these transporters are functional . Overall, these results indicate that although Muc4(+) cells express less MDR1 and MRP1, they are more resistant to drugs recognized by these transporters.

Biochemistry, 2003 May 13, 42(18), 5429 - 37
Multidrug resistance protein (MRP) 1 and MRP3 attenuate cytotoxic and transactivating effects of the cyclopentenone prostaglandin, 15-deoxy-Delta(12,14)prostaglandin J2 in MCF7 breast cancer cells; Paumi CM et al.; One of the most potent cyclopentenone prostaglandins, 15-deoxy-Delta(12,14)prostaglandin J(2) (15-d-PGJ(2)), has been shown to be cytotoxic in some tumor cells and, as a ligand of peroxisome proliferator activated receptor gamma (PPARgamma), to influence the transcriptional regulation of several genes . We examined whether a glutathione conjugate of 15-d-PGJ(2), 15-d-PGJ(2)-SG, is formed and if the glutathione conjugate efflux pumps, MRP1 and MRP3, could transport this conjugate, thereby attenuating the cytotoxicity and transactivating activity of 15-d-PGJ(2) in MCF7 breast cancer cells . Formation of 15-d-PGJ(2)-SG was demonstrated both in vitro and in cells, and its structure was determined by ESI/MS and NMR . Expression of MRP1 and MRP3 was achieved by stable transduction of parental MCF7 cells . Membrane vesicles derived from these cells supported efficient, ATP-dependent transport of 15-d-PGJ(2)-SG (K(M) 1.4 and 2.9 microM for MRP1 and MRP3, respectively) . When compared with parental, MRP-minus MCF7 cells, expression of MRP1 and MRP3 conferred approximately 2-fold protection from 15-d-PGJ(2) cytotoxicity . 15-d-PGJ(2)-mediated transcriptional activation was evaluated in cells transiently transfected with a reporter gene under the transcriptional control of a PPAR responsive element . Treatment of parental MCF7 cells with 15-d-PGJ(2) resulted in a time-dependent induction of reporter gene activity-induction that was measurable with concentrations of added 15-d-PGJ(2) as low as 100 nM . In contrast, expression of MRP1 or MRP3 abolished 15-d-PGJ(2)-dependent reporter gene induction . Depletion of intracellular glutathione reversed MRP1- and MRP3-mediated attenuation of 15-d-PGJ(2) cytotoxicity and transactivation . These data indicate that MRP1 and MRP3 can modulate the biological effects of 15-d-PGJ(2), and likely other cyclopentenone prostaglandins, in a glutathione-dependent manner . The results are consistent with a mechanism for the attenuation of the biological activities of 15-d-PGJ(2) that involves the formation and active efflux of its glutathione conjugate, 15-d-PGJ(2)-SG.

Biochemistry, 2003 May 13, 42(18), 5214 - 24
Functional and structural consequences of cysteine substitutions in the NH2 proximal region of the human multidrug resistance protein 1 (MRP1/ABCC1); Leslie EM et al.; The 190 kDa multidrug resistance protein 1 (MRP1; ABCC1) is comprised of three membrane spanning domains (MSDs) and two nucleotide binding domains (NBDs) configured MSD1-MSD2-NBD1-MSD3-NBD2 . MRP1 overexpression in tumor cells results in an ATP-dependent efflux of many oncolytic agents and arsenic and antimony oxyanions . MRP1 also transports GSSG and GSH as well as conjugated organic anions, including leukotriene C(4) and 17beta-estradiol 17-(beta-D-glucuronide) and certain xenobiotics in association with GSH . Previous studies have shown that portions of MSD1 and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function . In the present study, Cys residues at positions 43, 49, 85, 148, and 190 in MSD1 and positions 208 and 265 in CL3 were mutated to Ala and Ser, and the effects on protein expression, plasma membrane localization, trypsin sensitivity, organic anion transport, and drug resistance properties were investigated . Confocal microscopy showed that 11 of 14 mutants displayed significant levels of nonplasma membrane-associated MRP1 . Most mutant proteins were also more resistant to trypsin proteolysis than wild-type MRP1 . All Cys mutants transported organic anions (0.5-1.5-fold wild-type MRP1 activity), and cells expressing Ser-substituted but not Ala-substituted Cys43 and Cys265 MRP1 mutants exhibited a 2.5-fold decrease and a 3-fold increase in arsenite resistance, respectively; Cys43Ser MRP1 also conferred lower levels of vincristine resistance . These results indicate that certain Cys residues in the NH(2) proximal region of MRP1 can be important for its structure and selected transport activities.

J Gene Med, 2003 May, 5(5), 366 - 76
Drug-selected co-expression of P-glycoprotein and gp91 in vivo from an MDR1-bicistronic retrovirus vector Ha-MDR-IRES-gp91; Sugimoto Y et al.; BACKGROUND: Retroviral transduction of human hematopoietic stem cells is an attractive strategy in gene therapy; however, transduction efficiency and duration of transgene expression may not be satisfactory in current protocols . Co-expression of a human multidrug resistance gene (MDR1) with a therapeutic gene affords selectable growth advantage to genetically modified cells . METHODS: A bicistronic retrovirus vector, Ha-MDR-IRES-gp91, was constructed for the co-expression of MDR1 and gp91, a gene responsible for X-linked chronic granulomatous disease (X-CGD) . Drug-selected co-expression of P-glycoprotein and gp91 was evaluated in transduced cells . RESULTS: Epstein-Barr virus-transformed B cells from X-CGD patients transduced with Ha-MDR-IRES-gp91 co-expressed human P-glycoprotein and gp91, and acquired superoxide-generating activity . Human CD34-positive cells from an X-CGD patient were transduced with Ha-MDR-IRES-gp91 and subsequently treated with 2 ng/ml vincristine . After 13 days, 20% of Ha-MDR-IRES-gp91-transduced cells were P-glycoprotein- and gp91-positive by FACS analysis . The superoxide-generating activity of the transduced population was 27% of that of normal cells . Mice transplanted with Ha-MDR-IRES-gp91-transduced bone marrow cells showed co-expression of P-glycoprotein and gp91 in peripheral blood mononuclear cells . By administering paclitaxel, the proportions of P-glycoprotein- and gp91-positive cells were increased in all the four mice examined . When mice transplanted with Ha-MDR-IRES-gp91-transduced cells were repeatedly administered paclitaxel, the ratios of P-glycoprotein- and gp91-positive cells were maintained for over 1 year . CONCLUSIONS: These results suggest that MDR1-bicistronic vectors may be useful to select the transduced hematopoietic cells in vivo . This may lead to the sustained expression of transgenes in the blood cells of patients treated with stem cell gene therapy .

Toxicol Sci, 2003 Jul, 74(1), 174 - 81 Epub 2003 May 02.
Genomic analysis of the rat lung following elemental mercury vapor exposure; Liu J et al.; Elemental mercury (Hg0) is a highly toxic chemical with increasing public health concern . Although the lung receives the highest exposure to Hg0 vapor, it is resistant to Hg0 toxicity relative to the kidney and brain . In an earlier study, exposure of rats to 4 mg Hg0 vapor/m3, 2 h per day for 10 days, did not produce pathological alterations in the lung but increased metallothionein and glutathione S-transferase in the kidney . This study was undertaken to examine pulmonary gene expression associated with Hg0 vapor inhalation . Total RNA was extracted from lung tissues of rats, previously exposed to air or Hg0 vapor, and subjected to microarray analysis . Hg0 vapor exposure increased the expression of genes encoding inflammatory responses, such as chemokines, tumor necrosis factor-alpha (TNFalpha), TNF-receptor-1, interleukin-2 (IL-2), IL-7, prostaglandin E2 receptor, and heat-shock proteins . As adaptive responses, glutathione S-transferases (GST-pi, mGST1), metallothionein, and thioredoxin peroxidase were all increased in response to Hg exposure . Some transporters, such as multidrug resistance-associated protein (MRP), P-glycoprotein, and zinc transporter ZnT1, were also increased in an attempt to reduce pulmonary Hg load . The expression of transcription factor c-jun/AP-1 and PI3-kinases was suppressed, while the expression of protein kinase-C was increased . Expression of epidermal fatty acid-binding protein was also enhanced . Real-time RT-PCR and Western blot analyses confirmed the microarray results . In summary, genomic analysis revealed an array of gene alterations in response to Hg0 vapor exposure, which could be important for the development of pulmonary adaptation to Hg during Hg0 vapor inhalation.

Biochim Biophys Acta, 2003 May 2, 1612(1), 90 - 7
Escherichia coli lacking the AcrAB multidrug efflux pump also lacks nonproteinaceous, PHB-polyphosphate Ca2+ channels in the membrane; Jones HE et al.; PHB(polyP) complexes bind calcium and form calcium channels in the cytoplasmic membrane in Escherichia coli and are likely to be important in Ca(2+) homeostasis in this organism . E . coli N43, which lacks the AcrA component of a major multidrug resistance pump, was shown to be defective in calcium handling, with an inability to maintain submicromolar levels of free Ca(2+) in the cytoplasm . Therefore, using an N-phenyl-1-napthylamine (NPN)-dependent fluorescence assay, we measured temperature-dependent phase transitions in the membranes of intact cells . These transitions specifically depend on the presence of PHB(Ca(2+)polyP) complexes . PHB(Ca(2+)polyP) channel complexes, particularly in stationary phase cultures, were detected in wild-type strains; however, in contrast, isogenic acrA(-) strains had greatly reduced amounts of the complexes . This indicates that the AcrAB transporter may have a novel, hitherto undetected physiological role, either directly in the membrane assembly of the PHB complexes or the transport of a component of the membrane, which is essential for assembly of the complexes into the membrane . In other experiments, we showed that the particular defective calcium handling detected in N43 was not due to the absence of AcrA but to other unknown factors in this strain.

Bioorg Med Chem Lett, 2003 May 19, 13(10), 1777 - 81
Multidrug resistance reversal activity of key ningalin analogues; Soenen DR et al.; Key analogue derivatives of the ningalins, potent multidrug resistance (MDR) reversal compounds, were examined resulting in the discovery of a potent MDR reversal agent that hypersensitizes P-gp resistant tumor cell lines to front-line conventional therapeutic agents.

Int J Tuberc Lung Dis, 2003 Apr, 7(4), 394 - 8
Multidrug-resistant tuberculous meningitis in patients with AIDS; Daikos GL et al.; We present clinical manifestations, bacteriologic characteristics, and outcomes for eight patients with multidrug-resistant (MDR) tuberculous meningitis and AIDS . All developed meningitis as a terminal complication of previously diagnosed MDR-TB despite anti-tuberculosis therapy . Seven patients presented with fever, five with headache, four with altered mentation, two with focal deficits and one with seizures . CSF examination revealed pleocytosis, hypoglychorrhachia and elevated protein . Mycobacterium tuberculosis resistant to at least isoniazid and rifampin was isolated from all patients . Intracerebral mass lesions were detected in three patients, hydrocephalus in three, meningeal enhancement in five, and infarcts in two . Seven patients died 1-16 weeks after the diagnosis of meningitis; the eighth was lost to follow-up . MDR tuberculous meningitis is a difficult-to-treat infection with a high fatality rate.

Int J Tuberc Lung Dis, 2003 Apr, 7(4), 347 - 53
Peripheral neuropathy associated with treatment for multidrug-resistant tuberculosis; Shin SS et al.; OBJECTIVE: To review the incidence and management of peripheral neuropathy in patients receiving therapy for MDR-TB . METHODS: A case series with retrospective chart review of 75 patients who initiated individualized therapy for multidrug-resistant tuberculosis (MDR-TB) in Lima, Peru, between 1 August 1996 and 31 January 1999 . RESULTS: All patients had confirmed MDR-TB and were receiving individualized therapy, comprised of an average of six drugs . Ten (13%) of these patients presented with symptoms of peripheral neuropathy, confirmed by electromyography . All symptoms were reported in the lower extremities, and all were sensory in nature . Median time to presentation from initiation of MDR-TB therapy was 9.1 months . No significant risk factors associated with development of peripheral neuropathy were identified . Management strategies depended on the severity of symptoms and included the treatment of contributing co-morbidities, medications for neuropathic pain, and adjustment of doses of possible offending agents . All patients responded to management; three patients were left with mild residual symptoms . Patients whose neuropathy resolved had symptoms for a median of 7 months . CONCLUSIONS: Peripheral neuropathy was encountered in 13% of our cohort of MDR-TB patients . The diagnosis of peripheral neuropathy can be based on clinical presentation alone, and effective management of this side-effect is possible without sacrificing MDR-TB treatment efficacy.

Int J Tuberc Lung Dis, 2003 Apr, 7(4), 343 - 6
The role of the nurse in the community-based treatment of multidrug-resistant tuberculosis (MDR-TB); Palacios E et al.; SETTING: A community-based treatment program for multidrug-resistant tuberculosis (MDR-TB) in Lima, Peru . OBJECTIVES: To describe the activities carried out by the nurses working with the program . DESIGN: A qualitative study using a variety of ethnographic methods, including participant observation, focus groups, and key informant interviews over a 5-year period . RESULTS: Nurses were responsible for carrying out a wide variety of activities within the program . These included patient-focused activities such as identifying patients, evaluating patients prior to starting and during therapy, and managing emergencies; educational activities for both patients and health professionals managing MDR-TB; and coordination activities, including over-seeing health workers and communicating between team members . CONCLUSION: Nurses play a key role in the community-based management of MDR-TB.

Int J Tuberc Lung Dis, 2003 Apr, 7(4), 336 - 42
Resistance to anti-tuberculosis drugs and practices in drug susceptibility testing in Moldova, 1995-1999; Crudu V et al.; OBJECTIVE: To evaluate practices in initial drug susceptibility testing (DST) in Moldova, anti-tuberculosis drug resistance and the implications for tuberculosis control . METHODS: Retrospective record review in the national reference laboratory . RESULTS: Of 3463 cases, 57.1% were recorded as 'new' and 24.6% as 'retreatment' cases; previous treatment status was not recorded for 18.3% . Of the 'new' cases, 1655 were correctly classified according to international recommendations and 322 were misclassified . The number of cases increased from 443 in 1995 to 939 in 1999; the proportion of 'retreatment' increased from 17.4% to 35.5%, 'any drug resistance' from 20.3% to 41.6%, and 'multidrug resistance' from 2.7% to 11.2% . In 1998-1999, 'any drug resistance' and 'multidrug resistance' in 800 previously untreated cases were respectively 29.1% and 5.3%, and respectively 61.0% and 21.9% in 521 'retreatment' cases . Of a total of 216 'multidrug-resistant' cases in 1998-1999, 21.8% were reported resistant to ethambutol and 81.5% to streptomycin . CONCLUSIONS: Initial specimens for culture are frequently taken late, after the start of treatment, compromising their usefulness for case management or surveillance . Inadequate treatment has led to an increase in the number of cases, the proportion of previously treated cases and the prevalence of drug resistance . In 1998-1999, a high proportion of cases with 'multidrug resistance' were susceptible to ethambutol.

Cancer Res, 2003 May 1, 63(9), 2200 - 5
Molecular description of evolving paclitaxel resistance in the SKOV-3 human ovarian carcinoma cell line; Lamendola DE et al.; Ovarian cancer is currently the most lethal gynecological malignancy in the United States . Although effective therapies exist, the acquisition of multidrug resistance within persisting tumor cells renders curative therapies elusive for the majority of women with ovarian cancer . In an attempt to better define the evolution of paclitaxel resistance, three SKOV-3 sublines were selected during successive rounds of exposure to increasing paclitaxel concentrations . The sublines were selected to represent early (0.003 micro M), intermediate (0.03 micro M), and late (0.3 micro M) paclitaxel resistance . RNA from these cell lines, SKOV-3(0.003TR), SKOV-3(0.03TR), and SKOV-3(0.3TR), as well as the parent cell line SKOV-3, was analyzed by cDNA array to evaluate transcript expression profiles . Arrays were performed using Affymetrix HG-U95Av2 arrays, which contain probes for approximately 9600 known human genes . Signal intensities were calculated by Microarray Suite 5.0 (Affymetrix, Santa Clara, CA) . Expression patterns were analyzed by Affymetrix Data Mining Tool 3.0 with filtering of expression patterns for fold change in expression (maximum divided by minimum expression value/gene) and for variation of expression (maximum minus minimum expression value/gene) . This analysis dismissed approximately 11,000 of approximately 12,000 expression patterns . The remaining approximately 1000 expression patterns were normalized and segregated into 20 partitions of a self-organizing map (SOM) . The resulting SOM discriminates between genes, which are differentially expressed in early versus intermediate versus late paclitaxel resistance . For example, multidrug resistance 1 transcript expression is not elevated in SKOV-3(0.003TR) as compared with parental SKOV-3 but demonstrates elevated expression in SKOV-3(0.03TR) and SKOV-3(0.3TR) . In contrast, SOM analysis demonstrates early (SKOV-3(0.003TR)) transcriptional changes in a wide variety of genes, including gene families involved in cell growth/maintenance, cell structure, signal transduction, and inflammatory response . The use of array analysis with SOMs in sublines with progressive paclitaxel resistance can successfully define an evolution of resistance . Such an analysis may be useful at defining candidate gene families involved in the early-drug resistance phenotype.

J Exp Ther Oncol, 2003 Jan-Feb, 3(1), 14 - 26
Superinduction of P-glycoprotein messenger RNA in vivo in the presence of transcriptional inhibitors; Lee CH et al.; P-Glycoprotein (P-gp) is comprised of a small family of plasma membrane proteins, and its presence in high amounts often correlates with multidrug resistance in cultured cells . Dramatically increased levels of a single member of P-gp mRNA (pgp2) have been observed in experimental liver carcinogenesis models, during liver regeneration, upon culturing of hepatocytes and in the uterus of pregnant animals . In all cases, the increase in mRNA level appears to be the result of an increase in mRNA half-life (stability) . Previously, we have used transcriptional inhibitors alpha-amanitin and actinomycin D to measure P-gp mRNA half-life in normal liver and in liver tumors . We showed that the level of all three P-gp mRNAs decreased with time in the presence of transcriptional inhibitors, yielding measured half-lives of less than 2 h in liver but greater than 12 h in liver tumors . This observation raised the possibility that regulation of P-gp mRNA stability plays a role in liver carcinogenesis . In the present study, we measured P-gp mRNA half-life in other normal tissues to determine if a short P-gp mRNA half-life is unique to the liver . Our study reveals that in contrast to liver, measured P-gp mRNA half-lives in most tissues examined are greater than 12 h . Moreover, we observed an unexpected, marked increase in the level of pgp2 mRNA with time after injection of transcriptional inhibitors . This can only be explained if the transcriptional inhibitors directly or indirectly inhibit the normally high degradation rate of pgp2 mRNA, resulting in the superinduction of this mRNA . These findings have implications for our understanding of the regulation of P-gp gene expression and drug resistance in vivo.

Am J Physiol Cell Physiol, 2003 Sep, 285(3), C584 - 91 Epub 2003 Apr 30.
Role of MDR1 and MRP1 in trophoblast cells, elucidated using retroviral gene transfer; Atkinson DE et al.; Natural differences in expression and retroviral transduction techniques were used to test the hypothesis that MDR1 P-glycoprotein (P-gp) and MRP1 (multidrug resistance-related protein) contribute to xenobiotic handling by placental trophoblast . RT-PCR and Western blotting in placenta, primary cytotrophoblast cell cultures, and BeWo, JAr, and JEG choriocarcinoma cell lines showed that MRP1 was ubiquitously expressed, whereas MDR1 was absent or minimally expressed in BeWo and JEG cell lines . In syncytiotrophoblast, P-gp was localized predominantly to the microvillous, maternal facing plasma membrane, and MRP1 to the basal, fetal facing plasma membrane . Functional studies showed that cyclosporin A-sensitive accumulation of {3H}vinblastine by cells containing both transport proteins was significantly different from those expressing predominantly MRP1 . Retroviral gene transfer of MDR1 to BeWo cells confirmed that this difference was due to the relative expression of MDR1 . Therefore, both P-gp and MRP1 contribute to xenobiotic handling by the trophoblast . Localization of P-gp to the microvillous membrane suggests an essential role in preventing xenobiotic accumulation by the syncytiotrophoblast and, therefore, in protecting the fetus.

Gan To Kagaku Ryoho, 2003 Apr, 30(4), 468 - 77
{Gene therapy for breast cancer}; Takahashi S et al.; Breast cancer is sensitive to chemotherapy and endocrine therapy, but the prognosis of advanced or relapsed breast cancer is unsatisfactory . Gene therapy is promising as another useful therapeutic approach for advanced breast cancer . Strategies of gene therapy for breast cancer in ongoing clinical protocols can be divided into four: (1) suppression of oncogenes or transduction of tumor suppressors; (2) enhancement of immunological response to cancer cells; (3) transduction of suicide genes; and (4) protection of bone marrow using drug resistance genes . We have started a clinical study of gene therapy for breast cancer using multidrug resistance gene (MDR1), in which advanced or relapsed breast cancer patients received high dose chemotherapy and autologous peripheral blood stem cell transplantation (PBSCT) with MDR1-transduced hemopoietic cells, and then were treated with docetaxel . Two patients have been treated so far, and in vivo enrichment of MDR1-transduced cells with docetaxel treatment after PBSCT was seen in both cases . Both patients are in complete remission and have no apparent adverse effect from MDR1 gene transduction.

