Multidrug resistant

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 substrate binding pockets of As(GS)(3) and leukotriene C(4) . Finally, human MRP2 also transported As(GS)(3) . In conclusion, MRP1 transports inorganic arsenic as a tri-GSH conjugate, and GSTP1-1 may have a synergistic role in this process.

Anticancer Res, 2004 Mar-Apr, 24(2B), 935 - 41
Three-dimensional culture and multidrug resistance: effects on immune reactivity of MCF-7 cells by monocytes; Mougel L et al.; BACKGROUND: Multicellular spheroids are known to be the most adapted model to keep the in vitro resistance properties of cells . This in vivo-like tissue-culture representation was applied to investigate the immune reactivity of MCF-7 cells by monocytes . MATERIALS AND METHODS: Human blood monocytes, obtained by elutriation, were co-cultured with multicellular tumor spheroids of drug-sensitive (MCF-7S) and doxorubicin-resistant (MCF-7DXR) MCF-7 breast cancer cells . RESULTS: Tumor cells, according to their phenotype, induced differential recruitment and behavior of the immune cells towards the two types of spheroids . The secretion of various cytokines and the expression of several adhesion molecules were analysed . The MCF-7DXR/monocytes co-culture supernatant showed higher levels of IL-6 and IL-8 than the MCF-7S/monocytes co-culture supernatant . Cells from the MCF-7DXR spheroids expressed some adhesion molecules, CD-44 and CD-54, leading to a strong cellular cohesion in comparison with the sensitive spheroids . CONCLUSION: The two spheroid phenotypes represented an excellent model system for determining the precise tumor microenvironment in which cells move, the crucial molecular requirements and the mechanisms by which immunotherapeutic strategies could be developed to eradicate chemo-resistant tumors.

Anticancer Res, 2004 Mar-Apr, 24(2B), 865 - 71
New silicon compounds as resistance modifiers against multidrug-resistant cancer cells; Molnar J et al.; The efficiency of chemotherapy is often decreased by the development of resistance of cancer cells to cytostatic drugs . This phenomenon is in most cases caused by the activity of the various ABC transporters, multidrug-resistance (MDR) gene-encoded p-glycoproteins, that pump anticancer drugs out of the cells . The inhibition of the activities of the MDR proteins MDR1 and MRP was investigated via the administration of two new organosilicon compounds, alis-409 and alis-421 . The study was focused on the inhibition of MDR by blocking the ADR1 gene expression and through the inhibition of the pump-function of mdr-p-glycoprotein, in human breast cancer cell lines expressing mrp and prostate cancer cell line (PC-3) . Apoptosis induction and the interaction between epirubicin and the silicon-substituted compounds were studied in human MDR-1 gene-transfected mouse lymphoma and its parent cell line, Colo320/MDR-LRP and sensitive subline Colo205, by means of rhodamine 123 accumulation . The activity of MRP1 p-glycoprotein was studied in human breast cancer cell lines such as HTB-26/MRP1 and two MRP-negative breast cancer cell lines, T47D and MCF7, by carboxyfluorescein accumulation, and on a stomach cancer cell line . The activity of MRP in 257P/MDR and its drug-sensitive derivative were studied in human stomach cancer cells by daunorubicin accumulation in a flow cytometer . The two representative organosilicon derivatives, alis-409 and alis-421, showed antiproliferative effects without apoptosis induction . The drug accumulation in the human MDR1 gene-transfected mouse lymphoma cells was increased without down-regulation of the MDR1 gene expression tested by RT-PCR assay . The rhodamine uptake was increased in L5178/MDR1 and Colo320/MDR1-LRP, but not drug-sensitive human breast cancer MCF-7 and T47D, and L5178 mouse lymphoma parent cells in the presence of alis-409 and alis-421 . The MRP-mediated carboxyfluorescein accumulation in HTB-26/MRP human breast cancer cells and daunorubicin accumulation in human stomach cancer cells 257P/MDR were not modified by these alis compounds . A synergistic interaction between epirubicin and the silicon-substituted resistance modifiers was found only in MDR1-mediated MDR in the case of colo-320/MDR1-LRP cells and mouse lymphoma cells transfected with the human MDR1 gene . The results indicate that the organosilyl derivatives specifically act on MDR1 p-glycoprotein 170 . The alis compounds act on pgp170 in a way which is similar to verapamil isomers.

Anticancer Res, 2004 Mar-Apr, 24(2B), 859 - 64
Effect of cycloartanes on reversal of multidrug resistance and apoptosis induction on mouse lymphoma cells; Madureira AM et al.; The ability of fifteen cycloartanes, isolated from Euphorbia species, to reverse multidrug resistance (MDR) and apoptosis induction in L5178Y mouse lymphoma cells, including its multidrug-resistant subline, was studied by flow cytometry . Reversion of MDR was investigated using a standard functional assay with rhodamine 123 as a fluorescent substrate analogue . For the evaluation of apoptosis, the cells were stained with FITC-labeled annexin V and propidium iodide . The majority of the compounds were able to reverse MDR of the tested human MDR1 gene-transfected mouse lymphoma cells . Some of the compounds were able to induce moderate apoptosis in the PAR cell line, but this effect was less effective on multidrug-resistant cells . The results indicate that cycloartanes can be substrates of ABC transporters, which might compete with certain anticancer chemotherapeutics.

Leuk Lymphoma, 2004 Apr, 45(4), 821 - 4
Plasma cell leukemia occurring in a patient with thrombocythemia treated with hydroxyurea and busulphan; Candoni A et al.; Plasma cell leukemia (PCL) is a rare aggressive lymphoproliferative disease with a short median survival and a very poor prognosis . We report the case of a 63-year-old man who developed a PCL after 5 years of chemotherapy with hydroxyurea and busulphan for Essential thrombocythemia (ET) . The karyotype showed a deletion of chromosome 7 and the plasma cells cytofluorimetric examination revealed a high expression of Multidrug Resistance related P-glycoprotein (PGP) . After the second cycle of VAD chemotherapy the patient had a severe pneumonia and died with refractory PCL . This is a rare example of the coexistence of a chronic myeloproliferative and lymphoproliferative diseases in the same patient, and to the best of our knowledge, the first reported in the literature involving PCL and ET . Moreover, this case shows the possibility of secondary malignancies developing in patients treated with busulphan and hydroxyurea for chronic myeloproliferative disorders.

Leuk Lymphoma, 2004 Apr, 45(4), 649 - 54
Breast cancer resistance protein (BCRP) in acute leukemia; Plasschaert SL et al.; Multidrug resistance, cross-resistance to structurally and functionally unrelated drugs, is an important cause of treatment failure in acute leukemia . Multidrug resistance can result from the overexpression of ATP-dependent efflux pumps, such as P-glycoprotein and members of the multidrug resistance associated protein (MRP) family . Recently a novel transporter has been identified, which is called breast cancer resistance protein (BCRP), ABCG2 or mitoxantrone resistance protein . BCRP confers resistance to chemotherapeutic agents, such as mitoxantrone, doxorubicin and daunorubicin . This review describes BCRP detection techniques and the normal physiology of BCRP . The role of BCRP in the physiology of hematopoietic stem cells is addressed as well as the involvement of BCRP in multidrug resistance in acute leukemia . In AML and ALL, several studies showed that BCRP is expressed and functionally active at low, but variable levels . However, further studies are warranted to investigate its effect on clinical outcome, and explore whether patients could benefit from the combination of BCRP inhibitors and chemotherapy.

Int J Pharm, 2004 Jun 11, 277(1-2), 155 - 62
Combined cancer therapy by micellar-encapsulated drug and ultrasound; Rapoport N; A new modality of drug targeting to tumors that is under development in our lab is based on the drug encapsulation in polymeric micelles followed by a localized release at the tumor site triggered by focused ultrasound . The rationale behind this approach is that drug encapsulation in micelles decreases systemic concentration of drug and provides for a passive drug targeting to tumors via the enhanced penetration and retention (EPR) effect, thus, reducing unwanted drug interactions with healthy tissues . In addition, polymeric micelles sensitize multidrug resistant (MDR) cells to the action of drugs . Upon the accumulation of drug-loaded micelles at the tumor site, ultrasonic irradiation of the tumor is used to provide for the effective intracellular drug uptake . Ultrasound releases drug from micelles and enhances the intracellular uptake of both released and encapsulated drug . An important advantage of ultrasound is that it is noninvasive, can penetrate deep into the interior of the body, can be focused and carefully controlled . The results of the in vitro application of this technique for delivering anthracyclin drugs to ovarian carcinoma A2780 drug-sensitive and MDR cells are described.

Trends Cell Biol, 1996 May, 6(5), 174 - 8
Vaults are the answer, what is the question?
Kickhoefer VA, Vasu SK, Rome LH.
Vaults are large cytoplasmic ribonucleoprotein (RNP) particles of eukaryotic cells, whose considerable abundance and striking evolutionary conservation argue for an important general cellular function . Early studies on vaults focused on the structural features and cellular distribution of the particle and will only be summarized briefly here . In this article, we discuss the molecular characterization of vault components and describe genetic studies carried out in Dictyostelium . The recent finding that the major vault protein is elevated in non-P-glycoprotein multidrug resistant cancer cells has direct implications concerning the function of the vault particle and indicates a potential role for vaults in resistance of tumour cells to anticancer drugs.

Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2004 Apr, 12(2), 159 - 62
{Study on the relationship between {Ca2+}i and the MDR formation in K562/A02 cells}; Chen BA et al.; To explore the relationship of multidrug resistance formation in K562/A02 cells with the intracellular concentration of {Ca(2+)}i, the cytotoxicities of daunorubicin (DNR) were assayed by MTT method, the variations of {Ca(2+)}i of K562 cells and K562/A02 cells after treatment of Tet, DRL and DNR alone or in combination were detected by using Fura-2/AM . The results showed as follows: (1) The cytotoxicities of DNR to cell line K562/A02 were enhanced by 1 micro mol/L Tet or 5 micro mol/L DRL . Their IC(50) was (7.28 +/- 2.06) micro g/ml and (7.58 +/- 3.44) micro g/ml; multiple of their reversal effect was 2.94 and 2.82, but IC(50) of combined Tet and DRL was (1.66 +/- 0.41) micro g/ml . Its reverse effect distinctly increased by 12.9 times . (2) The {Ca(2+)}i in K562/A02 cells were higher than that in K562 cells . (3) One micro mol/L Tet and 5 micro mol/L DRL alone increased the {Ca(2+)}i in K562/A02 cells time-dependently and there was antagonism when both were used . It is concluded that high {Ca(2+)}i is supposed to be a reason of MDR in K562/A02 cells, the action of resistance modifying agents (RMA) in MDR reverse course, however, needs further research.

Am J Trop Med Hyg, 2004 May, 70(5), 461 - 6
Polymorphism of the Plasmodium falciparum multidrug resistance and chloroquine resistance transporter genes and in vitro susceptibility to aminoquinolines in isolates from the Peruvian Amazon; Huaman MC et al.; In vitro drug sensitivity to chloroquine (CQ), mefloquine (MQ) and quinine was investigated in 60 culture-adapted Plasmodium falciparum isolates from malaria patients in Padrecocha, a village in the Amazonian Department of Loreto, Peru . All isolates showed resistance to CQ, decreased susceptibility to quinine, and sensitivity to MQ . These isolates were examined for mutations in the P . falciparum multidrug resistance 1 (pfmdr1) and chloroquine resistance transporter (pfcrt) genes previously linked to CQ resistance . The mutations N86Y and D1246Y, two of the five mutations commonly observed in the pfmdr1 gene of CQ-resistant clones, were not found . The pfcrt mutation K76T, associated with CQ resistance, was identified in all the isolates tested . Sequence analysis of codons 72-76 in the pfcrt gene showed the haplotypes SVMNT and CVMNT.

Mol Pharmacol, 2004 Jun, 65(6), 1536 - 42
Function of the ABC signature sequences in the human multidrug resistance protein 1; Ren XQ et al.; Human multidrug resistance protein 1 (MRP1) is a membrane ATP-binding cassette transporter that confers multidrug resistance to tumor cells by effluxing intracellular drugs in an ATP-dependent manner . The mechanisms by which transport occurs and by which ATP hydrolysis is coupled to drug transport are not fully elucidated . In particular, the function of the signature sequences in the nucleotide binding domains (NBDs) of MRP1 is unknown . We therefore investigated the effect of mutation of the signature sequences (G771D and G1433D) and of the Walker A motifs (K684M and K1333M) in the NBDs on the 8-azido-{alpha-32P}ATP photolabeling and 8-azido-{alpha-32P}ADP vanadate trapping of MRP1 . Both mutations in the Walker A motif almost completely inhibited the labeling of the mutated NBD with 8-azido-{alpha-32P}ATP but not the labeling of the other intact NBD . In contrast, the G771D mutation in the signature sequence of NBD1 enhanced the labeling of NBD1 but slightly decreased the labeling of NBD2 . The G1433D mutation in the signature motif of NBD2 enhanced the labeling of NBD2 but did not affect the labeling of NBD1 . These effects were all substrate-independent . Photolabeling of NBD2 and a very slight photolableing of NBD1 were detectable under vanadate trapping conditions with 8-azido-{alpha-32P}ATP . Trapping at both NBD1 and NBD2 was almost completely inhibited by K684M and K1333M mutations and by the K684M/K1333M double mutation . The G771D mutation completely inhibited trapping at NBD2 and considerably inhibited trapping at NBD1 . However, whereas the G1433D mutation also considerably inhibited trapping at NBD1, it only partially inhibited trapping of NBD2, and the trapping could still be enhanced by leukotriene C4 . Our findings suggest that both signature sequences of MRP1 are involved in ATP hydrolysis and must be intact for the ATP hydrolysis and the transport by MRP1.

