Antimicrob Agents Chemother, 2004 Jul, 48(7), 2736 - 8 pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates in Portugal; Portugal I et al.; The nucleotide sequences of the pncA genes within 55 multidrug-resistant pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates were determined . Fifty-three out of the 55 isolates were pyrazinamidase (PZase) negative . Four strains contained a wild-type pncA gene, and PZase activity was undetectable in two of these strains . Seven of the 18 identified pncA mutations found have not been described in previous studies. Antimicrob Agents Chemother, 2004 Jul, 48(7), 2673 - 82 Active efflux of ciprofloxacin from J774 macrophages through an MRP-like transporter; Michot JM et al.; The accumulation and efflux kinetics of ciprofloxacin have been examined by using murine J774 macrophages . Accumulation (at equilibrium) was increased (three- to fourfold) (i) when cells were incubated with high extracellular drug concentrations (typically 200 mg/liter) as opposed to clinically meaningful concentrations (10 mg/liter or lower), (ii) during ATP- depletion and at acid pH, and (iii) during coincubation with probenecid, gemfibrozil and the preferential multidrug resistance-related protein (MRP) inhibitor MK571 . All these conditions were also associated with a marked decrease in ciprofloxacin efflux (half-lives increased from <2 min in controls to up to 10 min) . Monensin (a proton ionophore), verapamil, and the preferential P-glycoprotein (P-gp) inhibitor GF120918 had no or only minimal effect, while cyclosporin A, which is not specific for P-gp but also acts on MRP, had an intermediate effect . Short-term uptake studies showed that the influence of the modulators on the apparent drug influx was almost immediate (delay of < or =1 min) . Cells made resistant to probenecid and showing a marked overexpression of MRP1 (by Western blot analysis and confocal microscopy) accumulated ciprofloxacin to almost the same extent as did control cells, but efflux was inhibited less by probenecid, gemfibrozil, and MK571 . We conclude that ciprofloxacin is subject to constitutive efflux in J774 macrophages through the activity of an MRP-related transporter which is probably distinct from MRP1 . We also suggest that the cellular accumulation of ciprofloxacin in wild-type cells is constitutively impaired at therapeutically meaningful concentrations. Antimicrob Agents Chemother, 2004 Jul, 48(7), 2415 - 23 Efflux pump-mediated intrinsic drug resistance in Mycobacterium smegmatis; Li XZ et al.; The Mycobacterium smegmatis genome contains many genes encoding putative drug efflux pumps . Yet with the exception of lfrA, it is not clear whether these genes contribute to the intrinsic drug resistance of this organism . We showed first by reverse transcription (RT)-PCR that several of these genes, including lfrA as well as the homologues of Mycobacterium tuberculosis Rv1145, Rv1146, Rv1877, Rv2846c (efpA), and Rv3065 (mmr and emrE), were expressed at detectable levels in the strain mc(2)155 . Null mutants each carrying an in-frame deletion of these genes were then constructed in M . smegmatis . The deletions of the lfrA gene or mmr homologue rendered the mutant more susceptible to multiple drugs such as fluoroquinolones, ethidium bromide, and acriflavine (two- to eightfold decrease in MICs) . The deletion of the efpA homologue also produced increased susceptibility to these agents but unexpectedly also resulted in decreased susceptibility to rifamycins, isoniazid, and chloramphenicol (two- to fourfold increase in MICs) . Deletion of the Rv1877 homologue produced some increased susceptibility to ethidium bromide, acriflavine, and erythromycin . The upstream region of lfrA contained a gene encoding a putative TetR family transcriptional repressor, dubbed LfrR . The deletion of lfrR elevated the expression of lfrA and produced higher resistance to multiple drugs . Multidrug-resistant single-step mutants, independent of LfrA and attributed to a yet-unidentified drug efflux pump (here called LfrX), were selected in vitro and showed decreased accumulation of norfloxacin, ethidium bromide, and acriflavine in intact cells . Finally, use of isogenic beta-lactamase-deficient strains showed the contribution of LfrA and LfrX to resistance to certain beta-lactams in M . smegmatis. J Med Chem, 2004 Jul 1, 47(14), 3697 - 9 Total synthesis and anti-tubulin activity of epi-c3 analogues of cryptophycin-24; Buck SB et al.; Epi-C3-cryptophycin-24, epi-C3-m-chlorobenzyl-cryptophycin-24, and the corresponding styrenes were synthesized and tested in vitro against the MCF-7 and multidrug-resistant MCF-7/ADR breast cancer cell lines and in an in vitro tubulin assembly assay . The results demonstrate that the S configuration at the C3 stereocenter is not required to induce potent cytotoxicity and the m-Cl substituent present on the C10 side chain did not induce any large change in activity. Pharm Res, 2004 Jun, 21(6), 1055 - 64 Pharmacokinetic analysis of ramatroban using a recirculatory model with enterohepatic circulation by measuring portal and systemic blood concentration difference in Sprague-Dawley and Eisai hyperbilirubinemic rats; Moriwaki T et al.; PURPOSE: The aim of this study was to characterize the in vivo pharmacokinetics with the enterohepatic circulation (EHC) and identify the role of multidrug resistance-associated protein 2 (MRP2/Mrp2) in biliary excretion and absorption of ramatroban, a thromboxane A2 antagonist using a recirculatory model . METHODS: Ramatroban was intravenously or orally administered to Sprague-Dawley rats (SDR) and Eisai hyperbilirubinemic rats (EHBR) . Portal and systemic blood and bile samples were collected, and the drug concentrations were analyzed by high-performance liquid chromatography (HPLC) to estimate various global and local moments . RESULTS: The bioavailability (BA) of ramatroban was estimated at 21.0% in SDR and 61.9% in EHBR . The local absorption ratio for the dosage after oral administration (Fa(dosage)) and the single-pass local absorption ratio for EHC (Fa') in the rats were similar and nearly 100% . The hepatic recovery ratio (Fh) and the single-pass biliary excretion ratio through the liver for the sum of ramatroban and its glucuronides (Fb) in EHBR were 61.4% and 8.88%, respectively, which differed considerably from those in SDR (15.0% and 22.4%) . The difference in hepatic elimination between these strains would be caused, at least in part, by the reduced biliary excretion in EHBR, although the biliary excretion was not completely impaired . CONCLUSIONS: Ramatroban may be excreted by multiple transport systems, followed by efficient enterohepatic reabsorption in both strains . The results suggest that ramatroban may not be susceptible to drug-drug interaction involving MRP2/Mrp2 in biliary excretion and absorption. Birth Defects Res A Clin Mol Teratol, 2004 Jun, 70(6), 396 - 9 A common ABCC2 promoter polymorphism is not a determinant of the risk of spina bifida; Jensen LE et al.; BACKGROUND: There is compelling evidence that the risk of spina bifida, a malformation of the caudal neural tube, is associated with maternal and/or embryonic disturbances in folate/homocysteine metabolism . Hence, functional variants of genes that influence folate/homocysteine metabolism constitute a biologically plausible group of candidate risk factors for spina bifida and other neural tube defects . One such candidate is ABCC2, the gene encoding ABCC2, (a.k.a . canalicular multispecific organic anion transporter {cMOAT}, multidrug resistance related protein 2 {MRP2}), a member of the ABC transporter family that effluxes natural folates and anti-folate drugs such as methotrexate . METHODS: The association between the risk of spina bifida and both the maternal and embryonic ABCC2 C(-24)T genotype was evaluated by using the transmission disequilibrium test and log-linear modeling . RESULTS: These analyses provided no evidence that the risk of spina bifida was significantly related to either the maternal or embryonic ABCC2 C(-24)T genotype . CONCLUSIONS: The results of the present analyses suggest that the C(-24)T variant of the ABCC2 gene is not a major determinant of spina bifida risk . Am J Trop Med Hyg, 2004 Jun, 70(6), 635 - 7 Short report: High prevalence of multidrug-resistant Plasmodium falciparum malaria in the French territory of Mayotte; Pettinelli F et al.; A drug-resistance survey was conducted in the French territory of Mayotte in the Comorian Islands in the Indian Ocean where malaria is endemic . A high prevalence of resistant Plasmodium falciparum parasites was observed, not only to chloroquine (88%) and pyrimethamine (99%), but more surprisingly to quinine (17%), mefloquine (9%), and amodiaquine (24%) . This leaves few treatment alternatives other than artemisine-mefloquine combinations . However, despite notification to French Health authorities three years ago, inadequate treatment (chloroquine plus sulfadoxine-pyrimethamine) is still used in this locality . Thus, people still die of malaria in this remote territory of France. J Pharmacol Exp Ther, 2004 Nov, 311(2), 476 - 84 Epub 2004 Jun 21. Differential multidrug resistance-associated protein 1 through 6 isoform expression and function in human intestinal epithelial Caco-2 cells; Prime-Chapman HM et al.; Multidrug resistance-associated protein (MRP) isoforms 1 through 6 mRNA are expressed in the human intestine and Caco-2 cells . In Caco-2 cells, the rank order for mRNA expression was MRP2 > or = MRP6 > MRP4 > or = MRP3 > MRP1 = MRP5 . The functional expression of MRP-like activity was quantified as the efflux of the fluorescent probe calcein from confluent, polarized monolayers of Caco-2 cells . Calcein efflux was sensitive to temperature, energy depletion, and the MRP antagonist MK571 {3-{{3-{2-(7-chloroquinolin-2-yl)vinyl}phenyl}-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl} propionic acid} . Calcein efflux across the apical membrane of Caco-2 cells exceeded that across the basolateral by approximately 2-fold, correlating with the apical localization of MRP2 visualized by immunocytochemical staining . T84 cells do not express MRP2 and show a predominance of basolateral calcein efflux over apical efflux . MRP3 was localized by immunocytochemical staining to the basolateral membrane . MRP1 staining was not localized to either membrane domain and MRP5 staining was not detected . Thus, basolateral calcein efflux may reflect a function of MRP3 or MRP4 and 6 inferred by their basolateral localization in other tissues . Basolateral, but not apical, calcein efflux was sensitive to glutathione depletion with buthioninesulfoximine, indicating that whereas MRP2-mediated apical efflux is independent of glutathione, basolateral efflux is glutathione-dependent . Benzbromarone, probenecid, pravastatin, and diclofenac were able to inhibit both apical and basolateral calcein efflux . The apical calcein efflux in Caco-2 cells was selectively sensitive to indomethacin and propranolol, but not verapamil or erythromycin, whereas the converse was observed for basal efflux . The differential pharmacological sensitivity of apical (MRP2) and basolateral calcein efflux provides tools for dissecting MRP isoform functional roles. Aquat Toxicol, 2004 Jul 30, 69(1), 1 - 10 Regulation of expression of multixenobiotic resistance (MXR) genes by environmental factors in the blue mussel Mytilus edulis; Luedeking A et al.; Marine organisms and especially those living in tidal zones are confronted with dramatic changes in their environment such as temperature fluctuations on a daily and/or seasonal basis . In the present study, we investigated whether these parameters affect expression of multixenobiotic resistance (MXR)-related genes that serve as a first line of defense against a broad spectrum of natural and man-made toxicants . Expression of MXR-related genes seems to be an appropriate biomarker to determine hazardous effects of chemicals in contaminated marine habitats . The interference of natural environmental factors in the expression of biomarkers is an important issue with respect to the use of biomarkers in monitoring biological effects of pollutants, making interpretations difficult . We studied the effects of temperature, salinity and oxygen supply (anaerobiosis) on expression of MXR-related genes in gills and digestive gland of the blue mussel Mytilus edulis in order to differentiate between pollution-induced stress and responses to natural environmental variations . We found changes in expression levels of P-glycoprotein (pgp), major vault protein (mvp), topoisomerase II (topoII), heat shock protein 70 (hsp70), but not of the multidrug resistance-related protein (mrp2) genes, in laboratory experiments in relation to high temperature, low salinity and anaerobiosis but not low temperature . These effects of environmental factors have to be considered in sampling strategies for monitoring programmes to prevent false interpretation of results . Bioorg Med Chem, 2004 Jul 15, 12(14), 3923 - 30 Synthesis of 1-(omega-aminoalkyl)naphthoindolediones with antiproliferative properties; Shchekotikhin AE et al.; The preparation and cytotoxic properties of 4,11-dihydroxynaphtho{2,3-f}indole-5,10-dione derivatives carrying N-aminoalkyl substituents are described . The N-aminobutylnaphthoindolediones obtained were studied in National Cancer Institute Screening Program and demonstrated high antiproliferative activity against 60 human cancer cell lines . All N-(4-aminobutyl) derivatives have higher potency than adriamycin or mitoxantrone against adriamycin selected multidrug resistant breast cancer cell line NCI/ADR. Diabet Med, 2004 Jul, 21(7), 710 - 5 Diabetic foot ulcer and multidrug-resistant organisms: risk factors and impact; Hartemann-Heurtier A et al.; AIMS: The primary objective was to characterize factors allowing the colonization of diabetic foot wounds by multidrug-resistant organisms (MDRO), and the secondary objective was to evaluate the influence of MDRO colonization/infection on wound healing . METHODS: In 180 patients admitted to a specialized diabetic foot unit, microbiological specimens were taken on admission . Potential risk factors for MDRO-positive specimens were examined using univariate and multivariate analyses . Prospective follow-up data from 75 patients were used to evaluate the influence of MDRO colonization/infection on time to healing . RESULTS: Eighteen per cent of admission specimens were positive for MDRO . MDRO-positive status was not associated with patient characteristics (age, sex, type of diabetes, complications of diabetes), wound duration, or wound type (neuropathic or ischaemic) . In the multivariate analysis, the only factors significantly associated with positive MDRO status on admission were a history of previous hospitalization for the same wound (21/32 compared with 48/148; P = 0.0008) or the presence of osteomyelitis (22/32 compared with 71/148; P = 0.025) . In the longitudinal study of 75 wounds, MDRO-positive status on admission or during follow-up (6 months at least or until healing, mean 9 +/- 7 months) was not associated with time to healing (P = 0.71) . CONCLUSION: MDROs are often present in severe diabetic foot wounds . About one-third of patients with a history of previous hospitalization for the same wound, and 25% of patients with osteomyelitis, had MDRO-positive specimens . This suggests that hygiene measures, or isolation precautions in the case of admission of patients presenting with these characteristics, should be aggressively implemented to prevent cross-transmission . Positive MDRO status is not associated with a longer time to healing. Nucl Med Commun, 2004 Jul, 25(7), 711 - 20 99mTc glucarate high-resolution imaging of drug sensitive and drug resistant human breast cancer xenografts in SCID mice; Liu Z et al.; BACKGROUND AND AIM: Previous studies have showed that 99mTc labelled glucarate (GLA) might be an agent for non-invasive detection of breast tumours . In xenografted BT20 breast tumours, GLA was found to have higher uptake than 99mTc sestamibi (MIBI) . It is unclear whether GLA can localize in all cell line breast cancer xenografts, as well as breast tumours with multidrug resistance (MDR) . The present study aimed to investigate the properties of GLA in detecting drug sensitive and drug resistant MCF7 breast cancer xenografts in mice by using dynamic single photon emission computed tomography (SPECT) imaging . METHODS: MCF7/S cells are drug sensitive breast carcinoma cells . MCF7/D40 cells are 40-fold more resistant to doxorubicin compared to MCF7/S . Subcutaneous tumours were grown in SCID mice for 10-14 days after injection of 1 x 10(6) cells into the right thigh . Anaesthetized mice with MCF7/S (MIBI, n=9; GLA, n=8) and MCF7/D40 (MIBI, n=6; GLA, n=5) tumours were imaged using a high-resolution SPECT system called FASTSPECT . Dynamic images were acquired for 2 h after intravenous injection of GLA or MIBI . Expression of MDR P-glycoprotein (Pgp) in the tumours was demonstrated in the MCF7/D40 tumours by western blotting, not in the MCF7/S tumours . RESULTS: The xenografted tumours were visualized unequivocally within 10-30 min in GLA images and remained detectable for at least 2 h after injection . Drug resistant tumours, from which MIBI was rapidly expelled, retained GLA as readily as did drug sensitive tumours . The biodistribution data of GLA demonstrated significantly higher accumulation (%ID/g) compared to MIBI . CONCLUSION: MCF7 tumour xenografts can be detected by 99mTc glucarate imaging . More importantly, 99mTc glucarate can potentially localize drug resistant breast tumours. Plant Cell, 2004 Jul, 16(7), 1812 - 26 Epub 2004 Jun 18. A multidrug resistance-associated protein involved in anthocyanin transport in Zea mays; Goodman CD et al.; Anthocyanin biosynthesis is one of the most thoroughly studied enzymatic pathways in biology, but little is known about the molecular mechanisms of its final stage: the transport of the anthocyanin pigment into the vacuole . We have identified a multidrug resistance-associated protein (MRP), ZmMrp3, that is required for this transport process in maize (Zea mays) . ZmMrp3 expression is controlled by the regulators of anthocyanin biosynthesis and mirrors the expression of other anthocyanin structural genes . Localization of ZmMRP3 in vivo shows its presence in the tonoplast, the site at which anthocyanin transport occurs . Mutants generated using antisense constructs have a distinct pigmentation phenotype in the adult plant that results from a mislocalization of the pigment as well as significant reduction in anthocyanin content, with no alteration in the anthocyanin species produced . Surprisingly, mutant plants did not show a phenotype in the aleurone . This appears to reflect the presence of a second, highly homologous gene, ZmMrp4, that is also coregulated with the anthocyanin pathway but is expressed exclusively in aleurone tissue . This description of a plant MRP with a role in the transport of a known endogenous substrate provides a new model system for examining the biological and biochemical mechanisms involved in the MRP-mediated transport of plant secondary metabolites. J Biol Chem, 2004 Sep 10, 279(37), 38871 - 80 Epub 2004 Jun 18. Mutational analysis of ionizable residues proximal to the cytoplasmic interface of membrane spanning domain 3 of the multidrug resistance protein, MRP1 (ABCC1): glutamate 1204 is important for both the expression and catalytic activity of the transporter; Situ D et al.; The multidrug resistance protein MRP1 is an ATP-dependent transporter of organic anions and chemotherapeutic agents . A significant number of ionizable amino acids are found in or proximal to the 17 transmembrane (TM) helices of MRP1, and we have investigated 6 of these at the cytoplasmic interface of TM13-17 for their role in MRP1 expression and transport activity . Opposite charge substitutions of TM13 Arg(1046) and TM15 Arg(1131) did not alter MRP1 expression nor did they substantially affect activity . In contrast, opposite charge substitutions of TM16 Arg(1202) and Glu(1204) reduced protein expression by >80%; however, MRP1 expression was not affected when Arg(1202) and Glu(1204) were replaced with neutral or same-charge residues . In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1 . In contrast, organic anion transport by E1204L was substantially reduced, whereas transport by E1204D was comparable with wild-type MRP1, with the notable exception of GSH . Opposite charge substitutions of TM16 Arg(1197) and TM17 Arg(1249) did not affect MRP1 expression but substantially reduced transport . Mutants containing like-charge substitutions of Arg(1197) or Arg(1249) were also transport-inactive and no longer bound leukotriene C(4) . In contrast, substrate binding by the transport-compromised E1204L mutant remained intact . Furthermore, vanadate-induced trapping of azido-ADP by E1204L was dramatically increased, indicating that this mutation may cause a partial uncoupling of the catalytic and transport activities of MRP1 . Thus, Glu(1204) serves a dual role in membrane expression of MRP1 and a step in its catalytic cycle subsequent to initial substrate binding. Tuberculosis (Edinb), 2004, 84(5), 337 - 40 Mycobacterium tuberculosis, Beijing genotype strains not associated with radiological presentation of pulmonary tuberculosis; Borgdorff MW et al.; Mycobacterium tuberculosis strains of the Beijing genotype have been involved in various outbreaks of multidrug-resistant tuberculosis . Some studies suggest that the infection with the Beijing genotype is associated with a different host immune response . Since this might also lead to a different chest X-ray (CXR) presentation, we compared CXRs of patients with pulmonary tuberculosis, 33 of whom were infected with the Beijing genotype and 76 with other genotypes . No significant differences were detected . We conclude that the Beijing genotype is not associated with a different CXR presentation. Anticancer Drugs, 2004 Jul, 15(6), 641 - 6 Anticancer activity of methoxymorpholinyl doxorubicin (PNU 152243) on human hepatocellular carcinoma; Yuan S et al.; Methoxymorpholinyl doxorubicin (PNU 152243) is a morpholinyl analog possessing a methoxymorpholinyl group at the 3' position of the sugar moiety, which, compared with doxorubicin, appears to be less cardiotoxic and more cytotoxic against multidrug-resistant tumor cells . In this study, we report the anticancer activity of PNU 152243 on human hepatocellular carcinoma (HCC) in vitro and in vivo . The average IC50 value of PNU was 0.08 microM . In contrast, the average IC50 values of adriamycin (ADM), 4'-epidoxorubicin (EDR), mitomycin C (MMC), cisplatin and vepesid (VP-16) were 0.96, 0.74, 2.81, 7.27 and 26.66 microM, respectively . PNU 152243 was 13.7, 10.6, 40.1, 103.8 and 380.8 times more potent than ADM, EDR, MMC, cisplatin and VP-16 against HCC in vitro . In nude mice, the T/C (%) were 43.8 at the dose of 25 microg/kg and 41.2 at the dose of 50 microg/kg on BEL-7402 xenograft, the T/C (%) were 41.7 at the dose of 25 microg/kg and 54.6 at the dose of 50 microg/kg on Zip-177 xenograft . The results showed that PNU 152243 had growth inhibition of HCC in vitro and in vivo and may be useful in HCC chemotherapy. Anticancer Drugs, 2004 Jul, 15(6), 609 - 17 2-Pyrrolinodoxorubicin and its peptide-vectorized form bypass multidrug resistance; Castex C et al.; A well-known mechanism leading to the emergence of multidrug-resistant tumor cells is the overexpression of P-glycoprotein, which is capable of lowering intracellular drug concentrations . In the present study, we tested the capability of 2-pyrrolinodoxorubicin (p-DOX), a highly potent derivative of DOX, to bypass multidrug resistance . The accumulation, intracellular distribution and cytotoxicity of p-DOX were tested in two cell lines (K562 and A2780) and their DOX-resistant counterparts (K562/ADR and A2780/ADR) . Cellular accumulation and cytotoxicity were dramatically lowered for DOX in resistant cell lines, in comparison with non-resistant cells . In contrast, cellular accumulation, intracellular distribution and cytotoxicity of p-DOX were independent of the nature of the cell lines . The p-DOX showed potent dose-dependent inhibition of cell growth against resistant cells as compared with DOX . After treatment of resistant cells with verapamil, the intracellular levels of DOX were markedly increased and consequent cytotoxicity improved . In contrast, treatment of resistant cells with verapamil did not cause any further enhancement of cell uptake or an increase in the cytotoxic effect of the derivative p-DOX, indicating that the compound bypasses the P-glycoprotein . Finally, we show that vectorization of p-DOX by a peptide vector (SynB3) which has been shown to enhance the brain uptake of DOX and to decrease its heart accumulation does not affect this property . These results indicate that p-DOX and its vectorized form are potent and effective in overcoming multidrug resistance. Drug Metab Dispos, 2004 Jul, 32(7), 734 - 41 Lipopolysaccharide-mediated regulation of hepatic transporter mRNA levels in rats; Cherrington NJ et al.; The function of hepatic transporters is to move organic substances across sinusoidal and canalicular membranes . During extrahepatic cholestasis, transporters involved in the movement of substances from blood to bile, such as sodium/taurocholate-cotransporting polypeptide (Ntcp) and multidrug resistance protein 2 (Mrp2), are down-regulated, whereas others that transport chemicals from liver to blood, such as Mrp3, are up-regulated . Unlike extrahepatic cholestasis, where transporter expression responds to the stress of accumulating bile constituents, lipopolysaccharide (LPS)-induced intrahepatic cholestasis may be directly caused by alterations in transporter expression . The aim of this study was to quantitatively determine the effect of LPS on transporter expression and study the mechanism(s) by which LPS alters mRNA levels of major hepatic transporters in Sprague-Dawley rats . Hepatic mRNA levels of Mrp2, Mrp6, multiple drug resistance protein 1a (Mdr1a), organic anion-transporting polypeptide 1 (Oatp1), Oatp2, Oatp4, Ntcp, bile salt export pump, organic cation transporter 1 (Oct1), and organic anion transporter 3 (Oat3) were dramatically decreased, beginning approximately 6 h after LPS administration, whereas Mrp5 and Oat2 levels were unchanged . In contrast, LPS increased mRNA levels of Mrp1, Mrp3, and Mdr1b concurrently with the down-regulated transporters . Pretreatment with dexamethasone, which decreases the release of cytokines, reversed the reduction of Mdr1a, Oatp1, Oatp2, Oct1, and Ntcp mRNA following LPS administration . Furthermore, dexamethasone pretreatment also prevented the LPS-mediated increase in Mrp1, Mrp3, and Mdr1b, whereas pretreatment with aminoguanidine or gadolinium chloride, an inhibitor of inducible nitric oxide synthetase and a Kupffer cell toxicant, respectively, had no effect on the LPS-induced changes . The concurrent repression and induction of various transporters, as well as dexamethasone abatement of both LPS-mediated repression and induction, indicates that these responses may be mediated through similar pathways. Cancer Res, 2004 Jun 15, 64(12), 4346 - 52 Phytoestrogens/flavonoids reverse breast cancer resistance protein/ABCG2-mediated multidrug resistance; Imai Y et al.; Breast cancer resistance protein (BCRP), also called ABCG2, confers resistance to anticancer agents such as 7-ethyl-10-hydroxycamptothecin (SN-38), mitoxantrone, and topotecan . We found previously that sulfated estrogens are physiologic substrates of BCRP . Flavonoids with weak estrogenic activities are called phytoestrogens . In this study, we show that phytoestrogens/flavonoids, such as genistein, naringenin, acacetin, and kaempferol, potentiated the cytotoxicity of SN-38 and mitoxantrone in BCRP-transduced K562 (K562/BCRP) cells . Some glycosylated flavonoids, such as naringenin-7-glucoside, also effectively inhibited BCRP . These flavonoids showed marginal effect on the drug sensitivity of K562 cells . Genistein and naringenin reversed neither P-glycoprotein-mediated vincristine resistance nor multidrug resistance-related protein 1-mediated VP-16 resistance . Genistein and naringenin increased cellular accumulation of