Phytother Res, 2003 Apr, 17(4), 348 - 52
Cytotoxic and multidrug resistance reversal activity of a vegetable, 'Anastasia Red', a variety of sweet pepper; Motohashi N et al.; The vegetable, Anastasia Red, Capsicum annuum L . var . angulosum Mill . (Solanaceae) was successively extracted with hexane, acetone, methanol and 70% methanol, and the extracts were further separated into a total of 21 fractions by silica gel or octadecylsilane (ODS) column chromatography . The biological activities of extracts and fractions were determined . These extracts showed relatively higher cytotoxic activity against two human oral tumor cell lines (HSC-2, HSG) than against normal human gingival fibroblasts (HGF), suggesting a tumor-specific cytotoxic activity . The cytotoxic activity of these extracts was enhanced by fractionation on silica gel {H2, A2, M1-M3} or ODS column chromatography {70M} . Several fractions {H2, H4, H5, A1, A2, A3, A5, A6, A7, M2} reversed the multidrug resistance (MDR) phenotype with L5178 mouse lymphoma T cells, more efficiently than (+/-)-verapamil . The extracts and fractions did not show any detectable anti-human immunodeficiency virus (HIV) or anti-Helicobacter pylori activity . Thus, this study suggests the effective and selective antitumor potential of 'Anastasia Red' of sweet pepper for further phytochemical and biological investigation .

Semin Oncol, 2003 Apr, 30(2 Suppl 3), 11 - 4
Gemcitabine and anthracyclines in breast cancer; Jassem J; Anthracyclines are among the most active drugs in breast cancer . Gemcitabine is a novel agent that has also shown good antitumor activity in advanced breast cancer . This compound has a favorable toxicity profile, no apparent multidrug resistance, and retains its activity in anthracycline-pretreated patients . Therefore, it was logical to combine gemcitabine with anthracyclines . The combinations of gemcitabine and anthracyclines (doxorubicin, liposomal doxorubicin, epirubicin) have been tested in a few phase I and II studies . Generally, these combinations have been shown to be feasible and well tolerated . The pharmacokinetics of gemcitabine and anthracyclines was not affected by their combined use . Myelosuppression was the most common adverse event; neutropenia was easily reversible . Data from phase II clinical studies suggest that gemcitabine/anthracycline combinations are relatively effective in first- or second-line therapy for women with locally advanced and metastatic breast cancer . Future randomized clinical trials are warranted to further elucidate the role of these regimens in breast cancer care .

Cancer Chemother Pharmacol, 2003 Apr, 51(4), 297 - 305 Epub 2003 Mar 13.
Safety and efficacy of the MDR inhibitor Incel (biricodar, VX-710) in combination with mitoxantrone and prednisone in hormone-refractory prostate cancer; Rago RP et al.; PURPOSE: VX-710 (biricodar, Incel) restores drug sensitivity to cells expressing P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP1) . MRP1 is expressed in a high proportion of prostate tumors while P-gp expression is variable . Since mitoxantrone (M) and prednisone (P) are substrates for MDR transporters, we initiated a study to evaluate the safety, pharmacokinetics, and efficacy of VX-710 plus M/P in patients with hormone-refractory prostate cancer (HRPC) . PATIENTS AND METHODS: Eligible patients had progressive HRPC (defined as new lesions, new disease-related pain, or 50% increase in PSA within 6 weeks of entry), testosterone <30 ng/ml, no prior chemotherapy, ECOG performance status of 0-3, and adequate organ function . Patients received VX-710 (120 mg/m(2) per h) as a 72-h continuous intravenous infusion with intravenous bolus mitoxantrone (12 mg/m(2)) administered 4 h after VX-710 was started and prednisone (5 mg twice daily) administered throughout the study treatment . Endpoints included serum PSA response, PSA response duration, time to PSA progression, pain reduction, and quality of life measures . RESULTS: Enrolled in the study were 40 patients and 184 courses of VX-710 plus M/P were administered . Intensive pharmacokinetics, which were performed on six patients who received one cycle of M/P alone, followed by VX-710 plus M/P for all other cycles, showed that VX-710 did not alter mitoxantrone clearance . VX-710 blood concentration at the time of mitoxantrone administration averaged 4.52 microg/ml . VX-710 plus M/P was well tolerated . Transient nausea/vomiting and mild neutropenia were the principal treatment toxicities . Five patients experienced an uncomplicated febrile neutropenic episode (12%), three had severe nausea/vomiting, and two experienced transient moderate to severe ataxia . Of the 40 patients, 12 (30%, 95% confidence interval 16-44%) had a reduction in PSA of >/=50% and 9 of the 12 patients (23% overall, 95% CI 10-35%) achieved a reduction in PSA of >/=80% that was sustained for the duration of treatment with M/P plus VX-710 . The median time to PSA progression was 41 weeks (95% CI 34-68 weeks) . Of the 40 patients, 15 completed treatment with stable disease and 13 had progressive disease with increasing serum PSA during study treatment . Median survival was 48 weeks for the intent-to-treat population of 40 patients . CONCLUSIONS: The addition of VX-710 to M/P therapy did not appear to increase the proportion of patients with significant serum PSA reductions compared to M/P alone . However, the duration of PSA response observed for the 12 PSA responders suggests that MDR inhibition may benefit some patients with HRPC . In addition to MRP1 or P-gp expression, other mechanisms of drug resistance are probably associated with the relative insensitivity of HRPC to cytotoxic therapy.

Br J Pharmacol, 2003 Apr, 138(8), 1553 - 61
Localization of the GSH-dependent photolabelling site of an agosterol A analog on human MRP1; Ren XQ et al.; 1 . Human multidrug resistance protein 1 (MRP1) is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells . We recently demonstrated that glutathione (GSH) is required for the labelling of the C-terminal half of MRP1 with a photoanalog of agosterol A (azido AG-A) . In this study, we further characterized the GSH-dependent photolabelling site of azido AG-A on MRP1 . 2 . An epitope-inserted MRP1, MRP1 1222HA, which has two hemagglutinin A (HA) epitopes in the extracellular loop between transmembrane segment (TM) 16 and TM17 of the transporter, could bind azido AG-A in a GSH-dependent manner . 3 . Protease digestion of the photolabelled MRP1 1222HA, followed by immunoprecipitation with an anti-HA antibody suggested that the GSH-dependent azido AG-A photolabelling site on MRP1 resides in the region within TM14-17 and the cytoplasmic region proximate to the C-terminus of TM17 . 4 . Arg(1210) in human MRP2 that corresponds to Arg(1202) in human MRP1 has an important role in the transporting activity of MRP2 . Therefore, we replaced the Arg residue at position 1202 of MRP1 with Gly . Whereas photolabelling of the mutant MRP1 R1202G was greatly reduced, it retained leukotriene C(4) (LTC(4)) transport activity and conferred Vincristine resistance in LLC-PK1 cells . 5 . In summary, this study demonstrated that the GSH-dependent azido AG-A photolabelling site on MRP1 resides in the region within TM14-17 and the cytoplasmic region proximate to the C-terminus of TM17 . The charged amino acid Arg(1202) proximate to TM helix 16 is of critical importance for the GSH-dependent photolabelling of MRP1 with azido AG-A . Arg(1202) itself or the region nearby Arg(1202) may be involved in azido AG-A photolabelling.

World J Gastroenterol, 2003 May, 9(5), 894 - 8
Effect of ZNRD1 gene antisense RNA on drug resistant gastric cancer cells; Zhang YM et al.; AIM: To investigate the expression level of ZNRD1 gene in gastric cancer cells SGC7901 and gastric cancer MDR (multidrug resistant) cells SGC7901/VCR, and to observe the drug sensitizing and proliferation effect of ZNRD1 antisense nucleic acid transduction on SGC7901/VCR cells . METHODS: Amplification of sequences encoding ZNRD1 from SGC7901/VCR cDNA by PCR . The levels of ZNRD1 mRNA expression were demonstrated using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) . Eukaryotic expression vector pcDNA3.1-anti ZNRD1 was constructed and transfected into SGC7901/VCR cells by lipofectamine . Immunochemical method was used to detect the expression of protein in SGC7901/VCR cells and transfectants . The cell cycle alteration and the intracellular adriamycin (ADM) accumulation were observed by FACS . Growth curve and drug sensitization of cells for vincristine (VCR) were analyzed with MTT assay . RESULTS: We cloned the open reading frame of full-length ZNRD1 . The expression of ZNRD1 showed higher in SGC7901/VCR than in SGC7901 cells . The antisense ZNRD1 drug-resistant clones were selected after gene transfection . Immunochemical results showed that the expression level of ZNRD1 protein was lower in anti ZNRD1-SGC7901/VCR cells than that in non-transfectants . Comparing to SGC7901/VCR and pcDNA3.1-SGC7901/VCR, anti ZNRD1-SGC7901/VCR showed gradually accumulated in G(1) phase, with a concomitant decrease of cell population in S phase . FACS also suggested intracellular ADM accumulation increased 2fold in SGC7901/VCR cells after transfected with antisense ZNRD1 . MTT assay showed that transfectants cells proliferation was lagged and more sensitive to VCR than non-transfectants . CONCLUSION: ZNRD1 gene displayed highly expression in VCR resistant gastric cancer cells . Expression of ZNRD1 protein was effectively blocked in anti ZNRD1-SGC7901/VCR cells by gene transfection . ZNRD1 antisense nucleic acid transfection sensitized drug resistant gastric cancer cells to VCR, increased ADM accumulation and inhibited the cells proliferation . ZNRD1 antisense RNA transduction could reverse the MDR of human drug-resistant gastric cancer cell SGC7901/VCR to a degree.

J Neurosci, 2003 Apr 15, 23(8), 3394 - 406
Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress; Shih AY et al.; Astrocytes have a higher antioxidant potential in comparison to neurons . Pathways associated with this selective advantage include the transcriptional regulation of antioxidant enzymes via the action of the Cap'n'Collar transcription factor Nrf2 at the antioxidant response element (ARE) . Here we show that Nrf2 overexpression can reengineer neurons to express this glial pathway and enhance antioxidant gene expression . However, Nrf2-mediated protection from oxidative stress is conferred primarily by glia in mixed cultures . The antioxidant properties of Nrf2-overexpressing glia are more pronounced than those of neurons, and a relatively small number of these glia (< 1% of total cell number added) could protect fully cocultured naive neurons from oxidative glutamate toxicity associated with glutathione (GSH) depletion . Microarray and biochemical analyses indicate a coordinated upregulation of enzymes involved in GSH biosynthesis (xCT cystine antiporter, gamma-glutamylcysteine synthetase, and GSH synthase), use (glutathione S-transferase and glutathione reductase), and export (multidrug resistance protein 1) with Nrf2 overexpression, leading to an increase in both media and intracellular GSH . Selective inhibition of glial GSH synthesis and the supplementation of media GSH indicated that an Nrf2-dependent increase in glial GSH synthesis was both necessary and sufficient for the protection of neurons, respectively . Neuroprotection was not limited to overexpression of Nrf2, because activation of endogenous glial Nrf2 by the small molecule ARE inducer, tert-butylhydroquinone, also protected against oxidative glutamate toxicity.

Indian J Chest Dis Allied Sci, 2003 Apr-Jun, 45(2), 97 - 103
Clinical characteristics and treatment response among patients with multidrug-resistant tuberculosis: a retrospective study; Subhash HS et al.; BACKGROUND: This retrospective study was conducted to evaluate the characteristics and therapeutic response among patients with multidrug-resistant tuberculosis (MDR TB) . METHODS: One hundred subjects with isolates resistant to isoniazid and rifampicin were included over a three-year period (1997-1999) . There were 82% males with a mean age of 36 years, mean duration of symptoms of 29 months, and a previous history of tuberculosis in 85% (pulmonary 96% and extrapulmonary 4%) . RESULTS: HIV ELISA test was positive in two out of 28 (7%) patients while diabetes was diagnosed in 16 percent . Mean time to diagnose MDR TB was 5.5 months . Subjects had received a mean of 3.2 anti-TB drugs before the diagnosis of MDR TB was made . Forty-five patients were lost to follow-up . The rest had a median follow-up of 13.5 months (range 1-37 months) . Follow-up AFB smear and culture results were available in 49 out of 55 and 26 out of 55 patients, respectively . Sputum smear became negative for AFB in 26 out of 49 (53%) and culture converison occurred in 16 out of 26 (61.5%) patients . Clinical and radiological response was noted in 31 (56%) and 13 (32.5%) out of 40 patients respectively . A mean of 5.5 drugs was used in those who achieved sputum conversion . Combination therapy containing ofloxacin in the regimen was noted to have a favourable response . CONCLUSION: Only a limited number of patients with MDR tuberculosis have a favourable response.

Rev Soc Bras Med Trop, 2003 Jan-Feb, 36(1), 27 - 34 Epub 2003 Apr 22.
{Epidemiological features of multidrug-resistant tuberculosis in a reference service in São Paulo city}; de Melo FA et al.; In order to study certain epidemiological features of multidrug-resistant tuberculosis (MDR-TB) carriers and their influence on the control and treatment, a group of patients was evaluated over a four-year period, selected by: Mycobacterium tuberculosis isolation from sputum; resistance to Rifampin, Isoniazid and one more drug, or, failure of reserve regimen, all cases were from a tuberculosis reference unit in the City of S o Paulo . A total of 182 patients were reviewed, with a mean age of 35.7 +/- 6.8 years and 112 (61.5%) were male . These patients was classified according to therapeutic history, as: primary MDR-TB (with initial sensitivity test) 11 (6%); post primary MDR-TB (after irregular use previous treatment) 134 (74%), and indeterminate MDR-TB (failure after regular use of initial and reserve regimens) 37 (20%) . Contagion was identified in 41/170 patients, acquired through domiciliary rather than institutional transmission . There were four familial outbreaks and none were institutional . The most frequent condition associated with these cases was abandonment of therapy (45%) followed by alcoholism (27%), sequential failure in the treatment regimens (23%), MDR contagion (15%), drug reaction (6%), HIV positive (4%) and diabetes (3%) . There was resistance to Rifampin+Isoniazid in 100%, Streptomycin in 83% and Ethambutol in 47% . Conventional X-ray revealed cavities in all, though only 35 (19%) were unilateral . These cases are discussed and some suggestions presented.

Rev Soc Bras Med Trop, 2003 Jan-Feb, 36(1), 5 - 9 Epub 2003 Apr 22.
In vitro evaluation of verapamil and other modulating agents in Brazilian chloroquine-resistant Plasmodium falciparum isolates; Menezes CM et al.; Verapamil, was assayed to record its modulating effect upon Brazilian Plasmodium falciparum isolates resistant to chloroquine . Other cardiovascular drugs known to be modulating agents in resistant malaria and/or multidrug-resistant neoplasias, including nifedipine, nitrendipine, diltiazem and propranolol, were also evaluated . Concentrations similar to those for cardiovascular therapy were used in the in vitro microtechnique for antimalarial drug susceptibility . Intrinsic antiplasmodial activity was observed from the lowest concentrations without a significant modulating action . Other reported modulating agents, such as the antipsychotic drug trifluoperazine and the antidepressants desipramine and imipramine, demonstrated similar responses under the same experimental conditions . Results suggest a much higher susceptibility of Brazilian strains, as well as an indifferent behaviour in relation to modulating agents.

Q J Nucl Med, 2003 Mar, 47(1), 58 - 62
Tetrofosmin as predictors of tumour response; Fuster D et al.; Non-invasive imaging methods in the evaluation of chemotherapy response in malignant tumours are currently being explored . Standard Nuclear Medicine procedures seem to offer the clinician a promising tool in the management of those oncologic patients, who might benefit from chemotherapy . Early studies focused on the relationship between radionuclides used in tumour diagnosis and factors associated with multidrug resistance (MDR) . The tumour expression of P-glycoprotein (Pgp) and multidrug resistance-related protein-1 expression (MRP) have been suggested as important factors in the failure of chemotherapy . Most studies found an association between Pgp levels and (99m)Tc-sestamibi ((99m)Tc-MIBI) or (99m)Tc-Tetrofosmin uptake ((99m)Tc-TF) . Currently investigations in nuclear medicine oncology are focusing on the potential role of radionuclide imaging in the assessment of chemotherapy . Recent papers discuss the usefulness of radionuclides as (99m)Tc-MIBI and (99m)Tc-TF as non-invasive procedures to predict and to monitor therapy response in patients affected by malignant tumours treatable using chemotherapy . This chapter will review the latest development in (99m)Tc-TF, giving an overview of recent investigations carried out using this radiotracer in therapy oncology, with emphasis on its potential role as predictor of tumour response.

Q J Nucl Med, 2003 Mar, 47(1), 46 - 50
MIBI as prognostic factor in breast cancer; Del Vecchio S et al.; Evaluation of treatment response is of primary importance in the management of patients with cancer . Both positron- and g-emitting compounds have been used to monitor changes in tumor metabolism or viability after therapy . The use of (99m)Tc-labeled lipophilic cations raised the possibility to predict the tumor response to treatment and to identify patients who will become refractory to subsequent therapy . In particular, many studies have shown the prognostic value of (99m)Tc-MIBI scan in different types of malignancy including breast and lung cancer, lymphoma and sarcoma . The ability of (99m)Tc-MIBI to interact with P-glycoprotein, allowing the functional assessment of the multidrug resistant phenotype, is one of the mechanisms underlying its prognostic value . Additional mechanisms of cell resistance, mainly involving alterations of apoptosis, may affect (99m)Tc-MIBI uptake in tumors . Therefore, either an enhanced tracer clearance or a reduced early uptake of (99m)Tc-MIBI indicate a poor response to therapy . In both cases, (99m)Tc-MIBI scan may ensure that the further management strategy will be effective in individual cancer patients.

Chemotherapy, 2003 May, 49(1-2), 8 - 16
Differential effect of HIV-1 protease inhibitors on P-glycoprotein function in multidrug-resistant variants of the human CD4+ T lymphoblastoid CEM cell line; Dupuis ML et al.; BACKGROUND: P-glycoprotein causing multidrug resistance (MDR) and limiting the efficacy of antineoplastic drugs and protease inhibitors (PIs) is expressed in human CD4+ T lymphocytes, one of the main targets of HIV, in a range of pharmacological barriers and at varying degrees in non-Hodgkin's lymphoma and Kaposi's sarcoma . METHODS: The differential effect of PIs on P-glycoprotein function was studied by measuring drug efflux inhibition, MDR-reversing ability and MAb UIC2 epitope modulation in MDR variants of the human T lymphoblastoid CEM cell line . RESULTS: The treatment of MDR cells with PIs induces different UIC2 epitope modulations indicating a differential recognition and binding of these antiviral drugs by MDR1 P-glycoprotein . In fact, ritonavir, saquinavir and indinavir act differently to the P-glycoprotein blocker in CEM-VBL10 cells . The MDR level of these cells was markedly affected by ritonavir and saquinavir in the order, while the PI indinavir does not seem to compete with the P-glycoprotein drug transport function . In CEM-VBL100 cells, expressing a very high number of P-glycoprotein molecules, only ritonavir acts as an efficient drug efflux inhibitor and MDR-reversing agent . CONCLUSION: The HIV-1 PIs ritonavir and saquinavir even at different levels act as genuine P-glycoprotein substrates by inhibiting dye substrate efflux, modulating UIC2 epitope and reversing drug resistance . Conversely, at least in the in vitro system used in the present study, the PI indinavir does not significantly alter P-glycoprotein drug transport activities and function .

J Nat Prod, 2003 Apr, 66(4), 572 - 4
Absolute configuration and complete assignment of 13C NMR data for new sesquiterpenes from Maytenus chiapensis; Nunez MJ et al.; Five new dihydro-beta-agarofuran sesquiterpenes (1-5) were isolated from the leaves of Maytenus chiapensis . The structures of 1-5 were determined by means of 1D and 2D NMR techniques . A semiselective HMBC technique was applied in order to obtain complete (13)C NMR assignments . Absolute configurations were determined by CD studies and chemical correlations and on biogenetic grounds . Compound 4 showed weak activity against a multidrug-resistant Leishmania tropica line.

Int J Cancer, 2003 Jul 1, 105(4), 472 - 9
The role of RAD51 in etoposide (VP16) resistance in small cell lung cancer; Hansen LT et al.; Etoposide (VP16) is a potent inducer of DNA double-strand breaks (DSBs) and is efficiently used in small cell lung cancer (SCLC) therapy . However, acquired VP16 resistance remains an important barrier to effective treatment . To understand the underlying mechanisms for VP16 resistance in SCLC, we investigated DSB repair and cellular VP16 sensitivity of SCLC cells . VP16 sensitivity and RAD51, DNA-PK(cs), topoisomerase IIalpha and P-glycoprotein protein levels were determined in 17 SCLC cell lines . In order to unravel the role of RAD51 in VP16 resistance, we cloned the human RAD51 gene, transfected SCLC cells with RAD51 sense or antisense constructs and measured the VP16 resistance . Finally, we measured VP16-induced DSBs in the 17 SCLC cell lines . Two cell lines exhibited a multidrug-resistant phenotype . In the other SCLC cell lines, the cellular VP16 resistance was positively correlated with the RAD51 protein level . In addition, downregulation or overexpression of the RAD51 gene altered the VP16 sensitivity . Furthermore, the levels of the RAD51 and DNA-PK(cs) proteins were related to VP16-induced DSBs . The results suggest that repair of VP16-induced DSBs is mediated through both RAD51-dependent homologous recombination and DNA-PK(cs)-dependent nonhomologous end-joining and may be a determinant of the variation in clinical treatment effect observed in human SCLC tumors of identical histologic subtype . Finally, we propose RAD51 as a potential target to improve VP16 efficacy and predict tumor resistance in the treatment of SCLC patients .

Cancer Control, 2003 Mar-Apr, 10(2), 159 - 65
Overcoming multidrug resistance in cancer: an update on the clinical strategy of inhibiting p-glycoprotein; Thomas H et al.; BACKGROUND: Multidrug resistance (MDR) is a significant obstacle to providing effective chemotherapy to many patients . Multifactorial in etiology, classic MDR is associated with the overexpression of P-glycoprotein (P-gp), resulting in increased efflux of chemotherapy from cancer cells . Inhibiting P-gp as a method to reverse MDR in cancer patients has been studied extensively, but the results have generally been disappointing . METHODS: The development of P-gp inhibitors is reviewed, including a discussion of early agents that are no longer being developed and third-generation agents that are currently in clinical trials . RESULTS: First-generation agents (eg, cyclosporin, verapamil) were limited by unacceptable toxicity, whereas second-generation agents (eg, valspodar, biricodar) had better tolerability but were confounded by unpredictable pharmacokinetic interactions and interactions with other transporter proteins . Third-generation inhibitors (tariquidar XR9576, zosuquidar LY335979, laniquidar R101933, and ONT-093) have high potency and specificity for P-gp . Furthermore, pharmacokinetic studies to date have shown no appreciable impact on cytochrome P450 3A4 drug metabolism and no clinically significant drug interactions with common chemotherapy agents . CONCLUSIONS: Third-generation P-gp inhibitors have shown promise in clinical trials . The continued development of these agents may establish the true therapeutic potential of P-gp-mediated MDR reversal.