Mol Pharmacol, 2004 Jun, 65(6), 1485 - 95
High-affinity interaction of tyrosine kinase inhibitors with the ABCG2 multidrug transporter; Ozvegy-Laczka C et al.; Tyrosine kinase inhibitors (TKIs) are promising new agents for specific inhibition of malignant cell growth and metastasis formation . Because most of the TKIs have to reach an intracellular target, specific membrane transporters may significantly modulate their effectiveness . In addition, the hydrophobic TKIs may interact with so-called multidrug transporters and thus alter the cellular distribution of unrelated pharmacological agents . In the present work, we show that certain TKIs, already in the clinical phase of drug development, directly interact with the ABCG2 multidrug transporter protein with a high affinity . We found that in several in vitro assay systems, STI-571 (Gleevec; imatinib mesylate), ZD1839 (Iressa; gefitinib), and N-{4-{(3-bromophenyl)amino}-6-quinazolinyl}-2-butynamide (EKI-785) interacted with ABCG2 at submicromolar concentrations, whereas other multidrug transporters, human multidrug resistance protein (P-glycoprotein, ABCB1) and human multidrug resistance protein 1 (ABCC1), showed much lower reactivity toward these agents . Low concentrations of the TKIs examined selectively modulated ABCG2-ATPase activity, inhibited ABCG2-dependent active drug extrusion, and significantly affected drug resistance patterns in cells expressing ABCG2 . Our results indicate that multidrug resistance protein modulation by TKIs may be an important factor in the clinical treatment of cancer patients . These data also raise the possibility that an extrusion of TKIs by multidrug transporters, e.g., ABCG2, may be involved in tumor cell TKI resistance.

Mol Pharmacol, 2004 Jun, 65(6), 1375 - 85
Mutations of charged amino acids in or near the transmembrane helices of the second membrane spanning domain differentially affect the substrate specificity and transport activity of the multidrug resistance protein MRP1 (ABCC1); Haimeur A et al.; Multidrug resistance protein 1 (MRP1) belongs to the ATP-binding cassette superfamily of transport proteins . In addition to drugs, MRP1 mediates the active transport of many conjugated and unconjugated organic anions . MRP1 consists of two membrane-spanning domains (MSD2 and MSD3) each followed by a nucleotide binding domain plus a third NH2-terminal MSD1 . MSD2 contains transmembrane (TM) helices 6 through 11, and previously, we identified two charged residues in TM6 as having important but markedly different roles in MRP1 transport activity and substrate specificity by characterizing mutants containing nonconservative substitutions of Lys332 and Asp336 . We have now extended these studies and found that the same-charge TM6 mutant K332R, like the nonconservatively substituted Lys332 mutants, exhibits a selective decrease in leukotriene C4 (LTC4) transport, associated with substantial changes in both Km and Vmax and LTC4 binding . The overall organic anion transport activity of the same-charge mutant of Asp336 (D336E) also remained very low, as observed for D336R . In addition, nonconservative substitutions of TM6-associated Lys319 and Lys347 resulted in a selective decrease in GSH transport . Of eight other charged residues in or proximal to TM7 to TM11 that were investigated, nonconservative substitutions of three of them {Lys396 (TM7), Asp436 (TM8), and Arg593 (TM11)} caused a substantial and global reduction in transport activity . However, unlike TM6 Asp336, wild-type transport activity could be reestablished in these MRP1 mutants by conservative substitutions . We conclude that MSD2-charged residues in or proximal to TM6, TM7, TM8, and TM11 play critical but differential roles in MRP1 transport activity and substrate specificity.

Anticancer Res, 2004 Mar-Apr, 24(2C), 1097 - 104
Expression and prognostic value of the lung resistance-related protein (LRP) in germ cell testicular tumors; Mandoky L et al.; BACKGROUND: Lung resistance-related protein (LRP) was first detected in a non-P-glycoprotein-mediated multidrug-resistant lung cancer cell line and has been shown to be the major human vault protein . The aim of this study was to evaluate the expression of LRP in germ cell testicular tumors (GCT) and to determine the correlation between LRP expression and the clinical outcome of these tumors . PATIENTS AND METHODS: Seventy cases of primary testicular tumors were investigated . LRP protein was detected by immunohistochemistry and Western blotting methods . LRP mRNA was determined with RT-PCR . Patients' clinical parameters and tumor response to treatment were recorded . RESULTS: With immunohistochemistry, LRP was detected in 29 (41%) out of 70 primary testicular tumors . Twenty-two (63%) out of 35 tumors expressed LRP mRNA and LRP protein on examination by RT-PCR and Western blotting, respectively . Pure teratomas showed significantly higher LRP expression compared to other types of GCTs (p=0.0418) . No relationship was demonstrated between the LRP immunostaining and stage of disease (p=0.2263) . A significantly higher proportion of patients with LRP-negative tumors achieved complete response than those with LRP-positive tumors (p=0.0155) . Patients whose tumors showed expression of LRP had significantly shorter overall survival (p=0.0428) than LRP-negative patients . CONCLUSION: Immunohistochemistry is a reliable method to evaluate LRP expression in testicular germ cell tumors . A positive correlation was found between LRP immunostaining and pure teratomas . LRP expression was associated with an adverse clinical outcome and shorter overall survival . Our findings suggest that LRP has prognostic value in testicular germ cell tumors and can predict clinical outcome.

Anticancer Res, 2004 Mar-Apr, 24(2A), 449 - 55
Functional studies on the MRP1 multidrug transporter: characterization of ABC-signature mutant variants; Szentpetery Z et al.; BACKGROUND: MRP1 is a key multidrug resistance ATP-binding Cassette (ABC) transporter in tumor cells . A functionally important signature motif is conserved within all ABC domains . Our current studies aimed to elucidate the role of these motifs in the cooperation of MRP1 ABC domains . MATERIALS AND METHODS: We designed human MRP1 mutants based on a bacterial ABC structure . Conserved leucines (Leu) were replaced by arginines (Arg), while glycines (Gly) were substituted for aspartic acids (Asp) . The activity of these mutants was assayed by measuring ATPase activity and vesicular transport . ATP-binding and transition-state formation were studied by a photoreactive ATP analog . RESULTS: The Leu to Arg mutants retained both ATPase and transport activity, while the Gly to Asp mutants were inactive in all functional assays, while showing normal ATP-binding . CONCLUSION: Our results reinforce the notion that a single mutation in one of the ABC-signature regions affects the function of the whole protein . The relative role of the conservative leucines and glycines in MRP1 indicates a similar three-dimensional structure within the catalytic center of various ABC proteins.

Anticancer Res, 2004 Mar-Apr, 24(2A), 409 - 15
Structure-activity analysis of taxane-based broad-spectrum multidrug resistance modulators; Brooks TA et al.; BACKGROUND: Clinical drug resistance is frequently associated with overexpression of the multidrug resistance (MDR) proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP-1) and breast cancer resistance protein (BCRP) . Taxanes are substrates for Pgp and MRP-1, but not BCRP . Taxane-based reversal agents (tRAs) are non-cytotoxic MDR modulators previously examined for broad-spectrum modulation of Pgp, MRP-1 and BCRP . MATERIALS AND METHODS: Modulation by tRAs was studied by flow cytometry and resistance to taxanes was studied in cytotoxicity assays in the parental HL60/wt, 8226/wt and MCF7/S, and the resistant HL60/ADR, 8226/Dox6, 8226/MR20 and MCF7 AdVp3000 cell lines . Amino acid sequence (BLAST) alignments were performed using ClustalW . RESULTS: Structure-activity analysis demonstrated greatest alignment of BCRP with the transmembrane 7-12 region of Pgp and identified tRA side groups that contributed or were detrimental to modulation . CONCLUSION: Identification of tRA side groups contributing to modulation of Pgp, MRP-1 and BCRP will allow the design of a next generation of tRAs and will optimize their potential clinical applicability.

Exp Cell Res, 2004 Jun 10, 296(2), 337 - 46
Ribosomal proteins S13 and L23 promote multidrug resistance in gastric cancer cells by suppressing drug-induced apoptosis; Shi Y et al.; Ribosomal proteins (RP) S13 and RPL23 were previously identified as two upregulated genes in a multidrug-resistant gastric cancer cell line SGC7901/VCR compared to its parental cell SGC7901 by differential display PCR . The aim of this study was to explore the roles of RPS13 and RPL23 in multidrug resistance (MDR) in gastric cancer cells . RPS13 and RPL23 were genetically overexpressed in SGC7901 cells, respectively . Either RPS13 or RPL23 enhanced resistance of SGC7901 cells to vincristine, adriamycin, and 5-fludrouracil . RPL23 also enhanced resistance of SGC7901 cells to cisplatin . Overexpression of either RPS13 or RPL23 did not alter the population doubling time, {3H}leucine incorporation, and intracellular adriamycin accumulation of SGC7901 cells . However, either RPS13 or RPL23 could protect SGC7901 cells from undergoing vincristine-induced apoptosis . Western blot analysis revealed that both RPS13 and RPL23 significantly increased the expression level of Bcl-2 and Bcl-2/Bax ratio in SGC7901 cells . In addition, overexpression of RPL23 enhanced glutathione S-transferase (GST) activity and intracellular glutathione content in SGC7901 cells . Together, this work demonstrates that either RPS13 or RPL23 can promote MDR in gastric cancer cells by suppressing drug-induced apoptosis, and that RPL23 may also promote MDR in gastric cancer cells through regulation of glutathione S-transferase-mediated drug-detoxifying system.

Int J Cancer, 2004 Jul 10, 110(5), 627 - 32
Characterization of salvicine-resistant lung adenocarcinoma A549/SAL cell line; Miao ZH et al.; Salvicine is a diterpenoid quinone derived from a traditional Chinese medication that has been shown to possess potent in vitro and in vivo antitumor effects . This compound, which inhibits the activity of Topoisomerase II, was found to equipotently kill various multidrug-resistant tumor cells and their corresponding parental counterparts in vitro and to inhibit mdr1/P-gp expression in multidrug-resistant K562/A02 cells . To examine the features of tumor resistance to salvicine, we established a salvicine-resistant tumor cell subline of A549 lung adenocarcinoma cells . Compared with parental cells, A549/SAL cells displayed 8.91-fold resistance to salvicine and an average of 6.70-fold resistance to the antimetabolites . A549/SAL cells, however, were not resistant to alkylating agents, platinum compounds and other naturally-derived antineoplastics . RT-PCR analysis showed that the expression of mRNAs from the mdr-1, MRP, PCNA, topoisomerase II alpha and beta, GSTpi, p21 and GADD45 genes was not altered in the salvicine-resistant subline . In contrast, expression of p53 and bax mRNA was significantly lower, and expression of mdm2 mRNA was significantly higher, in A549/SAL cells compared to A549 cells . A549/SAL cells grew more slowly, and in a more scattered pattern, than A549 cells . In addition, the A549/SAL cells showed enhanced ability to migrate and invade in comparison to the parental cells . These results indicate that exposure to salvicine does not induce a tumor multidrug-resistant phenotype .

Gene, 2004 May 12, 332, 51 - 9
Analysis of gene network regulating yeast multidrug resistance by artificial activation of transcription factors: involvement of Pdr3 in salt tolerance; Onda M et al.; We established a strategy to constitutively activate Zn(2)Cys(6)-type protein by fusing its DNA-binding domain with the VP16 trans-activation domain . To explore gene network regulating yeast multidrug resistance, the strategy was applied to Pdr1, Pdr3 and Yrr1, known to regulate multidrug resistance, as well as three uncharacterized Yrr1-related transcription factors . DNA microarray analysis revealed that all of the six mutants induce typical drug transporter genes including SNQ2 and YOR1, suggesting redundancy in regulation . On the other hand, each displays a unique spectrum of targets, which is coincident with the phylogenetic tree of the transcription factors and presumably reflects their functional specification . Indeed, careful analysis of target genes specific to each transcription factor led us to reveal an unexpected role for Pdr3 in salt tolerance . The strategy would thus contribute not only to identify target genes but to reveal redundancy and specificity in complex gene regulatory networks.

Zhonghua Wai Ke Za Zhi, 2004 Apr 7, 42(7), 424 - 7
{Overcoming multi-drug resistance using anti-MDR1 ribozymes}; Wang H et al.; OBJECTIVE: To reverse multidrug resistance (MDR) of HepG2 by anti-MDR1 hammerhead ribozyme . METHODS: We developed an anti-MDR1 hammerhead ribozyme and delivered it to P-gp-overproducing human hepatocarcinoma cell line HepG2 by a retroviral vector containing RNA polymerase III promoter . We detected the expression of MDR1/Pgp and Rz in HepG2, HepG2 multidrug-resistant cell line and HepG2 Rz-tranduced cells by real-time RT-PCR, semi-quantitative RT-PCR and western blot methods . Moreover, MTT assay was tested to detect sensitivity of these ribozyme-tranduced cells, and Rhodamine123 (Rh123) applied to test the function of Pgp . RESULTS: The Rz-tranduced HepG2 cells became doxorubicin-sensitive, concomitant with the decreases in MDR1 expression, P-gp amounts and efflux pump function . CONCLUSIONS: The approaches using either retrovirus or liposome-mediated transfer of anti-MDR1 ribozyme may be selectively applicable to the treatment of MDR cells.

Tuberk Toraks, 2003, 51(4), 410 - 5
{The cost of treatment in new case and multidrug resistant case in pulmonary tuberculosis}; Kizkin O et al.; The treatment of multidrug-resistant pulmonary tuberculosis (MDR-Tbc) is quite difficult, and the disease has high morbidity and mortality rates . This study was designed to compare the costs of treatment in new tuberculosis (new-Tbc) cases and MDR-Tbc cases . Data base of the study was composed of the data from therapy principles of new-Tbc cases and MDR-Tbc, and official directives and price lists of Turkish Pharmocology Society in 2001 fiscal year regulating treatment costs . For new-Tbc cases, the treatment cost included expanses for 20 days of hospitalisation, one month work loss and six months drug supply and laboratory costs; for MDR-Tbc cases, it was comprised by expenses for seven months hospitalisation in average, 12 months work loss, 24 months drug supply and laboratory costs, and probable surgical interventions and post-operative intensive care . The service of hospital stuff and medical equipments provided was disregarded . The cost analyses was calculated as charge price of American Dollars ($) dated 14.09.2001 . It was found that the cost of therapy for new-Tbc cases and MDR-Tbc cases were 1134.89 $ and 17529.15 $, respectively . In MDR-Tbc cases, the costs of hospitalisation, work loss, drug therapy and laboratory procedures were 10.5, 12, 98.7 and 5.3 times higher respectively, when compared with those of new-Tbc . The cost of thoracotomy for one patient including the cost for 10 days period of post-operative care in intensive care unit was 391.93 $ . The treatment of MDR-Tbc has a high cost, and 16 new-Tbc cases can be treated with the same cost in our country . In conclusion, we think that successful treatment strategies for both new-Tbc cases and MDR-Tbc cases will lower the cost of tuberculosis treatment.