topotecan in K562/BCRP cells . K562/BCRP cells also accumulated less {(3)H}genistein than K562 cells . {(3)H}genistein transport in the basal-to-apical direction was greater in BCRP-transduced LLC-PK1 (LLC/BCRP) cells, which express exogenous BCRP in the apical membrane, than in parental cells . Fumitremorgin C abolished the increased transport of {(3)H}genistein in LLC/BCRP cells compared with parental cells . TLC analysis revealed that genistein was transported in its native form but not in its metabolized form . These results suggest that genistein is among the natural substrates of BCRP and competitively inhibits BCRP-mediated drug efflux . The results have two important clinical implications: (a) flavonoids and glycosylated flavonoids may be useful in overcoming BCRP-mediated drug resistance in tumor cells; and (b) coadministration of flavonoids with BCRP-substrate antitumor agents may alter the pharmacokinetics and consequently increase the toxicity of specific antitumor agents in cancer patients. Am J Physiol Gastrointest Liver Physiol, 2004 Nov, 287(5), G1008 - 16 Epub 2004 Jun 17. LPS-induced downregulation of MRP2 and BSEP in human liver is due to a posttranscriptional process; Elferink MG et al.; Endotoxin-induced cholestasis in rodents is caused by hepatic downregulation of transporters, including the basolateral Na+-dependent taurocholate transporter (ntcp) and the canalicular bile salt export pump (bsep) and multidrug resistance-associated protein 2 (mrp2) . Details about the regulation of the human transporter proteins during this process are lacking . We used precision-cut human and rat liver slices to study the regulation of transporter expression during LPS-induced cholestasis . We investigated the effect of LPS on nitrate/nitrite and cytokine production in relation to the expression of inducible nitric oxide synthase, NTCP, BSEP, and MRP2 both at the level of mRNA with RT-PCR and protein using immunofluorescence microscopy . In liver slices from both species, LPS-induced expression of inducible nitric oxide synthase was detected within 1-3 h and remained increased over 24 h . In rat liver slices, this was accompanied by a significant decrease of rat ntcp and mrp2 mRNA levels, whereas bsep levels were not affected . These results are in line with previous in vivo studies and validate our liver slice technique . In LPS-treated human liver slices, NTCP mRNA was downregulated and showed an inverse correlation with the amounts of TNF-alpha and Il-1beta produced . In contrast, MRP2 and BSEP mRNA levels were not affected under these conditions . However, after 24-h LPS challenge, both proteins were virtually absent in human liver slices, whereas marker proteins remained detectable . In conclusion, we show that posttranscriptional mechanisms play a more prominent role in LPS-induced regulation of human MRP2 and BSEP compared with the rat transporter proteins. Leuk Res, 2004 Aug, 28(8), 813 - 9 Modified VAD and PSC-833 in the treatment of resistant or relapsing chronic lymphocytic leukemia (E4996): a trial of the Eastern Cooperative Oncology Group; Friedenberg WR et al.; BACKGROUND & METHOD: The role of multidrug resistance (MDR) was investigated in patients with relapsed chronic lymphocytic leukemia (CLL) . PSC-833 was added to modified VAD (a 4-day infusion of vincristine, doxorubicin, with oral dexamethasone, every 3 weeks), in an attempt to improve the response rate (21%) in a prior study . Laboratory tests to determine MDR and apoptosis proteins were correlated with response . RESULTS: Two of the seven MDR-positive cases and one of the four MDR-negative patients achieved a partial response (no significant difference) . No significant correlation with response was found in any of the laboratory tests for apoptosis . CONCLUSION: VAD plus PSC-833 had the same (21%) partial response rate as a prior ECOG study without PSC-833 . No correlation of response with MDR or apoptosis testing was found . Other drug resistance factors must play a significant role in determining the response of relapsed patients with CLL. Hepatol Res, 2004 Jul, 29(3), 160 - 166 Mutations identified in the human multidrug resistance P-glycoprotein 3 (ABCB4) gene in patients with primary hepatolithiasis; Kano M et al.; Primary hepatolithiasis (HL), highly prevalent in the Far East, including Japan, is characterized clinically by chronic proliferative cholangitis with frequent recurrences . In HL patients, hepatic hyposecretion of phospholipid due to decreased multidrug resistance P-glycoprotein 3 (MDR3; now referred to as ABCB4) expression levels (Hepatology 2001;33:1194-1205) may contribute to the formation of aggressive ductular lesions through a decreased formation of mixed micelles . However, specified factors underlying the decreased expression levels of MDR3 have not been well defined . To determine whether the decreased MDR3 expression level is associated with the gene mutations, mutation analysis of cDNA of the MDR3 gene with focus on the coding region was performed using liver specimens . Heterozygous mutations were detected in only two of 16 HL patients . By sequence analysis of the gene, a 77-bp deletion at nucleotides 537-613 in exon 7 in transmembrane domain (TM) 3, which results in a frameshift at codon 179 and an early stop codon predicting a truncated protein, was found as a heterozygous mutation in two of the 16 patients . A 1-bp deletion at nucleotide 1015 in exon 10 in TM 6 was found as a heterozygous mutation in one of those two patients, and a 242-bp deletion at nucleotides 2683-2924 in exons 22-23 in TM 11 was found as a heterozygous mutation in the same patient . No other mutations were found in the other 14 patients . In real-time polymerase chain reaction (PCR), no significant difference was found between the mRNA levels of MDR3 in the two HL patients with mutations nor in the other 14 patients without mutations . Immunostaining of MDR3 protein was found in the bile canaliculi of liver sections from the two patients with mutations . The results suggest that in primary HL the decreased transcription levels of MDR3 in the liver are not due to the mutations detected in the coding region of the gene. J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Aug 5, 807(2), 203 - 8 Liquid chromatography method for the quantitation of the breast cancer resistance protein ABCG2 inhibitor fumitremorgin C and its chemical analogues in mouse plasma and tissues; Garimella TS et al.; Fumitremorgin C (FTC) was recently discovered to be a potent and selective inhibitor of the breast cancer resistance protein (BCRP/ABCG2) . FTC was shown to reverse multidrug resistance mediated by BCRP and to increase the cytotoxicity of several anticancer agents in vitro . To support in vivo studies a reverse phase HPLC method with ultraviolet detection was developed to quantitate FTC in mouse plasma and tissues . Further, assay method validation was performed for the determination of FTC in mouse plasma . Plasma standard curves ranged from 0.03 to 30 microg/ml, while the various tissue assay ranges differed to some extent . The sample preparation consisted of acetonitrile precipitation with separation accomplished with a C18 Novapak column and a C18 pre-column utilizing an isocratic mobile phase of ammonium acetate and acetonitrile . UV detection was set at 225 nm for FTC and at 312 nm for roquefortine, the internal standard . The retention times were approximately 9.5 min for FTC and 13.0 min for roquefortine . The recoveries for FTC and roquefortine from plasma were 90.8+/-5.8% and 111.6+/-13.6, respectively . The reported assay can be used for future study of BCRP resistance in vivo in different biological matrices . Further, we found that a more potent analogue of FTC, Ko143, was able to be extracted and detected, with a maximal UV absorbance at 320 nm under the conditions reported. Biotechnol Appl Biochem . 2004 Jun 17; {Epub ahead of print} Inhibition of P-glycoprotein and increasing of drug sensitivity of human carcinoma cell line by an antisense oligodeoxynucleotide-doxorubicin conjugate in vitro; Ren Y et al.; In order to improve the antisense activity of the antisense oligodeoxynucleotide (AS ODN), a conjugate which covalently linked with deoxorubicin (DOX) at 3'-end was synthesized and its antisense activity in human carcinoma DOX resistant cells (KB-A-1) was investigated in vitro . The intracellular DOX concentration was detected in KB-A-1 cells treatment with the conjugate in vitro by HPLC . The results showed that the intracellular DOX concentration was 6.4-fold increased in KB-A-1 cells treated with the conjugate compared to treatment with DOX alone . In contrast, 1.8-fold increasing was observed when treated with the AS ODN . RT-PCR and western blot analysis showed a significantly decrease in the amount of mdr1 mRNA and P- glycoprotein in KB-A-1 cells . Chemosensitivity of KB-A-1 cells to DOX were also investigated in vitro . When the cells were first exposed to the conjugate (0.5 muM) and then exposed to DOX for 24 h, the IC50 value of DOX decreased from 21.5 muM to 2.2 muM . In contrast, when treated with the mixture of the same concentration of the AS ODN with equivalent DOX, the IC50 value of DOX was 16.8 muM . These results suggest that the conjugate is effective in reversing multidrug resistance . Certainly, further studies are conducting to explore the antitumour effect of the conjugate in vivo. Di Yi Jun Yi Da Xue Xue Bao, 2004 Jun, 24(6), 646 - 9 {Expression of multidrug resistance gene in nasopharyngeal carcinoma cells after irradiation exposure}; Bu JG et al.; OBJECTIVE: To examine the changes in the function and expression of multidrug resistance gene (mdr1) and P-gly-coprotein (P-gp) in nasopharyngeal carcinoma (NPC) CNE1 cell following irradiation for determining the sequential order of radiotherapy and chemotherapy in the treatment of NPC . METHODS: The expressions of mdr1 gene and its protein P-gp as well as the function of P-gp efflux were examined in CNE1 cells before and after irradiation exposure by reverse transcriptional polymerase chain reaction (RT-PCR), Western blotting and flow cytometry, respectively . RESULTS: Irradiation of CNE1 cells induced a long-term overexpression of mdr1 gene and P-gp and reduction in intracellular daunorubicin accumulation . CONCLUSION: Irradiation decreases the chemotherapy sensitivity of CNE1 cell, and induction chemotherapy should be therefore performed before radiotherapy in the treatment of advanced NPC. Emerg Infect Dis, 2004 May, 10(5), 865 - 72 Multidrug-resistant tuberculosis in central Asia; Cox HS et al.; Multidrug-resistant tuberculosis (MDR-TB) has emerged as a major threat to TB control, particularly in the former Soviet Union . To determine levels of drug resistance within a directly observed treatment strategy (DOTS) program supported by Medecins Sans Frontieres in two regions in Uzbekistan and Turkmenistan, Central Asia, we conducted a cross-sectional survey of smear-positive TB patients in selected districts of Karakalpakstan (Uzbekistan) and Dashoguz (Turkmenistan) . High levels of MDR-TB were found in both regions . In Karakalpakstan, 14 (13%) of 106 new patients were infected with MDR-TB; 43 (40%) of 107 previously treated patients were similarly infected . The proportions for Dashoguz were 4% (4/105 patients) and 18% (18/98 patients), respectively . Overall, 27% of patients with positive smear results whose infections were treated through the DOTS program in Karakalpakstan and 11% of similar patients in Dashoguz were infected with multidrug-resistant strains of TB on admission . These results show the need for concerted action by the international community to contain transmission and reduce the effects of MDR-TB. AAPS PharmSci . 2004 Mar 09;6(1):E8. Development and characterization of a recombinant Madin-Darby canine kidney cell line that expresses rat multidrug resistance-associated protein 1 (rMRP1); Yang Z et al.; Multidrug resistance-associated protein 1 (MRP1) is one of the major proteins shown to mediate efflux transport of a broad range of antitumor drugs, glucuronide conjugates, and glutathione, in addition to endogenous substrates . Significant differences in substrate selectivity were reported for murine and human MRP1 . As preclinical drug disposition and pharmacokinetics studies are often conducted in rats, we have recently cloned the rat MRP1 (rMRP1) and demonstrated that rMRP1 expressed in transfected cells effluxes calcein, a commonly used fluorescence substrate for human MRP1 . To further characterize the rat ortholog of MRP1, we isolated a cell line stably expressing recombinant rMRP1 . These cells were tested for their ability to transport calcein and a range of chemotherapeutic drugs . Our results showed that cells expressing rMRP1 consistently efflux calcein at a rate 5-fold greater than control cells . The rMRP1 transfected cells, like their human ortholog, can confer drug resistance to vinca alkaloid (vinblastine and vincristine) and anthracycline drugs (daunorubcin and doxorubicin), and the resistance conferred by the MRP1 can be partially abolished by the MRP-specific inhibitors . The transepithelial permeability due to rMRP1 expression in differentiated Madin-Darby canine kidney cells (MDCK) cells was also investigated . The MRP1 transport activity is directional, as demonstrated by directional vinblastine transport . Collectively, our results demonstrate that the cellular expression of rMRP1, like its human ortholog, could confer resistance to anticancer drugs. Cancer Biol Ther, 2004 Jun, 3(6), 566 - 7 Epub 2004 Jun 24. Pharmacogenomics on gastric cancer; Katoh M et al.; Gastric cancer is one of most common malignancies in the world . 