J Biol Chem, 2003 Jun 6, 278(23), 20449 - 52 Epub 2003 Apr 23.
Permanent activation of the human P-glycoprotein by covalent modification of a residue in the drug-binding site; Loo TW et al.; The human multidrug resistance P-glycoprotein (ABCB1) transports a broad range of structurally diverse compounds out of the cell . The transport cycle involves coupling of drug binding in the transmembrane domains with ATP hydrolysis . Compounds such as verapamil stimulate ATPase activity . We used cysteine-scanning mutagenesis of the transmembrane segments and reaction with the thiol-reactive substrate analog of verapamil, methanethiosulfonate (MTS)-verapamil, to test whether it caused permanent activation of ATP hydrolysis . Here we report that one mutant, I306C(TM5) showed increased ATPase activity (8-fold higher than untreated) when treated with MTS-verapamil and isolated by nickel-chelate chromatography . Drug substrates that either enhance (calcein acetoxymethyl ester, demecolcine, and vinblastine) or inhibit (cyclosporin A and trans-(E)-flupentixol) ATPase activity of Cys-less or untreated mutant I306C P-glycoprotein did not affect the activity of MTS-verapamil-treated mutant I306C . Addition of dithiothreitol released the covalently attached verapamil, and ATPase activity returned to basal levels . Pretreatment with substrates such as cyclosporin A, demecolcine, verapamil, vinblastine, or colchicine prevented activation of mutant I306C by MTS-verapamil . The results suggest that MTS-verapamil reacts with I306C in a common drug-binding site . Covalent modification of I306C affects the long range linkage between the drug-binding site and the distal ATP-binding sites . This results in the permanent activation of ATP hydrolysis in the absence of transport . Trapping mutant I306C in a permanently activated state indicates that Ile-306 may be part of the signal to switch on ATP hydrolysis when the drug-binding site is occupied.

Biochem Biophys Res Commun, 2003 May 2, 304(2), 260 - 5
New phenothiazine-type multidrug resistance modifiers: anti-MDR activity versus membrane perturbing potency; Hendrich AB et al.; The phenothiazine multidrug resistance (MDR) modulators are chemically diversified but share the common feature to be hydrophobic cationic molecules . Molecular mechanisms of their action may involve interactions with either P-glycoprotein or membrane lipid matrix . In the present work we study the anti-MDR and biophysical membrane effects of new phenothiazine derivatives differing in the type of group substituting phenothiazine ring at position 2 (H-, Cl-, CF(3)-) and in the side chain group (NHCO(2)CH(3) or NHSO(2)CH(3)) . Within each phenothiazine subset we found that anti-MDR activity (determined by P-glycoprotein inhibition assessed by flow cytometry) correlates with the theoretically calculated hydrophobicity value (logP) and experimental parameters (determined by calorimetry and fluorescence spectroscopy) of lipid bilayers . It is concluded that the biological and biophysical activity of phenothiazine derivatives depends more on the type of ring substitution than on the nature of the side chain group.

Med Res Rev, 2003 Jul, 23(4), 519 - 34
Flavonoids: promising anticancer agents; Ren W et al.; Flavonoids are polyphenolic compounds that are ubiquitously in plants . They have been shown to possess a variety of biological activities at nontoxic concentrations in organisms . The role of dietary flavonoids in cancer prevention is widely discussed . Compelling data from laboratory studies, epidemiological investigations, and human clinical trials indicate that flavonoids have important effects on cancer chemoprevention and chemotherapy . Many mechanisms of action have been identified, including carcinogen inactivation, antiproliferation, cell cycle arrest, induction of apoptosis and differentiation, inhibition of angiogenesis, antioxidation and reversal of multidrug resistance or a combination of these mechanisms . Based on these results, flavonoids may be promising anticancer agents .

Antimicrob Agents Chemother, 2003 May, 47(5), 1719 - 26
Multiple resistance mechanisms among Aspergillus fumigatus mutants with high-level resistance to itraconazole; Nascimento AM et al.; A collection of Aspergillus fumigatus mutants highly resistant to itraconazole (RIT) at 100 micro g ml(-1) were selected in vitro (following UV irradiation as a preliminary step) to investigate mechanisms of drug resistance in this clinically important pathogen . Eight of the RIT mutants were found to have a mutation at Gly54 (G54E, -K, or -R) in the azole target gene CYP51A . Primers designed for highly conserved regions of multidrug resistance (MDR) pumps were used in reverse transcriptase PCR amplification reactions to identify novel genes encoding potential MDR efflux pumps in A . fumigatus . Two genes, AfuMDR3 and AfuMDR4, showed prominent changes in expression levels in many RIT mutants and were characterized in more detail . Analysis of the deduced amino acid sequence encoded by AfuMDR3 revealed high similarity to major facilitator superfamily transporters, while AfuMDR4 was a typical member of the ATP-binding cassette superfamily . Real-time quantitative PCR with molecular beacon probes was used to assess expression levels of AfuMDR3 and AfuMDR4 . Most RIT mutants showed either constitutive high-level expression of both genes or induction of expression upon exposure to itraconazole . Our results suggest that overexpression of one or both of these newly identified drug efflux pump genes of A . fumigatus and/or selection of drug target site mutations are linked to high-level itraconazole resistance and are mechanistic considerations for the emergence of clinical resistance to itraconazole.

Antimicrob Agents Chemother, 2003 May, 47(5), 1636 - 42
Shiga-like toxin II impairs hepatobiliary transport of doxorubicin in rats by down-regulation of hepatic P glycoprotein and multidrug resistance-associated protein Mrp2; Hidemura K et al.; We investigated the effect of Shiga-like toxin II (SLT-II), derived from Escherichia coli O157:H7, on the hepatobiliary excretion of doxorubicin, a substrate for P glycoprotein and the multidrug resistance-associated protein Mrp2, and on the expression of P glycoprotein and Mrp2 in rats . Histopathological examination did not show any liver injury in SLT-II-treated rats . A significant delay in the disappearance of doxorubicin from plasma after its intravenous administration (5 mg/kg of body weight) was observed in rats treated 24 h earlier with SLT-II (2 micro g/animal) . When rats received an infusion of doxorubicin (2.6 micro g/min) 24 h after intravenous injection of SLT-II, the steady-state concentration of doxorubicin in plasma increased and the bile flow decreased, whereas the concentration in liver did not alter . SLT-II significantly increased the unbound fraction of doxorubicin in plasma but did not alter the concentration in liver tissue . SLT-II significantly decreased the biliary excretion rate and biliary clearance of doxorubicin based on the total concentration and concentration of the unbound fraction in plasma and liver . Western blot analysis revealed that SLT-II down-regulated P glycoprotein and Mrp2 in the liver, which could explain the observed decrease in the biliary excretion of doxorubicin by SLT-II . A tumor necrosis factor alpha (TNF-alpha) production inhibitor, pentoxifylline, could not protect SLT-II-induced decreases in the biliary clearance of doxorubicin and down-regulation of both transporters . It is unlikely that TNF-alpha plays a major role in the SLT-II-induced decrease in the hepatobiliary transport of doxorubicin and the down-regulation of both transporters.

Cancer Sci, 2003 Jan, 94(1), 81 - 6
Effects of carvedilol on MDR1-mediated multidrug resistance: comparison with verapamil; Kakumoto M et al.; The reversing effects of carvedilol and other beta-adrenoceptor antagonists on multidrug resistance (MDR) were assessed in HeLa cells and the MDR1-overexpressing derivative Hvr100-6 cells, established by stepwise increases of vinblastine concentration in the culture medium . The inhibitory effects on the transcellular transport and intracellular accumulation of {3H}vinblastine and {3H}daunorubicin were also assessed using LLC-GA5-COL150 cell monolayers, established by transfection of human MDR1 cDNA into porcine kidney epithelial LLC-PK1 cells . The cytotoxic effects of vinblastine, paclitaxel, doxorubicin and daunorubicin in Hvr100-6 were reversed 1.4- to 7.1-fold by carvedilol at the realistic clinical concentration of 1 microM, whereas other beta-adrenoceptor antagonists had weaker or no such effects . Transport experiments using LLC-GA5-COL150 cell monolayers demonstrated that this effect of carvedilol was due to the inhibition of MDR1-mediated transport of vinblastine, paclitaxel, doxorubicin and daunorubicin . These MDR1-mediated reversing effects of carvedilol were similar to those of 1 microM verapamil, suggesting that carvedilol could be a candidate modulator of MDR in clinical use . Since other beta-adrenoceptor antagonists had no inhibitory effect on transport, the effects of carvedilol were not related to beta-adrenoceptors and might have been due to antioxidant activity.

Cancer Sci, 2003 Jan, 94(1), 15 - 21
Molecular targeting therapy of cancer: drug resistance, apoptosis and survival signal; Tsuruo T et al.; Recent progress in the development of molecular cancer therapeutics has revealed new types of antitumor drugs, such as Herceptin, Gleevec, and Iressa, as potent therapeutics for specific tumors . Our work has focused on molecular cancer therapeutics, mainly in the areas of drug resistance, apoptosis and apoptosis resistance, and survival-signaling, which is related to drug resistance . In this review, we describe our research on molecular cancer therapeutics, including molecular mechanisms and therapeutic approaches . Resistance to chemotherapeutic drugs is a principal problem in the treatment of cancer . P-Glycoprotein (P-gp), encoded by the MDR1 gene, is a multidrug transporter and has a major role in multidrug resistance (MDR) . Targeting of P-gp by small-molecular compounds and/or antibodies is an effective strategy to overcome MDR in cancer, especially hematologic malignancies . Several P-gp inhibitors have been developed and are currently under clinical phased studies . In addition to the multidrug transporter proteins, cancer cells have several drug resistance mechanisms . Solid tumors are often placed under stress conditions, such as glucose starvation and hypoxia . These conditions result in topo II poison resistance that is due to proteasome-mediated degradation of DNA topoisomerases . Proteasome inhibitors effectively prevent this stress-induced drug resistance . Glyoxalase I, which is often elevated in drug- and apoptosis-resistant cancers, offers another possibility for overcoming drug resistance . It plays a role in detoxification of methylglioxal, a side product of glycolysis, which is highly reactive with DNA and proteins . Inhibitors of glyoxalase I selectively kill drug-resistant tumors that express glyoxalase I . Finally, the susceptibility of tumor cells to apoptosis induced by antitumor drugs appears to depend on the balance between pro-apoptotic and survival (anti-apoptotic) signals . PI3K-Akt is an important survival signal pathway, that has been shown to be the target of various antitumor drugs, including UCN-01 and geldanamycin, new anticancer drugs under clinical evaluation . Our present studies provide novel targets for future effective molecular cancer therapeutics.

Gene, 2003 Mar 27, 307, 41 - 50
The Drosophila melanogaster multidrug-resistance protein 1 (MRP1) homolog has a novel gene structure containing two variable internal exons; Grailles M et al.; Drosophila melanogaster has a gene very similar to human MRP1 that encodes a full ABC-transporter containing three membrane-spanning domains and two nucleotide-binding domains . This 19 exon insect gene, dMRP (FBgn0032456), spans slightly more than 22 kb . The cDNA SD07655 representing this gene was sequenced and found to contain sequences from 12 exons including single copies of two exons having multiple genomic copies . The gene contains two variant copies of exon 4 and seven of exon 8 . While a cDNA contains only one version of each variable exon, all forms of these variable exons were detected in adult fly mRNA . These results predict that Drosophila could make 14 different MRP isoforms from a single gene by substituting different variable exons . This is the first report of any organism using differential splicing of alternative, internal exons, to produce such a large array of MRP isoforms having the same size, but with limited and defined internal variations . Defining the functional differences in the dMRP isoforms should provide clues to the structure/function relationships of the amino acids in these MRP domains, both for the insect enzyme and for those of other species.

Adv Drug Deliv Rev, 2003 Apr 29, 55(5), 653 - 65
Xenobiotic transporter expression and function in the human mammary gland; Ito S et al.; Xenobiotic transport in the mammary gland has tremendous clinical, toxicological and nutritional implications . Mechanisms such as passive diffusion, carrier-mediated transport, and transcytosis mediate xenobiotic transfer into milk . In vivo animal and human studies suggest the functional expression of both xenobiotic and nutrient transporters in the lactating mammary gland and the potential involvement of such systems in the significant accumulation of certain compounds in milk . In vitro cell culture systems provide further evidence for carrier-mediated transport across the lactating mammary epithelium . Additionally, molecular characterization studies indicate the expression of various members of the organic cation transporter, organic anion transporter, organic anion polypeptide transporter, oligopeptide transporter, nucleoside and nucleobase transporter, multidrug resistant transporter, and multidrug resistant-like protein transporter families at the lactating mammary epithelium . The in vivo relevance of the expression of such xenobiotic and nutrient transporters and their involvement in drug disposition at the mammary gland requires investigation.

Eur J Cancer, 2003 May, 39(7), 909 - 16
Expression of multidrug resistance proteins, P-gp, MRP1 and LRP, in soft tissue sarcomas analysed according to their histological type and grade; Komdeur R et al.; The biological behaviour of different histological types and grades of soft tissue sarcomas (STS) varies . This might result in a differing sensitivity to cytotoxic drugs . Cross-resistance to functionally and structurally distinct natural-product drugs, known as multidrug resistance (MDR), is associated with the overexpression of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and lung resistance-related protein (LRP) . The purpose of this study was to evaluate the expression of P-gp, MRP1 and LRP in STS according to their histological type and grade . In 141 chemotherapy-naive STS patients, the expression of the three MDR proteins was detected by immunohistochemistry . Nine histological types were documented . These were 19% grade 1, 34% grade 2 and 47% grade 3 tumours . Expression of P-gp and LRP was observed more frequently than the expression of MRP1 (P<0.0001) . P-gp expression was most pronounced in malignant fibrous histiocytoma (MFH), but was low in leiomyosarcomas . MRP1 was expressed in most malignant peripheral nerve sheath tumours (MPNST) . LRP was strongly expressed in MFH and unspecified sarcomas, but was low in liposarcomas . MRP1 and LRP expression was significantly more common in grades 2 and 3 compared with grade 1 tumours . P-gp expression was correlated with MRP1, especially in grade 3 STS . In conclusion, P-gp, MRP1 and LRP are expressed in the majority of STS, but this expression varies according to the histological type . MRP1 and LRP, but not P-gp expression, were found to be correlated to tumour grade . MDR might contribute to the observed differences in clinical behaviour within the heterogeneous group of STS.

Arch Biochem Biophys, 2003 May 1, 413(1), 32 - 40
Taurine supplementation induces multidrug resistance protein 2 and bile salt export pump expression in rats and prevents endotoxin-induced cholestasis; Muhlfeld A et al.; The effect of oral taurine supplementation on endotoxin-induced cholestasis was investigated in rat liver . At 12h following lipopolysaccharide (LPS) injection (4mg/kg body weight i.p.) bile flow and bromosulfophthalein (BSP) and taurocholate (TC) excretion were determined in the perfused liver and the expression of the canalicular transporters multidrug resistance protein 2 (Mrp2) and bile salt export pump (Bsep) was analyzed . Injection of LPS induced a significant decrease of bile flow ( 2.2+/-0.2 microl/g liver wet weight/min vs 3.3+/-0.1 microl/g liver wet weight in controls), biliary BSP excretion (10.8+/-2.2 nmol/g/min vs 21.0+/-3.8 nmol/g/min), and biliary TC excretion (114+/-23 nmol/g/min vs 228+/-8 nmol/g/min) . These effects were due to transporter retrieval from the canalicular membrane and downregulation of Mrp2 and Bsep expression . In taurine-supplemented rats bile flow was 30% higher than that in untreated rats and the expression of Mrp2 and Bsep protein was increased two- to threefold . In taurine-supplemented rats there was no significant reduction of bile flow or of BSP and TC excretion at 12h following LPS injection . This protective effect of taurine was due to higher Mrp2 and Bsep protein levels compared to nonsupplemented LPS-treated rats, whereas relative Mrp2 retrieval from the canalicular membrane induced by LPS was not significantly different . LPS-induced tumor necrosis factor alpha and interleukin-1beta release were lower in taurine-fed rats; however, downregulation of Mrp2 and Bsep expression by LPS was delayed but not prevented . The data show that oral supplementation of taurine induces Mrp2 and Bsep expression and may prevent LPS-induced cholestasis.

Brain Res, 2003 May 9, 971(2), 221 - 31
Characterisation of the brain multidrug resistance protein (BMDP/ABCG2/BCRP) expressed at the blood-brain barrier; Eisenblatter T et al.; The blood-brain barrier (BBB) plays the predominant role in controlling the passage of endogenous and xenobiotic substances between the circulating blood and the extracellular fluid environment of the brain . The transfer of compounds is strictly regulated by brain capillary endothelial cells (BCEC), which are interconnected to each other by well developed tight junctions, without fenestrations . Although hydrophobic molecules such as nicotine and ethanol readily cross the BBB by diffusion, the brain microvasculature shows a highly restrictive permeability to hydrophobic antitumor agents . So far, this multidrug resistance has been almost exclusively attributed to the most prominent member of the ATP-binding cassette (ABC) transporter family, P-glycoprotein located in the luminal membrane of brain capillary endothelial cells and to a minor extent to the multidrug resistance-associated proteins (MRPs) . The brain multidrug resistance protein (BMDP) has recently been discovered at the porcine BBB and was shown to be highly homologous to the human breast cancer resistance protein (BCRP/ABCG2) . Here, we demonstrate by northern blot and RT-PCR analysis that BMDP mRNA is more highly expressed in the capillary endothelial cells compared to other cell types of the brain . Immunocytochemistry of porcine BCEC showed a clear plasma membrane localisation of BMDP . Analysis of the total mRNA pool revealed that BMDP is more strongly expressed than P-glycoprotein and MRP1 . Consistently, first transport studies indicate that active exclusion of the chemotherapeutic drug daunorubicin from the central nervous system is mediated mainly by this new transporter compared to P-glycoprotein or MRP1 . Thus, we hypothesise that BMDP might play an important role in the exclusion of xenobiotics from the porcine brain.

Intern Med, 2003 Mar, 42(3), 237 - 43
Molecular cancer therapeutics: recent progress and targets in drug resistance; Tsuruo T; Recent progress in development of molecular cancer therapeutics revealed new types of antitumor drugs, such as Herceptin, Gleevec, and Iressa as potent therapeutics for each specific tumor . We have been working on molecular cancer therapeutics, and in particular, those related to drug resistance, Here, I describe several resistance mechanisms, including apoptosis regulation, cellular stress response and cellular survival signals which have show close relevance to drug resistance . P-glycoprotein (P-gp) is the key molecule in multidrug resistance (MDR) and a good target for chemotherapy . Proteasome is involved in the resistance mechanism to topo II-targeted chemotherapy in solid tumors . Apoptosis program in tumor cells plays a critical role in chemotherapy-induced tumor cell killing, and the blockade of the apoptosis-inducing pathway could be another mechanism for drug resistance . Glyoxalase I is a molecule involved in apoptosis resistance mechanism in tumors . Survival (antiapoptosis) signals are the good targets for various antitumor drugs to overcome innate drug resistance . Our present studies provide novel targets for effective molecular cancer therapeutics in future.

Schweiz Rundsch Med Prax, 2003 Mar 26, 92(13), 601 - 5
{25-year-old therapy naive patient with 3 class resistant HIV infection}; Hasse B et al.; The wide use of potent antiretroviral therapy has significantly reduced mortality and morbidity in patients infected with the HI-Virus . Good individual tolerance and regular intake of an adequate drug regimen often lead to a substantial recovery of impaired immunologic function . In a substantial fraction of patients drug resistant virus is emerging . In the last decade transmission of such viruses to newly infected patients has been reported in several countries . Especially transmission of multidrug-resistant virus is of major concern . We report a case of a 25 year-old Swiss man with a newly diagnosed HIV-infection with a multidrug-resistant virus . In connection to this case we discuss the importance of resistance testing in newly diagnosed patients.

J Biol Chem, 2003 Jun 27, 278(26), 23529 - 37 Epub 2003 Apr 19.
Differential modulation of the human liver conjugate transporters MRP2 and MRP3 by bile acids and organic anions; Bodo A et al.; The multidrug resistance proteins MRP2 (ABCC2) and MRP3 (ABCC3) are key primary active transporters involved in anionic conjugate and drug extrusion from the human liver . The major physiological role of MRP2 is to transport conjugated metabolites into the bile canaliculus, whereas MRP3 is localized in the basolateral membrane of the hepatocytes and transports similar metabolites back to the bloodstream . Both proteins were shown to interact with a large variety of transported substrates, and earlier studies suggested that MRPs may work as co-transporters for different molecules . In the present study we expressed the human MRP2 and MRP3 proteins in insect cells and examined their transport and ATPase characteristics in isolated, inside-out membrane vesicles . We found that the primary active transport of estradiol-17-beta-d-glucuronide (E217betaG), a major product of human steroid metabolism, was differently modulated by bile acids and organic anions in the case of human MRP2 and MRP3 . Active E217betaG transport by MRP2 was significantly stimulated by the organic anions indomethacin, furosemide, and probenecid and by several conjugated bile acids . In contrast, all of these agents inhibited E217betaG transport by MRP3 . We found that in the case of MRP2, ATP-dependent vesicular bile acid transport was increased by E217betaG, and the results indicated an allosteric cross-stimulation, probably a co-transport of bile acids and glucuronate conjugates through this protein . There was no such stimulation of bile acid transport by MRP3 . In conclusion, the different transport modulation of MRPs by bile acids and anionic drugs could play a major role in regulating physiological and pathological metabolite fluxes in the human liver.