Br J Haematol, 2004 May, 125(4), 477 - 9
A sensitive model for prediction of relapse in adult acute myeloid leukaemia with t(8;21) using white blood cell count, CD56 and MDR1 gene expression at diagnosis; Schaich M et al.; Acute myeloid leukaemia (AML) carrying t(8;21) has an overall favourable prognosis . However, relapse occurs and the impact of multidrug resistance gene (MDR1) expression on recurring disease in this group of patients is not known . We determined quantifiable MDR1 expression in the bone marrow of 28 AML patients with t(8;21) by a validated real-time polymerase chain reaction assay . Using MDR1 expression, white blood cell count and CD56 expression at diagnosis we observed complete concordance of predicted and observed relapses . A calculated logit out of these three variables was a strong independent prognostic factor for overall (P = 0.007) and disease-free survival (P = 0.002).

Int J Tuberc Lung Dis, 2004 Apr, 8(4), 465 - 72
Drug resistance trends among previously treated tuberculosis patients in a national registry in Peru, 1994-2001; Vasquez-Campos L et al.; SETTING: National mycobacteriology reference laboratory in Peru conducting routine testing of susceptibility to isoniazid, rifampin, ethambutol, pyrazinamide, and streptomycin, in Mycobacterium tuberculosis isolates from previously treated patients . OBJECTIVE: To determine the percentage of isolates resistant to each of five anti-tuberculosis agents and to ascertain in these data the presence of trends of clinical relevance . DESIGN: Retrospective study of a national registry of M . tuberculosis isolates from patients referred for drug susceptibility testing between 1994 and 2001 . RESULTS: Among 14,736 isolates tested, 10,837 (73.5%, 95%CI 72.8-74.3) demonstrated anti-tuberculosis resistance, and 8455 (57.4%, 95%CI 56.6-58.2) demonstrated resistance to at least both isoniazid and rifampin, by convention defined as multidrug-resistant tuberculosis (MDR-TB) . A significant increasing trend could be discerned for resistance to each of the drugs tested and in isolates classified as MDR-TB (P < 0.001 for trend) . Additional clinically relevant trends were found in polyresistance and multidrug resistance percentages . CONCLUSIONS: Data from a national reference laboratory can be used to inform the design of retreatment regimens.

Mol Cancer Ther, 2004 May, 3(5), 633 - 9
Overexpression of glucosylceramide synthase and P-glycoprotein in cancer cells selected for resistance to natural product chemotherapy; Gouaze V et al.; Resistance to natural product chemotherapy drugs is a major obstacle to successful cancer treatment . This type of resistance is often acquired in response to drug exposure; however, the mechanisms of this adverse reaction are complex and elusive . Here, we have studied acquired resistance to Adriamycin, Vinca alkaloids, and etoposide in MCF-7 breast cancer cells, KB-3-1 epidermoid carcinoma cells, and other cancer cell lines to determine if there is an association between expression of glucosylceramide synthase, the enzyme catalyzing ceramide glycosylation to glucosylceramide, and the multidrug-resistant (MDR) phenotype . This work shows that glucosylceramide levels increase concomitantly with increased drug resistance in the KB-3-1 vinblastine-resistant sublines KB-V.01, KB-V.1, and KB-V1 (listed in order of increasing MDR) . The levels of glucosylceramide synthase mRNA, glucosylceramide synthase protein, and P-glycoprotein (P-gp) also increased in parallel . Increased glucosylceramide levels were also present in Adriamycin-resistant KB-3-1 sublines KB-A.05 and KB-A1 . In breast cancer, detailed analysis of MCF-7 wild-type and MCF-7-AdrR cells (Adriamycin-resistant) demonstrated enhanced glucosylceramide synthase message and protein, P-gp message and protein, and high levels of glucosylceramide in resistant cells . Similar results were seen in vincristine-resistant leukemia, etoposide-resistant melanoma, and Adriamycin-resistant colon cancer cell lines . Cell-free glucosylceramide synthase activity was higher in lysates obtained from drug-resistant cells . Lastly, glucosylceramide synthase promoter activity was 15-fold higher in MCF-7-AdrR compared with MCF-7 cells . We conclude that selection pressure for resistance to natural product chemotherapy drugs selects for enhanced ceramide metabolism through glucosylceramide synthase in addition to enhanced P-gp expression . A possible connection between glucosylceramide synthase and P-gp in drug resistance biology is suggested.

Biochem J, 2004 Sep 1, 382(Pt 2), 711 - 6
Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding; Alqawi O et al.; ABCG2 {also known as BCRP (breast cancer resistance protein) or MXR} is an ABC (ATP-binding cassette) protein shown to confer multidrug resistance . ABCG2 was initially identified in resistant breast carcinoma cells (MCF-7/AdrVp1000) selected with doxorubicin and verapamil . Later studies demonstrated the presence of a point mutation (Arg482 to Thr) in ABCG2 in MCF-7/AdrVp1000 cells . This mutation was shown to modulate the transport of Rh123 (rhodamine 123) . In the present study, we have used a previously characterized photoreactive drug analogue of Rh123, IAARh123 (iodoaryl-azido-Rh123), to examine the effects of the Arg482Thr mutation on Rh123 binding and transport by ABCG2 . Our results show that both wild-type (ABCG2R482) and mutant (ABCG2T482) ABCG2 bound directly to IAARh123 . Surprisingly, however, wild-type ABCG2R482, which does not transport Rh123, was more intensely photolabelled than mutant ABCG2T482 . In addition, inhibition of IAARh123 photolabelling using various drug substrates of ABCG2 revealed some differences between wild-type and mutant ABCG2 . For example, a molar excess of mitoxantrone was more effective at inhibiting IAARh123 labelling of wild-type than of mutant ABCG2, while excess cisplatin, taxol and methotrexate showed significant inhibition of IAARh123 binding to both wild-type and mutant ABCG2 . Taken together, the results of this study provide the first demonstration of the direct binding of drugs to ABCG2.

Pharm Res, 2004 Apr, 21(4), 635 - 40
D-glucose triggers multidrug resistance-associated protein (MRP)-mediated secretion of fluorescein across rat jejunum in vitro; Legen I et al.; PURPOSE: To examine the transport characteristics of the multidrug resistance-associated protein (MRP) substrate fluorescein across the isolated rat small intestinal segments . METHODS: The transport of fluorescein was studied in side-by-side diffusion chambers under short-circuited conditions at physiological pH . RESULTS: The serosal-to-mucosal permeability of fluorescein significantly exceeded the permeability in the opposite direction in the jejunum, but not in the ileum . This asymmetry in transport in the jejunum was observed only when D-glucose was present at the mucosal side of the tissue, and not in the presence of D-galactose or D-mannitol . In the presence of D-glucose at the mucosal side, serosal-to-mucosal permeability of fluorescein in the jejunum can be divided into an active (Michaelis-Menten constant, KM = 1.07 mM; maximum flux of the substrate . Jmax = 14.0 nmol/h x cm2) and a passive component (passive permeability, Ppas = 2.51 x 10(-6) cm/s) . The polarization of fluorescein transport was almost completely abolished by MRP inhibitor, benzbromarone (50 or 100 microM, applied apically), and by MRP/P-glycoprotein inhibitor, verapamil (200 microM, applied apically) . CONCLUSIONS: D-glucose at the mucosal side activates fluorescein secretion across rat jejunum by an apical MRP, most probably by isoform 2 (MRP2), which could have an impact on the intestinal absorption of MRP substrates.

Pharm Res, 2004 Apr, 21(4), 625 - 34
Contribution of cholesterol and phospholipids to inhibitory effect of dimethyl-beta-cyclodextrin on efflux function of P-glycoprotein and multidrug resistance-associated protein 2 in vinblastine-resistant Caco-2 cell monolayers; Arima H et al.; PURPOSE: The purpose of this study is to reveal the contribution of membrane components to the inhibitory effect of 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CyD) on P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) function in vinblastine-resistant Caco-2 (Caco-2R) cell monolayers . METHODS: The transport of rhodamine-123 and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was studied in Caco-2R cell monolayers . P-gp and MRP2 residing in the monolayers and releasing in cell supernatants were detected by Western blotting . The mRNA levels of MDR1 and MRP2 were detected by reverse transcription-polymerase chain reaction (RT-PCR) method . Cholesterol, phospholipids, and proteins were mainly determined by each assay kit . RESULTS: Of various beta-cyclodextrin derivatives (beta-CyDs), DM-beta-CyD most significantly impaired the efflux function of P-gp and MRP2 without changing cell viability and membrane integrity . The treatment with CyDs did not change the mRNA levels of MDR1 and MRP2 . DM-beta-CyD lowered cholesterol content and P-gp level in caveolar membranes . In addition, DM-beta-CyD released not only cholesterol and phospholipids but also proteins including P-gp and MRP2 from apical membranes of the monolayers . CONCLUSIONS: DM-beta-CyD may impair P-gp and MRP2 function in Caco-2R cell monolayers, probably, at least in part, through the release of these transporters from the apical membranes of monolayers, and the exertion of the inhibitory effect of DM-beta-CyD may require the extraction of not only cholesterol but also phospholipids from the monolayers.

Int J Tuberc Lung Dis, 2004 Mar, 8(3), 361 - 8
Self-administered, standardized regimens for multidrug-resistant tuberculosis in South Korea; Park SK et al.; SETTING: National Masan Tuberculosis Hospital, Masan, South Korea, a 430-bed tertiary referral hospital specializing in tuberculosis . OBJECTIVE: To evaluate the treatment outcomes of standardized, empiric regimens for multidrug-resistant tuberculosis (MDR-TB) . DESIGN: A retrospective analysis of the hospital records of 142 patients with MDR-TB who had failed short-course chemotherapy . Between 1 January 1998 and 30 June 2000, patients were started on one of two standardized, empiric regimens based on previous treatment history . Drug susceptibility testing of the infecting strain was not used to modify the treatment regimen . Treatment was continued for at least 18 months after conversion to a negative culture . RESULTS: Sixty-three patients (44.1%) were cured and discharged from treatment after at least 18 months of negative cultures; 18 (12.7%) failed treatment, 41 (28.9%) defaulted, four died (2.8%), and 15 (10.6%) were transferred to another institution . One patient is still on treatment . Resistance to ofloxacin was the only risk factor related to poor outcome (death or failure) in univariate or multiple logistic regression analysis . CONCLUSIONS: High levels of resistance to second-line drugs are likely a cause of poor outcome of MDR-TB therapy in Korea . Directly observed therapy and other methods to increase patient compliance should be considered nationwide, as they may improve MDR-TB treatment outcomes.

Int J Tuberc Lung Dis, 2004 Mar, 8(3), 352 - 60
Identification of MDR-TB Beijing/W and other Mycobacterium tuberculosis genotypes in Nairobi, Kenya; Githui WA et al.; SETTING: Suspected tuberculosis (TB) patients in Nairobi, Kenya . OBJECTIVE: To identify the presence of multidrug-resistant (MDR) Beijing/W type and other genotypes of Mycobacterium tuberculosis . METHODS: Thirty-three isolates resistant to one or more drugs (resistance ratio method), including 15 MDR isolates and 40 susceptible isolates selected at random, were analysed by dot-blot hybridisation for mutations associated with resistance to isoniazid, rifampicin, streptomycin and ethambutol . All strains were genotypically classified using spoligotyping . RESULTS: Of the 33 drug-resistant isolates, 21 (64%) were from males and 12 (36%) were from females . Mutations associated with resistance to isoniazid (katG 315) and rifampicin (rpoB526, 531) were confirmed in 83.3% and 100% of the isolates, respectively, and in 87% of the MDR isolates . Mutations were detected in 25% and 71.5% of the isolates resistant to streptomycin (rpsL43) and ethambutol (embB306), respectively . No mutations were detected in drug-susceptible isolates . Spoligotyping grouped the isolates into 25 groups . Ten of these groups corresponded to previously identified strain groups, including seven families in the international database . One of these families (CAS1) comprised six (40%) of the 15 MDR isolates . Another family (Beijing) had six (8.3%) isolates, of which two (33.3%) were MDR (Beijing/W) . CONCLUSION: This study is the first in Kenya and the second in sub-Saharan Africa to report the presence of MDR Beijing/W type and other possible drug-resistant outbreak strains . Application of the molecular techniques and markers will allow us to monitor the spread of existing drug-resistant strains and the appearance of new ones.

Oncol Rep, 2004 Jun, 11(6), 1257 - 63
Differentially expressed genes in multidrug resistant variants of U-2 OS human osteosarcoma cells; Chano T et al.; Multidrug resistance (MDR) to anticancer agents is a major barrier to the successful treatment of human osteosarcomas . Current understanding of the genes that contribute to the features of MDR is limited, and the mechanisms remain unclear . Here we applied differential display reverse transcription-polymerase chain reaction (DDRT-PCR) to parental and MDR-variants of U-2 OS human osteosarcoma cells, to clarify the genes involved in the MDR cells, and identified five candidate genes . These are BCRP (breast cancer resistance protein) encoding a transmembrane efflux pump; RB1CC1 (RB1-inducible coiled-coil 1), a tumor suppressor regulating RB1 (retinoblastoma 1) expression; a novel transcriptional variant of dUTPase; SSR2 (beta-signal sequence receptor), which is associated with protein translocation across ER membrane; and HSP105 encoding high molecular mass heat shock proteins . Molecular and biological characterization of these genes will yield further insight into the features between MDR and tumor progression in human osteosarcomas.