5-fluoroyracil, CPT-11, TS-1, Paclitaxel and Docetaxel are used as chemotherapeutic agents for advanced gastric cancer, although endoscopic mucosal resection and surgical gastrectomy are potentially curative treatments . Most important problems associated chemotherapeutic agents are side effects and drug resistances . TSG101, implicated in membrane trafficking and transcriptional regulation, is upregulated in gastric cancer with multidrug resistant phenotype . Shen et al reported that transient transfection of TSG101 siRNA reduced the expression level of TSG101 protein as well as resistance to vincristine and adriamycin in gastric cancer cells . Bioinformatics, microarray analyses, SNP typing, and high-throughput functional analyses are applied for pharmacogenomics in the post-genome era . Therapeutics based on the genotyping of each patient is the future goal of personalized medicine (or tailor-made medicine) for gastric cancer. Mol Pharmacol . 2004 Jun 14; {Epub ahead of print} Regulation of the Stability of P-glycoprotein by Ubiquitination; Zhang Z et al.; Ubiquitination plays a crucial role in regulating protein turnover . Here we show that ubiquitination regulates the stability of the MDR1 gene product, P-glycoprotein, thereby affecting the functions of this membrane transporter that mediates multidrug resistance . We found that P-glycoprotein was constitutively ubiquitinated in drug-resistant cancer cells . Transfection of multidrug resistant cells with wild-type ubiquitin or treatment with an N-glycosylation inhibitor increased the ubiquitination of P-glycoprotein and increased P-glycoprotein degradation . MG-132, a proteasome inhibitor, induced accumulation of ubiquitinated P-glycoprotein, suggesting the involvement of the proteasome in the turnover of the transporter . Treatment of multidrug resistant cells with 12-O-tetradecanoylphorbol-13-acetate, a phorbol ester that increases the phosphorylation of P-glycoprotein through activation of protein kinase C, or substituting phosphorylation sites of P-glycoprotein by nonphosphorylatable residues did not affect the ubiquitination of the transporter . Enhanced ubiquitination of P-glycoprotein resulted in decrease of the function of the transporter, as demonstrated by increased intracellular drug accumulation and increased cellular sensitivity to drugs transported by P-glycoprotein . Our results indicate that the stability and function of P-glycoprotein can be regulated by the ubiquitin-proteasome pathway, and suggest that modulating the ubiquitination of P-glycoprotein might be a novel approach to reversal of drug resistance. Genome Res, 2004 Jul, 14(7), 1333 - 44 Epub 2004 Jun 14. Identifying candidate causal variants responsible for altered activity of the ABCB1 multidrug resistance gene; Soranzo N et al.; The difficulty of fine localizing the polymorphisms responsible for genotype-phenotype correlations is emerging as an important constraint in the implementation and interpretation of genetic association studies, and calls for the definition of protocols for the follow-up of associated variants . One recent example is the 3435C>T polymorphism in the multidrug transporter gene ABCB1, associated with protein expression and activity, and with several clinical conditions . Available data suggest that 3435C>T may not directly cause altered transport activity, but may be associated with one or more causal variants in the poorly characterized stretch of linkage disequilibrium (LD) surrounding it . Here we describe a strategy for the follow-up of reported associations, including a Bayesian formalization of the associated interval concept previously described by Goldstein . We focus on the region of high LD around 3435C>T to compile an exhaustive list of variants by (1) using a relatively coarse set of marker typings to assess the pattern of LD, and (2) resequencing derived and ancestral chromosomes at 3435C>T through the associated interval . We identified three intronic sites that are strongly associated with the 3435C>T polymorphism . One of them is associated with multidrug resistance in patients with epilepsy (chi2 = 3.78, P = 0.052), and sits within a stretch of significant evolutionary conservation . We argue that these variants represent additional candidates for influencing multidrug resistance due to P-glycoprotein activity, with the IVS 26+80 T>C being the best candidate among the three intronic sites . Finally, we describe a set of six haplotype tagging single-nucleotide polymorphisms that represent common ABCB1 variation surrounding 3435C>T in Europeans . Zhonghua Zhong Liu Za Zhi, 2004 Mar, 26(3), 139 - 42 {The mechanism of topotecan resistance in ovarian cancer cell line}; Jia P et al.; OBJECTIVE: To study the mechanism of topotecan (TPT) resistance in ovarian cancer cell line . METHODS: A TPT-resistant ovarian cancer cell line A2780/TPT established in this laboratory was used in this study . Intracellular rhodamine fluorescence intensity of the TPT-resistant cells and parental cells were measured by flow cytometry . The gene expression of membrane protein transporter such as transporter P-glycoprotein (P-gp), multidrug resistance associated protein (MRP), breast cancer resistance protein (BCRP) was evaluated by RT-PCR . The antisense-phosphorothioate oligonucleotide (ASODN) including a translation initiation site of BCRP mRNA was transferred into resistant cells by liposome . RESULTS: Intracellular rhodamine fluorescence intensity of the resistant cells was 31.19% of that in the parental cells (P < 0.01) . No expression of P-gp was demonstrated, and that of MRP was very weak in the TPT-resistant cells (relative expression value = 0.057) . BCRP was overexpressed in the TPT-resistant cells (relative expression = 0.66), but not in the parental cells . Transfer of ASODN into resistant cells resulted in a 59.42% reduction of BCRP gene expression (P < 0.05) and an obviously increased intracellular rhodamine fluorescence intensity from 5.42 to 16.63 (P < 0.05) . CONCLUSION: The overexpression of BCRP which mediated drug efflux may play an important role in the induction of TPT-resistance in ovarian cancer. Am J Physiol Gastrointest Liver Physiol, 2004 Jul, 287(1), G42 - 9 Mrp2 is involved in benzylpenicillin-induced choleresis; Ito K et al.; Benzylpenicillin (PCG; 180 micromol/kg), a classic beta-lactam antibiotic, was intravenously given to Sprague-Dawley (SD) rats and multidrug resistance-associated protein 2 (Mrp2)-deficient Eisai hyperbilirubinemic rats (EHBR) . A percentage of the {(3)H}PCG was excreted into the bile of the rats within 60 min (SD rats: 31.7% and EHBR: 4.3%) . Remarkably, a transient increase in the bile flow ( approximately 2-fold) and a slight increase in the total biliary bilirubin excretion were observed in SD rats but not in the EHBR after PCG administration . This suggests that the biliary excretion of PCG and its choleretic effect are Mrp2-dependent . Positive correlations were observed between the biliary excretion rate of PCG and bile flow (r(2) = 0.768) and more remarkably between the biliary excretion rate of GSH and bile flow (r(2) = 0.968) . No ATP-dependent uptake of {(3)H}PCG was observed in Mrp2-expressing Sf9 membrane vesicles, whereas other forms of Mrp2-substrate transport were stimulated in the presence of PCG . GSH efflux mediated by human MRP2 expressed in Madin-Darby canine kidney II cells was enhanced in the presence of PCG in a concentration-dependent manner . In conclusion, the choleretic effect of PCG is caused by the stimulation of biliary GSH efflux as well as the concentrative biliary excretion of PCG itself, both of which were Mrp2 dependent. Biochem Biophys Res Commun, 2004 Jul 9, 319(4), 1124 - 31 Pyronaridine, a novel modulator of P-glycoprotein-mediated multidrug resistance in tumor cells in vitro and in vivo; Qi J et al.; One of the major mechanisms of multidrug resistance (MDR) in cancer therapy is the overexpression of P-glycoprotein (Pgp) . We previously reported that pyronaridine (PND), a synthetic quinoline derivative in the clinic for the treatment of malaria infections, was capable of reversing MDR phenotype in Pgp-overexpressing tumor cells . Here we further evaluated the reversal activity of PND using two Pgp-overexpressing human tumor cell lines: K562/A02 and MCF-7/ADR . PND significantly enhanced the sensitivity of K562/A02 and MCF-7/ADR cells to doxorubicin (DOX), but had no such effect on the parent K562 and MCF-7 cells . The MDR-modulating effect of PND persisted for longer than 24h after removal of the agent from the culture . In nude mice bearing K562/A02 xenografts PND significantly enhanced the antitumor activity of DOX when given intraperitoneally or orally without increasing the toxicity of DOX . Our observations suggest that PND represents a promising agent for overcoming MDR in cancer therapy. Biomed Pharmacother, 2004 Jun, 58(5), 320 - 4 Multidrug resistance-1 (MDR-1) in autoimmune disorders IV . P-glycoprotein overfunction in lymphocytes from myasthenia gravis patients; Richaud-Patin Y et al.; Multidrug resistance (MDR) mechanisms have been widely studied in cancer . Among them, P-glycoprotein (P-gp) overfunction has been associated with resistance to several antineoplastic agents . The physiological role of P-gp involves hormone and metabolite secretion, bacterial product detoxification, and transport of several drugs to the extracellular space, thus inhibiting their toxic or therapeutic effects . The study of MDR-1 in diseases of autoimmune origin has just recently emerged . Corticosteroids remain the mainstay therapy for autoimmune diseases . As prednisone (PDN) is transported by P-gp, the aim of this study was to evaluate the P-gp function in lymphocytes from myasthenia gravis (MG) patients . Thirty MG patients and 25 healthy controls were studied . Peripheral blood mononuclear cells were isolated by gradient centrifugation and incubated with daunorubicin (DNR) (a fluorescent drug extruded by P-gp) . Functional activity of P-gp was analyzed by flow cytometry . Results were expressed as percentage of gated lymphocytes able to efflux DNR . Overall, MG patients showed increased numbers of lymphocytes with functional P-gp activity when compared with controls (x = 4.92 +/- 5.26% vs . x = 0.7 +/- 0.48%, respectively) (P < 0.0001) . When patients were classified as responders (n = 21) or refractory (n = 9) to treatment, the latter group exhibited higher values of functional P-gp (x = 10.18 +/- 6.39%) when compared to the responder group (x = 2.66 +/- 2.45%) (P = 0.0076) . These data suggest, on the one hand, that drug resistance may be induced by long-term treatment or by high PDN doses and, on the other, emphasize the need for the study of P-gp antagonists in order to improve the current therapeutical schemes for the treatment of MG . Biochem Pharmacol, 2004 Jul 15, 68(2), 253 - 61 Increased chloride efflux in colchicine-resistant airway epithelial cell lines; Dragomir A et al.; Colchicine has been proposed as a treatment to alleviate chronic lung inflammation in cystic fibrosis patients and clinical trials are ongoing . Our aim was to investigate whether chronic exposure of cystic fibrosis cells to colchicine can affect their ability to transport chloride in response to cAMP . Colchicine-resistant cells were selected by growing in medium containing nanomolar concentrations of the drug . While microtubuli were affected by acute exposure to colchicine, they appeared normal in colchicine-resistant cells . Colchicine-resistant clones had higher expression of multidrug resistance proteins compared to untreated cells . Cystic fibrosis transmembrane conductance regulator (CFTR) labelling by immunocytochemistry showed no significant changes . The intracellular chloride concentration and basal chloride efflux of the cystic fibrosis treated cells increased significantly compared with untreated cells, while for the cAMP-stimulated Cl-efflux there was no significant change . The results suggest that colchicine promotes chloride efflux via alternative chloride channels . Since this is an accepted strategy for pharmacological treatment of cystic fibrosis, the results strengthen the notion that colchicine would be beneficial to these patients. Ai Zheng, 2004 Jun, 23(6), 631 - 4 {Arsenic trioxide induced cell apoptosis by mitochondria dependent pathway in KB and KBv200 cells}; Li YF et al.; BACKGROUND & OBJECTIVE: Arsenic trioxide (As2O3) is a new drug used to treat the patients with solid tumor,but the mechanism is still unclear . This study was designed to investigate the effect of mitochondrial dependent pathway in apoptosis induced by arsenic trioxide in multidrug resistant KBv200 cells and their parental sensitive KB cells . METHODS: The cytotoxic effect of As2O3 on KB and KBv200 cells was measured by MTT assay . KB and KBv200 cells were treated respectively with As2O3 for 12, 24, 48 hours . Cell apoptosis was determined by Annexin V FITC staining . Mitochondrial membrane potential was labeled by DiOC6 and examined by flow cytometry . RESULTS: Arsenic trioxide showed the inhibition of KB and KBv200 cells proliferation in vitro . The IC(50)s of As2O3 to KB and KBv200 cells were (0.22+/-0.02)microg/ml and (0.20+/-0.01)microg/ml, respectively . As2O3 induced cell apoptosis in time- dependent manner . The apoptosis rates were 20.2%+/-3.1% and 52.2%+/-11.0% for KB cells and 15.8%+/-1.3% and 36.4%+/-5.9% for KBv200 cells under 2.5 microg/ml As2O3 treating 24 h and 48 h, respectively . The levels of mitochondrial membrane potential were concentration-dependently decreased after treating with arsenic trioxide for 12, 24, and 48 hours . CONCLUSION: The decrease of mitochondrial membrane potential maybe plays an important role in apoptosis induced by As2O3. Hua Xi Kou Qiang Yi Xue Za Zhi, 2004 Apr, 22(2), 115 - 6, 151 {Detection of P-glycoprotein and glutathine S-transferase in mucoepidermoid carcinoma of salivary gland}; He J et al.; OBJECTIVE: The aim of this study was to investigate the mechanism(MDR) of multidrug resistance(MDR) of mucoepidermoid carcinoma in salivary gland . METHODS: 40 cases of mucoepidermoid carcinoma in salivary gland were examined the MDR gene product P-glycoprotein using a monoclonal antibody JSB-1 . And 10 of them were also investigated by detecting the expression of GST-pi . All the cases had not been accepted any therapy before the samples were collected . RESULTS: 1 . Positive expression of JSB-1 was observed in 27 of the 40 specimens . The positive expression was related not only with clinical stage, but also with differentiation degree . 