Ai Zheng, 2003 Apr, 22(4), 441 - 4
{Drug-resistant proteins in breast cancer: recent progress in multidrug resistance}; Wu DL et al.; Breast cancer resistance protein (BCRP) is a new multidrug resistance-related transmembrane transporter . BCRP is a 655-amino acid, 72.6 kDa protein, localized in the plasma membrane . As a member of the ATP-binding cassette family of drug transporters, BCRP has only one ATP-binding cassette and six putative transmembrane domains, suggesting that BCRP is a half-transporter, which may function as a homodimer or heterodimer . The BCRP-overexpressing tumor cells are resistant to mitoxantrone, adriamycin, daunorubicin, etoposide, topotecan and irinotecan, but lack resistance to paclitaxel and vincristine . Fumitremorgin C and GF120918 can effectively reverse multidrug resistance in BCRP-overexpressing tumor cells, associated with an increase in drug accumulation . In normal human tissues, low to high expressions of BCRP in placental syncytiotrophoblasts, in the epithelium of the small intestine and colon, in the liver canalicular membrane, in ducts of the breast, in endothelium of the blood vessel and in stem cells were reported . This expression profile allows speculation on a role of BCRP in protection of the fetus and in the regulation of transport of chemicals through the epithelium of the gastrointestinal tract . BCRP can account for chemoresistance of some clinical cancers such as acute myeloid leukemia, non-small cell lung cancer,and breast cancer.

An Med Interna, 2003 Feb, 20(2), 91 - 100
{Current treatment of tuberculosis}; Garcia Ramos R et al.; Tuberculosis (TB) is a growing national and international health concern today . A revision of pharmacological treatment of TB is done in this issue . Regimens for TB disease and latent TB treatment are described . Common adverse reactions and drug interactions of first and second-line antituberculosis drug are summarized in tables . Management strategies to improve treatment adherence and TB control, treat special situations like immunodeficiences, pregnancy, hepatic and renal impairment and multidrug resistant TB are presented . In a next future the prospects for using more effective and shorter new treatments to fight against this disease are not very promising.

J Biol Chem, 2003 Jun 27, 278(26), 23538 - 44 Epub 2003 Apr 17.
Evidence for two interacting ligand binding sites in human multidrug resistance protein 2 (ATP binding cassette C2); Zelcer N et al.; Multidrug resistance protein 2 (MRP2) belongs to the ATP binding cassette family of transporters . Its substrates include organic anions and anticancer drugs . We have used transport assays with vesicles derived from Sf9 insect cells overproducing MRP2 to study the interactions of drugs, organic anions, and bile acids with three MRP2 substrates: estradiol-17-beta-d-glucuronide (E217betaG), methotrexate, and glutathione-S-dinitrophenol . Complex inhibition and stimulation patterns were obtained, different from those observed with the related transporters MRP1 and MRP3 . In contrast to a previous report, we found that the rate of E217betaG transport by MRP2 increases sigmoidally with substrate concentration indicative of homotropic cooperativity . Half-maximal transport was obtained at 120 microm E217betaG, in contrast to values < 20 microm for MRP1 and 3 . MRP2 stimulators, such as indomethacin and sulfanitran, strongly increased the affinity of MRP2 for E217betaG (half-maximal transport rates at 65 and 16 microm E217betaG, respectively) and shifted the sigmoidal dependence of transport rate on substrate concentration to a more hyperbolic one, without substantially affecting the maximal transport rate . Sulfanitran also stimulated MRP2 activity in cells, i.e . the transport of saquinavir through monolayers of Madin-Darby canine kidney II cells . Some compounds that stimulate E217betaG transport, such as penicillin G or pantoprazole, are not detectably transported by MRP2, suggesting that they allosterically stimulate transport without being cotransported with E217betaG . We propose that MRP2 contains two similar but nonidentical ligand binding sites: one site from which substrate is transported and a second site that regulates the affinity of the transport site for the substrate.

Cancer Res, 2003 Apr 15, 63(8), 1838 - 45
HTI-286, a synthetic analogue of the tripeptide hemiasterlin, is a potent antimicrotubule agent that circumvents P-glycoprotein-mediated resistance in vitro and in vivo; Loganzo F et al.; Hemiasterlin is a natural product derived from marine sponges that, like other structurally diverse peptide-like molecules, binds to the Vinca-peptide site in tubulin, disrupts normal microtubule dynamics, and, at stoichiometric amounts, depolymerizes microtubules . Total synthesis of hemiasterlin and its analogues has been accomplished, and optimal pharmacological features of the series have been explored . The biological profile of one analogue, HTI-286, was studied here . HTI-286 inhibited the polymerization of purified tubulin, disrupted microtubule organization in cells, and induced mitotic arrest, as well as apoptosis . HTI-286 was a potent inhibitor of proliferation (mean IC(50) = 2.5 +/- 2.1 nM in 18 human tumor cell lines) and had substantially less interaction with multidrug resistance protein (P-glycoprotein) than currently used antimicrotubule agents, including paclitaxel, docetaxel, vinorelbine, or vinblastine . Resistance to HTI-286 was not detected in cells overexpressing the drug transporters MRP1 or MXR . In athymic mice implanted with human tumor xenografts, HTI-286 administered i.v . in saline inhibited the growth of numerous human tumors derived from carcinoma of the skin, breast, prostate, brain, and colon . Marked tumor regression was observed when used on established tumors that were >1 gram in size . Moreover, HTI-286 inhibited the growth of human tumor xenografts (e.g., HCT-15, DLD-1, MX-1W, and KB-8-5) where paclitaxel and vincristine were ineffective because of inherent or acquired resistance associated with P-glycoprotein . Efficacy was also achieved with p.o . administration of HTI-286 . These data suggest that HTI-286 has excellent preclinical properties that may translate into superior clinical activity, as well as provide a useful synthetic reagent to probe the drug contact sites of peptide-like molecules that interact with tubulin.

Am J Physiol Gastrointest Liver Physiol, 2003 Aug, 285(2), G449 - 59 Epub 2003 Apr 17.
Estradiol-17beta-D-glucuronide induces endocytic internalization of Bsep in rats; Crocenzi FA et al.; Endocytic internalization of the multidrug resistance-associated protein 2 (Mrp2) was previously suggested to be involved in estradiol-17beta-D-glucuronide (E217G)-induced cholestasis . Here we evaluated in the rat whether a similar phenomenon occurs with the bile salt export pump (Bsep) and the ability of DBcAMP to prevent it . E217G (15 micromol/kg i.v.) impaired bile salt (BS) output and induced Bsep internalization, as assessed by confocal microscopy and Western blotting . Neither cholestasis nor Bsep internalization occurred in TR- rats lacking Mrp2 . DBcAMP (20 micromol/kg i.v.) partially prevented the decrease in bile flow and BS output and substantially prevented E217G-induced Bsep internalization . In hepatocyte couplets, E217G (50 microM) diminished canalicular accumulation of a fluorescent BS and decreased Bsep-associated fluorescence in the canalicular membrane; DBcAMP (10 microM) fully prevented both effects . In conclusion, our results suggest that changes in Bsep localization are involved in E217G-induced impairment of bile flow and BS transport and that DBcAMP prevents this effect by stimulating insertion of canalicular transporter-containing vesicles . Mrp2 is required for E217G to induce its harmful effect.

Am J Physiol Gastrointest Liver Physiol, 2003 Aug, 285(2), G316 - 24 Epub 2003 Apr 17.
Mechanisms by which cAMP increases bile acid secretion in rat liver and canalicular membrane vesicles; Misra S et al.; Bile acid secretion induced by cAMP and taurocholate is associated with recruitment of several ATP binding cassette (ABC) transporters to the canalicular membrane . Taurocholate-mediated bile acid secretion and recruitment of ABC transporters are phosphatidylinositol 3-kinase (PI3K) dependent and require an intact microtubular apparatus . We examined mechanisms involved in cAMP-mediated bile acid secretion . Bile acid secretion induced by perfusion of rat liver with dibutyryl cAMP was blocked by colchicine and wortmannin, a PI3K inhibitor . Canalicular membrane vesicles isolated from cAMP-treated rats manifested increased ATP-dependent transport of taurocholate and PI3K activity that were reduced by prior in vivo administration of colchicine or wortmannin . Addition of a PI3K lipid product, phosphoinositide 3,4-bisphosphate, but not its isomer, phosphoinositide 4,5-bisphosphate, restored ATP-dependent taurocholate in these vesicles . Addition of a decapeptide that activates PI3K to canalicular membrane vesicles increased ATP-dependent transport above baseline activity . In contrast to effects induced by taurocholate, cAMP-stimulated intracellular trafficking of the canalicular ABC transporters was unaffected by wortmannin, and recruitment of multidrug resistance protein 2, but not bile salt excretory protein (bsep), was partially decreased by colchicine . These studies indicate that trafficking of bsep and other canalicular ABC transporters to the canalicular membrane in response to cAMP is independent of PI3K activity . In addition, PI3K lipid products are required for activation of bsep in the canalicular membrane . These observations prompt revision of current concepts regarding the role of cAMP and PI3K in intracellular trafficking, regulation of canalicular bsep, and bile acid secretion.

J Biol Chem, 2003 Jun 20, 278(25), 22908 - 17 Epub 2003 Apr 07.
Identification of domains participating in the substrate specificity and subcellular localization of the multidrug resistance proteins MRP1 and MRP2; Konno T et al.; The human multidrug resistance protein MRP1 and its homolog, MRP2, are both thought to be involved in cancer drug resistance and the transport of a wide variety of organic anions, including the cysteinyl leukotriene C4 (LTC4) (Km = 0.1 and 1 microm) . To determine which domain of these proteins is associated with substrate specificity and subcellular localization, we constructed various chimeric MRP1/MRP2 molecules and expressed them in polarized mammalian LLC-PK1 cells . We examined the kinetic properties of each chimeric protein by measuring LTC4 and methotrexate transport in inside-out membrane vesicles, sensitivity to an anticancer agent, etoposide, and subcellular localization by indirect immunofluorescence methods . The following results were determined in these studies: (i) when the NH2-proximal 108 amino acids of MRP2, including transmembrane (TM) helices 1-3, were exchanged with the corresponding region of MRP1, Km(LTC4) values of the chimera decreased approximately 4-fold and Km(methotrexate) values increased approximately 5-fold relative to those of wild-type MRP2 and MRP1, respectively, whereas resistance to etoposide increased approximately 3-fold; (ii) when the NH2-proximal region up to TM9 of MRP2 was exchanged with the corresponding region of MRP1, a further increase in etoposide resistance was observed, and subcellular localization moved from the apical to the lateral membrane; (iii) when two-thirds of MRP2 at the NH2 terminus were exchanged with the corresponding MRP1 region, the chimeric protein transported LTC4 with an efficiency comparable with that achieved by the wild-type MRP1; and (iv) exchange of the COOH-terminal 51 amino acids between MRP1 and MRP2 did not affect the localization of either of the proteins . These results provide a strong framework for further studies aimed at determining the precise domains of MRP1 and MRP2 with affinity for LTC4 and anticancer agents.

Neuroscience, 2003, 118(2), 417 - 29
Expression and cellular distribution of multidrug transporter proteins in two major causes of medically intractable epilepsy: focal cortical dysplasia and glioneuronal tumors; Aronica E et al.; The cell-specific distribution of multidrug resistance extrusion pumps was studied in developmental glioneuronal lesions, including focal cortical dysplasia (15 cases) and ganglioglioma (15 cases) from patients with medically intractable epilepsy . Lesional, perilesional, as well as normal brain regions were examined for the expression of the multidrug resistance gene 1 encoded P-glycoprotein (P-gp) and the multidrug resistance-associated protein 1 (MRP1) by immunocytochemistry . In normal brain MRP1 expression was below detection, whereas P-gp staining was present only in blood vessels . MRP1 and P-gp immunoreactivity was observed in dysplastic neurons of 11/15 cases of focal cortical dysplasia, as well as in the neuronal component of 14/15 ganglioglioma . Glial cells with astrocytic morphology within the lesion showed multidrug-resistant protein immunoreactivity (P-gp>MRP1) . Moderate to strong MRP1 and P-gp immunoreactivity was observed in a population of large ballooned neuroglial cells . P-gp appeared to be most frequently expressed in glial fibrillary acidic protein-positive balloon cells (glial type), whereas MRP1 was more frequently expressed in microtubule-associated protein 2-positive balloon cells (neuronal type) . In both types of lesions strong P-gp immunoreactivity was found in lesional vessels . Perilesional regions did not show increased staining in vessels or in neuronal cells compared with normal cortex . The predominant intralesional cell-specific distribution of multidrug transporter proteins supports the hypothesis of a constitutive overexpression as common mechanism underlying the intrinsic pharmaco-resistance to antiepileptic drugs of both malformative and neoplastic glioneuronal developmental lesions.

J Med Chem, 2003 Apr 24, 46(9), 1764 - 8
Reduction of peptide character of HIV protease inhibitors that exhibit nanomolar potency against multidrug resistant HIV-1 strains; Tamamura H et al.; Novel HIV protease inhibitors containing a hydroxyethylamine dipeptide isostere as a transition state-mimic king structure were synthesized by combining substructures of known HIV protease inhibitors . Among them, TYA5 and TYB5 were proven to be not only potent enzyme inhibitors (K(i) = 0.12 nM and 0.10 nM, respectively) but also strong anti-HIV agents (IC(50) = 9.5 nM and 66 nM, respectively), even against viral strains with multidrug resistance . Furthermore, insertion of an (E)-alkene dipeptide isostere at the P(1)-P(2) position of TYB5 led to development of a purely nonpeptidic protease inhibitor, TYB1 (K(i) = 0.38 nM, IC(50) = 160 nM).

J Med Chem, 2003 Apr 24, 46(9), 1706 - 15
Synthesis and biological evaluation of N-heterocyclic indolyl glyoxylamides as orally active anticancer agents; Li WT et al.; A series of N-heterocyclic indolyl glyoxylamides were synthesized and evaluated for in vitro and in vivo anticancer activities . They exhibited a broad spectrum of anticancer activity not only in murine leukemic cancer cells but also in human gastric, breast, and uterus cancer cells as well as their multidrug resistant sublines with a wide range of IC(50) values . They also induced apoptosis and caused DNA fragmentation in human gastric cancer cells . Among the compounds studied, 7 showed the most potent activity of growth inhibition (IC(50) = 17-1711 nM) in several human cancer cells . Given orally, compounds 7 and 13 dose-dependently prolonged the survival of animals inoculated with P388 leukemic cancer cells . N-Heterocyclic indolyl glyoxylamides may be useful as orally active chemotherapeutic agents against cancer and refractory cancerous diseases of multidrug resistance phenotype.

Curr Drug Targets, 2003 May, 4(4), 297 - 304
Vascular and parenchymal mechanisms in multiple drug resistance: a lesson from human epilepsy; Marroni M et al.; Long term treatment with antiepileptic drugs (AEDs) is the standard therapeutic approach to eradicate seizures . However, a small but significant number of patients fail AED treatment . Intrinsic drug resistance may depend on two main and not necessarily mutually exclusive mechanisms: 1) Loss of pharmacological target (e.g., GABAA receptors); 2) poor penetration of the drug into the central nervous system (CNS) . The latter is due to the action of multiple drug resistance proteins capable of active CNS extrusion of drugs . These include MDR1 (P-glycoprotein, PgP), the multidrug resistance related proteins MRP1-5, and lung-resistance protein (LRP) . Overexpression of MDR1 occurs in human epileptic brain . It has therefore been proposed that MDR1/PgP may contribute to multiple drug resistance in epilepsy . In addition to MDR1/PgP, other genes such as MRP2, MRP5, and human cisplatin resistance-associated protein are also overexpressed in drug-resistant epilepsy . In normal brain tissue MDR1/PgP is expressed almost exclusively by endothelial cells (EC), while in epileptic cortex both EC and perivascular astrocytes express MDR1/PgP . The underlying causes for tissue differences may be genomic (i.e., at the DNA level), or MDR1/PgP could be induced by seizures, previous drug treatment, or a combination of the above . We will present evidence showing that expression of multiple drug resistance genes in epilepsy is a complex phenomenon and that glial cells are involved . This second line of defense for xenobiotics may have profound implications for the pharmacokinetic properties of antiepileptic drugs and their capacity to reach neuronal targets.

Br J Cancer, 2003 Apr 22, 88(8), 1327 - 34
Reduced endocytosis and altered lysosome function in cisplatin-resistant cell lines; Chauhan SS et al.; We isolated human KB adenocarcinoma cisplatin-resistant (CP-r) cell lines with multidrug-resistance phenotypes because of reduced accumulation of cisplatin and other cytotoxic compounds such as methotrexate and heavy metals . The uptake of horseradish peroxidase (HRPO) and Texas Red dextran was decreased several-fold in KB-CP-r cells, indicating a general defect in fluid-phase endocytosis . In contrast, although EGF receptors were decreased in amount, the kinetics of EGF uptake, a marker of receptor-mediated endocytosis, was similar in sensitive and resistant cells . However, 40-60% of the (125)I-EGF released into the medium after uptake into lysosomes of KB-CP-r cells was TCA precipitable as compared to only 10% released by sensitive cells . These results indicate inefficient degradation of internalised (125)I-EGF in the lysosomes of KB-CP-r cells, consistent with slower processing of cathepsin L, a lysosomal cysteine protease . Treatment of KB cells by bafilomycin A(1), a known inhibitor of the vacuolar proton pump, mimicked the phenotype seen in KB-CP-r cells with reduced uptake of HRPO, (125)I-EGF, (14)C-carboplatin, and release of TCA precipitable (125)I-EGF . KB-CP-r cells also had less acidic lysosomes . KB-CP-r cells were crossresistant to Pseudomonas exotoxin, and Pseudomonas exotoxin-resistant KB cells were crossresistant to cisplatin . Since cells with endosomal acidification defects are known to be resistant to Pseudomonas exotoxin and blocking of endosomal acidification mimics the CP-r phenotype, we conclude that defective endosomal acidification may contribute to acquired cisplatin resistance.

Zhonghua Xue Ye Xue Za Zhi, 2003 Mar, 24(3), 141 - 3
{Overexpression of S3 ribosomal protein gene is involved in drug resistance in K562/DOX cells}; Zhu NX et al.; OBJECTIVE: To investigate the effect of overexpression of S3 ribosomal protein (S3rp) gene on the resistance of leukemia cell to antitumor drugs . METHODS: Both sense and antisense cDNA recombinants of S3rp gene were constructed with pcDNA3.1 expression vector . Subsequently, the sense S3rp cDNA recombinant was transfected into K562 cells while the antisense one into K562/DOX cells (a multidrug resistant cell line) . In addition, empty pcDNA3.1 vector was transfected into the corresponding cells as negative controls . The chemosensitivity of cells was evaluated by MTT assay . RESULTS: Sense S3rp cDNA transfected K562 cells were 5.8 times more resistant to doxorubicin than control cells did, whereas antisense S3rp cDNA transfected K562/DOX cells were 3.2 times less resistant to doxorubicin than control cells did . CONCLUSION: Overexpression of S3rp gene plays an important role in the development of drug resistance in K562/DOX cells.

BMC Cancer . 2003 Mar 26;3(1):10.
A protein kinase Cbeta inhibitor attenuates multidrug resistance of neuroblastoma cells; Svensson K et al.; BACKGROUND: The acquisition of drug resistance is a major reason for poor outcome of neuroblastoma . Protein kinase C (PKC) has been suggested to influence drug resistance in cancer cells . The aim of this study was to elucidate whether inhibition of PKCbeta isoforms influences drug-resistance of neuroblastoma cells . METHODS: The effect of the PKCbeta inhibitor LY379196 on the growth-suppressing effects of different chemotherapeutics on neuroblastoma cells was analyzed with MTT assays . The effect of LY379196 on the accumulation of {3H}vincristine was also investigated RESULTS: The PKCbeta inhibitor LY379196 suppressed the growth of three neuroblastoma cell lines . LY379196 also augmented the growth-suppressive effect of doxorubicin, etoposide, paclitaxel, and vincristine, but not of carboplatin . The effect was most marked for vincristine and for the cell-line (SK-N-BE(2)) that was least sensitive to vincristine . No effect was observed on the non-resistant IMR-32 cells . Two other PKC inhibitors, Go6976 and GF109203X, also enhanced the vincristine effect . The PKC inhibitors caused an increased accumulation of {3H}vincristine in SK-N-BE(2) cells . CONCLUSIONS: This indicates that inhibition of PKCbeta could attenuate multidrug resistance in neuroblastoma cells by augmenting the levels of natural product anticancer drugs in resistant cells.

Lab Invest, 2003 Apr, 83(4), 507 - 17
Expression profiling of osteosarcoma cells transfected with MDR1 and NEO genes: regulation of cell adhesion, apoptosis, and tumor suppression-related genes; Sanchez-Carbayo M et al.; The expression patterns of the osteosarcoma cell line U-2 OS, and three derived subclones containing stably transfected MDR1, NEO and MDR1/NEO genes were compared using cDNA microarrays comprising 8976 known genes and expressed sequenced tags . Data provided new insights into three critical issues . First, MDR1 overexpression was associated with altered expression of genes related to several cellular pathways, including (a) . drug influx/efflux (eg, dynamin 3), (b) . metabolic enzymes (eg, monoamine oxidase A), (c) . cell adhesion (eg, EPCAM), (d) . apoptotic signaling (eg, I-TRAF), (e) . senescence (eg, telomerase RNA binding protein staufen), (f) . tumor suppression-related genes (eg, KISS-1 and ephrin B3), and (g) . immune system receptors (eg, LENG2) . MDR1, EPCAM, and ephrin B3 expression was confirmed by immunohistochemistry . Second, MDR1 transfected cells selected with either doxorubicin or neomycin showed distinct expression profiles that could be related to differential selection . Moreover, hierarchical clustering indicated that cells transfected with MDR1 alone, or cotransfected with NEO, displayed more closely related expression profiles than cells transfected only with NEO . Third, transfection with NEO and selection with neomycin produced a considerable number of expression changes within the cell . This study further elucidates the genetic events associated with MDR1 expression and identifies novel targets associated with multidrug resistance.