Hepatobiliary Pancreat Dis Int, 2004 May, 3(2), 307 - 10
Detection of multidrug resistant gene 1 in pancreatic cancer; Zhao YP et al.; BACKGROUND: The results are conflicting in detecting P-glycoprotein (P-gp) in pancreatic cancer . The aim of this study was to detect the expression of multidrug resistant gene 1 (MDR1) in pancreatic cancer cell lines . METHODS: MDR1 mRNA and P-gp were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical assay (IHC) in three pancreatic cancer cell lines SW1990, CAPAN-1 and P3 . P-gp functions were evaluated through the rhodamine extrusion test . RESULTS: Two of the three cell lines expressed MDR1 positively at different levels . The rhodamine extrusion test showed that the percentage of positive cells in MDR(+) cells was significantly lower than that in MDR1(-) cells . The results of IHC, RT-PCR and the rhodamine extrusion test were consistent with each other . CONCLUSION: All of these methods are reliable in the detection of MDR1 in pancreatic cancer tissue, thus providing a guide for clinical chemotherapy of pancreatic cancer.

Hepatobiliary Pancreat Dis Int, 2004 May, 3(2), 296 - 9
Clinical relationship between MDR1 gene and gallbladder cancer; Wang BL et al.; BACKGROUND: The most common mechanisms of multidrug resistance (MDR) in cancer cells is the expression of an energy-dependent exfflux pump . P-glycoprotein (P-gp) encoded by MDR1 gene and multidrug associated protein (MRP) are well known proteins associated with MDR . In human cancers, the MDR1 gene expression is common in patients with intrinsic and acquired MDR . It is a major therapeutic problem in cancer chemotherapy . Previously we found that the MDR of HCC is related to MRP gene expression and initiates the intrinsic MDR . The aim of this study is to study the expression of MDR1 gene encoding P-gp and MDRl mRNA in primary gallbladder carcinoma, and analyze its clinical significance . METHODS: Immunohistochemistry (IHC) S-P method and in situ polymerase chain reaction (ISPCR) were used to detect the expression of P-gp and MDR1 mRNA in 53 cases of untreated primary gallbladder carcinoma and 12 cases of cholecystitis (archival paraffin-embedded tissues) . RESULTS: The positive expression rates of P-gp and MDR1 mRNA in the 53 cases and 12 cases were 60.38%, 71.69% and 25.00%, 33.33%, respectively . There was a significant difference between the two groups (P<0.05).The positive expression rate of P-gp and MDR1mRNA were 69.44%, 83.33% and 41.18%, 47.06% respectively in tissues in stage of Nevin I-III against Nevin IV, V (P<0.05) . In well, moderately differentiated gallbladder carcinoma tissues, their expressions were 79.49%, 69.23% against 50.00%, 35.71% in low, undifferentiated tissues (P<0.05) . CONCLUSIONS: MDR to gallbladder carcinoma is closely related to the intrinsic MDR and it provides an important evidence to reverse the MDR by detection of the MDR1 gene . Meanwhile, MDR1 gene expression in gallbladder carcinoma is correlated with some biological characteristics, takes part in the carcinogenesis of gallbladder tissues, and acts as a valuable biomarker of prognosis.

Int J Tuberc Lung Dis, 2004 May, 8(5), 574 - 8
Surveillance of drug-resistant childhood tuberculosis in Bangui, Central African Republic; Kassa-Kelembho E et al.; SETTING: Bangui, the capital of the Central African Republic, where overall drug resistance and multidrug resistance among adult new tuberculosis (TB) cases were respectively 16.4% and 1.1% in 1998 . OBJECTIVE: To determine the prevalence of drug resistance among children with tuberculosis and to compare the epidemiological and clinical features of TB in children with drug-resistant and drug-susceptible TB . METHODS: All strains of Mycobacterium tuberculosis obtained from children aged 0-15 years at Bangui Paediatric Hospital were prospectively collected from April 1998 to June 2000, and susceptibility testing was performed for each specimen . The children's epidemiological and clinical data were recorded . RESULTS: Susceptibility results were available for 165/190 children with M . tuberculosis . Overall drug resistance and multidrug resistance were 15.2% and 0.6%, respectively . Isoniazid and streptomycin were the only drugs associated with TB monoresistance . No significant difference was found in the epidemiological or clinical data of children infected with a resistant strain and those infected with a susceptible strain . CONCLUSION: The prevalence of drug resistance in childhood is similar to that observed in adult new TB cases in the same period . Surveillance will continue to be performed in Bangui periodically to assess the trend of true drug resistance among new TB cases.

Int J Tuberc Lung Dis, 2004 May, 8(5), 560 - 7
Results of a standardised regimen for multidrug-resistant tuberculosis in Bangladesh; Van Deun A et al.; SETTING: Individualised regimens based on drug susceptibility test results, generally used to treat multidrug-resistant tuberculosis (MDR-TB), require often unavailable expertise and resources . OBJECTIVE: To evaluate a standardised regimen based on the susceptibility profiles of locally prevalent MDR-TB strains . DESIGN: The activities of a successful DOTS programme in Bangladesh were complemented by offering treatment with a standardised 21-month regimen to patients with laboratory-confirmed MDR-TB disease . The regimen contained kanamycin, ofloxacin, prothionamide, pyrazinamide, ethambutol, isoniazid and clofazimine . Clinical and bacteriological progress was monitored quarterly until treatment completion, then 6 monthly for 2 years . RESULTS: The status at the end of treatment of this cohort of 58 documented MDR-TB patients was as follows: eight (14%) deaths, seven (12%) defaults, three (5%) failures and 40 (69%) cures . One bacteriologically-confirmed relapse was recognised . Frequent and sometimes serious side effects proved to be the main problem, suggesting the need for a better tolerated but equally effective regimen . CONCLUSION: A standardised approach may provide a reasonable alternative to individualised treatment of MDR-TB in resource-poor settings . However, DOTS-plus programmes in resource-poor settings may confront significant difficulties in the enrolment, diagnosis and management of MDR-TB patients.

Hepatol Res, 2004 May, 29(1), 60 - 66
Modulation of organic anion transporting polypeptide 1 and multidrug resistance protein 3 expression in the liver and kidney of Gunn rats; Higuchi K et al.; Background: Gunn rat is an animal model of Crigler-Najjar syndrome (CNS) type I that develops jaundice due to defect of bilirubin conjugation . Bilirubin UDP-glucuronosyltransferase (UGT1A1), which plays a critical role in bilirubin glucuronidation, has been reported to be deficient in CNS type 1 . On the other hand, little is known about the expression of organic anion transporters in Gunn rats . In the present study, we evaluated expressions of organic anion transporting polypeptide (oatp) 1 and 2, multidrug resistance-associated protein (mrp) 2 and mrp3 in the liver and kidney of Gunn rats . Methods: Serum samples, liver and kidney tissues were obtained from Gunn rats and normal SD rats ( {Formula: see text}, in each group) . Semi-quantitative mRNA expression of oatp1, oatp2, mrp2, and mrp3 mRNA was evaluated by constructed reverse transcription-polymerase chain reaction (RT-PCR) . Protein expressions were determined by Western blotting and by immunohistochemistry . Results: Marked elevation of serum unconjugated bilirubin concentration ( {Formula: see text} micromol/l) was observed in Gunn rats . Hepatic expression of oatp1 and oatp2 mRNA was 44% ( {Formula: see text} ) and 35% ( {Formula: see text} ) lower in Gunn rats than in SD rats, respectively . Hepatic oatp1 protein expression was 37% ( {Formula: see text} ) lower in Gunn rats than in SD rats . In contrast to oatp1, hepatic expression of mrp3 mRNA and protein was 76% ( {Formula: see text} ) and 557% ( {Formula: see text} ) higher in Gunn rats than in SD rats, respectively . Hepatic expression of oatp2 and mrp2 protein was not significantly different between Gunn rats and SD rats . Like the Western blot analysis, immunohistochemical staining disclosed decrease of oatp1 and increase of mrp3 protein expressions in the liver of Gunn rats . Decrease of oatp1 and increase of mrp3 expressions were also observed in the kidney of Gunn rats . Conclusion: Decreased expression of oatp1 and increased expression of mrp3 were observed in the liver and kidney of Gunn rats . Deficient UGT1A1 activity-associated retention of unconjugated bilirubin in the hepatocytes may modulate the expressions of these transporters in Gunn rats.

Curr Pharm Des, 2004, 10(13), 1493 - 503
PET Studies on P-glycoprotein function in the blood-brain barrier: how it affects uptake and binding of drugs within the CNS; Elsinga PH et al.; Permeability of the blood-brain barrier (BBB) is one of the factors determining the bioavailability of therapeutic drugs . The BBB only allows entry of lipophilic compounds with low molecular weights by passive diffusion . However, many lipophilic drugs show negligible brain uptake . They are substrates for transporters such as P-glycoprotein (P-gp), multidrug-resistance associated protein (MRP) and organic anion transporting polypeptides (OATPs) . The action of these carrier systems results in rapid efflux of xenobiotics from the central nervous system (CNS) . Classification of candidate drugs as substrates or inhibitors of such carrier proteins is of crucial importance in drug development . Positron emission tomography (PET) can play an important role in the screening process by providing in vivo information, after the putative drug has passed in vitro tests . Although radiolabeled probes for MRP and OATP function are not yet available, many radiotracers have been prepared to study P-glycoprotein function in vivo with PET . These include alkaloids ((11)C-colchicine), antineoplastic agents ((11)C-daunorubicin, (18)F-paclitaxel), modulators of L-type calcium channels ((11)C-(+/-)verapamil, (11)C-R(+)-verapamil), beta-adrenoceptor antagonists ((11)C-(S)-carazolol, (18)F-(S)-1'-fluorocarazolol, (11)C-carvedilol), serotonin 5-HT(1A) receptor antagonists ((18)F-MPPF), opioid receptor antagonists ((11)C-loperamide, (11)C-carfentanyl), and various (64)Cu-labeled copper complexes . Studies in experimental animals have indicated that it is possible to assess P-glycoprotein function in the BBB and its effect on the uptake and binding of drugs within the intact CNS, using suitable P-gp modulators labeled with positron emitters . Provided that radiopharmaceuticals (and P-gp modulators) can be developed for human use, several exciting fields of study may be explored, viz . (i) direct evaluation of the effect of modulators on the cerebral uptake of therapeutic drugs; (ii) assessment of mechanisms underlying drug resistance in epilepsy; (iii) examination of the role of the BBB in the pathophysiology of neurodegenerative and affective disorders; and (iv) exploration of the relationship between polymorphisms of transporter genes and the pharmacokinetics of test compounds within the CNS.

Curr Med Chem, 2004 May, 11(10), 1265 - 84
Recent advances in artemisinin and its derivatives as antimalarial and antitumor agents; Jung M et al.; Artemisinin, the first and last naturally occurring 1, 2, 4-trioxane originated from Artemisia annua, L . and its derivatives are a potent class of antimalarial drugs . The clinical efficacy of these drugs is characterized by an almost immediate onset and rapid reduction of parasitemia, and it is high in such areas as well where multidrug-resistance is rampant . Furthermore, artemisinin and many of its analog possess not only antiparasitic effect against Plasmodium falciparum, Schistosoma japonicum and Clonorchis sinensi but also immuno-modulation effects, and antitumor activities . This review covers the chemistry of artemisinin including synthesis of acetal-, non acetal-type C-12 analogs, C-11- and C-13 derivatives from artemisitene, ring-contracted derivatives, dimers, and trimers . Modes of biological action of artemisinin - derived analogs are also reviewed . The main objective of this article is to review the literatures of recent progress taken place in chemistry, mode of biological actions of artemisinin, and its derivatives as antimalarial and antitumor agents during the last three years (1999-2001).

Curr Pharm Des, 2004, 10(12), 1295 - 312
ABC transporters and the blood-brain barrier; Begley DJ; The blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) form a very effective barrier to the free diffusion of many polar solutes into the brain . Many metabolites that are polar have their brain entry facilitated by specific inwardly-directed transport mechanisms . In general the more lipid soluble a molecule or drug is, the more readily it will tend to partition into brain tissue . However, a very significant number of lipid soluble molecules, among them many useful therapeutic drugs have lower brain permeability than would be predicted from a determination of their lipid solubility . These molecules are substrates for the ABC efflux transporters which are present in the BBB and BCSB and the activity of these transporters very efficiently removes the drug from the CNS, thus limiting brain uptake . P-glycoprotein (Pgp) was the first of these ABC transporters to be described, followed by the multidrug resistance-associated proteins (MRP) and more recently breast cancer resistance protein (BCRP) . All are expressed in the BBB and BCSFB and combine to reduce the brain penetration of many drugs . This phenomenon of "multidrug resistance" is a major hurdle when it comes to the delivery of therapeutics to the brain, not to mention the problem of cancer chemotherapy in general . Therefore, the development of strategies for bypassing the influence of these ABC transporters and for the design of effective drugs that are not substrates and the development of inhibitors for the ABC transporters becomes a high imperative for the pharmaceutical industry.

Biol Chem, 2004 Mar-Apr, 385(3-4), 331 - 9
Enhanced expression of basolateral multidrug resistance protein isoforms Mrp3 and Mrp5 in rat liver by LPS; Donner MG et al.; Lipopolysaccharide (LPS) induces hepatocellular down-regulation and endocytic retrieval of multidrug resistance protein 2 (Mrp2, Abcc2) . Basolateral Mrp isoforms may compensate for the intracellular metabolic changes in cholestasis . Therefore, the effect of LPS on the zonal localization of Mrp2 and Mrp3 and the expression of Mrp3, Mrp4, Mrp5, and Mrp6 mRNA were investigated in rat liver . In normal rat liver Mrp3 was found in pericentral hepatocytes also expressing glutamine synthetase . In LPS-treated rat liver the decrease in Mrp2 protein was most pronounced in pericentral hepatocytes, with only minor down-regulation in periportal hepatocytes . Conversely, induction of Mrp3 was found in pericentral hepatocytes with a low expression of Mrp2 . Furthermore, we found a strong induction of Mrp5 mRNA . Likewise, Mrp6 mRNA was up-regulated, however Mrp6 protein expression was not significantly altered . It is concluded that Mrp3 is inversely regulated to Mrp2 in a zonal pattern and may compensate for the LPS-induced loss of Mrp2 in the perivenous area . Induction of pericentral Mrp3 and up-regulation of Mrp5 mRNA may play an important role in the hepatocellular clearance of cholephilic substances and cyclic nucleotides accumulating after LPS treatment.

