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