Mol Pharmacol, 2003 May, 63(5), 1094 - 103
Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5; Reid G et al.; The human multidrug resistance proteins MRP4 and MRP5 are organic anion transporters that have the unusual ability to transport cyclic nucleotides and some nucleoside monophosphate analogs . Base and nucleoside analogs used in the chemotherapy of cancer and viral infections are potential substrates . To assess the possible contribution of MRP4 and MRP5 to resistance against these drugs, we have investigated the transport mediated by MRP4 and MRP5 . In cytotoxicity assays, MRP4 conferred resistance to the antiviral agent 9-(2-phosphonomethoxyethyl)adenine (PMEA) and high-performance liquid chromatography analysis showed that, like MRP5, MRP4 transported PMEA in an unmodified form . MRP4 also mediated substantial resistance against other acyclic nucleoside phosphonates, whereas MRP5 did not . Apart from low-level MRP4-mediated cladribine resistance, the cytotoxicity of clinically used anticancer nucleosides was not influenced by overexpression of MRP4 or MRP5 . In contrast, MRP5 mediated efflux of the pyrimidine-based antiviral 2',3'-dideoxynucleoside 2',3'-didehydro-2',3'-dideoxythymidine 5'-monophosphate (d4TMP) and its phosphoramidate derivative alaninyl-d4TMP from cells loaded with the 2',3'-didehydro-2',3'-dideoxythymidine prodrugs cyclosaligenyl-d4TMP and aryloxyphosphoramidate d4TMP (So324), respectively . Moreover, only inside-out membrane vesicles derived from MRP5-overexpressing cells accumulated alaninyl-d4TMP . Cellular efflux and vesicular uptake studies were carried out to further compare transport mediated by MRP4 and MRP5 and showed that dipyridamole, dilazep, nitrobenzyl mercaptopurine riboside, sildenafil, trequinsin and MK571 inhibited MRP4 more than MRP5, whereas cyclic nucleotides and monophosphorylated nucleoside analogs were equally poor inhibitors of both pumps . These results strongly suggest that the affinity of MRP4 and MRP5 for nucleotide-based substrates is low.

Eur J Haematol, 2003 May, 70(5), 296 - 303
Validation and clinical implication of a quantitative real-time PCR determination of MDR1 gene expression: comparison with semi-quantitative PCR in 101 patients with acute myeloid leukemia; Olesen LH et al.; INTRODUCTION: The multidrug resistance protein 1 (MDR1) has the capacity to extrude chemotherapeutics and has been implicated in treatment failure in acute myeloid leukemia (AML) . Previous methods for determination of MDR1 expression have included dye exclusion, demonstration of P-glycoprotein by flow cytometry and/or immunohistochemistry, and molecular polymerase chain reaction (PCR)-based assays for RNA expression . However, these assays have either proven difficult to standardize or tedious to perform . We have therefore designed a real-time quantitative (RQ)-PCR based assay measuring MDR1 gene expression and validated it in AML patients by direct comparison with a competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay . PATIENTS AND METHODS: Bone marrow or peripheral blood from 101 AML patients diagnosed (1987-96) at our department were assessed for quantitative expression of MDR1 employing TaqMan RQ-PCR . These data were compared with results obtained by a semi-quantitative competitive PCR assay employing an artificial internal RNA construct . RESULTS: While the RQ-PCR method was able to determine MDR1 gene expression in a continuous fashion over five logs, the semi-quantitative PCR only yielded data in a discontinuous fashion and over four logs at best . Compared with the MDR1 positive and negative cell lines 8226 DOX40 and REH AML cells exhibited variation of 10 PCR cycles, equivalent to a 1000-fold difference . A significant correlation was observed between the two methods, Spearman's correlation coefficient = -0.502, P-value = 10-5 . CONCLUSION: We conclude that, RQ-PCR is a novel methodology, which enables sensitive and quantitative measurement of MDR1 gene expression . This assay is moreover suitable because of its high throughput for longitudinal follow-up and large number of patients.

Gen Physiol Biophys, 2002 Dec, 21(4), 471 - 8
Carbonyl group of aliphatic side chain of pentoxifylline does not play role for P-glycoprotein antagonizing effect of pentoxifylline; Kupsakova I et al.; Previously we have found that pentoxifylline (PTX), but not caffeine, theophylline, or 1-methyl-3-isobutylxanthine, affects sensitivity of L1210/VCR cells, a line with multidrug resistance mediated by P-glycoprotein (P-gp) to vincristine (VCR) and doxorubicine . Comparison of chemical structure of PTX with other above xanthines has revealed only one marked difference . PTX contains extended aliphatic chain containing reactive electrophilic carbonyl group in the position N1 . The investigation of possibility that this group is crucial for PTX-induced MDR reversal represents the aim of the current paper . To prove this hypothesis, we used the new synthesized PTX derivative in which the carbonyl group is modified by a substance containing amino-group and the product of reaction is the respective Schiff base (SB) . Successful reaction was observed when PTX reacted with 3,5-diaminobenzenesulfonyl acid (DABS) . The product of reaction of DABS with carbonyl group of aliphatic part of PTX was proved using NMR and IR spectroscopy . We found that the resulting PTX derivative PTX-SB revealed higher cytotoxicity on both sensitive L1210 and multidrug resistant L1210/VCR cells than PTX . Moreover, PTX-SB exerts more pronounced MDR reversal effect on L1210/VCR cells than PTX . These results indicate that electrophilic carbonyl group on aliphatic chain located in position N1 of PTX is not essential for MDR reversal effects of PTX.

Gen Physiol Biophys, 2002 Dec, 21(4), 381 - 404
Malignant gliomas display altered plasma membrane potential and pH regulation--interaction with Tc-99m-MIBI and Tc-99m-Tetrofosmin uptakes; Perek N et al.; Two radiopharmaceuticals, Tc-99m-MIBI (MIBI) and Tc-99m-Tetrofosmin (Tfos), are currently used for in vivo non-invasive monitoring of the MultiDrug Resistant (MDR) status of tumours . As gliomas are highly multidrug resistant, it is expected that the tracers would be poorly retained in those cells, but the in vivo and in vitro studies to date have shown that Tfos was highly retained in malignant gliomas . The high degree of malignancy of tumour cells is linked to alterations of physiological parameters as plasma membrane potential and intracellular pH . In order to elucidate the contribution of those parameters to Tfos and MIBI uptakes in malignant gliomas, we used several glioma cell lines--G111, G5, G152, and 42 MG-BA . These cells showed to be chemoresistant with a high level of expression and activity of the Multidrug Resistant associated Protein 1 (MRP1) . They also had an alkaline intracellular pH (pHi) related to the Na+/H+ antiporter (NHE-1) expression and depolarised plasma membranes (-45 to -55 mV) . In spite of their chemoresistance, we have found a high accumulation of both radiotracers in gliomas, more important for Tfos than MIBI, related to the presence and activity of NHE-1 . In conjunction, the uptakes of the tracers were only partially dependent upon the plasma membrane potential of the glioma cell lines, again Tfos uptake being less dependent on this parameter than MIBI uptake . In conclusion, the evidence accumulated in this study suggests that Tfos could be a suitable glioma marker in vivo.

Southeast Asian J Trop Med Public Health, 2002 Sep, 33(3), 570 - 4
Multidrug-resistant tuberculosis at Srinagarind Hospital, Khon Kaen, Thailand; Reechaipichitkul W; Pulmonary tuberculosis is a very common infectious disease in Thailand . Multidrug-resistant tuberculosis (MDR-TB) is the most serious form of the disease . Failure to control resistant tuberculosis is associated with its resurgence . The objective of this study was to analyze the drug susceptibility pattern of M . tuberculosis and to study the clinical characteristics and outcome of patients diagnosed with MDR-TB at Srinagarind Hospital . Between January 1995 and December 2000, 899 isolates of M . tuberculosis were recovered . Rifampicin (RIF) resistance was the most common finding (8.2%) . Twenty-two patients (2.4%) were infected with MDR-TB . Other susceptibility results showed resistance to isoniazid (INH) (4.2%), ethambutol (EMB) (4.3%), streptomycin (SM) (3.7%), kanamycin (Kana) (3.0%), and ofloxacin (Oflox) (2.3%) . Twenty MDR-TB patients were retrospectively reviewed . The mean age was 37 years (range: 17 to 64) . The male to female ratio was 3:1 . The mean duration of symptoms before treatment was 3.8 months (range: 3 days to 2 years) . The commonest comorbidity was HIV-infection (7 patients) . Eleven patients (55%) had a past history of treatment with anti-TB drugs . In addition to INH and RIF resistance, many organism also resisted EMB (35%), SM (30%), Oflox (30%), and Kana (10%) . Only five patients (25%) responded to medical treatment . Seven patients (35%) died, and the other eight were unavailable for an evaluation of clinical outcome . Although the prevalence of MDR-TB was not high in Srinagarind Hospital, the treatment was costly and the outcomes were poor . Preventing new cases of MDR-TB by using effective treatment strategies for patients with drug-sensitive TB is a priority.

Southeast Asian J Trop Med Public Health, 2002 Sep, 33(3), 525 - 31
A comparative clinical trial of combinations of dihydroartemisinin plus azithromycin and dihydroartemisinin plus mefloquine for treatment of multidrug resistant falciparum malaria; Krudsood S et al.; With the deteriorating situation of multidrug resistant falciparum malaria, a new drug or drugs in combinations are urgently needed . We conducted a study comparing a combination of dihydroartemisinin 240 mg and mefloquine 1,250 mg given over 3 days (Group 1) and a combination of dihydroartemisinin 240 mg and azithromycin 1,500 mg given over 3 days (Group 2), to determine safety, efficacy and tolerability . All of the patients stayed in a non-malaria endemic area during the study . By the third day after drug administration, most patients were free of parasites and none had serious adverse events . The cure rates at day 28 were 100% and 69.7% in Group 1 and Group 2, respectively (p<0.01) . We conclude that a combination of dihydroartemisnin and azithromycin was safe and effective and may be another interesting regimen of the treatment of uncomplicated multidrug resistant Plasmodium falciparum malaria in Thailand.

Southeast Asian J Trop Med Public Health, 2002 Sep, 33(3), 519 - 24
An open randomized clinical trial of Artecom vs artesunate-mefloquine in the treatment of acute uncomplicated falciparum malaria in Thailand; Wilairatana P et al.; The efficacy and safety of Artecom were assessed in an open randomized trial in adults presenting with acute, uncomplicated Plasmodium falciparum malaria in Thailand . Three hundred and fifty-two patients were randomly enroled at the ratio of 2:1 into group A:B and received Artecom (group A) and the standard combination of artesunate and mefloquine (group B) respectively . All patients had rapid initial clinical and parasitological responses . There were no significant differences in fever clearance time and parasite clearance time between the two groups . The 28-day cure rates were high as 97% in both groups . Artecom was effective and well-tolerated as artesunate-mefloquine, the current treatment in this area of multidrug-resistant P . falciparum malaria.

Mol Cancer Res, 2003 Apr, 1(6), 420 - 7
Overexpression of extracellular matrix metalloproteinase inducer in multidrug resistant cancer cells; Yang JM et al.; Multidrug resistant (MDR) cancer cells overexpressing P-glycoprotein (P-gp) display variations in invasive and metastatic behavior . We previously reported that these properties of MDR cancer cell lines overexpressing P-gp could be altered by chemotherapeutic drugs or MDR modulators (R . S . Kerbel et al., Cancer Surv., 7: 597-629, 1988) . To attempt to clarify the mechanism(s) underlying these observations, we studied the expression of extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein enriched on the surface of tumor cells that can stimulate the production of matrix metalloproteinases (MMPs), in sensitive and MDR cancer cells . Using immunofluorescence staining and fluorescence-activated cell sorting analysis, we found that EMMPRIN expression was increased in MDR carcinoma cell lines, MCF-7/AdrR, KBV-1, and A2780Dx5, as compared to their parental counterparts . The MDR cell lines produced more matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-9 (MMP-9), as determined by zymography, Western blot, and reverse transcription-PCR . Treatment of MDR cells with an anti-EMMPRIN antibody inhibited the activity of MMP-1, MMP-2, and MMP-9 . In MDR cell line MCF-7/AdrR, an increased in vitro invasive ability was observed as compared with the sensitive line MCF-7, and EMMPRIN antibody could inhibit the in vitro invasion in drug-resistant cells . In addition, the expression and activity of MMP-1, MMP-2, and MMP-9 in MDR cells were decreased by treatment with U-0126, an inhibitor of mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/Erk) . Our results suggest that during the development of MDR, the expression of EMMPRIN is responsible for the increased activity of MMP in MDR cell lines.

Gut, 2003 May, 52(5), 759 - 66
Multidrug resistance 1 gene (P-glycoprotein 170): an important determinant in gastrointestinal disease?
Ho GT, Moodie FM, Satsangi J.
The interface between luminal contents and intestinal epithelium constitutes the largest area of interaction between the host and the environment . There is now strong evidence that the gene product of the multidrug resistant pump (MDR) plays a critical role in host-bacterial interactions in the gastrointestinal tract and maintenance of intestinal homeostasis . This review highlights the efflux mechanism in the intestinal epithelium which is mediated by the multidrug resistant pump, also known as P-glycoprotein 170 . Current studies promise to provide further insights into the contribution of the MDR1 gene in the pathogenesis of inflammatory and malignant disorders of the gastrointestinal tract.

Leuk Lymphoma, 2003 Jan, 44(1), 85 - 95
High functional P-glycoprotein activity is more often present in T-cell acute lymphoblastic leukaemic cells in adults than in children; Plasschaert SL et al.; There is a distinct difference in prognosis between childhood versus adult acute lymphoblastic leukaemia (ALL) . To define whether multidrug resistance (MDR) genes might contribute to this distinction, the expression and functional activity of P-glycoprotein (P-gp) and MDR associated proteins (MRP) were determined with RT-PCR (MDR-1, MRP1, MRP2, MRP3) and flow cytometry (P-gp and MRP) . Patient samples were obtained from 36 children and 35 adults with de novo ALL . Of these patients, 38 showed a T-lineage and 33 showed a B-lineage immunophenotype . In the samples, large variability in P-gp activity (0.8-4.9) and MRP activity (1.1-13.9) was observed . Most T-ALL patients with high P-gp activity were adults (89%) . The mRNA expression of MDR-1 correlated weakly with P-gp activity . In contrast, MRP activity did not correlate with the mRNA expression of MRP1, MRP2 and MRP3 . In T-ALL, a worse overall survival and event-free survival was observed with increasing P-gp activity . P-gp activity had no prognostic impact in B-lineage ALL . In addition, high MRP activity did not influence treatment outcome in either T- or B-lineage ALL . Multivariate Cox regression analysis, showed P-gp activity to be the only unfavourable prognostic factor for overall survival in T-ALL . In conclusion, this study demonstrates the prognostic relevance of P-gp activity in T-ALL . Since the majority of the patients with high P-gp activity were adults, P-gp might contribute to the poor prognosis of adult T-ALL.

Blood, 2003 Aug 15, 102(4), 1466 - 73 Epub 2003 Apr 10.
Multidrug resistance protein attenuates gemtuzumab ozogamicin-induced cytotoxicity in acute myeloid leukemia cells; Walter RB et al.; Gemtuzumab ozogamicin (GO) is a novel immunoconjugate therapy for acute myeloid leukemia (AML) . P-glycoprotein (Pgp) confers resistance to GO and is associated with a worse clinical response . To address whether multidrug resistance protein (MRP) affects GO susceptibility, we characterized Pgp, MRP1, and MRP2 expression in CD33+ cell lines and CD33+ AML samples and analyzed the effect of the Pgp inhibitor cyclosporine (CSA) and the MRP inhibitor MK-571 on GO-induced cytotoxicity . MRP1, but not MRP2, expression correlated with MRP activity . MK-571 enhanced GO-induced cytotoxicity in Pgp-negative/MRP-positive NB4 and HL-60 cells . CSA, but not MK-571 alone, restored GO susceptibility in Pgp-positive/MRP-positive TF1 cells; however, MK-571 enhanced cytotoxicity in the presence of CSA . All patient samples exhibited MRP activity, and 17 of 23 exhibited Pgp activity . CSA increased GO-induced cytotoxicity in 12 Pgp-positive samples, whereas MK-571 alone was effective in only one sample with minimal Pgp activity . In 3 Pgp-positive/MRP-positive samples, MK-571 enhanced GO-induced cytotoxicity in the presence of CSA . Thus, MRP1 may attenuate susceptibility to GO . This effect was comparatively less than that for Pgp and required the inhibition of Pgp for detection in cells that coexpressed both transporters . Because MK-571 and CSA failed to affect cytotoxicity in a portion of Pgp-positive/MRP-positive AML samples, additional resistance mechanisms are likely important.

Curr Opin Pulm Med, 2003 May, 9(3), 186 - 92
Current thinking on the management of tuberculosis; Bastian I et al.; High-income countries are moving toward tuberculosis (TB) elimination . Sophisticated diagnostic tests and effective treatment regimens are readily available . The range of available resources even makes effective treatment of multidrug-resistant tuberculosis (MDRTB) possible . The introduction of highly active antiretroviral therapy and specific TB control measures has reduced the incidence of HIV-associated TB disease . Unfortunately, the situation in low-income countries that carry 95% of the global TB burden is less positive . TB diagnosis still relies upon sputum smear microscopy . The management of MDRTB remains problematic though guidelines for DOTS-plus programs have been developed, and cheaper second-line drugs are becoming available . The HIV epidemic continues to confound TB control efforts, particularly in sub-Saharan Africa . The appropriate package of interventions for controlling HIV/TB disease remains undefined and unimplemented . The international community must provide the funding and technical support to address the alarming dichotomy in TB control that exists between rich and poor countries.

J Clin Microbiol, 2003 Apr, 41(4), 1520 - 4
Drug-susceptible Mycobacterium tuberculosis Beijing genotype does not develop mutation-conferred resistance to rifampin at an elevated rate; Werngren J et al.; The Mycobacterium tuberculosis Beijing genotype has drawn attention because it is often strongly associated with multidrug-resistant tuberculosis (MDR-TB) . A possible reason is that the Beijing strains may have an enhanced capacity to develop drug resistance . In this study, we used the Luria-Delbruck fluctuation test to investigate whether strains of Beijing and non-Beijing genotypes exhibit differences in the acquisition of drug resistance . The M . tuberculosis reference strain H37Rv and 12 fully drug-susceptible clinical isolates, 6 of which were of the Beijing genotype, were examined . To determine the distribution of rifampin-resistant mutants, 25 independent cultures were made for each strain . The average mutation frequencies for the non-Beijing (H37Rv included) and Beijing genotypes were estimated to be 4.4 x 10(-8) and 3.6 x 10(-8), respectively . The corresponding average mutation rates for the non-Beijing and Beijing strains were 1.3 x 10(-8) and 1.1x 10(-8) mutations per cell division, respectively . The results suggest that the association of the Beijing genotype with MDR-TB is not due to an altered ability to develop resistance.

Pharmacotherapy, 2003 Apr, 23(4), 436 - 42
Evaluation of P-glycoprotein-mediated renal drug interactions in an MDR1-MDCK model; Karyekar CS et al.; STUDY OBJECTIVE: To evaluate P-glycoprotein (P-gp)-mediated renal drug interactions in an in vitro model of tubular secretion . DESIGN: In vitro experiment . SETTING: University-affiliated pharmacokinetics laboratory . CELL LINES: Madin-Darby canine kidney (MDCK), multidrug-resistant-1 (MDR1)-MDCK, and human colon carcinoma (Caco-2) cells . INTERVENTION: Transepithelial transport (basolateral-to-apical and apical-to-basolateral) of cimetidine was assessed in the absence and presence of various concentrations of the P-gp inhibitors itraconazole and PSC-833 in a renal P-gp cell culture model (MDR1-MDCK) . MEASUREMENTS AND MAIN RESULTS: Apparent permeability of cimetidine was characterized, and level of P-gp expression was determined by Western blot analysis, in MDCK (wild type), MDR1-MDCK, and Caco-2 cells (for relative comparison) . In the presence of PSC-833, cimetidine's apparent permeability value for basolateral-to-apical transport decreased from 2.96 to 1.15 x 10(-6) cm/second, coupled with a decrease in efflux ratio from 2.36 to 1.80 . The effect of itraconazole was concentration dependent, with cimetidine's apparent permeability value for basolateral-to-apical transport decreasing from 3.96 to 1.92 x 10(-6) cm/second (p < 0.05), resulting in a 50% decrease in efflux ratio . Expression of P-gp was negligible in MDCK (wild-type) cells, but high-level expression was confirmed in both MDR1-MDCK and Caco-2 cells . CONCLUSION: P-glycoprotein plays a significant role in the renal tubular secretion of organic cations such as cimetidine, and the high level of P-gp expression in MDR1-MDCK cells makes this a well-suited model for evaluating mechanisms of renal drug interactions.