Curr Drug Targets, 2004 May, 5(4), 389 - 406
Molecular targeting of drug delivery systems to cancer; Minko T et al.; This review presents molecular targeting approaches in anticancer drug delivery systems (DDS) and identifies new developments in these systems . Targeting approaches include passive targeting (enhanced permeability and retention effect), targeting specific tumor conditions, topical delivery and active targeting, namely, targeting organs, cells, intracellular organelles and molecules, sandwich targeting, promoter targeting, indirect targeting and targeting by external stimuli . A novel advanced proapoptotic anticancer DDS that utilizes several molecular targets will be considered . Experimental data suggest that this DDS can simultaneously: (1) induce cell death, (2) prevent adverse effects on healthy tissues; (3) suppress and prevent multidrug resistance; and (4) inhibit cellular antiapoptotic defense.

Curr Drug Targets, 2004 May, 5(4), 375 - 82
Sphingolipid metabolism enzymes as targets for anticancer therapy; Kok JW et al.; Treatment with anti-cancer agents in most cases ultimately results in apoptotic cell death of the target tumor cells . Unfortunately, tumor cells can develop multidrug resistance, e.g., by a reduced propensity to engage in apoptosis by which they become insensitive to multiple chemotherapeutics . Ceramide . the central molecule in cellular sphingolipid metabolism, has been recognized as an important mediator of apoptosis . Moreover, an increased cellular capacity for ceramide glycosylation has been identified as a novel multidrug resistance mechanism . Indeed, virtually all multidrug resistant cell types exhibit a deviating sphingolipid composition, most typically an increased level of glucosylceramide . Thus, the enzyme glucosylceramide synthase, which converts ceramide into glucosylceramide, has emerged as a potential target to increase apoptosis and decrease drug resistance of tumor cells . In addition, several other steps in the pathways of sphingolipid metabolism arc altered in multidrug resistant cells, opening a perspective on additional sphingolipid metabolism enzymes as targets for anti-cancer therapy . In this article, we present an overview of the current understanding concerning drug resistance-related changes in sphingolipid metabolism and how interference with this metabolism can be exploited to over come multidrug resistance.

Curr Drug Targets, 2004 May, 5(4), 333 - 46
Intractable cancers: the many faces of multidrug resistance and the many targets it presents for therapeutic attack; Stein WD et al.; Some types of cancer respond far less favorably to treatment than do others . A quantitative estimate of this intuition can be obtained from the SEER (Surveillance, Epidemiology and End-Results) Cancer Statistics Review . Of particular interest, from a drug resistance perspective, are the five-year survival data for patients presenting with tumors that were diagnosed as "distant" . A good correlation can be found between those numbers and an estimate of treatment successes obtained from a survey of current literature on chemotherapy applied to cancers originating from these various tissues . These two measures, considered together, define "the axis of intractability", a parameter that characterizes the (possibly) inherent, physiological basis of the tissue-by-tissue intractability of cancers . Exploring the basis of this intractability, it appears that factors other than the classical ABC transporter-based, multidrug resistance systems probably play a major role . An ineffective DNA repair system, coupled to reduced apoptosis, is the basis for the inherent tractability of testicular cancer . For other tissues, important contributions to resistance arise from cell adhesion-mediated drug resistance, which is overcome when cells are released from tissues during anoikis . Making a direct comparison between gene expression in solid tumors and their corresponding cell lines, genes controlling the extracellular matrix and cell-cell communication appear among the genes that are over-expressed in the solid tumors, while genes coding for the protein biosynthesis system are over-expressed in the cell lines . The more tractable cancers are closer to the cell lines in their expression profiles of these sets of genes.

Mol Biol Cell, 2004 Jul, 15(7), 3393 - 405 Epub 2004 May 07.
Arabidopsis immunophilin-like TWD1 functionally interacts with vacuolar ABC transporters; Geisler M et al.; Previously, the immunophilin-like protein TWD1 from Arabidopsis has been demonstrated to interact with the ABC transporters AtPGP1 and its closest homologue, AtPGP19 . Physiological and biochemical investigation of pgp1/pgp19 and of twd1 plants suggested a regulatory role of TWD1 on AtPGP1/AtPGP19 transport activities . To further understand the dramatic pleiotropic phenotype that is caused by loss-of-function mutation of the TWD1 gene, we were interested in other TWD1 interacting proteins . AtMRP1, a multidrug resistance-associated (MRP/ABCC)-like ABC transporter, has been isolated in a yeast two-hybrid screen . We demonstrate molecular interaction between TWD1 and ABC transporters AtMRP1 and its closest homologue, AtMRP2 . Unlike AtPGP1, AtMRP1 binds to the C-terminal tetratricopeptide repeat domain of TWD1, which is well known to mediate protein-protein interactions . Domain mapping proved that TWD1 binds to a motif of AtMRP1 that resembles calmodulin-binding motifs; and calmodulin binding to the C-terminus of MRP1 was verified . By membrane fractionation and GFP-tagging, we localized AtMRP1 to the central vacuolar membrane and the TWD1-AtMRP1 complex was verified in vivo by coimmunoprecipitation . We were able to demonstrate that TWD1 binds to isolated vacuoles and has a significant impact on the uptake of metolachlor-GS and estradiol-beta-glucuronide, well-known substrates of vacuolar transporters AtMRP1 and AtMRP2.

Cell Death Differ, 2004 Sep, 11(9), 1028 - 37
Mutational analysis of P-glycoprotein: suppression of caspase activation in the absence of ATP-dependent drug efflux; Tainton KM et al.; P-glycoprotein (P-gp) can induce multidrug resistance (MDR) through the ATP-dependent efflux of chemotherapeutic agents . We have previously shown that P-gp can inhibit nondrug apoptotic stimuli by suppressing the activation of caspases . To determine if this additional activity is functionally linked to ATP hydrolysis, we expressed wild-type and ATPase-mutant P-gp and showed that cells expressing mutant P-gp could not efflux chemotherapeutic drugs but remained relatively resistant to apoptosis . CEM lymphoma cells expressing mutant P-gp treated with vincristine showed a decrease in the fraction of cells with apoptotic morphology, cytochrome c release from the mitochondria and suppression of caspase activation, yet still accumulated in mitosis and showed a loss of clonogenic potential . The loss of clonogenicity in vincristine-treated cells expressing mutant P-gp was associated with accumulation of cells in mitosis and the presence of multinucleated cells consistent with mitotic catastrophe . The antiapoptotic effect of mutant P-gp was not affected by antibodies that inhibit the efflux function of the protein . These data are consistent with a dual activity model for P-gp-induced MDR involving both ATPase-dependent drug efflux and ATPase-independent inhibition of apoptosis . The structure-function analyses described herein provide novel insight into the mechanisms of action of P-gp in mediating MDR.

Pediatr Infect Dis J, 2004 May, 23(5), 390 - 8
Potential role of fluoroquinolone therapy in childhood otitis media; Dagan R et al.; Increased resistance of pneumococci and other pathogens to available antibiotics raises concerns about bacteriologic and clinical failure in children with acute otitis media . Few therapeutic options exist for patients with recurrent infections or recent treatment failure . The limited experience from the compassionate use of fluoroquinolones in pediatrics and pediatric studies has not been linked unequivocally with arthrotoxicity, the primary safety concern in children . Newer 8-methoxyfluoroquinolones may have a role in selected cases associated with multidrug-resistant pathogens.

J Pharmacol Exp Ther, 2004 Aug, 310(2), 800 - 7 Epub 2004 May 06.
Nonsteroidal anti-inflammatory drugs potentiate 1-methyl-4-phenylpyridinium (MPP+)-induced cell death by promoting the intracellular accumulation of MPP+ in PC12 cells; Morioka N et al.; In this study, we investigated the effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on 1-methyl-4-phenylpyridinium (MPP(+))-induced cell death in PC12 cells . Coincubation of PC12 cells with indomethacin, ibuprofen, ketoprofen, or diclofenac, but not aspirin or N-{2-(cyclohexyloxy)-4-nitrophenyl}methanosulfonamide (NS-398), significantly potentiated the MPP(+)-induced cell death . In contrast, these NSAIDs had no effect on rotenone-induced cell death . The potentiating actions of these NSAIDs were not suppressed by treatment with phenyl-N-butyl-nitrone, a radical scavenger; N-acetyl-l-cysteine, an antioxidant; Ac-DEVD-CHO, a selective caspase-3 inhibitor; or 2-chloro-5-nitro-N-phenylbenzamide (GW9662), a selective antagonist of peroxisome proliferator-activated receptor gamma . Furthermore, we observed that DNA fragmentation, which is one of the hallmarks of apoptosis, was not induced by coincubation with MPP(+) and NSAIDs . We confirmed that coincubation of PC12 cells with 30 microM MPP(+) and 100 microM indomethacin, ibuprofen, ketoprofen, or diclofenac led to a significant increase in the accumulation of intracellular MPP(+) compared with incubation with 30 microM MPP(+) alone . In addition, these NSAIDs markedly reduced the efflux of MPP(+) from PC12 cells . (3-(3-(2-(7-Chloro-2-quinolinyl) ethenyl) phenyl ((3-dimethyl amino-3oxo-propyl) thio) methyl) propanoic acid (MK 571), which is an inhibitor of multidrug resistance proteins (MRPs), mimicked the NSAIDs-induced effects, increasing cell toxicity and promoting the accumulation of MPP(+) . Moreover, some types of MRPs' mRNA were detected in PC12 cells . These results suggest that some NSAIDs might cause a significant increase in the intracellular accumulation of MPP(+) via the suppression of reverse transport by the blockade of MRP, resulting in the potentiation of MPP(+)-induced cell death.

Clin Cancer Res, 2004 May 1, 10(9), 3179 - 88
Arsenic trioxide-induced death of neuroblastoma cells involves activation of Bax and does not require p53; Karlsson J et al.; PURPOSE: On the basis of clinical studies showing that arsenic trioxide (As(2)O(3)), via an apoptotic mechanism, and with minimal toxicity induces complete remission in patients with refractory acute promyelocytic leukemia and that multidrug-resistant and p53-mutated neuroblastoma cells are sensitive to As(2)O(3) both in vitro and in vivo, we searched for molecular mechanisms involved in the As(2)O(3)-induced neuroblastoma cell death . EXPERIMENTAL DESIGN: We have studied the effect of As(2)O(3) on the expression and cellular localization of proteins involved in drug-induced death in two neuroblastoma cell lines with intact p53 and two with mutated p53, the latter two displaying multidrug resistance . RESULTS: As(2)O(3) provoked Bax expression in all tested neuroblastoma cell lines, including SK-N-BE(2) cells with mutated p53 and LA-N-1 cells, which have a deleted p53 . In all cell lines exposed to As(2)O(3), p21 Bax was proteolytically cleaved in a calpain-dependent way into the more proapoptotic p18 Bax, which was detected exclusively in a mitochondria-enriched subcellular fraction . As(2)O(3) also caused an increase of cytoplasmic cytochrome c, translocation of antiapoptosis-inducing factor to the nuclei, and a slight activation of caspase 3 . However, inhibition of caspase 3 did not prevent cell death, whereas inhibition of Bax cleavage was associated with a decreased As(2)O(3)-induced cell death . CONCLUSIONS: We show that multidrug-resistant neuroblastoma cells die after exposure to As(2)O(3), independent of functional p53, suggesting activation of a cytotoxic pathway different from that induced by conventional chemotherapeutic agents . We further propose that proteolytic activation of Bax is an important event in As(2)O(3)-induced cell death.

Clin Cancer Res, 2004 May 1, 10(9), 3156 - 68
Decreased nucleotide excision repair activity and alterations of topoisomerase IIalpha are associated with the in vivo resistance of a P388 leukemia subline to F11782, a novel catalytic inhibitor of topoisomerases I and II; Kruczynski A et al.; PURPOSE: The purpose of the study was to investigate the mechanisms associated with antitumor activity and resistance to F11782, a novel dual catalytic inhibitor of topoisomerases with DNA repair-inhibitory properties . EXPERIMENTAL DESIGN: For that purpose, an F11782-resistant P388 leukemia subline (P388/F11782) has been developed in vivo and characterized . RESULTS: Weekly subtherapeutic doses of F11782 (10 mg/kg) induced complete resistance to F11782 after 8 weekly passages . This resistant P388/F11782 subline retained some in vivo sensitivity to several DNA-topoisomerase II and/or I complex-stabilizing poisons and showed marked collateral sensitivity to cisplatin, topotecan, colchicine, and Vinca alkaloids, while proving completely cross-resistant only to merbarone and doxorubicin . Therefore, resistance to F11782 did not appear to be associated with a classic multidrug resistance profile, as further reflected by unaltered drug uptake and no overexpression of resistance-related proteins or modification of the glutathione-mediated detoxification process . In vivo resistance to F11782 was, however, associated with a marked reduction in topoisomerase IIalpha protein (87%) and mRNA (50%) levels, as well as a diminution of the catalytic activity of topoisomerase IIalpha . In contrast, only minor reductions in topoisomerases IIbeta and I levels were recorded . However, of major interest, nucleotide excision repair activity was decreased 3-fold in these P388/F11782 cells and was more specifically associated with a decreased (67%) level of XPG (human xeroderma pigmentosum group G complementing protein), an endonuclease involved in this DNA repair system . CONCLUSIONS: These findings suggest that both topoisomerase IIalpha and XPG are major targets of F11782 in vivo and further demonstrate the original mechanism of action of this novel compound.

Blood, 2004 Aug 15, 104(4), 1174 - 82 Epub 2004 May 06.
Regulation of monocyte migration by amphoterin (HMGB1); Rouhiainen A et al.; Amphoterin (HMGB1) is a 30-kD heparin-binding protein involved in process extension and migration of cells by a mechanism involving the receptor for advanced glycation end products (RAGE) . High levels of amphoterin are released to serum during septic shock . We have studied the expression of amphoterin in monocytes and the role of amphoterin and RAGE in monocyte transendothelial migration . Un-activated monocytes in suspension did not reveal amphoterin on their surface, but adherent monocytes exported amphoterin to the cell surface . Immunohistochemical staining of arterial thrombi in vivo revealed amphoterin in mononuclear cells and in surrounding extracellular matrix . Amphoterin was secreted from phorbol ester and interferon-gamma (IFN-gamma)-activated macrophages, and the secretion was inhibited by blocking the adenosine 5'-triphosphate (ATP)-binding cassette transporter-1, a member of the multidrug resistance protein family . Amphoterin was specifically adhesive for monocytes in peripheral blood leukocyte adhesion assay . Adhesion caused an extensive spreading of cells, which was inhibited by the dominant-negative RAGE receptor (soluble ectodomain of RAGE), and adhesion up-regulated chromogranin expression in monocytes, also suggesting a RAGE-dependent interaction . Monocyte transendothelial migration was efficiently inhibited by anti-amphoterin and anti-RAGE antibodies and by the soluble RAGE . We suggest that amphoterin is an autocrine/paracrine regulator of monocyte invasion through the endothelium.