2 . The GST-pi positive expression was found in 9 of 10 cases . There was no significant different between the positive expression of JSB-1 and GST-pi . CONCLUSION: JSB-1 and GST-pi play an important role in MDR of mucoepidermoid carcinoma. Drug Metab Dispos . 2004 Jun 9; {Epub ahead of print} Kinetic Characterization of P-Glycoprotien Mediated Efflux of Rhodamine 6G in the Intact Rabbit Lung; Roerig DL et al.; P-glycoprotein (P-gp) is an ATP dependent drug efflux transporter involved in multidrug resistance and drug disposition in many organ systems . A majority of P-gp substrates are lipophilic amine drugs which also exhibit rapid extensive accumulation in lung tissue . P-gp is expressed in lung tissue and the very nature of this drug efflux mechanism suggests a moderating role in pulmonary drug disposition . Little is known about P-gp mediated efflux out of lung tissue or its kinetic characteristics as they may relate to P-gp impact on pulmonary drug accumulation . The present study develops an experimental and kinetic model to characterize the kinetics of P-gp mediated efflux of rhodamine 6G dye (R6G) out of the intact rabbit lung . The perfusate concentration of R6G with time during recirculation through an isolated perfused rabbit lung was measured and 66.6+/-2.6% (SE) of the perfusate R6G was taken up by the lung . In the presence of P-gp inhibitors, R6G uptake increased significantly to 87.5+/-1.1% (P<0.002) indicating a functional pulmonary P-gp efflux transporter . Fractional lung accumulation of R6G increased with increasing R6G perfusate concentration, a result consistent with saturation of an efflux transporter . A parsimonious three compartment kinetic model of R6G pulmonary disposition was used to interpret data sets from experiments with different perfusion variables and to estimate parameters descriptive of the dominant kinetic processes involved in R6G pulmonary accumulation . The estimated value of the kinetic parameter, kpgp, rate constant for P-gp mediated R6G efflux, indicates this transporter plays a significant role in moderating R6G pulmonary disposition. Chest, 2004 Jun, 125(6), 2146 - 55 Regional deposition of aerosolized interferon-gamma in pulmonary tuberculosis; Condos R et al.; STUDY OBJECTIVES: Aerosol interferon-gamma (IFN-gamma) is a potential immunomodulator in the treatment of pulmonary tuberculosis (TB) . Previous investigations demonstrated conversion of sputum smears in five patients with multidrug-resistant TB after 12 treatments over 1 month, and induction of signaling molecules in 10 of 11 drug-sensitive TB patients using BAL . The objective of the current study was to evaluate particle size and deposition pattern in patients with TB receiving aerosol IFN-gamma treatment . DESIGN: Particle size was determined with a cascade impactor, and deposition of IFN-gamma mixed with (99m)Tc-labeled human serum albumin was assessed using a gamma camera . Local levels of IFN-gamma were measured in BAL using enzyme-linked immunosorbent assays . Study patients/intervention: Fourteen patients with pulmonary TB received IFN-gamma aerosol (500 micro g) for 12 treatments in addition to antimycobacterial therapy with BAL before and after IFN-gamma aerosol treatment . Eight patients with minimal-to-moderate parenchymal involvement underwent deposition studies . Deposited (99m)Tc-labeled IFN-gamma aerosol was partitioned between upper airways and lungs using attenuation correction measurements . (133)Xe equilibrium scanning, (133)Xe washout, and (99m)Tc- macroaggregate injection defined regional lung volume, ventilation, and perfusion . RESULTS: Upper airway deposition was significant often exceeding lung deposition (53.9 +/- 7.09 micro g vs 35.8 +/- 2.73 micro g, respectively {mean +/- SE}) . IFN-gamma levels measured in BAL fluid were significantly increased with aerosol treatment (0.83 +/- 0.43 micro g before vs 24.76 +/- 8.71 micro g after, p </= 0.017), and IFN-gamma levels correlated with regional deposition of IFN-gamma aerosol (r = 0.823) . Four-quadrant analysis of regional lung deposition best correlated with regional perfusion (r = 0.422, p = 0.013) with penetration of aerosol into areas of obvious radiographic infiltration on chest radiograph . CONCLUSIONS: Aerosol therapy with IFN-gamma in patients with pulmonary TB is widely distributed and results in significant enhancement of IFN-gamma levels in the lower respiratory tract . In patients without lung destruction, IFN-gamma aerosol may be an adjuvant to enhance the local immune response. Eur J Pharmacol, 2004 Jun 16, 493(1-3), 57 - 64 Avermectins inhibit multidrug resistance of tumor cells; Korystov YN et al.; The modification of the sensitivity of Hep-2 and P388 tumor cells to taxol and vincristine, substrates of multidrug resistance proteins, by naturally occurring avermectins and the effect of avermectins on the accumulation of calcein in cells and the efflux of rhodamine 123 were studied . While avermectins did not affect the sensitivity of tumor cells to hydrogen peroxide and cisplatin, they significantly enhanced the sensitivity of cells of both wild-type and resistant strains to taxol and vincristine . The coefficients of modification for resistant strains were substantially higher . Avermectins suppressed the efflux of rhodamine 123 from cells and increased the accumulation of calcein in cells . The relative inhibitory activity of avermectins depended on the cell type and on the substrate of multidrug resistance proteins whose transport they suppressed (vincristine, taxol, rhodamine 123, calcein acetoxymethyl ester) . The least active was avermectin B1 or ivermectin; the most active avermectins varied depending on the substrate and the cell type . In the case of vincristine transport, the most active avermectin was almost by one order of magnitude more effective than the traditional inhibitor of multidrug resistance cyclosporin A . This property of avermectins can be used in tumor therapy by combining application of avermectins with antitumor preparations, the substrates of multidrug resistance proteins . J Med Chem, 2004 Jun 17, 47(13), 3455 - 61 Novel pyridazino{4,3-b}indoles with dual inhibitory activity against Mycobacterium tuberculosis and monoamine oxidase; Velezheva VS et al.; Tuberculosis is one of the most common infectious diseases known to man . About 37% of the world's population (about 1.86 billion people) are infected with Mycobacterium tuberculosis . According to the World Health Organization, every year approximately 8 million people develop active tuberculosis and almost 2 million of those die from the disease . The incidence of multidrug-resistant tuberculosis (MDR-TB) is increasing . The present drug regimen for treating tuberculosis has been in existence for 30 years . New drugs that will shorten total treatment duration, improve the treatment of MDR-TB, and address latent tuberculosis are the most urgent need of tuberculosis control programs . A new series of synthetic 3-amino-4-arylpyridazino{4,3-b}indoles (pyridazinoindoles) were identified as inhibitors of Mycobacterium tuberculosis . The design, synthesis, and antimycobacterial activity of these compounds are described . While the most active compounds are still not comparable to the front-line drugs rifampicin and isoniazid, they do show promise . Most of the pyridazinoindoles with appreciable antituberculosis activity also inhibit monoamine oxidase, suggestive of a novel inhibitory effect on mycobacterial redox reactions. World J Gastroenterol, 2004 Jun 15, 10(12), 1722 - 5 Reversal of multidrug resistance in drug-resistant human gastric cancer cell line SGC7901/VCR by antiprogestin drug mifepristone; Li DQ et al.; AIM: To explore the reversal effect of mifepristone on multidrug resistance (MDR) in drug-resistant human gastric cancer cell line SGC7901/VCR and its mechanisms . METHODS: Expression of multidrug resistance-associated protein(MRP) was detected using reverse transcription-polymerase chain reaction(RT-PCR) . Flow cytometry was used to assay the expression of P-glycoprotein(P-gp), Bcl-2, Bax, and the mean fluorescent intensity of intracellular rhodamine 123 in the cells . Meanwhile, the protein levels of Bcl-2 and Bax were also detected by Western blotting analysis . The sensitivity of cells to the anticancer agent, vincrimycin(VCR), and the intracellular {(3) H}VCR accumulation were determined by tetrazolium blue (MTT) assay and a liquid scintillation counter, respectively . RESULTS: Expression of MRP and P-gp in SGC7901/VCR cells was 6.04-and 8.37-fold higher as compared with its parental SGC7901 cells, respectively . After treatment with 1, 5, 10, and 20 micromol/L mifepristone, SGC7901/VCR cells showed a 1.34-, 2.29-, 3.11-, and 3.71-fold increase in the accumulation of intracellular VCR, a known substrate of MRP, and a 1.03-, 2.04-, 3.08-, and 3.68-fold increase in the retention of rhodamine 123, an indicator of P-gp function, respectively . MTT assay revealed that the resistance of SGC7901/VCR cells to VCR was 11.96-fold higher than that of its parental cells . The chemosensitivity of SGC7901/VCR cells to VCR was enhanced by 1.02-, 7.19-, 12.84-, and 21.17-fold after treatment with mifepristone at above-mentioned dose . After 96 h of incubation with mifepristone 10 micromol/L, a concentration close to plasma concentrations achievable in human, the expression of Bcl-2 protein was decreased to (9.21+/-0.65)% from (25.32+/-1.44)%, whereas the expression of Bax protein was increased to (19.69+/-1.13)% from (1.24+/-0.78)% (P<0.01) . Additionally, the effects of mifepristone on the expression of Bcl-2 and Bax proteins in SGC7901/VCR cells were further demonstrated by Western blotting analysis . CONCLUSION: Mifepristone has potent reversal effect on MDR in SGC7901/VCR via inhibiting the function of MRP and P-gp, modulating the expression of Bcl-2 and Bax proteins, and enhancing the sensitivity to anticancer agent VCR. Arch Pharm (Weinheim), 2004 Jun, 337(6), 317 - 27 Lead identification for modulators of multidrug resistance based on in silico screening with a pharmacophoric feature model; Langer T et al.; Considerable effort has been devoted to the characterization of P-glycoprotein - drug interaction in the past . Systematic quantitative structure-activity relationship (QSAR) studies identified both predictive physicochemical parameters and pharmacophoric substructures within homologous series of compounds . Comparative molecular field analysis (CoMFA) led to distinct 3D-QSAR models for propafenone and phenothiazine analogs . Recently, several pharmacophore models have been generated for diverse sets of ligands . Starting from a training set of 15 propafenone-type MDR-modulators, we established a chemical function-based pharmacophore model . The pharmacophoric features identified by this model were (i) one hydrogen bond acceptor, (ii) one hydrophobic area, (iii) two aromatic hydrophobic areas, and (iv) one positive ionizable group . In silico screening of the Derwent World Drug Index using the model led to identification of 28 compounds . Substances retrieved by database screening are diverse in structure and include dihydropyridines, chloroquine analogs, phenothiazines, and terfenadine . On the basis of its general applicability, the presented 3DQSAR model allows in silico screening of virtual compound libraries to identify new potential lead compounds. Cancer Biother Radiopharm, 2004 Apr, 19(2), 165 - 70 Functional imaging of multidrug resistant phenotype by 99mTc-MIBI scan in patients with multiple myeloma; Fonti R et al.; Overexpression of P-glycoprotein (Pgp) is one of the primary mechanisms of multidrug resistance (MDR) in several diseases, including multiple myeloma . The aim of this study was to investigate whether the washout of 99mTc-MIBI, a transport substrate of Pgp, is enhanced in the bone marrow of patients with multiple myeloma overexpressing Pgp . Seventeen (17) patients were i.v . injected with 555 MBq of 99mTc-MIBI, and whole-body scans were performed at 10 and 60 minutes . A region of interest (ROI) was drawn over the thoracic spine of each scan, and the washout of 99mTc-MIBI was calculated, after decay correction, as: (10-minute counts/pixel minus 60-minute counts/pixel) divided by 10-minute counts/pixel . Pgp expression was determined in 17 bone marrow samples obtained from the same patients immediately before the 99mTc-MIBI scan . Following centrifugation over the Ficoll-Hypaque gradient, cytospins were obtained and immunostained with C219 monoclonal antibody . The immunostaining of Pgp was graded as 1, 2, or 3 when a faint, moderate, or intense reaction, respectively, was observed in infiltrating plasma cells . Washout of 99mTc-MIBI ranged between 5% and 26% . A statistically significant direct correlation was found between the washout of the tracer and Pgp expression (Spearman rank correlation coefficient r = 0.74, p < 0.001) . A partial overlap of washout values was observed in different classes of Pgp expression, thus preventing the discrimination of individual patients . Washout of 99mTc-MIBI, expressed as the percentage of radioactivity cleared from the bone marrow over a 1-hour period, may be used as a noninvasive tool for in vivo whole-body imaging of Pgp expression and function in multiple myeloma patients. Malar J . 