Cancer Chemother Pharmacol, 2003 May, 51(5), 407 - 14 Epub 2003 Apr 10.
Ketotifen reverses MDR1-mediated multidrug resistance in human breast cancer cells in vitro and alleviates cardiotoxicity induced by doxorubicin in vivo; Zhang Y et al.; PURPOSE: To investigate the effect of the antihistamine ketotifen on multidrug resistance in human breast cancer cells and doxorubicin toxicity in mice . METHODS: Clonogenicity assays were used to test the effect of ketotifen on human multidrug resistant breast cancer cell lines exposed to chemotherapeutic agents . Flow cytometry was used to measure accumulation of doxorubicin in cells . Fluorimetry was used to measure accumulation of doxorubicin in cardiac tissues . Histological analysis and toxicity studies in mice were used to test the effect of ketotifen on doxorubicin-induced toxicity . RESULTS: Ketotifen was found to restore the sensitivity of P-glycoprotein-overexpressing multidrug-resistant MCF-7/adr cells to doxorubicin, mitoxantrone, VP-16 and vinblastine, but not to methotrexate or camptothecin . Ketotifen, however, was unable to restore sensitivity of BCRP-overexpressing MCF-7/mx cells or MRP-overexpressing MCF-7/vp cells to mitoxantrone or VP-16, respectively . In vivo, pretreatment of mice with ketotifen caused an increased accumulation of doxorubicin in cardiac tissue, consistent with a block in drug clearance . However, unlike verapamil, ketotifen pretreatment did not enhance doxorubicin toxicity but in fact provided protection, both at the level of cardiac tissue damage and in terms of survival . CONCLUSIONS: Taken together, these observations show that ketotifen is unique in its ability both to reverse multidrug resistance due to P-glycoprotein overexpression and to provide cardioprotection to doxorubicin.

Can J Clin Pharmacol, 2003 Spring, 10(1), 17 - 23
The kidney--the body's playground for drugs: an overview of renal drug handling with selected clinical correlates; Perri D et al.; A greater understanding of transport mechanisms contributing to renal drug handling may be useful in predicting drug clearance and drug interactions . Renal clearance is a dynamic process expressed as the sum of the rates of glomerular filtration and tubular secretion minus the rate of tubular reabsorption . Because the transport of drugs is often against a concentration gradient, renal secretion is mostly an active process involving a variety of transporter mechanisms . Discoveries from molecular biology techniques and gene 'knock-out' experiments have identified a variety of renal tubular proteins responsible for the transport of organic cations, organic anions, neutral and cationic hydrophobic compounds, anionic conjugates and specific agents such as prostaglandins . The discovery of a P-glycoprotein (P-gp) transporter at the apical membrane of renal tubular cells is particularly important . By elucidating compounds that act as substrates, inhibitors or inducers of transport proteins, pharmacologists and clinicians may better understand renal drug clearance . This paper provides a brief overview of several identified renal transport proteins including organic anion transporters, organic cation transporters, ATP-dependent transporters (multidrug resistance {P-gp} and multi-drug resistance associated protein), nucleoside transporters (sodium-dependent purine nucleoside transporter and concentrative nucleoside transporter 1) and peptide transporters . A special focus on known P-gp-mediated drug interactions is included to demonstrate the clinical relevance of transporter protein science . At the patient level, this may lead to novel approaches to alter in vivo pharmacokinetics and improve drug safety through a greater understanding of toxic substrate clearance and drug interactions.

N Engl J Med, 2003 Apr 10, 348(15), 1442 - 8
Association of multidrug resistance in epilepsy with a polymorphism in the drug-transporter gene ABCB1; Siddiqui A et al.; BACKGROUND: One third of patients with epilepsy have drug-resistant epilepsy, which is associated with an increased risk of death and debilitating psychosocial consequences . Because this form is resistant to multiple antiepileptic drugs, the mode of resistance must be nonspecific, involving drug-efflux transporters such as ATP-binding cassette sub-family B member 1 (ABCB1, also known as MDR1 and P-glycoprotein 170) . We hypothesized that the CC genotype at the ABCB1 C3435T polymorphism, which is associated with increased expression of the protein, influences the response to antiepileptic-drug treatment . METHODS: ABCB1 3435 was genotyped in 315 patients with epilepsy, classified as drug-resistant in 200 and drug-responsive in 115, and 200 control subjects without epilepsy . Recently devised methods were used to control for population stratification, and linkage disequilibrium was calculated across the gene . RESULTS: As compared with patients with drug-responsive epilepsy, patients with drug-resistant epilepsy were more likely to have the CC genotype at ABCB1 3435 than the TT genotype (odds ratio, 2.66; 95 percent confidence interval, 1.32 to 5.38; P=0.006) . There was no genetic stratification between the two groups of patients . The polymorphism fell within an extensive block of linkage disequilibrium spanning much or all of the gene, implying that the polymorphism may not itself be causal but rather may be linked with the causal variant . CONCLUSIONS: These pharmacogenomic results identify a genetic factor associated with resistance to antiepileptic drugs .

J Neurophysiol, 2003 Apr, 89(4), 1870 - 7
ATP released from astrocytes during swelling activates chloride channels; Darby M et al.; ATP release from astrocytes contributes to calcium ({Ca(2+)}) wave propagation and may modulate neuronal excitability . In epithelial cells and hepatocytes, cell swelling causes ATP release, which leads to the activation of a volume-sensitive Cl(-) current (I(Cl,swell)) through an autocrine pathway involving purinergic receptors . Astrocyte swelling is counterbalanced by a regulatory volume decrease, involving efflux of metabolites and activation of I(Cl,swell) and K(+) currents . We used whole cell patch-clamp recordings in cultured astrocytes to investigate the autocrine role of ATP in the activation of I(Cl,swell) by hypo-osmotic solution (HOS) . Apyrase, an ATP/ADP nucleotidase, inhibited HOS-activated I(Cl,swell), whereas ATP and the P2Y agonists, ADPbetaS and ADP, induced Cl(-) currents similar to I(Cl,swell) . Neither the P2U agonist, UTP nor the P2X agonist, alpha,beta-methylene ATP, were effective . BzATP was less effective than ATP, suggesting that P2X7 receptors were not involved . P2 purinergic antagonists, suramin, RB2, and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) reversibly inhibited activation of I(Cl,swell), suggesting that ATP-activated P2Y1 receptors . Thus ATP release mediates I(Cl,swell) in astrocytes through the activation of P2Y1-like receptors . The multidrug resistance protein (MRP) transport inhibitors probenicid, indomethacin, and MK-571 all potently inhibited I(Cl.swell) . ATP release from astrocytes in HOS was observed directly using luciferin-luciferase and MK-571 reversibly depressed this HOS-induced ATP efflux . We conclude that ATP release via MRP and subsequent autocrine activation of purinergic receptors contributes to the activation of I(Cl,swell) in astrocytes by HOS-induced swelling.

Scand J Infect Dis, 2003, 35(1), 47 - 51
Multidrug-resistant strains of Mycobacterium tuberculosis isolated from patients in Tehran belong to a genetically distinct cluster; Feizabadi MM et al.; Restriction fragment length polymorphism (RFLP) analysis was used to study the molecular epidemiology of tuberculosis (TB) in certain areas of Tehran . 120 isolates of Mycobacterium tuberculosis, including drug-resistant strains (n = 23), were analysed using polymorphic GC-rich sequence (PGRS) and IS6110 probes . There was considerable diversity among the strains cultured from patients from certain areas . The results of RFLP showed that multidrug resistant (MDR) isolates of M . tuberculosis in Tehran belong to a group of strains with low copies of IS6110 and PGRS . The degree of clustering was higher for the drug-resistant strains than for the susceptible ones (65% vs 20%) . Based on the demographic data and results of RFLP, it appears that recent transmissions of TB from old patients have occurred in Tehran . However, drug-resistant TB in the city is mainly caused by strains that look different from those cultured from such patients . The majority of MDR isolates (85%) in this study contained a low copy number of IS6110 and PGRS in RFLP, and were mostly recovered from immigrants and refugees.

Clin Infect Dis, 2003 Apr 15, 36(8), 996 - 1003 Epub 2003 Apr 04.
Treatment of multidrug-resistant tuberculosis during pregnancy: a report of 7 cases; Shin S et al.; Multidrug-resistant tuberculosis (MDR-TB) is a global public health problem affecting women of childbearing age . Little is known, however, about the safety of the drugs used to treat MDR-TB during pregnancy . We describe 7 patients who were treated for MDR-TB during pregnancy . These patients had chronic tuberculosis that had caused extensive parenchymal damage and had high-grade resistance to antituberculous drugs . All patients received individualized antituberculous therapy prior to delivery of healthy term infants . Neither obstetrical complications nor perinatal transmission of MDB-TB was observed . One patient experienced treatment failure, and another abandoned therapy . The other 5 patients are currently cured or in treatment and have culture-negative status . In each of these 7 cases, excellent treatment outcomes were obtained for the women and their children . Under certain circumstances, MDR-TB can be successfully treated during pregnancy.

Int J Oncol, 2003 May, 22(5), 1057 - 64
Molecular determinants of intrinsic resistance to doxorubicin in human cancer cell lines; Gariboldi MB et al.; Intrinsic or acquired drug resistance poses a major challenge to the success of chemotherapy in the clinical management of human cancers . While acquired multidrug resistance (MDR), whereby cells become refractory to multiple drugs, has been extensively investigated, the mechanistic basis for intrinsic resistance remains elusive, so that this condition is largely unmanageable in the clinical setting . To address this issue, we have assessed the effects of the anticancer agent doxorubicin (DX) on a panel of human tumor cell lines originally derived from untreated patients and tried to establish a correlation between cell response and a number of parameters, including drug accumulation and/or drug efflux; differences in expression and/or subcellular distribution of proteins involved in the apoptotic process (e.g., p53, Bcl-2, Bax) and intracellular signal transducers (PKCalpha); changes in key detoxification processes . Based on our results, 'classic' multispecific drug transporters (P-glycoprotein, MDR-related proteins) only seem to play a minor role in the intrinsically resistant phenotype, whereas LRP may contribute to resistance in non-small cell lung carcinoma (NSCLC) cells . No relationship was observed between drug response and expression and/or subcellular localization of apoptosis-related proteins; however, increased PKCalpha levels are associated with poor drug response, suggesting that one or more substrates of this enzyme may be relevant to the resistant phenotype . Finally, overactive glutathione-recycling pathways may contribute to DX resistance.

Biochem Biophys Res Commun, 2003 Apr 18, 303(4), 1135 - 41
The multidrug resistance protein ABCC1 drug-binding domains show selective sensitivity to mild detergents; Alqawi O et al.; The multidrug resistance protein (ABCC1 or MRP1) causes resistance to multiple drugs through reduced drug accumulation . We have previously demonstrated direct interaction between MRP1 and unmodified drugs using photoreactive drug analogues . In this study, we describe the use of {125I}iodoaryl azido-rhodamine123 (IAARh123)-a photoactive drug analogue of rhodamine 123, to study the effects of mild detergents and denaturing agents on MRP1-drug binding in membrane vesicles prepared from HeLa cells transfected with the MRP1 cDNA . Our results show that the zwitterionic detergent CHAPS and a nonionic detergent Brij35 inhibited the photolabeling of MRP1 with IAARh123 . Sodium deoxycholate (SDC) and octyl-beta-glucoside (OG), structurally similar to CHAPS and Brij35 and disrupting the lipid bilayer, showed a modest increase of MRP1 photolabeling with IAARh123 . Proteolytic digestion of IAARh123 photolabeled MRP1 labeled in the presence or absence of various detergents (CHAPS, SDC, or OG) revealed identical photolabeled peptides . Consistent with the drug-binding results, non-toxic concentrations of CHAPS and Brij35 reversed vincristine and etoposide (VP16) toxicity in MRP1 expressing cells . Taken together, the results of this study show that MRP1-drug interaction can occur outside the lipid bilayer environment . However, this interaction is inhibited with certain mild detergents.

Anticancer Drugs, 2003 Apr, 14(4), 281 - 7
Up-regulation of caveolin expression by cytotoxic agents in drug-sensitive cancer cells; Belanger MM et al.; Caveolin 1 expression is down-regulated 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 the multidrug-resistance phenotype . Here, we show, in drug-sensitive lung cancer cells (A549, Calu-6 or NCI-H69), that exposure to cytotoxic drugs (taxol, doxorubicin or etoposide) is sufficient to strongly up-regulate caveolin 1 and 2 protein levels . This up-regulation is sustained even 1 week after drug removal . Our results suggest that caveolin up-regulation is an early cellular response to a cytotoxic stress taking place before drug resistance.

Eur J Pharmacol, 2003 Apr 11, 466(1-2), 223 - 4
Expression of multidrug resistance protein 4 and 5 in the porcine coronary and pulmonary arteries; Mitani A et al.; Guanosine 3',5'-cyclic monophosphate (cGMP) has an important role in regulating vascular smooth muscle tone . We examined whether mRNA for multidrug resistance protein (MRP) 4 and MRP5, which were recently identified as ATP-dependent export pumps for cyclic nucleotides, is expressed in the porcine coronary and pulmonary arteries . The results showed that both arteries express mRNA for MRP4 and MRP5, and thus these proteins may be novel targets for the prevention and/or treatment of various cardiovascular diseases.

Zhonghua Xue Ye Xue Za Zhi, 2003 Jan, 24(1), 28 - 31
{Arsenic trioxide inhibits P-glycoprotein expression in multidrug-resistant human leukemia K562/ADM cell line that overexpresses mdr-1 gene and enhances their chemotherapeutic sensitivity}; Wei HL et al.; OBJECTIVE: To investigate the effects of arsenic trioxide (As(2)O(3)) on the apoptosis and P-glyco-protein (P-gp) expression of multidrug-resistant human leukemia K562/ADM cells, and the combined effects of As(2)O(3) with conventional chemotherapeutic agents . METHODS: Multidrug-resistant human leukemia cell line K562/ADM that overexpresses mdr-1 gene was used as the target cells . The cell proliferating activity was assessed with a MTT assay . Cell morphology was examined by light microscopy, confocal microscopy and electron-microscopy . P-gp expression, cell-cycle status were determined by flow cytometry . RESULTS: K562/ADM cells were highly resistant to adriamycin, and cross-resistant to daunorubicin and etoposide . As(2)O(3) at concentrations of 0.5 to 20 micromol/L inhibited the proliferation of K562/ADM cells, and K562/ADM cells were more sensitive to As(2)O(3) than their parent K562 cells did . As(2)O(3) induced marked apoptosis of K562/ADM cells showed by typical apoptotic morphological changes and the appearance of high sub-G(1) cell population . As(2)O(3) significantly inhibited the P-gp expression in K562/ADM cells, and exerted a synergistic effect on the enhancement of the cell sensitivity to adriamycin, daunorubicin and etoposide . CONCLUSION: As(2)O(3) induces growth-inhibition and apoptosis of multidrug-resistant K562/ADM cells, and augments synergistically the sensitivity of the cells to conventional chemotherapeutic agents via down-regulation of P-gp expression.

Zhonghua Xue Ye Xue Za Zhi, 2003 Jan, 24(1), 14 - 7
{Identification of multidrug resistance related genes in leukemia by suppression subtractive hybridization}; Zhu NX et al.; OBJECTIVE: To clone and screen genes related to multidrug resistance (MDR) in leukemia . METHODS: Suppression subtractive hybridization (SSH) was performed to profile differentially expressed genes between a MDR leukemia cell line (K562/DOX, as tester) and its parent cell line (K562, as driver) . Reverse Northern dot blot was carried out to further screen the subtracted cDNA library . The overexpressed cDNA fragments in K562/DOX cells were sequenced and compared with known genes in Genbank . RT-PCR and Northern blot were employed to confirm the differential expression of some identified genes . RESULTS: Eleven genes were identified being overexpressed in K562/DOX, including S3 ribosomal protein (S3rp) gene, NADH dehydrogenase subunit 2 (ND2) gene and My023 gene, which have not been reported to be related to MDR in cancer . CONCLUSION: Several genes, which might be involved in MDR were identified, indicating novel mechanisms of MDR in leukemia.

Curr Med Chem, 2003 Jul, 10(13), 1137 - 50
Perspective in antimalarial chemotherapy; Kumar A et al.; Antimalarial chemotherapy has become more complex and challenging because of multidrug resistant strains of Plasmodium falciparum . Due to resistance of malarial parasite against well known drugs, the chemotherapy of malaria has become complicated . In this review we have discussed brief introduction followed by life cycle of malaria parasite . The list of commercially available antimalarial drugs along with there action on different stages of parasite have been discussed . A brief description of their mechanism of action and advantages and disadvantages were reported . The natural products as antimalarial have been discussed in the review . On the basis of chemical classes the natural products were divided in the following categories; Quinoline alkaloids, Iso-quinoline alkaloids, Indoloquinoline alkaloids, Carbolines, Bis-isoquinoline, 4-Quinazole derivatives, Trioxanes, Terpenes, Naphthoquinone, Anthraquinones, Chalcones, Hydroxy flavanones, Coumarins and phenolic glycoside . The combination chemotherapy has been highlighted in the review . The Biochemical and Immunological changes in malarial infection are discussed along with complications of malarial chemotherapy due to resistance . In the conclusion section, the future strategies for the chemotherapy of malaria have been discussed.

Curr Med Chem Anti-Canc Agents, 2001 May, 1(1), 95 - 112
A new concept for the design of antisense oligonucleotides based on nucleic acid thermostability; Sugimoto N et al.; The antisense method is one of the most promising anti-cancer methods, however, the design of antisense oligonucleotides is difficult because many factors affecting their activitiy and stability must be considered . Recently, the oligonucleotide stabilities related to the antisense effects were quantitatively investigated based on nearest-neighbor parameters . We demonstrated that DeltaG(o) (37, hyb), a free energy change for the hybridization of antisense oligodeoxynucleotides (ODNs) with target RNAs is related to the RNase H cleavage of TAg (SV40 large T antigen) mRNA, the expression of a rabbit globin mRNA, and the protein function encoded by hMDR1 (human multidrug resistance-1) mRNA, while DeltaG(o) (37, hp), a free-energy change for hairpin formations of the antisense ODNs significantly affected the arrest efficiency of the DHFR (dihydrofolate reductase) mRNA transcription, the expression of the proalpha1(I) chain of human, and the hybridization extent for HIV-1 alpha-1 . For ras RNA (Ha-ras mRNA), DeltaG(o) (37, sc), a free energy change for the conformational change of the mRNA required for antisense ODN binding showed the best correlation with the equilibrium constants for the hybridization with their target RNA . On the other hand, the antisense effects ifor the HSV-1 IE5 (herpes simplex virus type 1 immediate early pre-mRNA5) showed less of a relationship to the hybridization stability of the antisense ODNs with the target pre-mRNA, because the antisense ODNs targeting the pre-mRNA must collapse its secondary structure around the splicing site to cancel out the expected antisense effects . Based on these results, we illustrate a new concept for the design of antisense ODNs based on DeltaG(o) (37, hyb), DeltaG(o) (37, hp), and DeltaG(o) (37, sc).

Curr Med Chem Anti-Canc Agents, 2001 Aug, 1(2), 163 - 74
Modulation of P-glycoprotein (PGP) mediated multidrug resistance (MDR) using chemosensitizers: recent advances in the design of selective MDR modulators; Krishna R et al.; Over the past two decades, a number of chemical entities have been investigated in the continuing quest to reverse P-glycoprotein (PGP) mediated multidrug resistance (MDR) in cancer . The complexity of interactions between these agents and the proteins responsible for MDR in conjunction with the challenges associated with developing SAR/QSAR relationships for MDR modulators has hampered our ability to develop agents that modulate MDR with enhanced specificity of target, increased efficacy, and minimized toxicity when coadministered with anticancer drugs . With an increased understanding of the molecular interaction, target-mediated SAR and combinatorial chemistry approaches, newer more selective inhibitors have been recently reported . These agents have shown remarkable promise in preclinical trials; although their ultimate clinical therapeutic utility remains to be established . The emphasis of this review is placed on the current understanding of modulator-drug transport protein interactions and to review the advances in the structure-based design, synthetic efforts and the cellular pharmacology of MDR modulating activity of a number of known PGP inhibitors.

Curr Med Chem Anti-Canc Agents, 2002 Jan, 2(1), 123 - 48
The biology and medicinal chemistry of epothilones; Wartmann M et al.; Epothilones are naturally occurring 16-membered macrolides with the ability to promote tubulin polymerization in vitro and to stabilize preformed microtubules against Ca(2+)- or cold-induced depolymerization . At the cellular level, interference with microtubule functionality results in potent inhibition of cancer cell proliferation at nM to even sub-nM concentrations . Most significantly, epothilones, unlike paclitaxel (Taxol), are equally active against drug-sensitive and multidrug-resistant cell lines in vitro and epothilone B has also shown potent in vivo antitumor activity in Taxol-resistant human tumor models . Epothilone B is currently undergoing Novartis-sponsored Phase II clinical trials . In addition to naturally occurring epothilones, numerous synthetic and semi-synthetic analogs have been prepared since the absolute stereochemistry of epothilone B was first disclosed in mid-1996 and their in vitro biological activity has been determined . These studies have generated a wealth of SAR data in a remarkably short period of time, given the complexity of the synthetic targets pursued . One of these analogs, BMS-247550, is presently in Phase II clinical trials by Bristol-Myers Squibb . In a first part this review is intended to provide a summary of the basic features of the in vitro biological profile of epothilones A and B, including emerging data on potential cellular epothilone resistance mechanisms . The second and third part will feature a comprehensive discussion of the epothilone SAR as it has emerged from the work of various (industrial and academic) laboratories across the world, including our own, with regard to effects on tubulin polymerization, in vitro antiproliferative activity, and in vivo antitumor activity.