Am J Respir Crit Care Med, 2004 Aug 1, 170(3), 288 - 95 Epub 2004 May 06.
T cell-based tracking of multidrug resistant tuberculosis infection after brief exposure; Richeldi L et al.; Molecular epidemiology indicates significant transmission of Mycobacterium tuberculosis after casual contact with infectious tuberculosis cases . We investigated M . tuberculosis transmission after brief exposure using a T cell-based assay, the enzyme-linked-immunospot (ELISPOT) for IFN-gamma . After childbirth, a mother was diagnosed with sputum smear-positive multidrug-resistant tuberculosis . Forty-one neonates and 47 adults were present during her admission on the maternity unit; 11 weeks later, all underwent tuberculin skin testing (TST) and ELISPOT . We correlated test results with markers of exposure to the index case . The participants, who were asymptomatic and predominantly had no prior tuberculosis exposure, had 6.05 hours mean exposure (range: 0-65 hours) to the index case . Seventeen individuals, including two newborns, were ELISPOT-positive, and ELISPOT results correlated significantly with three of four predefined measures of tuberculosis exposure . For each hour sharing room air with the index case, the odds of a positive ELISPOT result increased by 1.05 (95% CI: 1.02-1.09, p = 0.003) . Only four adults were TST-positive and TST results did not correlate with exposure . Thus, ELISPOT, but not TST, suggested quite extensive nosocomial transmission of multidrug-resistant M . tuberculosis after brief exposure . These results help to explain the apparent importance of casual contact for tuberculosis transmission, and may have implications for prevention.

Biochem Pharmacol, 2004 May 15, 67(10), 1933 - 46
Effect of stereo and regiochemistry towards wild and multidrug resistant HIV-1 virus: viral potency of chiral PETT derivatives; Venkatachalam TK et al.; Chiral derivatives of several substituted halopyridyl and thiazolyl PETT compounds were synthesized as non-nucleoside inhibitors of the reverse transcriptase (RT) enzyme of the human immunodeficiency virus (HIV-1) . Molecular modeling studies indicated that because of the asymmetric geometry of the non-nucleoside inhibitors (NNRTI) binding pocket, the "R" stereoisomers would fit the NNRTI binding pocket of the HIV-1 RT much better than the corresponding "S" stereoisomers, as reflected by their 10(4)-fold lower K(i) values . The "R" stereoisomers of several PETT derivatives inhibited the recombinant RT in vitro with lower IC(50) values than their enantiomers . The active compounds were further evaluated for their ability to inhibit HIV-1 replication in human peripheral blood mononuclear cells (PBMCs) . All the "R" isomers again showed potent anti-HIV activity and inhibited the replication of the HIV-1 strains HTLV(IIIB) in PBMCs at nanomolar concentrations whereas their enantiomers were less potent . The lead compounds for the respective groups were further tested against A17 (NNRTI-resistant, Y181C mutant RT), and A17Var (NNI-resistant Y181C +/- K103N mutant RT) as well as multidrug resistant viral strains . The results indicated that the lead compounds were several logs more potent than the standard NNRTI drug nevirapine . Structure-activity relationship among the derivatives showed preference of pyridyl unit with halo substitutions primarily at 5-position demonstrating the importance of both the stereochemistry as well as regiochemistry . Our data provides experimental evidence that the stereochemistry and the regiochemistry of non-nucleoside inhibitors can profoundly affect their anti-HIV activity.

Toxicol In Vitro, 2004 Aug, 18(4), 403 - 9
Effect of chlorpyrifos on efflux transporter gene expression and function in Caco-2 cells; Agarwala S et al.; The effect of chlorpyrifos (CPF) and its metabolite, chlorpyrifos-oxon (CPO), on multidrug resistance-1 (MDR1) gene expression and efflux transporter function in Caco-2 cells was determined . The effect of CPF and CPO on gene expression in Caco-2 cells was tested as a function of time using RT-PCR and competitive PCR (compPCR) techniques . The RT-PCR results depicted a maximal effect of CPF exposure on MDR1 expression at 8 h, which decreased at 24 h . Studies with CPO displayed an initial increase in expression at 4 h only . The compPCR assays were conducted with the CPF-treated group to quantify the changes in gene expression levels . The compPCR data confirmed and quantitated the results from the time-course study using semiquantitative RT-PCR . In addition to the gene expression studies, changes in efflux transporter function were investigated using Caco-2 cells grown on semipermeable membranes in Transwell plates . The permeability of verapamil was determined in cells treated for 8 h with CPF . Efflux ratios demonstrated that verapamil was effluxed at a higher rate from the CPF-treated cells as compared to the control group, confirming the inductive action of CPF on transporter function . These results suggest that CPF has the potential to modulate the bioavailability of drugs via changes in expression and function of membrane efflux transporters.

Zhonghua Yi Xue Za Zhi, 2004 Apr 17, 84(8), 663 - 6
{Effects of hypoxia on expression of P-glycoprotein and multidrug resistance protein in human lung adenocarcinoma A549 cell line}; Xia S et al.; OBJECTIVE: To study the effects of hypoxia on expression of P-gp and multidrug resistance protein in human lung adenocarcinoma A549 cell line, and to explore the probable mechanism of hypoxia in tumor cell of MDR . METHODS: Culturing human lung adenocarcinoma A549 cell in hypoxia (2%O2) for 24 h, the expression of hypoxia inducible factor-1alpha, P-gp and multidrug resistance protein was detected by using immunohistochemistry, and after action of adriamycin or cisplatin in hypoxia (2%O2) for 24, the cell survival rate was detected by MTT . RESULTS: The expression of hypoxia inducible factor-1alpha, P-gp and multidrug resistance protein in hypoxia were higher than the expression in anoxia, and correlation between the expression of HIF-1alpha and P-gp or multidrug resistance protein was observed (P < 0.05) . The resistance of adriamycin of A549 cell was enhanced in hypoxia . CONCLUSION: The resistance of tumor chemotherapy is enhanced in hypoxia . The expression of HIF-1alpha is obviously correlated with the expression of P-gp and MRP in A549 cell.

Neuroreport, 2004 May 19, 15(7), 1183 - 6
Multidrug resistance protein 1-mediated transport of saquinavir by microglia; Dallas S et al.; Regulation of CNS distribution of the human immunodeficiency virus (HIV) protease inhibitor saquinavir may involve ATP-dependent membrane-bound efflux transport proteins that are expressed in several brain cellular compartments . We recently characterized molecular and functional expression of one such transporter, multidrug resistance protein-1 (MRP1) in microglia, the primary brain cellular target of HIV . In the present study, we further examine subcellular localization of MRP1 in a microglia cell line (MLS-9) using immunogold cytochemistry and directly demonstrate MRP1-mediated export of saquinavir . MRP1 localized primarily to the plasma membrane of the MLS-9 cells . {14C}Saquinavir efflux by MLS-9 monolayers was inhibited by well-established MRP1 inhibitors . These results indicate that MRP1 contributes, in part, to the overall low permeation of protease inhibitors in the brain .

Cancer Res, 2004 May 1, 64(9), 3296 - 301
Expression, up-regulation, and transport activity of the multidrug-resistance protein Abcg2 at the mouse blood-brain barrier; Cisternino S et al.; The breast cancer resistance protein (BCRP/ABCG2) is, like P-glycoprotein (P-gp), a member of the ABC family of drug transporters . These proteins actively transport various anticancer drugs from cells, causing multidrug resistance . The physiological expression of P-gp/ABCB1 at the blood-brain barrier (BBB) effectively restricts the brain uptake of many antitumor drugs by mediating their active efflux from the brain to the blood vessel lumen . However, little is known about the function of Abcg2 at the BBB in vivo . We used in situ brain perfusion to measure the uptake of two known Abcg2 substrates, prazosin and mitoxantrone, and the nonsubstrate vinblastine by the brains of wild-type and P-gp-deficient mutant mdr1a(-/-) mice with or without the P-gp/Abcg2 inhibitor GF120918 or the P-gp inhibitor PSC833 . P-gp had no effect on the brain transport of prazosin and mitoxantrone at the mouse BBB, but wild-type and P-gp-deficient mouse brains perfused with GF120918 or a high concentration of prazosin showed carrier-mediated effluxes of prazosin and mitoxantrone from the brain that did not involve P-gp . In contrast, the brain uptake of vinblastine was restricted only by P-gp and not by Abcg2 at the BBB . The amounts of abcg2 mRNA in cortex homogenates and capillary-enriched fractions of wild-type and mdr1a(-/-) mouse brains were measured by real-time quantitative reverse transcription-PCR . There was approximately 700-times more abcg2 mRNA in brain microvessels than in the cortex of the wild-type mice, confirming that Abcg2 plays an important role at the BBB . There was also approximately 3 times more abcg2 mRNA in the microvessels from P-gp-deficient mutant mouse brains than in the microvessels of wild-type mouse brains . These findings confirm that Abcg2 is a physiological transporter at the BBB that restricts the permeability of the brain to its substrates in vivo . Lastly, the defective P-gp in the mutant mdr1a(-/-) mice was associated with increased abcg2 mRNA at the BBB and a greater export of prazosin and mitoxantrone from the brain, as measured in the P-gp-deficient mice versus the wild-type mice.

Ann Intern Med, 2004 May 4, 140(9), 709 - 13
Early diagnosis of subclinical multidrug-resistant tuberculosis; Richeldi L et al.; BACKGROUND: Tuberculosis control hinges on prompt diagnosis of active cases and screening of contacts by tuberculin skin testing . Rapid blood tests for Mycobacterium tuberculosis infection are a new alternative to the tuberculin skin test, but whether they improve clinical outcomes is unknown . OBJECTIVE: To describe how a novel T-cell-based test for M . tuberculosis infection helped diagnose tuberculosis in an asymptomatic, immunosuppressed adult with a negative result on a tuberculin skin test . DESIGN: Case report . SETTING: Household contact . PATIENTS: Asymptomatic man receiving maintenance azathioprine therapy for Crohn disease whose wife had multidrug-resistant pulmonary tuberculosis . MEASUREMENTS: Enzyme-linked immunospot (ELISPOT) assay, computed tomography, and bronchoalveolar lavage cultures . RESULTS: The man had a negative tuberculin skin test result and a positive ELISPOT assay result . High-resolution computed tomography of the chest showed consolidation with early cavitation . Bronchoalveolar lavage and culture confirmed multidrug-resistant tuberculosis . LIMITATIONS: This single case report is a proof of concept and is not a formal evaluation of clinical utility . CONCLUSIONS: A positive ELISPOT assay result helped diagnose subclinical active tuberculosis in an immunosuppressed patient with a false-negative tuberculin skin test result . Large prospective studies that compare benefits and costs of this alternative to tuberculin skin testing are needed.

BMC Med . 2004 May 4;2(1):16.
Membrane transport of camptothecin: facilitation by human P-glycoprotein (ABCB1) and multidrug resistance protein 2 (ABCC2); Lalloo AK et al.; BACKGROUND: The purpose of the present study was to continue the investigation of the membrane transport mechanisms of 20-(S)-camptothecin (CPT) in order to understand the possible role of membrane transporters on its oral bioavailability and disposition . METHODS: The intestinal transport kinetics of CPT were characterized using Caco-2 cells, MDCKII wild-type cells and MDCKII cells transfected with human P-glycoprotein (PGP) (ABCB1) or human multidrug resistance protein 2 (MRP2) (ABCC2) . The effects of drug concentration, inhibitors and temperature on CPT directional permeability were determined . RESULTS: The absorptive (apical to basolateral) and secretory (basolateral to apical) permeabilities of CPT were found to be saturable . Reduced secretory CPT permeabilities with decreasing temperatures suggests the involvement of an active, transporter-mediated secretory pathway . In the presence of etoposide, the CPT secretory permeability decreased 25.6% . However, inhibition was greater in the presence of PGP and of the breast cancer resistant protein inhibitor, GF120918 (52.5%) . The involvement of additional secretory transporters was suggested since the basolateral to apical permeability of CPT was not further reduced in the presence of increasing concentrations of GF120918 . To investigate the involvement of specific apically-located secretory membrane transporters, CPT transport studies were conducted using MDCKII/PGP cells and MDCKII/MRP2 cells . CPT carrier-mediated permeability was approximately twofold greater in MDCKII/PGP cells and MDCKII/MRP2 cells than in MDCKII/wild-type cells, while the apparent Km values were comparable in all three cell lines . The efflux ratio of CPT in MDCKII/PGP in the presence of 0.2 microM GF120918 was not completely reversed (3.36 to 1.49) . However, the decrease in the efflux ratio of CPT in MDCKII/MRP2 cells (2.31 to 1.03) suggests that CPT efflux was completely inhibited by MK571, a potent inhibitor of the Multidrug Resistance Protein transporter family . CONCLUSIONS: The current results provide evidence that PGP and MRP2 mediate the secretory transport of CPT in vitro . However, the involvement of other transporters cannot be ruled out based on these studies . Since these transporters are expressed in the intestine, liver and kidney variations in their expression levels and/or regulation may be responsible for the erratic oral absorption and biliary excretion of CPT observed in human subjects.