2004 Jun 08;3(1):14. Malarone treatment failure not associated with previously described mutations in the cytochrome b gene; Wichmann O et al.; Malarone (atovaquone-proguanil) is an effective drug for the treatment and prophylaxis of multidrug-resistant falciparum malaria . However, first cases of resistance have been reported, which are associated with mutations at codon 268 of the parasite's cytochrome b gene . We report the first case of Malarone treatment failure from Central Africa.Drug concentration was well within curative range . Pre- and post-treatment Plasmodium falciparum isolates revealed codon 268 wild-type alleles, and no other mutations of the putative atovaquone-binding domain.These findings illustrate the spread of atovaquone-proguanil-resistance in Africa and question the usefulness of codon 268 as the only target for the surveillance of its emergence. Hepatology, 2004 Jun, 39(6), 1574 - 82 Interleukin-1beta represses MRP2 gene expression through inactivation of interferon regulatory factor 3 in HepG2 cells; Hisaeda K et al.; The human multidrug resistance protein 2 (MRP2/ABCC2), expressed on the bile canalicular membrane, mediates the multispecific efflux of several organic anions, including conjugates of glucuronate, sulfate, and glutathione . Expression of MRP2 can be altered in response to environmental stimuli such as cholestasis and jaundice . We previously reported that MRP2 mRNA expression levels are decreased in the nontumorous part of hepatitis C virus-infected human liver tissues, and that inflammatory cytokines inhibit MRP2 expression in human hepatic (HepG2) cells . We investigated the molecular mechanisms by which inflammatory cytokines modulate MRP2 gene expression in hepatic cells . Treatment of human hepatic cells with interleukin-1beta (IL-1beta) or tumor necrosis factor alpha resulted in a decrease in the protein and mRNA levels of MRP2 . IL-1beta inhibited the transcriptional activity of MRP2 promoter constructs by 40%, and this inhibition of MRP2 promoter activity was mediated through the interferon stimulatory response element (ISRE) . Electrophoretic mobility shift assays with IL-1beta-treated nuclear extracts showed a decrease in the formation of DNA protein complexes, specifically those including interferon regulatory factor 3 (IRF3) . Expression of recombinant human IRF3 increased MRP2 promoter activity . Treatment with a specific extracellular signal-regulated kinase inhibitor relieved IL-1beta-induced MRP2 mRNA downregulation and abrogated the binding of IRF3 to the ISRE element . In conclusion, IL-1beta induces downregulation of the MRP2 gene by inactivating IRF3 binding to ISRE on the MRP2 promoter in human hepatic cells; this inactivation is accomplished via interference with the extracellular signal-regulated kinase pathway. Int J Tuberc Lung Dis, 2004 Jun, 8(6), 778 - 84 Treatment and follow-up of HIV-negative multidrug-resistant tuberculosis patients in an infectious diseases reference hospital, Buenos Aires, Argentina; Palmero DJ et al.; SETTING: An Argentinean reference hospital specialising in infectious diseases . OBJECTIVE: To assess the outcomes of all human immunodeficiency virus (HIV) negative multidrug-resistant tuberculosis (MDR-TB) patients referred to or diagnosed at Hospital Muniz . DESIGN: Clinical study for the period 1996-1999, with follow-up until June 2002 . RESULTS: One hundred and forty-one adult patients (52.5% female) with resistance to two to seven drugs were studied . Fifty patients (35.5%) had not been treated previously . The most frequently used second-line drugs were 5-F-quinolones, cycloserine and ethionamide in susceptibility based individually tailored three- to five-drug regimens . Hospital admission was associated with treatment success . Forty-five episodes of severe toxicity occurred . Treatment was successful in 51.8% of cases, but follow-up of 73 patients yielded 11.9% relapse . The mortality rate was 19.1% and default was 19.9% . Logistic regression analysis was statistically significant for treatment success in relation to patient admission, residence and resistance pattern . CONCLUSION: The burden of MDR-TB in this setting--prolonged infection, treatment cost and difficulties, low rates of cure and treatment adherence and high rates of fatality and relapse--can be improved by strengthening TB control programme activities and fighting against poverty and HIV/AIDS. Int J Tuberc Lung Dis, 2004 Jun, 8(6), 767 - 71 Residual lung damage after completion of treatment for multidrug-resistant tuberculosis; de Valliere S et al.; SETTING: Limpopo Province, South Africa . OBJECTIVE: To assess the residual lung damage of patients who completed treatment for multidrug-resistant tuberculosis (MDR-TB) . DESIGN: Chest radiograph and lung function tests were performed at the end of treatment . The radiographs were read by two independent observers who attributed a zonal score of between 0 and 18, depending on the extent of radiographic abnormalities (opacification or cavitation), counted the number of visible cavities and measured the diameter of the largest cavity . RESULTS: The mean zonal score was 6.5 . Cavitation was present in more than half of the patients . Of 33 patients, 31 (94%) had abnormal lung function tests . The median FEV1 was 63% and FVC was 57% of the predicted value . Restrictive and combined restrictive-obstructive lung function patterns were the predominant abnormalities . CONCLUSIONS: Residual lung damage in MDR-TB patients who completed treatment is common and extensive . This may increase the risk of relapse of tuberculosis and reduce the quality of life and life expectancy of these patients . Additional efforts are warranted to diagnose MDR-TB early to reduce the extent of residual lung damage . Close follow-up of MDR-TB patients completing treatment will have to be ensured to detect relapses. Int J Tuberc Lung Dis, 2004 Jun, 8(6), 760 - 6 Surveillance of Mycobacterium tuberculosis susceptibility to second-line drugs in Hong Kong, 1995-2002, after the implementation of DOTS-plus; Kam KM et al.; OBJECTIVE: To determine the trend in changes in susceptibility of Mycobacterium tuberculosis strains, including to second-line drugs, from patients with a history of previous anti-tuberculosis (TB) treatment in a 'DOTS-Plus' programme . METHODS: A retrospective survey of centralised M . tuberculosis laboratory records of all culture-positive cases over an 8-year period . The drug susceptibility of the isolates was determined using the absolute concentration method . Isolates obtained from patients with a history of previous treatment were further analysed for trends of changes in susceptibility to first- and second-line drugs . RESULTS: Of 1921 patients with a previous history of treatment and positive cultures, 1425 (74.2%) had isolates susceptible to all four first-line drugs, while 176 (9.2%) were multidrug-resistant (MDR-TB) . For the MDR-TB group, 101 (57.4%) isolates were sensitive to all second-line drugs, while 30 (17.0%) were resistant to three or more second-line drugs . CONCLUSION: In a DOTS-Plus programme environment where there is strict control on use of second-line drugs, the prevalence of MDR-TB is low amongst retreatment cases and the prudent use of second-line drugs in a population with well functioning DOTS-Plus programme does not generate super-resistant strains . In circumstances where most retreatment strains are still susceptible and good laboratory support for detection of MDR cases is available, retreatment using first-line drugs is feasible. Int J Tuberc Lung Dis, 2004 Jun, 8(6), 749 - 59 Psychiatric issues in the management of patients with multidrug-resistant tuberculosis; Vega P et al.; INTRODUCTION: Psychiatric issues present a challenge in the treatment of patients with multidrug-resistant tuberculosis (MDR-TB) . Both baseline psychiatric disorders and development of psychiatric complications related to anti-tuberculosis drugs and psychosocial factors require aggressive management . SETTING: A community-based non-governmental health organization in Lima, Peru . OBJECTIVE: To review the literature for psychiatric complications associated with anti-tuberculosis medications, to describe the incidence and prevalence of depression, anxiety and psychosis among individuals receiving MDR-TB therapy, and to detail the management approach used in this cohort . METHODS: A retrospective case series was performed among the first 75 patients to receive individualized MDR-TB therapy in Lima, Peru, between 1996 and 1999 . RESULTS: Baseline depression and baseline anxiety were observed in respectively 52.2% and 8.7% of this cohort . Most individuals with baseline depression experienced improvement of depressive symptoms during the course of TB therapy . The incidence of depression, anxiety and psychosis during MDR-TB treatment was 13.3%, 12.0% and 12.0%, respectively . While the majority of individuals with depression, anxiety and psychosis required psychiatric pharmacotherapy, cycloserine was successfully continued in all but one case . CONCLUSION: Psychiatric comorbidities are not a contra-indication to MDR-TB therapy . Management of psychiatric complications is possible without compromising anti-tuberculosis treatment. Int J Tuberc Lung Dis, 2004 Jun, 8(6), 730 - 6 Retrospective descriptive study of adult tuberculosis in Wuhan, China; Chamla DD et al.; OBJECTIVE: To determine the rate and associated factors of adult tuberculosis (TB) in the central Chinese city of Wuhan . DESIGN: A retrospective descriptive study of 417 patients registered for TB treatment from 1 January to 31 December 2001 . RESULTS: The mean age of admission was 38.47 (median 35) years, with males aged 20-40 years mostly affected; 191 (45.8%) TB patients were classified as smear-positive, 221 (53%) smear-negative and for five (1.2%) the sputum results were not known . Of all admissions, 43 (10.32%) were retreatment cases and 50 (11.99%) were diagnosed as extra-pulmonary TB . All patients were treated under the DOTS strategy, with 391 (93.76%) cures, five (1.2%) treatment completed, five (1.2%) treatment failures, four (0.96%) deaths, three (0.72%) defaults and nine (2.16%) transfers out . Cure was associated with age (chi2 = 3.92, P < 0.05), but not with sex, retreatment TB, extra-pulmonary TB, type of treatment regimen, BCG status or delay in treatment (P > 0.05) . CONCLUSION: DOTS provides high TB cure rates . The reasons for the low detection rates, high retreatment rates and the increasing number of young adults affected by TB need further elucidation . For these purposes, routine human immunodeficiency virus screening and sputum culture for multidrug-resistant tuberculosis and case detection may be required. J Med Liban, 2003 Jan-Mar, 51(1), 4 - 8 Molecular fingerprinting of multidrug-resistant Mycobacterium tuberculosis strains in Beirut reveals genetic diversity and father to daughter transmission; Ahmad S et al.; The typing of six consecutive multidrug-resistant Mycobacterium tuberculosis strains isolated from patients with tuberculosis (TB) at the American University of Beirut Medical Center, was performed by touchdown double-repetitive-element (DRE)-PCR . The isolates exhibited four distinct patterns in DRE-PCR with three isolates exhibiting unique patterns and three isolates yielded similar DNA fragment patterns (cluster pattern) . Only two of the three cluster isolates exhibited identical patterns as revealed by restriction fragment length polymorphism (RFLP) targeting specific mutations in the rpoB and katG genes that confer resistance to rifampin and isoniazid, respectively . A direct epidemiological linkage for the two isolates exhibiting genotypic relatedness was also established as the isolates were recovered from a 33-year-old man and his 8-year-old daughter . The data show that transmission of multidrug-resistant M . tuberculosis strains is contributing to the emergence of drug-resistant TB in Beirut . Combining DRE-PCR with RFLP at the rpoB and katG genes could provide a powerful means for investigating the spread of multidrug-resistant M . tuberculosis strains in Lebanon. J Biol Chem, 2004 Jul 30, 279(31), 32367 - 72 Epub 2004 Jun 03. Weak base permeability characteristics influence the intracellular sequestration site in the multidrug-resistant human leukemic cell line HL-60; Duvvuri M et al.; A number of organelles contained within mammalian cells have been implicated in the selective sequestration of chemical entities including drug molecules . Specifically, weakly basic molecules have been shown to selectively associate with either the mitochondrial compartment or lysosomes; however, the structural basis for this differentiation has not been understood . To investigate this, we have identified a series of seven weakly basic compounds, all with pK(a) near neutrality, which have different sequestration sites within the multidrug-resistant HL-60 human leukemic cell line . Three of the compounds were selectively sequestered into the mitochondria of the cells, whereas the remainder were predominantly localized within lysosomes . Using specific chemical inhibitors to disrupt either mitochondrial or lysosomal accumulation capacity, we demonstrated that accumulation of these compounds into respective organelles are not competitive processes . Comparison of the permeability characteristics of these compounds as a function of pH revealed striking differences that correlate with the intracellular sequestration site . Only those compounds with significantly reduced permeability in the ionized state relative to the un-ionized state had the capacity to accumulate within lysosomes . Alternatively, those compounds with relatively pH-insensitive permeability selectively accumulated into mitochondria . Using novel quantitative assays for assaying drug accumulation into subcellular organelles, we demonstrated a correlation between these permeability characteristics and the lysosomal versus mitochondrial accumulation capacity of these compounds . Together, these results suggest that the selective accumulations of weakly basic compounds in either lysosomes and mitochondria occur via exclusive pathways governed by a unique permeability parameter. BJU Int, 2004 Jun, 93(9), 1333 - 8 Identification of multidrug resistance-associated protein 1 and glutathione as multidrug resistance mechanisms in human prostate cancer cells: chemosensitization with leukotriene D4 antagonists and buthionine sulfoximine; van Brussel JP et al.; OBJECTIVE: To assess the involvement of the multidrug resistance-associated protein 1 (MRP1) and the glutathione pathway in the multidrug resistant (MDR) phenotype of prostate cancer in vitro . MATERIALS AND METHODS: Chemoselection of human prostate cancer cell lines PC3 and DU145 with etoposide resulted in the resistant cell lines PC3-R and DU-R . Resistance against etoposide, doxorubicin and vincristine, and its reversal with leukotriene D4 antagonists MK-571 and zafirlukast, and buthionine sulfoximine (BSO), was assessed using tetrazolium-dye viability assays . Western blot analysis of MRP1 expression and glutathione content