Curr Med Chem Anti-Canc Agents, 2002 May, 2(3), 419 - 39
Recent progress in the development of anticancer agents; Eckhardt S; Cancer chemotherapy started with the discovery of the cytostatic effect of N-mustard and its derivatives more than five decades ago . This observation opened the way for the synthesis of various alkylating agents, antimetabolites and antimitotics expliciting antitumour activity against several human malignancies . The considerable toxicity of these drugs however, limited their application and only hormone-active products were relatively well tolerated . Besides, the majority of human malignant tumours proved to be chemoresistant . Consequently, there was still an urgent need for finding less toxic compounds possessing broader antitumour spectrum . Therefore, it became obvious that better understanding of the cellular metabolism - due to revolution in molecular biology - yielded new targets for cancer chemotherapeutic agents . Key enzymes active in signal transduction pathways could be blocked by new substances . Cell cycle control could be influenced by apoptosis inducers . Mitotic division could be inhibited by antitubulin agents . Multidrug resistance (MDR) could be modified by revertants . New concepts also emerged: a) chemoprevention, which is based on the principle, that since carcinogenesis is a genetically determined, progressive multistep process it can timely be reconverted into the direction of normal cellular metabolism by redifferentiating agents; b) antimetastatic therapy: originally performed postoperatively as an adjuvant therapy nowadays before surgical intervention, in order to block vascular dissemination of tumor cells (neoadjuvant therapy); c) antiangiogenic therapy: substances capable to hinder the vessel production essential for the development of metastasis; d) antitelomerase molecules inhibiting the immortal division capacity of DNA in malignant cells . All these new research approaches necessitate to review the existing drugs which are in clinical use or are investigational agents against human malignancies.

Curr Med Chem Anti-Canc Agents, 2002 May, 2(3), 387 - 401
Drug resistance and apoptosis in cancer treatment: development of new apoptosis-inducing agents active in drug resistant malignancies; Tolomeo M et al.; Modulation of multidrug resistance (MDR) has been extensively studied in vitro and in vivo . However, several clinical trials have failed to show any important benefits in terms of response to chemotherapy or the length of survival using MDR reversing agents . This may be due to the expression or co-expression of other drug resistance mechanisms in malignant cells . Several studies have shown that most, if not all, chemotherapeutic agents exert their anticancer activity by inducing apoptosis; therefore, resistance to apoptosis may be a major factor limiting the effectiveness of anticancer therapy . In the last few years, effort has been made to understand the biochemical alterations of apoptotic pathways in cancer . Many of these alterations confer a multidrug resistant phenotype to malignant cells . In this context, the new recently developed anticancer therapies based on drugs that modulate apoptosis may have importance for the treatment of tumors that are scarcely responsive to the conventional anticancer chemotherapy . In this review, we discuss the current knowledge about drug resistance, apoptosis and cancer and report the recently developed apoptosis modulating strategies that have potential therapeutic implications for the drug resistant tumors.

Curr Med Chem Anti-Canc Agents, 2002 Jul, 2(4), 441 - 63
Green tea catechins as novel antitumor and antiangiogenic compounds; Demeule M et al.; The concept of cancer prevention by use of naturally occuring substances that could be included in the diet is under investigation as a practical approach towards reducing cancer incidence, and therefore the mortality and morbidity associated with this disease . Tea, which is the most popularly consumed beverage aside from water, has been particularly associated with decreased risk of various proliferative diseases such as cancer and atherosclerosis in humans . Various studies have provided evidence that polyphenols are the strongest biologically active agents in green tea . Green tea polyphenols (GTPs) mainly consist of catechins (3-flavanols), of which (-)-epigallocatechin gallate is the most abundant and the most extensively studied . Recent observations have raised the possibility that green tea catechins, in addition to their antioxidative properties, also affect the molecular mechanisms involved in angiogenesis, extracellular matrix degradation, regulation of cell death and multidrug resistance . This article will review the effects and the biological activities of green tea catechins in relation to these mechanisms, each of which plays a crucial role in the development of cancer in humans . The extraction of polyphenols from green tea, as well as their bioavailability, are also discussed since these two important parameters affect blood and tissue levels of the GTPs and consequently their biological activities . In addition, general perspectives on the application of dietary GTPs as novel antiangiogenic and antitumor compounds are also presented.

Curr Cancer Drug Targets, 2003 Apr, 3(2), 89 - 107
Energy dependent transport of xenobiotics and its relevance to multidrug resistance; Sharma R et al.; Transport mechanisms for the exclusion of toxic xenobiotics and their metabolites from cellular environment are crucial for living organisms . Accumulation of these toxins may affect a number of regulatory and other functions, ultimately leading to cell death . This trafficking of toxins and their metabolites is an energy dependent, primary active process, involving the hydrolysis of nucleotide triphosphates (ATP or GTP), while transferring substrate molecules across the cell membrane, against a concentration gradient of the substrate . Therefore, specific membrane associated proteins, known as efflux pumps, are required to remove these undesirable molecules from the cellular environment . These transport proteins have diverse structural characteristics with molecular weights ranging from 28 kDa to 190 kDa and a broad substrate specificity ranging from anionic to weakly cationic compounds . While these transport mechanisms constitute an important part of the cellular defense machinery, they also pose a formidable threat to the efficacy of chemotherapy against pathogenic bacteria and cancer cells . In cancer cells, the over expression of these proteins may confer a multidrug resistance (MDR) phenotype . This problem of MDR in cancer cells has so far been attributed to the two major families of efflux pumps, P-glycoprotein (Pgp) and multidrug resistance associated proteins (MRP), which belong to the ATP-binding cassette (ABC) super family . However, the existence of these pumps has not been able to explain all types of acquired MDR . Therefore, the importance of transport mechanisms other than these ABC-transporters cannot be ruled out . One such transporter is DNP-SG ATPase, whose identity has recently been established with RLIP76, a Ral binding GTPase activating protein known to be involved in the Ras-Rho-Ral mediated signaling mechanism . In the present article, we review the comparative functional, structural, and molecular characteristics of some transporters and discuss their role in xenobiotic transport and multidrug resistance.

J Chemother, 2003 Feb, 15(1), 66 - 70
Long-term tolerance and effectiveness of moxifloxacin therapy for tuberculosis: preliminary results; Valerio G et al.; The resurgence of tuberculosis is a major problem . Increasing multiple resistance to current drugs used for therapy, non-compliance to therapy or co-morbidity are challenging problems that do not allow use of standard therapy in all patients . Quinolones are claimed to be active drugs in TB infection . Moxifloxacin shows the highest intracellular concentration in vitro and in experimental animals, but long-term tolerability is unknown . Our aim was to observe in compliant patients, not eligible for standard therapy, the effect of 6 months of therapy with moxifloxacin, isoniazid and rifampin . Nineteen patients, a control group, were observed for the same period under therapy with streptomycin, pirazinamide, rifampin, isoniazid . The patients were affected by indolent miliary pattern and concomitant lymphoma or leukemia in 3 cases; rare nodular involvement with genitourinary diseases in 3 others; segmental to lobar involvement in 4 others with concomitant multidrug resistance, bone localization, hepatitis . The control group was more uniform and showed segmental to lobar nodular involvement with pleuritis in 3 patients, together with hepatitis in 3 . Monthly checks of blood gas analysis, chest X-ray, functional testing, serum titers of antibodies against antigen 60, sputum slides and complete chemical analysis were performed . A follow-up visit was performed 1 month after therapy . Patients under moxifloxacin therapy experienced no toxicity, almost complete sterilization and remission of the disease . Sterilization was obtained in 15 days . Patients under standard therapy also had a good clinical outcome, although therapy was delayed in 3 cases because of increased transaminases within the first 15 days of therapy . Moxifloxacin seems to be well tolerated and combination therapy including moxifloxacin for TB seems to be as active as the standard therapy in patients with complex illness.

Planta Med, 2003 Mar, 69(3), 235 - 40
Reversal of P-glycoprotein-mediated multidrug resistance by protopanaxatriol ginsenosides from Korean red ginseng; Choi CH et al.; The overexpression of P-glycoprotein (Pgp) or the multidrug resistance-associated protein (MRP) confers multidrug resistance (MDR) to cancer cells . MDR cells can be sensitized to anticancer drugs when treated concomitantly with an MDR modulator . In this study, we investigated whether or not ginseng saponins could reverse MDR mediated by Pgp or MRP . The chemosensitization and drug accumulation effects of ginseng saponins such as the total saponin, protopanaxadiol ginsenosides (PDG), protopanaxatriol ginsenosides (PTG), ginsenosides-Rb 1, -Rb 2, -Rc, -Rg 1 and -Re were tested on the daunorubicin- and doxorubicin-resistant acute myelogenous leukemia sublines (AML-2/D100 and AML-2/DX100), which overexpress Pgp and MRP, respectively . PTG showed cytotoxicity in both sublines and was able to reverse resistance in the AML-2/D100 subline in a concentration-dependent manner . Conversely, other ginseng saponins at concentrations less than 300 microg/mL showed neither cytotoxicity nor chemosensitizing activity in both resistant sublines . Flow cytometry analysis showed that the effect of PTG (100 microg/mL) on drug accumulation of daunorubicin in the AML-2/D100 subline was 2-fold higher than that observed in the presence of verapamil (5 microg/mL) and 1.5 times less than cyclosporin A (3 microg/mL) . The maximum non-cytotoxic concentrations of PTG did not appear to increase the Pgp levels, which is in contrast to verapamil and cyclosporin A . PTG at 200 microg/mL or more completely inhibited the azidopine photolabeling of Pgp . The results suggest that PTG has a chemosensitizing effect on Pgp-mediated MDR cells by increasing the intracellular accumulation of drugs through direct interaction with Pgp at the azidopine site . In addition, PTG may have a beneficial effect on cancer chemotherapy with respect to the possibility of long-term use without the concern of Pgp activation.

J Pharmacol Exp Ther, 2003 Jul, 306(1), 310 - 6 Epub 2003 Apr 03.
The site-specific transport and metabolism of tacrolimus in rat small intestine; Tamura S et al.; The objective of this study was to evaluate the absorption of tacrolimus by means of simultaneous perfusion of intestinal lumen and blood vessels in rats . In our previous report, the permeability of tacrolimus was found to be higher in the jejunum than in the ileum or colon, suggesting the site-dependent absorption after oral administration . However, in this article, simultaneous perfusion technique revealed that the extent of absorption into blood vessels was similar in the jejunum and the ileum regardless of the site difference in permeability as the absorption rate . In addition to the multidrug resistance-associated protein-mediated efflux, cytochrome P450 (P450)-mediated metabolism could be a possible mechanism to explain the inconsistencies in the site dependence of tacrolimus absorption . Two enzyme inhibitors, ketoconazole and midazolam, were coperfused in rat intestinal lumen with tacrolimus to specify the effect of P-gp and P450 . In the jejunum, both inhibitors significantly enhanced the absorbed amount of tacrolimus, whereas the permeability was not affected . It was suggested that both inhibitors mainly suppress P450-mediated metabolism in the upper region of the intestine . In contrast, in the ileum, ketoconazole significantly enhanced both the absorbed amount and the permeability of tacrolimus . However, midazolam failed to enhance the absorption of tacrolimus, indicating the dominant role of P-glycoprotein (P-gp)-mediated efflux in the lower region . From these findings, it is concluded that the site-dependent differences in P-gp and/or P450 activity could be the prime cause of large intra- and interindividual variability in the oral absorption of tacrolimus.

Toxicol Lett, 2003 Apr 11, 140-141, 465 - 76
Interaction of ochratoxin A with human intestinal Caco-2 cells: possible implication of a multidrug resistance-associated protein (MRP2); Berger V et al.; Ochratoxin A (OTA), a nephrotoxic mycotoxin, is absorbed from small intestine and, in plasma, binds to serum albumin . Prolonged half-live results from reabsorption by proximal tubules and enterohepatic circulation . The mechanism whereby OTA crosses intestine was investigated by means of a cell culture system consisting of Caco-2 cells, as in vitro model of human intestinal epithelium . Cytotoxicity assays on proliferating Caco-2 cells showed that 0.4 microM OTA inhibits MTT reduction by 50% . Transepithelial transport and intracellular accumulation of OTA were studied in Caco-2 cells, differentiated in bicameral inserts . At pH 7.4, OTA is transported preferentially in basolateral (BL) to apical (AP) direction, suggesting a net secretion . Conditions closer to in vivo situation in duodenum (AP pH 6.0, BL pH 7.4) increase intracellular accumulation and transepithelial transport . AP to BL transport becomes higher than BL to AP transport, suggesting OTA absorption . Addition of serum albumin in BL compartment further increases OTA absorption across Caco-2 cells and suggests that in vivo OTA transport from serosal to luminal side of enterocytes is prevented, due to its binding to plasma proteins . Competition experiments showed that carrier systems for large neutral amino acids, H(+)/dipeptides cotransporter, organic anion (p-aminohippurate) carrier and organic anion transporter (oatp) are not implicated in OTA transport across Caco-2 cells, in contrast to what was reported in kidney and liver . AP and BL transport and intracellular accumulation of OTA are increased in the presence of non specific inhibitors of MRPs (indomethacin, genistein and probenecid) and of 1-chloro-2,4-dinitrobenzene (biotransformed into 2,4-dinitrophenyl-gluthatione, a specific inhibitor of MRPs), but are affected by verapamil, an inhibitor of P-gp . This suggests that the multidrug resistance-associated protein (MRP2) could be implicated in transepithelial transport . Therefore, absorption of OTA across the intestinal mucosa would be limited thanks to its excretion through MRP2 at the apical pole of enterocytes.

Toxicol Lett, 2003 Apr 11, 140-141, 133 - 43
The role of multidrug transporters in drug availability, metabolism and toxicity; Bodo A et al.; Multidrug resistance is frequently observed when treating cancer patients with chemotherapeutic agents . A variety of ATP binding cassette (ABC) transporters, localized in the cell membrane, cause this phenomenon by extruding a variety of chemotherapeutic agents from the tumor cells . However, the major physiological role of the multidrug transporters is the protection of our cells and tissues against xenobiotics, and these transporters play a key role in drug availability, metabolism and toxicity . Three major groups of ABC transporters are involved in multidrug resistance: the classical P-glycoprotein MDR1, the multidrug resistance associated proteins (MRP1, MRP2, and probably MRP3, MRP4 and MRP5), and the ABCG2 protein, an ABC half-transporter . All these proteins were shown to catalyze an ATP-dependent active transport of chemically unrelated compounds . MDR1 (P-glycoprotein) and ABCG2 preferentially extrude large hydrophobic, positively charged molecules, while the members of the MRP family can extrude both hydrophobic uncharged molecules and water-soluble anionic compounds . By examining the interactions of the multidrug transporters with pharmacological and toxic agents, a prediction for the cellular and tissue distribution of these compounds can be achieved . Oral bioavailability, entering the blood-brain and blood-CSF barrier, reaching the fetus through the placenta, liver and kidney secretion, cellular entry for affecting intracellular targets, are all questions, which can be addressed by basic in vitro studies on the multidrug resistance proteins . Investigation of the substrate interactions and modulation of multidrug transporters may pave the way for predictive toxicology and pharmacogenomics . Here we show that by using in vitro assay systems it is possible to measure the interactions of multidrug transporters with various drugs and toxic agents . We focus on the characterisation of the MRP1 and MRP3 proteins, their relevance in chemoresistance of cancer and in drug metabolism and toxicity.

Hua Xi Kou Qiang Yi Xue Za Zhi, 2003 Feb, 21(1), 70 - 3
{Induction of multidrug resistance in Tca8113 cells by transient exposure to different chemotherapeutic drugs}; Zhang P et al.; OBJECTIVE: The purpose of this study was to determine the effect of transient exposure to chemotherapeutic drugs on multidrug resistance of Tca8113 cells . METHODS: The MDR1 and MRP gene expressions in Tca8113 and K562/ADM cells lines were analyzed using reverse transcription polymerase chain reaction (RT-PCR), after the cells were treated with different cytotoxic drugs . The function and expressions of P-glycoprotein 170 and multidrug resistant associated protein were studied using fluorescence photometric assays . RESULTS: The inhibitive rate of Tca8113 cells was higher than that of K562/ADM, after exposure to chemotherapeutic drugs . The transient exposure to cytotoxic drugs weakly induced MDR1 and multidrug resistant associated protein expression in Tca8113 cells . The intracellular drug concentration in K562/ADM was lower than that in Tca8113 cells . CONCLUSION: Induction of MDR1 and multidrug resistant associated protein gene expression response to cytotoxic drugs may be related with the increased multidrug resistance in drug-treated human tumor cells.

Biol Chem, 2003 Jan, 384(1), 139 - 42
Identification by phage display of the linear continuous MRPr1 epitope in the multidrug resistance-associated protein (MRP1); Flego M et al.; In order to study the structure of the multidrug resistance-associated protein (MRP1), which is one of the most important members of the ATP-binding cassette (ABC) protein family acting as drug-efflux systems, we have developed an epitope mapping-based strategy . By means of the mAb MRPr1, we have immunoselected clones from two distinct random peptide libraries displayed on phages and have identified several peptide sequences mimicking the internal conformation of this 190 kDa multidrug transporter protein . Phage clones able to block the immunolabeling of the MRPr1 antibody to MRP1-overexpressing multidrug resistance (MDR) H69/AR cells were isolated and, after sequencing the corresponding inserts, their amino acid sequence was compared to that of MRP1 . This analysis led to the identification of the consensus sequence L.SLNWED, corresponding to the MRP1 segment LWSLNKED (residues 241-248) . This MRP1 sequence is partially overlapping with the MRPr1 epitope GSDLWSLNKE (residues 238-247) previously mapped using peptide scanning techniques . These results demonstrate the high reliability of phage display technology to study not only the topography of complex integral membrane proteins such as MRP1, but also to help identify critical residues participating in the formation of the epitope structure.

Cancer, 2003 Apr 15, 97(8), 1999 - 2005
Multidrug resistance proteins in rhabdomyosarcomas: comparison between children and adults; Komdeur R et al.; BACKGROUND: Pediatric rhabdomyosarcomas (RMS) have a more advantageous prognosis after multimodality treatment compared with adult RMS, which might be related to a decreased sensitivity to chemotherapy in adults . Resistance to chemotherapy might be conveyed by the multidrug resistance (MDR)-associated proteins P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), and lung resistance-related protein (LRP) . It was therefore suggested that these proteins were expressed differently in pediatric and adult patients . METHODS: The expression of P-gp, MRP1, and LRP was assessed immunohistochemically in 45 specimens of untreated RMS: 29 were obtained from children younger than 16 years old and 16 were obtained from adults . All children had an embryonal or botryoid RMS . Among the adults, there were 10 embryonal, 3 alveolar, and 3 pleomorphic RMS . Samples were scored as negative or positive according to the percentage of immunoreactive tumor cells: 0.5 (1-5%), 1 (5-25%), 2 (26-50%), 3 (51-75%), or 4 (> 75%) . RESULTS: Expression of LRP was more pronounced in embryonal and pleomorphic RMS in adults compared with RMS in children . In addition, LRP expression correlated with age at diagnosis . Alveolar RMS had remarkably low LRP expression . Expression of P-gp and MRP1 did not differ significantly between children and adults . CONCLUSIONS: In this series of embryonal and pleomorphic RMS, an increased LRP expression was observed in adults, which may explain their worse response to chemotherapy reported in other studies . In alveolar RMS, a low LRP expression was observed, suggesting that other mechanisms are responsible for the resistant phenotype in most of these tumors .

Gastroenterology, 2003 Apr, 124(4), 1037 - 42
A multidrug resistance 3 gene mutation causing cholelithiasis, cholestasis of pregnancy, and adulthood biliary cirrhosis; Lucena JF et al.; We describe a 47-year-old patient who developed cholelithiasis in adolescence, followed by recurrent intrahepatic cholestasis of pregnancy, and finally biliary cirrhosis in adulthood . In our patient, the consecutive presentation of the 3 mentioned disorders raised the suspicion of a defect of MDR3, the canalicular protein involved in the transport of phospatidylcholine to bile . Mutational analysis in our patient showed a heterozygous missense mutation of the MDR3 gene that has not been described previously, which occurs in exon 14 at codon 535, and results in the substitution of glycine for aspartic acid . Further analysis of 7 members of the family showed the same mutation in her daughter who, on follow-up, developed cholestasis of pregnancy and persisting high serum levels of gamma-glutamyl transpeptidase and alkaline phosphatase after delivery . Although biliary cirrhosis associated with MDR3 deficiency typically appears before the age of 25 years, in our case, the relatively mild MDR3 dysfunction allowed for a slower progression of the disease with established, well-advanced cirrhosis in the fifth decade of life . The present case, which accumulates the 3 clinical disorders assocaited with MDR3 deficiency, shows that this condition should be suspected not only in children or young people with high gamma-glutamyl transpeptidase cholestasis but also in middle-aged or older patients with chronic idiopathic cholestasis, especially when there is a previous history of cholestasis of pregnancy or juvenile cholelithiasis.

HIV Clin Trials, 2003 Mar-Apr, 4(2), 92 - 8
Use of bDNA testing in the immunologically nonresponding patient who has a low or undetectable viral load by RT-PCR testing; Grimes RM et al.; BACKGROUND: Studies have shown that reverse transcription-polymerase chain reaction (RT-PCR) technology underquantifies viral loads in patients with non-B clades of HIV-1 . Testing with bDNA technology gave higher viral loads in these subtypes . A study was conducted to determine whether virologically responding patients on HAART who were not immunologically responding would have higher viral loads using bDNA technology and whether these differences were due to non-B clades . METHOD: Forty-eight patients receiving HAART for more than 6 months who were having inappropriate immunologic responses in spite of undetectable or very low viral loads determined by RT-PCR (<3000 copies by Roche Amplicor 1.0) were studied . These patients had bDNA viral loads performed . All patients who had bDNA viral loads equivalent to >3000 by RT-PCR had clade and genotypic studies performed . RESULTS: Fifteen patients had viral loads by bDNA that were equivalent to >3000 copies by RT-PCR . Four of these were found to have non-B clades (one D clade and three AG clade) . The D clade patient had multidrug resistance; none of the AG clade patients had resistance . Of the remaining 11 patients, virus could not be recovered from 2 and 9 had a B clade . Six of these nine had genotypic resistance to HAART drugs . CONCLUSION: bDNA testing may be useful in the immunologically nonresponding patient.