Ann Hematol, 2004, 83 Suppl 1, S121 - 3
Evolution of BFM trials for childhood ALL; Schrappe M; Up to 80% of pediatric patients with acute lymphoblastic leukemia (ALL) can be cured if intensive therapy is applied . Severe side effects are encountered in all patients of which, however, only the minority is life-threatening . The leading cause of failure in childhood ALL is still recurrence of disease . To reduce the rate of relapses, but also to limit treatment morbidity, the ALL-BFM group has aimed to improve the risk-adaptation of therapy . The most important addition to clinical factors (e.g . age, WBC, extramedullary involvement), and biological characteristics (such as immunphenotype and cytogenetics), was the recognition of early in vivo treatment response as the strongest predictor for relapse . The determination of leukemic blasts in peripheral blood after exposure to 7 days of prednisone (PRED) and one dose of intrathecal methotrexate (prednisone response) as developed by BFM identified multidrug resistant patients: Such patients had still more than 1,000 blasts per microL at day 8 of therapy (defined as PRED poor responders, 10% of all patients) . Prognosis for these was only approximately 35% as compared to approximately 80% in patients with adequate PRED response . Patient characteristics at relapse reveal that most of them were originally comprised in "good risk" patient subgroups: e.g., in trial ALL-BFM 90, 50% of the relapses were noted in patients with c-ALL even though that group had an EFS of 82% (SE 1%) . 70% of the recurrences are found among patients with good response to PRED indicating the lack of specificity in the definition of that subgroup . Therefore, the more refined way of determining in vivo response based on the detection of minimal residual disease (MRD) at defined timepoints by identifying clone-specific T-cell receptor- (TCR) or immunglobuline (Ig) gene rearrangements appears to be able to define the patient at high risk to relapse more specifically . In the current ALL-BFM strategy, the high sensitivity of the method is utilized to apply treatment reduction in patients with fast clearance of leukemia . Persistent disease in contrast is an indication for treatment modification and intensification . Logistics and quality controls are demanding but essential for the introduction of this new technology into clinical practice.

J Biol Chem, 2004 Jul 2, 279(27), 27855 - 60 Epub 2004 Apr 27.
Complex interplay among regulators of drug resistance genes in Saccharomyces cerevisiae; Akache B et al.; The Gal4p family of yeast zinc cluster proteins comprises regulators of multidrug resistance genes . For example, Pdr1p and Pdr3p bind as homo- or heterodimers to pleiotropic drug response elements (PDREs) found in promoters of target genes . Other zinc cluster activators of multidrug resistance genes include Stb5p and Yrr1p . To better understand the interplay among these activators, we have performed native co-immunoprecipitation experiments using strains expressing tagged zinc cluster proteins from their natural chromosomal locations . Interestingly, Stb5p is found predominantly as a Pdr1p heterodimer and shows little homodimerization . No interactions of Stb5p with Pdr3p or Yrr1p could be detected in our assays . In contrast to Stb5p, Yrr1p is only detected as a homodimer . Similar results were obtained using glutathione S-transferase pull-down assays . Importantly, the purified DNA binding domains of Stb5p and Pdr1p bound to a PDRE as heterodimers in vitro . These results suggest that the DNA binding domains of Pdr1p and Stb5p are sufficient for heterodimerization . Our data demonstrate a complex interplay among these activators and suggest that Pdr1p is a master drug regulator involved in recruiting other zinc cluster proteins to fine tune the regulation of multidrug resistance genes.

Int J Cancer, 2004 Jul 1, 110(4), 511 - 22
MRP1 and glucosylceramide are coordinately over expressed and enriched in rafts during multidrug resistance acquisition in colon cancer cells; Klappe K et al.; Previously we have described a novel multidrug-resistant cell line, HT29(col), which displayed over expression of the multidrug-resistance protein 1 (MRP1) and an altered sphingolipid composition, including enhanced levels of glucosylceramide (GlcCer; Kok JW, Veldman RJ, Klappe K, Koning H, Filipeanu C, Muller M . Int J Cancer 2000;87:172-8) . In our study, long-term screening revealed that, during colchicine-induced acquisition of multidrug resistance in a new HT29(col) cell line, increases in GlcCer occurred concomitantly with upregulation of MRP1 expression . Both MRP1 and GlcCer were found enriched in Lubrol-insoluble membrane domains . The expression of MRP1 and GlcCer were tightly correlated, as indicated also by a reversal of both at the later stage of colchicine consolidation . Resistance to colchicine was determined by MRP1, while glucosylceramide synthase (GCS) did not contribute: 1) . Resistance was fully inhibited by MK571 . 2) . GCS expression and activity were not upregulated in HT29(col) cells . 3) . Inhibition of GCS did not affect MRP1-mediated efflux function or sensitivity to colchicine . Instead, overall sphingolipid metabolism was upregulated through an increased rate of ceramide biosynthesis . In conclusion, upregulation of MRP1 occurs in concert with upregulation of GlcCer during multidrug-resistance acquisition, and both are enriched in rafts . The increased GlcCer pool does not directly modulate MRP1 function and cell survival .

Epilepsy Res, 2004 Feb, 58(2-3), 85 - 91
Inhibition of multidrug transporters by verapamil or probenecid does not alter blood-brain barrier penetration of levetiracetam in rats; Potschka H et al.; Overexpression of multidrug efflux transporters such as P-glycoprotein (Pgp; ABCB1) or multidrug resistance proteins (MRPs; ABCC) in the blood-brain barrier has recently been suggested to explain, at least in part, pharmacoresistance in epilepsy, which affects about 30% of all patients with this common brain disorder . The novel antiepileptic drug (AED) levetiracetam (LEV) is an effective and well tolerated drug in many patients with otherwise AED-refractory epilepsy . One explanation for the favorable efficacy of LEV in pharmacoresistant patients would be that LEV is not a substrate for Pgp or MRPs in the BBB . In the present study, we used in vivo microdialysis in rats to study whether the concentration of LEV in the extracellular fluid of the cerebral cortex can be modulated by inhibition of Pgp or MRPs, using the Pgp inhibitor verapamil and the MRP1/2 inhibitor probenecid . Local perfusion with verapamil or probenecid via the microdialysis probe did not increase the extracellular brain concentration of LEV, which is in contrast to various other AEDs which have been studied previously by the same experimental protocol in this model . The data indicate that brain uptake of LEV is not affected by Pgp or MRP1/2 which may be an important reason for its antiepileptic efficacy in patients whose seizures are poorly controlled by other AEDs.

Cancer Chemother Pharmacol, 2004 Aug, 54(2), 131 - 8 Epub 2004 Apr 30.
In vivo imaging of hepatobiliary transport function mediated by multidrug resistance associated protein and P-glycoprotein; Hendrikse NH et al.; Multidrug resistance associated proteins (MRPs) and P-glycoprotein (P-gp) are involved in hepatobiliary transport of various compounds . Our aim was (1) to define transporter specificity of the cholescintigraphic agents 99mTc-HIDA and 99mTc-MIBI, which are used clinically for myocardial perfusion measurements; and (2) to deduce MRP and P-gp functions in vivo from hepatic 99mTc kinetics . Accumulation of radioactivity was measured in the human tumor cell lines GLC4, GLC4/ADR150x (MRP1-overexpressing/P-gp-negative) and GLC4/P-gp (P-gp-overexpressing) . Bile secretion was quantified in untreated and in glutathione-depleted control and MRP2-deficient (GY/TR-) rats . Hepatobiliary transport was measured using a gamma camera in both types of rats . 99mTc-HIDA accumulated 5.8-fold less in GLC4/ADR150x calls than in GLC4 or GLC4/P-gp cells . In GLC4/ADR150x, the cellular 99mTc-HIDA content was increased 3.4-fold by the MRP1,2 inhibitor MK571 (50 microM), while MK571 had no measurable effect in GLC4 and GLC4/P-gp cells . 99mTc-MIBI accumulated less in GLC4/P-gp and GLC4/ADR150x cells than in GLC4 cells . Bile secretion of 99mTc-HIDA was impaired in GY/TR- compared to control rats and not affected by glutathione depletion in GY/TR- rats . Hepatic secretion of 99mTc-HIDA was slower in GY/TR- (t1/2 40 min) than in control rats (t1/2 7 min) . Bile secretion of 99mTc-MIBI was similar in both rat strains and impaired by glutathione depletion in control rats only, indicating compensatory activity of additional transporter(s) in GY/TR- rats . 99mTc-HIDA is transported only by MRP1,2 only, while 99mTc-MIBI is transported by P-gp and MRP1,2 . The results indicate that hepatic P-gp and MRP1,2 function can be assessed in vivo by sequential use of both radiopharmaceuticals.

Ann Hepatol, 2004 Jan-Mar, 3(1), 11 - 7
Co-regulation of expression of phase II metabolizing enzymes and multidrug resistance-associated protein 2; Catania VA et al.; Treatment of experimental animals with prototypical enzyme inducers represents a useful tool to characterize the role of different isozymes in drug metabolism and to improve our knowledge on factors regulating their synthesis at the transcriptional level . The effect of model enzyme inducers on phase II (conjugating) enzyme families, including UDP-glucuronosyltransferase's and glutathione-S-transferase's, has been well characterized in rodent liver . More recently, the effect of inducers on the expression of canalicular multidrug resistance-associated protein 2 (Mrp2) has been focused upon . The identification of a number of conjugated drugs as Mrp2 substrates suggests that both the conjugation and transport systems act coordinately to improve drug elimination from the body . We provide evidence about circumstances resulting in the simultaneous upregulation of phase II enzymes and Mrp2 in hepatic and extrahepatic tissues, most likely involving activation of common nuclear receptors (e.g . FXR, PXR) . Additionally, we provide an analysis of examples of drug-induced toxicity leading to the simultaneous downregulation of both systems . Potential therapeutic strategies based on the modulation of expression of these systems are also briefly commented upon.

Leukemia, 2004 Jul, 18(7), 1246 - 51
Valproic acid inhibits proliferation and induces apoptosis in acute myeloid leukemia cells expressing P-gp and MRP1; Tang R et al.; The multidrug resistance (MDR) phenotype, induced by the overexpression of several ABC transporters or by antiapoptotic mechanisms, has been identified as the major cause of drug resistance in the treatment of patients with acute myeloid leukemia (AML) . In this study, we have shown that valproic acid (VPA) (a histone deacetylase inhibitor) can inhibit the proliferation of both P-glycoprotein (P-gp)- and MDR-associated protein 1 (MRP1)-positive and -negative cells . VPA also induced apoptosis of P-gp-positive cells . VPA induced apoptosis in K562 cells led to decrease in Flip (FLICE/caspase-8 inhibitory protein) expression with Flip cleavage, which could not be observed in HL60 cells . In HL60/MRP cell line, which proved to be resistant to apoptosis by VPA, we observed an abnormal expression of apoptotic regulatory proteins, overexpression of Bcl-2 and absence of Bax . Also, the Bcl-2 antagonist HA14-1 rapidly restored apoptosis in this cell line . Cotreatment with cytosine arabinoside induced very strong apoptosis in both K562/DOX and HL60/DNR cell lines . VPA also induced apoptosis in AML patient cells expressing P-gp and/or MRP1 . Our findings show VPA as an interesting drug that should be tested in clinical trials for overcoming the MDR phenotype in AML patients.

Clin Pharmacol Ther, 2004 May, 75(5), 422 - 33
CYP3A5 and MDR1 genetic polymorphisms and cyclosporine pharmacokinetics after renal transplantation; Anglicheau D et al.; BACKGROUND: The immunosuppressive drug cyclosporine (INN, ciclosporin), whose pharmacokinetic characteristics vary greatly among individuals, is a substrate for cytochrome P450 (CYP) 3A and P-glycoprotein, the product of the multidrug resistance 1 (MDR1) gene . Some of the single nucleotide polymorphisms (SNPs) in these genes are associated with deficient protein expression and reduced in vivo activity . We postulated that, in renal transplant recipients, these SNPs could be associated with interindividual variations in cyclosporine pharmacokinetics . PURPOSE: In 106 renal transplant patients, we evaluated retrospectively the effects of 4 MDR1 SNPs {T-129C, C1236T, G2677(T,A), and C3435T} and of the CYP3A5*1/*3 SNP on cyclosporine pharmacokinetic parameters and exposure indices . RESULTS: The CYP3A5*1 allele was present in 8.5% of patients . The MDR1 C1236T, G2677(T,A), and C3435T SNPs were frequent (17.9%, 18.9%, and 33%, respectively, for the variant homozygous genotype) and exhibited incomplete linkage disequilibrium . None of the cyclosporine pharmacokinetic parameters were associated with the CYP3A5 genetic polymorphism . Patients with the wild-type genotype in MDR1 C1236T SNP had slightly but significantly lower dose-adjusted peak drug concentrations (-16%) (P <.02) and dose-adjusted area under the concentration-time curve (AUC) values over the first 4 hours (-14%) (P <.05) as compared with mutated allele carriers . Haplotype analysis including MDR1 C1236T, G2677(T,A), and C3435T SNPs showed no significant association between haplotypes and cyclosporine pharmacokinetics or systemic exposure, although there was a nonsignificant trend toward higher dose-adjusted AUC values over the first 4 hours and AUC over the 12-hour administration interval for the T-T-T haplotype . CONCLUSION: The presence of the CYP3A5 SNP does not explain the high variability of cyclosporine pharmacokinetics in stable renal transplant patients . Despite the weak association found for the MDR1 C1236T SNP, MDR1 SNPs are unlikely to be useful for cyclosporine dose optimization in clinical practice.

Pharmacogenetics, 2004 May, 14(5), 309 - 18
Variable expression of P-glycoprotein in the human placenta and its association with mutations of the multidrug resistance 1 gene (MDR1, ABCB1); Hitzl M et al.; The MDR1 gene product P-glycoprotein in the human placenta is important for protecting the fetus from unintended, harmful drug exposure, but also for limiting the access of therapeutic drugs to the fetus after maternal drug intake . A polymorphism in exon 26 of the MDR1 gene (C3435T) has previously been shown to be associated with reduced P-glycoprotein expression in the small intestine, kidney and lymphocytes . In the present study, we examined systematically whether MDR1 polymorphisms also have an impact on P-glycoprotein expression in the human placenta . MDR1 mRNA and P-glycoprotein were analysed in 73 full-term human placentas of Caucasians, as well as respective MDR1 genotypes/haplotypes, for the C3435T and G2677T/A polymorphisms of mothers and infants . MDR1 mRNA levels were not different between these genotype groups . However, P-glycoprotein expression was significantly lower when both mother and infant were homozygous for the 3435T allele (TT/tt) compared to maternal and fetal homozygotes for the C-allele (0.40 +/- 0.18 a.u . for TT/tt versus 0.66 +/- 0.30 a.u . for CC/cc, P = 0.01) . Moreover, placentas from mothers carrying both polymorphisms (3435T and 2677T; TT/TT) also had a significantly lower P-glycoprotein expression (0.31 +/- 0.12 a.u.) compared to placentas of wild-type individuals (CC/GG, 0.71 +/- 0.31 a.u., P = 0.02) . Taken together, the MDR1 polymorphisms C3435T and G2677T are associated with altered P-glycoprotein expression in the human placenta, and may have clinical consequences due to genetically determined, variable drug exposure of the fetus .