were measured, and MRP1 function assessed in fluorescence assays . RESULTS: MRP1 was increased in the MDR models; the glutathione content was significantly higher in PC3-R but there was no increase in glutathione in DU-R . Adding non-toxic doses of MK-571, zafirlukast or BSO significantly increased the sensitivity of the MDR models to cytotoxic drugs . MRP1 function was inhibited with MK-571 in the MDR models . CONCLUSION: MRP1 and glutathione mediate MDR in newly developed prostate cancer models. Pharm Res, 2004 May, 21(5), 742 - 8 Characterization of the cellular localization, expression level, and function of SNP variants of MRP2/ABCC2; Hirouchi M et al.; PURPOSE: The presence of single nucleotide polymorphisms (SNPs) has been reported for multidrug resistance-associated protein 2 (MRP2/ABCC2) . The purpose of the current study was to characterize the localization, expression level, and function of MRP2 variants . METHODS: The expression and cellular localization of the wild-type and three kinds of reported SNP variants of MRP2 molecules were analyzed in LLC-PK1 cells after infection with the recombinant Tet-off adenoviruses . Their function was determined by using the isolated membrane vesicles from the infected LLC-PK1 cells . RESULTS: The transport activity for E217betaG, LTC4, and DNP-SG, normalized by the expression level of MRP2, was similar between the wild-type, V417I, and A1450T MRP2s . The transport activity of S789F MRP2 was slightly higher than that of wild-type MRP2 . However, the expression level of S789F and A1450T MRP2 proteins was significantly lower compared with the wild-type and V417I MRP2 . In addition, although the wild-type and V417I MRP2 were exclusively localized in the apical membrane, S789F and A1450T MRP2 were located in the apical membrane and also in the intracellular compartment . CONCLUSIONS: These results suggest that the most frequently observed V417I substitution may not affect the in vivo function of MRP2, whereas the much less frequently observed S789F and A1450T may be associated with the reduced in vivo function. Pharm Res, 2004 May, 21(5), 719 - 35 The complexities of hepatic drug transport: current knowledge and emerging concepts; Chandra P et al.; Recently, hepatic transport processes have been recognized as important determinants of drug disposition . Therefore, it is not surprising that characterization of the hepatic transport and biliary excretion properties of potential drug candidates is an important part of the drug development process . Such information also is useful in understanding alterations in the hepatobiliary disposition of compounds due to drug interactions or disease states . Basolateral transport systems are responsible for translocating molecules across the sinusoidal membrane, whereas active canalicular transport systems are responsible for the biliary excretion of drugs and metabolites . Several transport proteins involved in basolateral transport have been identified including the Na(+)-taurocholate co-transporting polypeptide {NTCP (SLC10A1)}, organic anion transporting polypeptides {OATPs (SLCO family)}, multidrug resistance-associated proteins {MRPs (ABCC family)}, and organic anion and cation transporters {OATs, OCTs (SLC22A family)} . Canalicular transport is mediated predominantly via P-glycoprotein (ABCB1), MRP2 (ABCC2), the bile salt export pump {BSEP (ABCB11)}, and the breast cancer resistance protein {BCRP (ABCG2)} . This review summarizes current knowledge regarding these hepatic basolateral and apical transport proteins in terms of substrate specificity, regulation by nuclear hormone receptors and intracellular signaling pathways, genetic differences, and role in drug interactions . Transport knockout models and other systems available for hepatobiliary transport studies also are discussed . This overview of hepatobiliary drug transport summarizes knowledge to date in this rapidly growing field and emphasizes the importance of understanding these fundamental processes in hepatic drug disposition. Scand J Gastroenterol, 2004 May, 39(5), 464 - 9 Expression patterns of cell cycle and apoptosis-related genes in a multidrug-resistant human colon carcinoma cell line; Fan CW et al.; BACKGROUND: An in vitro multidrug resistance (MDR) system from a human colonic cancer cell line (SW620-MDR) has been established . To further study the mechanisms at molecular level and prevention of multidrug resistance in clinical practice, it was demonstrated that the expressions of several apoptosis-related and cell cycle regulator genes were changed in the cells . METHODS: A multidrug-resistant colonic cell line (SW620-MDR) was established, and the Atlas human cDNA expression array was used for studying the pattern of gene expression in this cell line . Furthermore, Northern hybridization or real-time PCR analysis confirmed the pattern of gene expression . RESULTS: In the SW620-MDR cell line the pro-apoptosis genes, CASP4, BIK, PDCD2, and TACE were expressed with decreased levels, and the antiapoptosis genes CD27-L and IGFBP2 were over-expressed . Furthermore, the cell cycle regulator genes such as CDK6, CCND1, CDC27HS, CDC16HS, Wee1Hu, MAPKK1, and IGFBP6 were expressed with decreased levels in the drug-resistant cell line . CONCLUSIONS: It is worthwhile investigating whether the differentially expressed pattern of the aforementioned genes exists in the drug-resistant cancer specimens, and to further understand their functions in the cancer drug-resistance mechanism. Mar Environ Res, 2004 Aug-Dec, 58(2-5), 199 - 204 Identification of the multidrug resistance-associated protein (mrp) related gene in red mullet (Mullus barbatus); Sauerborn R et al.; Multixenobiotic resistance mechanism (MXR) in aquatic organisms is mediated by the activity of the P-glycoprotein (Pgp) transporter that binds and actively effluxes different chemicals out of cell . In addition to the Pgp, several other, non-Pgp transport proteins have been recently identified in different human and animal tissues . Given their characteristics and tissue distribution we hypothesized that members of the so-called multidrug resistance-associated protein (MRP) family may be expressed in aquatic organisms . This study attempted to identify MRP related genes in different tissues of several marine and freshwater bivalves (Mytilus galloprovincialis, Dreissena polymorpha, Anodonta cygnea) and fish species (Mullus barbatus, Cyprinus carpio, Salmo trutta) . Following an alignment of known MRP1 and MRP2 human sequences, as well as the GenBank available mrp2 sequences from different animals, we determined highly conserved regions and used them to design three pairs of consensus primers . Total RNA was isolated, reverse transcribed to cDNA and the obtained cDNAs were PCR amplified with the corresponding primers . The amplified PCR products were sequenced and their homology compared with Pgp and MRP protein sequences from different species . The expression of MRP related mRNA was clearly identified only in liver tissue isolated from red mullet, with homologies at the protein level ranging from 75% to 76% . Described results clearly pointed at the possibility that at least in the red mullet MXR as a general defense mechanism may be mediated by the activities of at least two different types of transport proteins. J Org Chem, 2004 Jun 11, 69(12), 4126 - 34 A practical total synthesis of hapalosin, a 12-membered cyclic depsipeptide with multidrug resistance-reversing activity, by employing improved segment coupling and macrolactonization; Palomo C et al.; A practical total synthesis of hapalosin, a compound with multidrug resistance-reversing activity, has been carried out using an unprecedented macrolactonization strategy . One of the features of the new approach is the straightforward and fully stereocontrolled access to the key gamma-amino beta-hydroxy carboxylic acid subunit via an efficient acetate aldol addition reaction with N-methyl alpha-aminoaldehydes, which relies on a camphor-derived chiral lithium acetate enolate reagent . The scope of this aldol reaction is investigated and its potential application to the synthesis of other structurally related, biologically relevant compounds illustrated . Remarkably, the chiral tether in the resulting gamma-amino aldol adducts sterically protect the carbonyl group, thus avoiding intramolecular cyclization during the amino group deprotection and the subsequent segment coupling event . After successful segment coupling and smooth, clean release of the chiral auxiliary, a new macrolactonization protocol, based on the principle of double activation of both reactive sites, is applied, which leads to the 12-membered macrolactone hapalosin in unprecedented chemical efficiency. J Pharm Sci, 2004 Jul, 93(7), 1901 - 11 Effects of benzyl-, phenethyl-, and alpha-naphthyl isothiocyanates on P-glycoprotein- and MRP1-mediated transport; Hu K et al.; The objective of this investigation was to evaluate the effects of two dietary isothiocyanates (ITCs), benzyl- (BITC) and phenethyl isothiocyanate (PEITC), and one synthetic ITC, alpha-naphthyl isothiocyanate (1-NITC), on the P-glycoprotein (P-gp)- and multidrug-resistance protein 1 (MRP1)-mediated efflux of daunomycin (DNM), determine whether PEITC is a substrate of P-gp and/or MRP1, and elucidate the mechanism(s) involved in the inhibition of transport . BITC, PEITC, and 1-NITC significantly increased the 2-h accumulation of DNM in MCF-7/ADR (P-gp overexpression), PANC-1 (MRP1 overexpression), and human colon adenocarcinoma Caco-2 cells (except for 1-NITC) . The accumulation of (14)C-PEITC was not changed in Caco-2, human breast cancer MDA435/LCC6 and MDA435/LCC6MDR1 (P-gp overexpression) cells in the absence and presence of the P-gp inhibitor verapamil, but significantly increased with the MRP inhibitor MK571 in PANC-1 cells . The isocyanate and amine metabolites had no effect on DNM accumulation in any cell line . After 2- and 24-h ITC treatments, cellular concentrations of glutathione (GSH) in PANC-1 and Caco-2 cells were depleted by BITC and PEITC, but not by 1-NITC; glutathione-S-transferase activity exhibited small changes . Our results suggest that (1) BITC, PEITC, and 1-NITC inhibit the P-gp- and MRP1-mediated efflux of DNM; (2) PEITC and/or its conjugates do not represent P-gp substrates; (3) BITC and PEITC, but not 1-NITC, inhibit MRP1 through the depletion of intracellular GSH, which acts as a cosubstrate for DNM efflux via MRP1; and (4) PEITC and/or its conjugates are MRP1 substrates so binding interactions with DNM represent a second potential mechanism involved in MRP1 inhibition . J Cell Physiol, 2004 Aug, 200(2), 223 - 34 Drug-resistant breast carcinoma (MCF-7) cells are paradoxically sensitive to apoptosis; Chen JS et al.; The purpose of this study was to determine whether expression of tissue transglutaminase (TG2) and caspase-3 proteins in drug-resistant breast carcinoma MCF-7/DOX cells would render these cells selectively susceptible to apoptotic stimuli . Despite high resistance to multidrug resistance (MDR)-related drug, doxorubicin (> or =150-fold), the MCF-7/DOX cells were extremely sensitive to apoptotic stimuli . Thus, calcium ionophore, A23187 (A23187) and the protein kinase C inhibitor staurosporine (STS) each induced rapid and time-dependent apoptosis in MCF-7/DOX cells . The apoptosis induced by either agent was accompanied by caspase-3 activation and other downstream changes that are typical of cells undergoing apoptosis . The alterations upstream of caspase-3 activation, however, such as loss in mitochondrial membrane potential (DeltaPsi), release of cytochrome c, and activation of caspase-8, and caspase-9, were detected only in STS-treated cells . The A12387 failed to induce any of the caspase-3 upstream changes, implying that A23187-induced apoptosis may utilize one or more novel upstream pathways leading to the activation of caspase 3 . In summary, these data demonstrate that MCF-7/DOX cells are much more sensitive to apoptotic stimuli than previously thought and that A23187-induced apoptosis may involve some novel, yet unidentified, upstream pathway that leads to the activation of caspase-3 and other downstream events . Clin Cancer Res, 2004 Jun 1, 10(11), 3788 - 93 Usefulness of 99mTc-sestamibi scintigraphy in suggesting the therapeutic effect of chemotherapy against gastric cancer; Kawata K et al.; PURPOSE: Imaging with (99m)Tc-sestamibi ((99m)Tc-MIBI) has been used to assess 170-kDa P-glycoprotein (P-gp) expression and predict chemotherapy responses in several types of malignancy, such as breast and lung cancers . The purpose of this study was to evaluate the relationship between (99m)Tc-MIBI accumulation in tumors and sensitivity to chemotherapy in gastric cancer patients . EXPERIMENTAL DESIGN: Thirty-six patients with advanced gastric cancer underwent (99m)Tc-MIBI scintigraphy before chemotherapy . Patients also underwent endoscopic biopsy, and the expression of P-gp or multidrug resistance-associated protein was analyzed by immunohistochemical staining . The relationship between the accumulation of (99m)Tc-MIBI in tumors and responses to chemotherapy with 5-fluorouracil/cis-diamminedichloroplatinum(II) or epirubicin was examined . RESULTS: Higher accumulation of (9m)Tc-MIBI in tumors was observed in 25 and 23 of 36 gastric cancer patients at the early (30 min) and delayed (120 min) images, respectively . Accelerated accumulation of (99m)Tc-MIBI negatively correlates with increased expression of P-gp, but not of multidrug resistance-associated protein, as determined by immunohistochemistry in gastric cancer tissues . The response rate to 5-fluorouracil/cis-diamminedichloroplatinum(II) chemotherapy in patients with high (99m)Tc-MIBI accumulation (15.4%) was much lower than that in patients with low (99m)Tc-MIBI accumulation (54.5%) . In contrast, patients with high (99m)Tc-MIBI accumulation show a higher response rate (41.7%) to chemotherapy with epirubicin, which is known to be a substrate of P-gp transporter . CONCLUSIONS: (99m)Tc-MIBI scintigraphy is useful to suggest the responses to chemotherapy of patients with advanced gastric cancer. Exp Parasitol, 2004 Mar-Apr, 