Cancer Res, 2003 Apr 1, 63(7), 1515 - 9
Small interfering RNA-induced suppression of MDR1 (P-glycoprotein) restores sensitivity to multidrug-resistant cancer cells; Wu H et al.; Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells . Clinically, MDR is one of the major causes for chemotherapeutic treatment failure in cancer patients . To explore a new approach to circumventing MDR, we adopted RNA interference to target MDR1 gene expression . RNA interference is a conserved biological response to double-stranded RNA, which results in sequence-specific gene silencing {G . J . Hannon, Nature (Lond.), 418: 244-251, 2002} . We report that introduction of an MDR1-targeted small interfering RNA duplex into drug-resistant cancer cells markedly inhibited the expression of MDR1 mRNA and P-gp, as determined by reverse transcription-PCR and Western blot . Inhibition of P-gp expression by small interfering RNA enhanced the intracellular accumulation of and selectively restored sensitivity to drugs transported by P-gp . These studies indicate that RNA interference can modulate MDR in preclinical models.

Pharm Res, 2003 Mar, 20(3), 351 - 9
Simultaneous modulation of multidrug resistance and antiapoptotic cellular defense by MDR1 and BCL-2 targeted antisense oligonucleotides enhances the anticancer efficacy of doxorubicin; Pakunlu RI et al.; PURPOSE: To enhance the anticancer efficacy of an established drug by the simultaneous suppression of pump and nonpump cellular resistance . METHODS: Multidrug resistant human ovarian (A2780/AD) and breast (MCF-7/AD) cancer cells were used . Doxorubicin (DOX) and antisense oligonucleotides (ASO) targeted to MDR1 and BCL-2 mRNA were combined in a solution within one liposomal drug delivery system (LDDS) in different combination series . Ten series of experiments were performed . In each series cells were incubated with 12 to 45 concentrations of free DOX and different liposomal formulations over a period of 6 to 48 h . Cytotoxicity, apoptosis induction, caspases, MDR1., BCL-2, and APAF-1 genes, P-glycoprotein, and BCL-2 protein were studied . RESULTS: The combination of DOX and ASO targeted to MDR1 and BCL-2 mRNA in one LDDS exhibited a dramatic increase in the anticancer action of DOX . As a result of the simultaneous suppression of pump and nonpump cellular resistance by the inhibition of P-glycoprotein and BCL-2 protein synthesis, a significant increase in the activation of caspases and apoptosis was observed . CONCLUSIONS: The simultaneous suppression of multidrug resistance and antiapoptotic cellular defense significantly enhanced the anticancer activity of DOX . Therefore, the proposed DDS combination may potentially be used in the treatment of multidrug-resistant ovarian and breast cancers.

Prescrire Int, 2003 Apr, 12(64), 46 - 8
Linezolid: new preparation . In severe gram-positive infections; Effects of a series of dihydroanthracene derivatives on drug efflux in multidrug resistant cancer cells; GERCTOP-UMR CNRS 6009, faculte de pharmacie, Universite de la Mediterranee, 27, bd Jean Moulin, 13385 cedex 05, Marseille, FranceA set of 9,10-dihydro-9,10-ethano and ethenoanthracene derivatives was tested with the aim to quantify the effect observed on drug efflux . Structure activity relationships and molecular modeling studies allowed to define topological display of pharmacophoric groups for these reversal agents.

Zhonghua Zhong Liu Za Zhi, 2002 Nov, 24(6), 557 - 60
{In situ hybridization of tight junction molecule occludin mRNA in gastric cancer}; Yin F et al.; OBJECTIVE: To analyze the distribution and significance of occludin mRNA expression in human gastric cancer, as well as its relationship with gastric cancer pathology and multidrug resistance (MDR) in vivo . METHODS: In situ hybridization (ISH) technique was used to evaluate the expression of occludin mRNA in 42 gastric carcinoma specimens obtained by surgery and 23 relatively normal gastric mucosa obtained by gastric endoscopy . All specimens had been stored in cryostatic section . RESULTS: Occludin mRNA was found positive in the cytoplasm of gastric glandulous epithelia as blue particles with intensive stain in 14 of 42 gastric carcinomas (33.3%), 23 of 42 paracancerous gastric tissues (54.8%), 14 of 23 relatively normal gastric tissues (60.9%), 9 of 16 well differentiated carcinomas (56.3%), 4 of 14 moderately differentiated carcinomas (28.6%), 1 of 10 poorly differentiated carcinomas (10.0%) and none of 2 mucosal carcinomas . There were significant differences in occludin mRNA positive rate between relatively normal gastric tissue and gastric cancer as well as between paracancerous gastric tissue and gastric cancer . The expression of occludin mRNA in moderately and poorly differentiated groups was gradually reduced when compared with well differentiated group, which suggests that there be a significant correlation between tumor differentiation and the expression of occludin mRNA . Furthermore, the positive signals of occludin mRNA distributed extensively in the cytoplasm of SGC7901/VCR cell, being vincristine resistant, derived from parental gastric cell line SGC7901 . The positive signals of SGC7901/VCR were stronger than those of SGC7901 cells . CONCLUSION: Occludin mRNA, being mainly located in epithelial cells and its expression correlated with tumor differentiation, may be involved in the development of multi-drug resistance in gastric cancer.

Zhonghua Zhong Liu Za Zhi, 2002 Nov, 24(6), 529 - 32
{Adenovirus-mediated transfer of anti-MDR1 ribozyme in the treatment of multidrug-resistant human lymphoma in SCID mice}; Xu D et al.; OBJECTIVE: To evaluate the effect of adenovirus-mediated transfer of anti-MDR1 ribozyme on restoring drug sensitivity of multidrug-resistant human lymphoma both in vitro and in SCID mice . METHODS: A recombinant adenovirus expressing ribozyme against codon 196 of MDR1 mRNA (Ad-196MDR1-Rz) was developed through cotransfection of shuttle vector pCA14 containing 196MDR1-Rz and rescue vector pJM17 into human embryonic kidney cell line 293 . In vitro Daudi/MDR20, a MDR1-mediated drug-resistant human lymphoma cell line, was transduced by Ad-196MDR1-Rz at MOI of 400 pfu/cell . RT-PCR and FACS analyses were used to evaluate the MDR1 expression in both transcriptional and translational levels . MTT assay was used for analysis of drug resistance . In vivo, SCID mice were inoculated subcutaneously by 5 x 10(6) Daudi/MDR20 or parental Daudi/wt cells . Adenovirus was injected locally . Vincristine (VCR) was given intraperitoneally . RESULTS: In vitro transduction of Ad-196MDR1-Rz to Daudi/MDR20 cells was able to interrupt MDR1 transcription, inhibit P-gp expression and restore drug sensitivity to VCR . Of SCID mice bearing Daudi/MDR20 cells, tumor free rate and long term survival were 66.7% (6/9) and > 120 days in the therapeutic group of Ad-196MDR1 + VCR vs 12.5% (7/8) and none survived > 120 days in the control groups of Ad-Mock + VCR or VCR alone . The difference was very statistically significant . CONCLUSION: Ad-mediated transfer of 196MDR1-Rz can revert drug resistance of MDR tumor cells both in vitro and in vivo . Ad-196MDR1-Rz may be helpful as an adjuvant in the chemotherapy of P-gp mediated MDR human tumor.

Zhonghua Zhong Liu Za Zhi, 2002 Nov, 24(6), 526 - 8
{Effect of tetrandrine and droloxifene on the reversion of drug resistance of K562/A02 cell line and induction of apoptosis}; Chen B et al.; OBJECTIVE: To study the reversal effect of tetrandrine (Tet) and droloxifene (Drol) on multidrug resistant cell line K562/A02 and apoptosis induction . METHODS: The cytotoxicity of daunorubicin (DNR) was assayed by MTT method . The effects of Tet and DRL, alone or combined were detected through the apoptosis of K562/A02 by agarose gel electrophoresis . RESULTS: The cytotoxicity of DNR to K562/A02 was enhanced by 0.62 microg/ml Tet or 1.94 microg/ml Drol with IC(50) 7.28 +/- 2.06 microg/ml, 7.58 +/- 3.44 microg/ml, giving a reversal effect of 2.94 and 2.82 . But IC(50) of combined Tet and Drol was 1.66 +/- 0.41 microg/ml with the reversal effect markedly increased to 12.9 . Neither 0.62 microg/ml Tet nor 1.94 microg/ml Drol could induce apoptosis of K562/A02 cells . CONCLUSION: Multidrug resistance (MDR) can be partially reversed by Tet or Drol, of which the combination shows a great synergistic reversal effect . The mechanism of Tet and Drol reversing multidrug resistance is not correlated with the apoptosis of K562/A02 cells.

Fundam Clin Pharmacol, 2003 Apr, 17(2), 147 - 53
The mechanisms of resistance to antimalarial drugs in Plasmodium falciparum; Le Bras J et al.; Drug-resistant malaria is primarily caused by Plasmodium falciparum, a species highly prevalent in tropical Africa, the Amazon region and South-east Asia . It causes severe fever or anaemia that leads to more than a million deaths each year . The emergence of chloroquine resistance has been associated with a dramatic increase in malaria mortality among inhabitants of some endemic regions . The rationale for chemoprophylaxis is weakening as multiple-drug resistance develops against well-tolerated drugs . Plasmodium falciparum drug-resistant malaria originates from chromosome mutations . Analysis by molecular, genetic and biochemical approaches has shown that (i) . impaired chloroquine uptake by the parasite vacuole is a common characteristic of resistant strains, and this phenotype is correlated with mutations of the Pfmdr1, Pfcg2 and Pfcrt genes; (ii) . one to four point mutations of dihydrofolate reductase (DHFR), the enzyme target of antifolates (pyrimethamine and proguanil) produce a moderate to high level of resistance to these drugs; (iii) . the mechanism of resistance to sulfonamides and sulfones involves mutations of dihydropteroate synthase (DHPS), their enzyme target; (iv) . treatment with sulphadoxine-pyrimethamine selects for DHFR variants Ile(51), Arg(59), and Asn(108) and for DHPS variants Ser(436), Gly(437), and Glu(540); (v) clones that were resistant to some traditional antimalarial agents acquire resistance to new ones at a high frequency (accelerated resistance to multiple drugs, ARMD) . The mechanisms of resistance for amino-alcohols (quinine, mefloquine and halofantrine) are still unclear . Epidemiological studies have established that the frequency of chloroquine resistant mutants varies among isolated parasite populations, while resistance to antifolates is highly prevalent in most malarial endemic countries . Established and strong drug pressure combined with low antiparasitic immunity probably explains the multidrug-resistance encountered in the forests of South-east Asia and South America . In Africa, frequent genetic recombinations in Plasmodium originate from a high level of malaria transmission, and falciparum chloroquine-resistant prevalence seems to stabilize at the same level as chloroquine-sensitive malaria . Nevertheless, resistance levels may differ according to place and time . In vivo and in vitro tests do not provide an adequate accurate map of resistance . Biochemical tools at a low cost are urgently needed for prospective monitoring of resistance.

Physiol Rev, 2003 Apr, 83(2), 633 - 71
Bile salt transporters: molecular characterization, function, and regulation; Trauner M et al.; Molecular medicine has led to rapid advances in the characterization of hepatobiliary transport systems that determine the uptake and excretion of bile salts and other biliary constituents in the liver and extrahepatic tissues . The bile salt pool undergoes an enterohepatic circulation that is regulated by distinct bile salt transport proteins, including the canalicular bile salt export pump BSEP (ABCB11), the ileal Na(+)-dependent bile salt transporter ISBT (SLC10A2), and the hepatic sinusoidal Na(+)- taurocholate cotransporting polypeptide NTCP (SLC10A1) . Other bile salt transporters include the organic anion transporting polypeptides OATPs (SLC21A) and the multidrug resistance-associated proteins 2 and 3 MRP2,3 (ABCC2,3) . Bile salt transporters are also present in cholangiocytes, the renal proximal tubule, and the placenta . Expression of these transport proteins is regulated by both transcriptional and posttranscriptional events, with the former involving nuclear hormone receptors where bile salts function as specific ligands . During bile secretory failure (cholestasis), bile salt transport proteins undergo adaptive responses that serve to protect the liver from bile salt retention and which facilitate extrahepatic routes of bile salt excretion . This review is a comprehensive summary of current knowledge of the molecular characterization, function, and regulation of bile salt transporters in normal physiology and in cholestatic liver disease and liver regeneration.

J Pharmacol Exp Ther, 2003 Jul, 306(1), 124 - 31 Epub 2003 Mar 27.
Multidrug resistance protein MRP2 contributes to blood-brain barrier function and restricts antiepileptic drug activity; Potschka H et al.; The blood-brain barrier (BBB) is a physical and metabolic barrier between the brain and the systemic circulation, which functions to protect the brain from circulating drugs, toxins, and xenobiotics . ATP-dependent multidrug transporters such as P-glycoprotein (Pgp; ABCB1), which are found in the apical (luminal) membranes of brain capillary endothelial cells, are thought to play an important role in BBB function by limiting drug penetration into the brain . More recently, the multidrug resistance protein MRP2 (ABCC2) has been found in the luminal surface of brain capillary endothelium of different species, including humans . In endothelial cells from patients with drug-resistant epilepsy, MRP2 was shown to be overexpressed, indicating that it may be critically involved in multidrug resistance of such patients . However, the role of MRP2 in drug disposition into the brain is defined poorly . Herein, we used different strategies to study the contribution of MRP2 to BBB function . First, the MRP inhibitor probenecid was shown to increase extracellular brain levels of the major antiepileptic drug phenytoin in rats, indicating that phenytoin is a substrate of MRP2 in the BBB . This was substantiated by using MRP2-deficient TR- rats, in which extracellular brain levels of phenytoin were significantly higher compared with the normal background strain . In the kindling model of epilepsy, coadministration of probenecid significantly increased the anticonvulsant activity of phenytoin . In kindled MRP2-deficient rats, phenytoin exerted a markedly higher anticonvulsant activity than in normal rats . These data indicate that MRP2 substantially contributes to BBB function.

Blood, 2003 Aug 15, 102(4), 1202 - 10 Epub 2003 Mar 27.
Quinine as a multidrug resistance inhibitor: a phase 3 multicentric randomized study in adult de novo acute myelogenous leukemia; Solary E et al.; Based on our previous demonstration that quinine could be used clinically to reverse P-glycoprotein-mediated resistance, we designed a multicenter, randomized trial aiming to determine whether quinine would improve the survival of adult patients (15-60 years old) with de novo acute myelogenous leukemia (AML) . These patients randomly received (n = 213) or did not receive (n = 212) a 30 mg/kg/day continuous intravenous infusion of quinine in combination with induction chemotherapy combining idarubicine and cytarabine and, depending on bone marrow examination at day 20, an additional course of cytarabine and mitoxantrone . The mean steady-state quinine concentration was 7.8 mg/L and the mean multidrug resistance reversing activity of serum was 1.96 . Complete remission (CR) was obtained in 344 patients (80.9%) without significant influence of quinine . Of the patients in complete remission, 82 were assigned to receive HLA-matched bone marrow transplants, whereas 262 were assigned to 2 courses of intensive consolidation chemotherapy, with or without quinine, depending on initial randomization . The 4-year actuarial overall survival (OS) of the 425 eligible patients was 42.0% +/- 2.5%, without significant influence of quinine . Of 160 patients who could be studied, 54 demonstrated rhodamine 123 efflux . In these patients, quinine significantly improved the CR rate from 12 of 25 (48.0%) to 24 of 29 (82.8%) (P =.01) . However, there was no significant difference in OS . Neither mdr1 gene nor P-glycoprotein expression influenced the outcome . We conclude that quinine does not improve the survival of adult patients with de novo AML, even though it improves CR rate in a small subgroup of patients defined by rhodamine 123 efflux.

Toxicol Appl Pharmacol, 2003 Mar 15, 187(3), 168 - 77
Polychlorinated biphenyls are not substrates for the multidrug resistance transporter-1; Tampal NM et al.; The multidrug resistance (MDR) transporter is a phosphorylated glycoprotein (P-gp) that has been implicated in the efflux of a large variety of xenobiotics, thereby protecting vital organs . This study examines the hypothesis that the multidrug resistance transporter is involved in restricting the entry of polychlorinated biphenyls (PCBs) into the brain . Three test systems were used . First, the ATPase activity of the human P-gp was measured as an indicator of the interaction of PCBs with the MDR transporter . PCB congeners and metabolites included in the study were PCB 153, PCB 169, PCB 77, and the 4-hydroxy and 4,4'-dihydroxy metabolites of PCB 77 . An increase in ATPase activity was observed for all the PCBs tested except the 4-hydroxy metabolite of PCB 77 . Second, we studied the transport of (14)C-PCB 77 and (14)C-PCB153 in a cell-culture model using porcine kidney cells expressing the human MDR1 or the mouse mdr1a gene and compared it to the transport in control cells . No difference in directional transport due to P-gp was observed with either of the congeners in any of the cell lines . Finally, the distribution pattern of (14)C-PCB 77 in mdr1a knockout mice and genetically matched wild-type mice was measured . No significant differences in tissue distribution, especially in the brain tissue, were observed between wild-type and mdr1a knockout mice . These results suggest that some PCB congeners can bind to the MDR1 transporter; however, they may not be transported by it.

Int J Tuberc Lung Dis, 2003 Mar, 7(3), 289 - 97
Pharmaceutical formulation of a fixed-dose anti-tuberculosis combination; Danckwerts MP et al.; SETTING: Department of Pharmacy and Pharmacology, University of the Witwatersrand . Despite the availability of highly effective treatment regimens for tuberculosis (TB), the cure rate still remains relatively low . This may be attributed to the high incidence of patient non-compliance, which subsequently leads to the emergence of multidrug-resistant TB (MDR-TB) . To avoid the problem of further creation and propagation of MDR-TB, it may be proposed that patients should be given fixed-dose combinations of anti-tuberculosis drugs whenever self-administration is permitted . OBJECTIVE: To optimise an anti-tuberculosis extemporaneous powder formulation for suspension in order to develop a fixed combination of rifampicin, isoniazid, pyrazinamide and ethambutol hydrochloride as a powder to be reconstituted with water by the patient prior to administration . METHODS: Different suspending agents were evaluated for their influence on powder flow properties, and sediment volume on the powder blends . Sodium starch glycolate was selected as the suspending agent because of its favourable powder flow properties and sediment volume produced . The dissolution characteristics of the extemporaneous powder for suspension were also compared to the dissolution profiles of commercially available anti-tuberculosis tablet dosage forms . RESULTS: The powder for suspension for rifampicin, isoniazid, pyrazinamide and ethambutol hydrochloride all compared favourably to the dissolution rate from the commercially available tablet dosage forms.

Biochim Biophys Acta, 2003 Apr 1, 1611(1-2), 107 - 14
Interactions between verapamil and neutral and acidic liposomes: effects of the ionic strength; Castaing M et al.; Patients with cancer often develop major electrolyte disorders, which are aggravated by radiation therapy and chemotherapy and by the concomitant impairment of the renal function and the development of drug resistance . In addition, tumour cells have membranes with more negative charges than normal eukaryotic cells . This study was designed to test the hypothesis that the ability of the Ca(2+) blocker verapamil to mediate the reversal of multidrug resistance (MDR) by interacting with the membrane phospholipids may be correlated with the ionic strength and membrane surface potential in resistant tumours . The permeation properties of verapamil, which is the best-known MDR-modulator, were therefore studied by quantifying its ability to induce the leakage of carboxyfluorescein through unilamellar liposomes containing various mole fractions of phosphatidic acid (x(EPA)=0, 0.1 and 0.3), at four different ionic strengths (I=0.052, 0.124, 0.204 and 0.318 M) . The dye leakage induced by verapamil varied greatly with I, depending on x(EPA) . The permeation process was a co-operative one (1.3<Hill coefficient<3.5) and the permeation doses inducing 50% dye leakage (PD(50)) ranged between 0.2 and 1.8 mM . A highly significant multiple correlation was found to exist between the variations of log(1/PD(50)) with those of 1/ radical I and x(EPA) (dlog(1/PD(50))/d(1/ radical I)=0.15+/-0.01, dlog(1/PD(50))/dx(EPA)=2.07+/-0.08, y-intercept=2.46+/-0.03, P<0.000001) . Kinetic studies on the permeation process showed that it involved two steps . The apparent rate constants of the slow and fast kinetic steps, which were driven by electrostatic and hydrophobic interactions, respectively, increased with the verapamil concentrations, depending on x(EPA) . The results provide evidence that in resistant tumours (high negative membrane surface potential), the MDR reversal by verapamil might be enhanced by favourable drug-membrane interactions in patients with severe hypo-electrolytic (Na(+) and K(+)) disorders, whereas the MDR reversal might be reduced by unfavourable drug-membrane interactions in patients with severe hyper-electrolytic (Ca(2+), Na(+) and K(+)) disorders.

Photochem Photobiol Sci, 2002 Jan, 1(1), 71 - 8
Photosensitizer accumulation in spontaneous multidrug resistant cells: a comparative study with Rhodamine 123, Rose Bengal acetate and Photofrine; Croce AC et al.; The influence both of overexpression of multidrug transporter proteins and of phenotype changes occurring in cells developing spontaneous resistance on the accumulation of photosensitizer molecules was studied on two tumor-derived cell lines (B16, A2780) expressing the MDR-1 phenotype . Rhodamine 123, Rose Bengal acetate (a fluorogenic substrate that is restored to the native active molecule by specific enzyme activity inside cells) and Photofrin were considered . The two resistant variants accumulate Rhodamine 123 to a lesser extent than the respective wild types . Treatment with verapamil markedly enhances Rhodamine 123 accumulation in resistant cells, blocking the drug's extrusion . The amount of Rose Bengal is larger in resistant cells than in wild type cells . Verapamil does not affect drug accumulation, although it significantly impairs the efflux process . The results are explained by the enhancement of both membrane traffic and esterase activity resulting in intracellular Rose Bengal production that counterbalances the increased ability in the outward transport of resistant cells . Photofrin is accumulated to a lower degree in resistant than in wild type cells . Verapamil does not alter the drug accumulation, although the release process is somewhat affected . Different intracellular turnovers of Photofrin take place in the cell variants, and the release of the monomeric fluorescent fractions is greater in resistant than in wild type cells.

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