106(3-4), 126 - 34 Cryptosporidium parvum: effect of multi-drug reversing agents on the expression and function of ATP-binding cassette transporters; Bonafonte MT et al.; In the present study, the gene expression of three multidrug resistance (MDR) and resistance-associated protein (MRP) transport proteins or efflux pumps was characterized and the phenotypic evidence for such pumps was demonstrated in cultured Madin-Darby canine kidney (MDCK) cells . A gradient for the fluorescent probe calcein was established between parasite and host cell suggestive of a parasite extrusion pump at the parasite-host interface . This gradient was decreased in a glucose-free medium containing 2-deoxyglucose or 3-O-methylglucose, by probenecid, and by the isoflavonoid, narigenin, suggesting that the calcein extrusion was energy-dependent and involved an MRP-like pump . While neither MDR or MRP inhibiters significantly affected transcript levels of any of the ABC transporters, transcript levels of the Cryptosporidium parvum ABC protein (CpABC1), an MRP transporter, were consistently expressed 4 logs higher than either CpABC3 or CpABC2, suggesting a prominent role in the intracellular stages of the parasite. Pediatr Blood Cancer, 2004 Jul, 43(1), 46 - 54 Differential responsiveness among "high risk" pediatric brain tumors in a pilot study of dose-intensive induction chemotherapy; Jennings MT et al.; BACKGROUND: These factors have been predictive for progressive disease on therapy (PDOT) among pediatric brain tumors: >1.5 cm(2) unresectable tumor, glioblastoma, supratentorial primitive neuroectodermal tumor, and metastatic medulloblastoma (MBL) . This pilot study sought to correlate cytoreductive response with progression free survival . PROCEDURES: Four courses of cisplatinum, cyclophosphamide, etoposide, and vincristine preceded hyperfractionated radiotherapy (RT) . Maintenance chemotherapy consisted of eight cycles of carboplatin, etoposide, and vincristine . Biopsy specimens were immunohistochemically studied for labeling index, hypoxia, and multidrug resistance proteins . RESULTS: Twenty newly diagnosed patients {nine primitive neuroectodermal tumors/MBL, one choroid plexus carcinoma, eight malignant gliomas, and two anaplastic ependymomas} were treated . Ten patients, who required neuraxis irradiation, constituted the "PNET" group . These demonstrated five complete and one partial response (PR), with an estimated median progression free survival of 44 months and median survival in excess of 53 months . Patients treated with involved field irradiation were designated the "Glioma" group . Induction chemotherapy produced partial and minor responses (MRs) among 5/10 . Their estimated median progression free survival was 6.9 months (P = 0.035 relative to the PNET) with a median survival of 10.7 months (P = 0.04) . Age, labeling index, the presence of hypoxia, and Pgp/MDR1 expression failed to discriminate between the two groups . CONCLUSIONS: This induction regimen produced a cytoreductive response in 6/10 and achieved a significant improvement in progression free survival among 7/10 in the PNET group . Unfortunately, responses among Glioma patients did not translate into durable control . Expression of the biologic factors was similar between both groups and did not correlate with diagnosis or response . Int J Cancer, 2004 Jul 20, 110(6), 882 - 90 Targeting an extracellular epitope of the human multidrug resistance protein 1 (MRP1) in malignant cells with a novel recombinant single chain Fv antibody; Binyamin L et al.; Inherent and acquired multidrug resistance (MDR) is characterized by a simultaneous resistance to diverse anticancer drugs and is a major impediment towards curative chemotherapy of cancer . Hence one important goal is to develop strategies aimed at specific targeting of major anticancer drug efflux transporters of the ATP-binding cassette (ABC) superfamily including multidrug resistance protein 1 -MRP1 (ABCC1) . To date, no monoclonal antibody has been isolated that can target an extracellular MRP1 epitope . Using a phage display approach, we have isolated a recombinant single-chain Fv (scFv) antibody that specifically reacts with the extracellular N-terminus of the human MRP1 . Flow cytometric analysis revealed that this scFv fragment binds specifically to various viable human tumor cells that display variable MRP1 expression levels but not to MRP1 null cells . Furthermore, this scFv antibody failed to react with tumor cells that overexpress other members of the MRP family that have an extracellular N-terminus (MRP2 and MRP3) as well as with MRP4, MRP5, and breast cancer resistance protein . Flow cytometric analysis also showed a good correlation between the fluorescence intensity of the anti-MRP1 scFv antibody and MRP1 levels in viable tumor cells . These findings constitute the first successful isolation of a small recombinant scFv antibody directed to an extracellular epitope of the MRP1 in viable malignant cells . These novel small Fv-based recombinant antibodies that possess superior tumor penetration capabilities may possibly be used to selectively target drugs or tumor cells that express MRP-1 . Pharmacogenetics, 2004 Mar, 14(3), 155 - 64 Genetic polymorphisms in the multidrug resistance-associated protein 3 (ABCC3, MRP3) gene and relationship to its mRNA and protein expression in human liver; Lang T et al.; AIMS: To determine the genetic variability of multidrug resistance protein 3 (MRP3) . METHODS: Genomic DNA samples from 103 Caucasians were systematically screened for genetic variations to find a potential relationship with hepatic MRP3 expression . Sequencing comprised all 31 exons, approximately 100 bp of the flanking intronic regions and 2 kb of the 5' UTR . RESULTS: In total, 51 mutations were identified . Fifteen SNPs were located in the coding exons of MRP3, six of which are nonsynonymous mutations . SNPs 39G>C (allele frequency: 0.5%, located in exon 1), 202C>T (1.6%, exon 2), 1037C>T (0.5%, exon 9), 1537C>A (0.5%, exon 12), 3890G>A (5.2%, exon 27) and 4267G>A (0.6%, exon 29) resulted in Lys13Asn, His68Tyr, Ser346Phe, Gln513Lys, Arg1297His and Gly1423Arg amino acid substitutions, respectively . A splice site mutation (1339-1G>T) was found at the intron 10-exon 11 boundary . To evaluate, whether mutations in the MRP3 gene correlate with human hepatic MRP3 expression, we analyzed the genetic variants in Caucasian liver samples, whose MRP3 mRNA (n = 84) and protein (n = 50) expression has been determined by real time quantitative PCR and Western Blot, respectively . We found a significant correlation of a polymorphism in the 5' promoter region (-211C>T) of MRP3 with mRNA expression . Individuals homozygous and heterozygous for the -211C>T promoter polymorphism had significantly lower MRP3 transcript levels compared to wild-type individuals (P < 0.05) . Accordingly, electrophoretic mobility shift assay demonstrated that -211C>T polymorphism affected the binding of nuclear factors . CONCLUSIONS: Multiple genetic polymorphisms of MRP3 exist in Caucasians . The -211C>T promoter polymorphism appears to be associated with altered hepatic MRP3 mRNA expression. J Acquir Immune Defic Syndr, 2004 Jun 1, 36(2), 649 - 658 Transport of HIV Protease Inhibitors Through the Blood-Brain Barrier and Interactions With the Efflux Proteins, P-Glycoprotein and Multidrug Resistance Proteins; Gimenez F et al.; HIV protease inhibitors (HPIs) have limited penetration into the brain . This poor transport through the blood-brain barrier is mainly due to active efflux by proteins such as P-glycoprotein (P-gp) preventing drugs from clearing the brain of the virus . The present paper focuses on cerebral uptake of HPIs and interactions between HPIs and efflux proteins, either as substrates or modulators . Most of the studies described HPIs as P-gp substrates . Studies are more controversial when investigating HPIs as inhibitors of P-gp . HPIs seem to be able to inhibit efflux proteins of in vitro cell models but with limited consequences in vivo . Moreover, after repeated administrations of HPIs, most of them are also able to induce the expression and functionality of P-gp . For these reasons, certain combinations of HPIs may not efficiently increase brain uptake of HPIs as would combinations of more potent efflux inhibitors. FEBS Lett, 2004 Jun 1, 567(1), 116 - 20 ABCG2 -- a transporter for all seasons; Sarkadi B et al.; The human ABCG2 (ABCP/MXR/BCRP) protein is a recently recognized ABC half-transporter, which forms homodimers in the plasma membrane and actively extrudes a wide variety of chemically unrelated compounds from the cells . This protein protects our cells and tissues against various xenobiotics, with a crucial role in the intestine, liver, placenta, and the blood-brain barrier . Moreover, ABCG2 seems to have a key function in stem cell protection/regulation, and also in hypoxic defense mechanisms . Widely occurring single nucleotide polymorphisms in ABCG2 may affect absorption and distribution, altering the effectiveness and toxicity of drugs in large populations . At the clinics, overexpression of ABCG2 in tumor cells confers cancer multidrug resistance to a variety of newly developed anticancer agents . On the other hand, specific substrate mutants of ABCG2 are advocated for use as selectable markers in stem-cell based gene therapy. Gene Ther, 2004 Jul, 11(14), 1170 - 4 Complete reversal of multidrug resistance by stable expression of small interfering RNAs targeting MDR1; Yague E et al.; Overexpression of P-glycoprotein, encoded by the MDR1 gene, confers multidrug resistance (MDR) on cancer cells and is a frequent impediment to successful chemotherapy . Recent developments in the use of small interfering RNAs to inhibit specific protein expression have highlighted their potential use as therapeutic agents . We have expressed two different short hairpin RNAs from stably integrated plasmids in doxorubicin-resistant K562 leukaemic cells . The MDR1-targeted RNA interference (RNAi) resulted in decreased MDR1 mRNA, abolished P-glycoprotein expression, and completely reversed the MDR phenotype to that of the drug-sensitive K562 parental line . This study demonstrates that MDR, which is solely due to overexpression of P-glycoprotein, can be reversed by RNAi . These target sequences can in the future be integrated into gene therapy vectors with potential clinical application. Int J Clin Oncol, 2004 Feb, 9(1), 13 - 24 Systemic chemotherapy as a new conservative treatment for intraocular retinoblastoma; Yanagisawa T; Retinoblastoma is the most common malignant intraocular tumor in childhood . With advances in the methods for early detection of this disease, the survival rate is over 90% in developed countries . The management of intraocular retinoblastoma has gradually changed over the past few decades . Every effort has been made to save life, with the preservation of the eye and sight, if possible . External beam radiotherapy has been a standard treatment for medium and large, or visually threatening, intraocular retinoblastoma, but it markedly increases the risk of cosmetic deformities and secondary cancer in children with germline RB mutations . For the past decade, primary systemic chemotherapy called "chemoreduction" has been employed to avoid radiotherapy and enucleation . This article gives an overview of the results of current trials of primary chemoreduction for intraocular retinoblastoma, and discusses its role and its limitations in conservative treatment . The article also discusses future directions to expand the indications for this treatment . Many children with advanced intraocular retinoblastoma could be spared external beam radiotherapy and enucleation, mostly as a result of chemoreduction and focal methods . Chemoreduction combined with focal treatments will continue to play an important role in the conservative management of children with intraocular retinoblastoma, possibly even in children with advanced disease, with the combined use of multidrug-resistance modulators. J Biol Chem, 2004 Jul 30, 279(31), 32700 - 8 Epub 2004 May 25. Arsenic transport by the human multidrug resistance protein 1 (MRP1/ABCC1) . Evidence that a tri-glutathione conjugate is required; Leslie EM et al.; Inorganic arsenic is an established human carcinogen, but its metabolism is incompletely defined . The ATP binding cassette protein, multidrug resistance protein (MRP1/ABCC1), transports conjugated organic anions (e.g . leukotriene C(4)) and also co-transports certain unmodified xenobiotics (e.g . vincristine) with glutathione (GSH) . MRP1 also confers resistance to arsenic in association with GSH; however, the mechanism and the species of arsenic transported are unknown . Using membrane vesicles prepared from the MRP1-overexpressing lung cancer cell line, H69AR, we found that MRP1 transports arsenite (As(III)) only in the presence of GSH but does not transport arsenate (As(V)) (with or without GSH) . The non-reducing GSH analogs L-gamma-glutamyl-L-alpha-aminobutyryl glycine and S-methyl GSH did not support As(III) transport, indicating that the free thiol group of GSH is required . GSH-dependent transport of As(III) was 2-fold higher at pH 6.5-7 than at a more basic pH, consistent with the formation and transport of the acid-stable arsenic triglutathione (As(GS)(3)) . Immunoblot analysis of H69AR vesicles revealed the unexpected membrane association of GSH S-transferase P1-1 (GSTP1-1) . Membrane vesicles from an MRP1-transfected HeLa cell line lacking membrane-associated GSTP1-1 did not transport As(III) even in the presence of GSH but did transport synthetic As(GS)(3) . The addition of exogenous GSTP1-1 to HeLa-MRP1 vesicles resulted in GSH-dependent As(III) transport . The apparent K(m) of As(GS)(3) for MRP1 was 0.32 microM, suggesting a remarkably high relative affinity . As(GS)(3) transport by MRP1 was osmotically sensitive and was inhibited by several conjugated organic anions (MRP1 substrates) as well as the metalloid antimonite (K(i) 2.8 microM) . As(GS)(3) transport experiments using MRP1 mutants with substrate specificities differing from wild-type MRP1 suggested a commonality in the substrat