J Med Chem, 2004 May 6, 47(10), 2523 - 33
Structure-function relationships of multidrug resistance P-glycoprotein; Pajeva IK et al.; The direct structure-function relationships of P-glycoprotein (P-gp) are presently unknown . In this paper two P-gp models are described: a homology model based on the Escherichia coli MsbA lipid transporter and a model based on the cross-linking results of Loo and Clarke . The pharmacophore pattern for the H-site (Hoechst 33342) is derived and binding sites on the transmembrane domains TM5 and TM11 are identified . Binding sites of rhodamines are also proposed on TM6 and TM12 in accordance with the published data . Location of the binding sites is opposite in both models, suggesting that TMs undergo rotation exposing the substrate bound from the membrane to the pore . It has been concluded that the models derived represent two different functional states of P-gp corresponding to nucleotide-free and nucleotide-bound P-gp . A qualitative correspondence to the P-gp crystallographic structure at 20 A resolution is found . A hypothesis is proposed about rearrangement of TMs upon state transition.

In Vivo, 2004 Mar-Apr, 18(2), 237 - 44
Modulation of multidrug resistance and apoptosis of cancer cells by selected carotenoids; Molnar J et al.; The multidrug resistance (MDR) proteins that belong to the ATP-binding casette superfamily are present in a majority of human tumors and are an important final cause of therapeutic failure . Therefore, compounds which inhibit the function of the MDR-efflux proteins may improve the cytotoxic action of anticancer chemotherapy . The effects of carotenoids were studied on the activity of the MDR-1 gene-encoded efflux pump system . The carotenoids, isolated from paprika and other vegetables, were tested on the rhodamine 123 accumulation of human MDR-1 gene-transfected L1210 mouse lymphoma cells and human breast cancer cells MDA-MB-231 (HTB-26) . Capsanthin and capsorubin enhanced the rhodamine 123 accumulation 30-fold relative to nontreated lymphoma cells . Lycopene, lutein, antheraxanthin and violaxanthin had moderate effects, while alfa- and beta-carotene had no effect on the reversal of MDR in the tumor cells . Apoptosis was induced in human MDR1 transfected mouse lymphoma cells and human breast cancer MDA-MB-231 (HTB-26) cell lines in the presence of lycopene, zeaxanthin and capsanthin . The data suggest the potential of carotenoids as possible resistance modifiers in cancer chemotherapy.

J Antibiot (Tokyo), 2004 Feb, 57(2), 143 - 50
Carminomycin, 14-hydroxycarminomycin and its novel carbohydrate derivatives potently kill human tumor cells and their multidrug resistant variants; Tevyashova AN et al.; The new hydrophilic derivatives of 14-hydroxycarminomycin were obtained using 13-dimethyl ketal of 14-bromocarminomycin (6) as the starting compound . The reductive alkylation of 6 with melibiose or D-galactose followed by hydrolysis of the corresponding intermediate bromoketals 9 and 11 produced 3'-N-{-alpha-D-(galactopyranosyl-(1 --> 6)-O-D-1-desoxyglucit-1-yl}-14-hydroxycarminomycin (10) and 3'-N-(1-desoxy-D-galactit-1-yl)-14-hydroxycarminomycin (12), respectively . These novel derivatives 10 and 12 were less toxic than carminomycin or 14-hydroxycarminomycin for leukemia (K562) and breast carcinoma (MCF-7) cells . Importantly, carminomycin, 14-hydroxycarminomycin and compounds 10 and 12 were similarly active for wild type cells and their multidrug resistant (MDR) sublines, K562i/S9 and MCF-7Dox.

World J Gastroenterol, 2004 May 1, 10(9), 1281 - 5
Correlation of expression of multidrug resistance protein and messenger RNA with 99mTc-methoxyisobutyl isonitrile (MIBI) imaging in patients with hepatocellular carcinoma; Wang H et al.; AIM: To explore whether P-glycoprotein (Pgp) and other pumps, multidrug resistance-associated protein (MRP) and lung resistance protein (LRP), could affect tumor accumulation and efflux of 99mTc-MIBI in liver cancer . METHODS: Surgically treated 78 liver cancer patients were included in this study . Before surgery, 99mTc-MIBI SPECT was performed 15 min and 120 min after injection of 20 mCi 99mTc-MIBI, respectively . Early uptake, delayed uptake (L/Nd), and washout rate (L/Nwr) of 99mTc-MIBI were obtained . Expressions of Pgp, MRP and LRP were investigated with Western blotting and immunohistochemistry . Messenger RNA (mRNA) level of Pgp, MRP and LRP was determined by RT-PCR . RESULTS: No 99mTc-MIBI uptakes in tumor lesions of 68 of 78 (87.2%) patients with hepatocellular carcinoma were found on 99mTc-MIBI SPECT . P-gp expression was observed in tumor tissues of the patients with no uptake of 99mTc-MIBI (P<0.017) . No appreciable correlation was found between liver cancer 99mTc-MIBI images and expression of MRP or LRP on the level of protein or mRNA . CONCLUSION: 99mTc-MIBI SPECT is noninvasive, and useful in predicting the presence of MDR1 gene-encoded Pgp in patients with hepatocellular carcinoma.

Biophys Chem, 2004 Jun 1, 109(3), 399 - 412
Presence of anionic phospholipids rules the membrane localization of phenothiazine type multidrug resistance modulator; Wesolowska O et al.; Substances able to modulate multidrug resistance (MDR), including antipsychotic phenothiazine derivatives, are mainly cationic amphiphiles . The molecular mechanism of their action can involve interactions with transporter proteins as well as with membrane lipids . The interactions between anionic phospholipids and MDR modulators can be crucial for their action . In present work we study interactions of 2-trifluoromethyl-10-(4-{methanesulfonylamid}buthyl)-phenothiazine (FPhMS) with neutral (PC) and anionic lipids (PG and PS) . Using microcalorimetry, steady-state and time-resolved fluorescence spectroscopy we show that FPhMS interacts with all lipids studied and drug location in membrane depends on lipid type . The electrostatic attraction between drug and lipid headgroups presumably keeps phenothiazine derivative molecules closer to surface of negatively charged membranes with respect to neutral ones . FPhMS effects on bilayer properties are not proportional to phosphatidylserine content in lipid mixtures . Behavior of equimolar PC:PS mixtures is similar to pure PS bilayers, while 2:1 or 1:2 (mole:mole) PC:PS mixtures resemble pure PC ones .

Autoimmun Rev, 2004 Mar, 3(3), 188 - 92
P-glycoprotein in autoimmune diseases; Richaud-Patin Y et al.; Multidrug resistance-1 (MDR-1) is characterized by overfunction of P-glycoprotein (P-gp), a pump molecule that decreases intracellular drug concentration by effluxing them from the intracellular space . Broad ranges of structurally unrelated compounds are transported by P-gp, including antineoplastic agents, HIV protease inhibitors, prednisone, gold salts, methotrexate, colchicine as well as several antibiotics . In contrast, many other compounds such as calcium channel blockers (verapamil) and immunosupressors (cyclosporine-A) are able to inhibit P-gp function . The P-gp role in therapeutic failures has been extensively studied in cancer; however, there is little information regarding MDR-1 phenotype in autoimmune disorders . It has been reported that an increased number of lymphocytes are able to extrude P-gp substrates in rheumatoid arthritis, immune thrombocytopenic purpura and systemic lupus erythematosus, the patients with poor response to treatment being the ones that exhibit the highest values . This may be due, at least in part, to a simultaneous long-term usage of several drugs that induce P-gp function . Since abnormally activated cell compartments characterize autoimmune diseases, it is possible that those cells are the ones that exhibit drug resistance . The study of drug resistance mechanisms in autoimmunity may be helpful for the optimization of the current therapeutic schemes through their combination with low doses of P-gp inhibitors.

Food Chem Toxicol, 2004 Jun, 42(6), 995 - 1002
St . John's Wort (Hypericum perforatum) induces overexpression of multidrug resistance protein 2 (MRP2) in rats: a 30-day ingestion study; Shibayama Y et al.; St . John's Wort (Hypericum perforatum, SJW) has been used as a herbal medicine for the treatment of depression in oral doses of 900-1050 mg/day in humans . However, the ingestion of SJW was reported to cause interactions with drugs . In the present study, we examined the effects of SJW treatment on the induction of drug transporters and enzymes in rats . An immunoblot analysis was performed to quantify the expression of the transporters and enzymes . SJW was given at a dose of 400 mg/kg/day, since it was reported that 400 mg/kg/day is antidepressant effective dose in rats . When SJW was administered for 10 days, the amounts of multidrug resistance protein 2 (MRP2), glutathione S-transferase-P (GST-P) and cytochrome P450 1A2 (CYP1A2) in the liver were increased to 304%, 252% and 357% of controls, respectively, although the amounts of P-glycoprotein and multidrug resistance protein 1 were not changed . Under the same conditions, an increase of MRP2 in the kidney was not observed . The increase in the levels of each protein was maximal at 10 days after SJW treatment and lasted for at least 30 consecutive days . These results suggest that SJW induces hepatic MRP2, GST-P and CYP1A2 overexpressions, and thus, it could affect drug metabolism, conjugation and disposition.

Cytometry B Clin Cytom, 2004 May, 59B(1), 46 - 53
A flow cytometric assay for simultaneous assessment of drug efflux, proliferation, and apoptosis; Barbier M et al.; BACKGROUND: Proliferative status and multidrug resistance play a key role in determining cell response to chemotherapy . There is a need to develop multiple labeling method that allows simultaneous assessment of multidrug resistant (MDR) phenotype, proliferative status, apoptosis related changes in mitochondrial potential, in chemosensitive and chemoresistant tumor cell populations . METHODS: A three-color labeling was performed using Hoechst 33342 (DNA), JC1 (mitochondrial potential), and a far red fluorescent membrane intercalating dye: PTIR271 (proliferation) . RESULTS: Combined staining of DNA and mitochondrial potential allows identification of subpopulations expressing and MDR phenotype mediated by P-glycoprotein (Pgp), and, in Pgp negative subpopulations, identification of apoptotic cells and evaluation of cell cycle status in viable cells . Addition of a far red fluorescent membrane intercalating dye, PTIR271, allows simultaneous monitoring of cell division status by dye dilution in both drug sensitive and drug resistant populations . CONCLUSION: This triple labeling is an interesting method to study the proliferation status of drug sensitivity and drug resistance in viable tumor cells .

Cytometry B Clin Cytom, 2004 May, 59B(1), 40 - 5
Antibody binding capacity for evaluation of MDR-related proteins in acute promyelocytic leukemia: Onset versus relapse expression; Damiani D et al.; BACKGROUND: Multidrug resistance (MDR) remains a major obstacle for successful treatment in cancer, in particular in acute leukemia . In acute promyelocytic leukemia (APL), the high sensitivity to anthracyclines appears to be attributable to the low frequency of MDR proteins overexpression at onset even if 30% of patients still relapse and become resistant to therapy . In attempt to explain different blast cell sensitivity, we studied the expression of PGP, MRP1, MRP2, and LRP in 45 cases of APL, comparing onset of disease with relapse . METHODS: PGP, LRP, and MRP on bone marrow or peripheral blood blast cells were evaluated by flow cytometry using the MRK-16, LRP-56, MRP-m6, and MRP2 antibodies and results expressed by the mean fluorescence index (MFI) . The antibody binding capacity (ABC) for each MDR protein was also calculated . RESULTS: At diagnosis, only 2 of 45 patients overexpressed PGP and 1 overexpressed LRP . PGP and LRP overexpressing cases significantly grew up during disease progression and at second relapse mean PGP MFI and mean LRP MFI were significantly higher than at onset (P = 0.001 and P = 0.008, respectively) . By analyzing ABC, the same trend was more evident because a significant increment of PGP and LRP was observed at second (P = 0.002 and P = 0.002, respectively), but even at first relapse (P = 0.018 and P = 0.002, respectively) . No changes were demonstrated in MRP1 and MRP2 expression in any phase of disease considered . CONCLUSIONS: Our data confirm the low expression at diagnosis of proteins related to development of drug resistance in APL . The evidence of a relative easy induction of PGP and LRP, but not of MRP, can be useful in choosing drugs to employ for consolidation or rescue therapy .

Antimicrob Agents Chemother, 2004 May, 48(5), 1889 - 91
Differential expression levels of MRP1, MRP4, and MRP5 in response to human immunodeficiency virus infection in human macrophages; Jorajuria S et al.; Multidrug resistance proteins (MRPs) have been reported to be involved in the efflux of some anti-human immunodeficiency virus (HIV) drugs . We show here that MRP1, MRP4, and MRP5 are expressed at the mRNA level in human monocyte-derived macrophages . HIV infection caused increased transcription of these MRPs; however, temporal differences in stimulation are reported.

Biochem Pharmacol, 2004 Mar 1, 67(5), 927 - 35
Disorazol A1, a highly effective antimitotic agent acting on tubulin polymerization and inducing apoptosis in mammalian cells; Elnakady YA et al.; Disorazol A1, a macrocyclic polyketide compound that is produced by the myxobacterium Sorangium cellulosum showed a remarkably high cytostatic activity . It inhibited the proliferation of different cancer cell lines including a multidrug-resistant KB line at low picomolar levels . In presence of disorazol A1, the nuclei of the cells increased in size and the cells often became multinucleate . Low concentrations of disorazol (<100 pM) induced an apoptotic process, characterized by enhanced capase-3 activity and DNA laddering, and abnormal, multipolar mitotic spindles . Low concentrations also induced an accumulation of p53 protein in the nucleus . At higher concentrations, we observed an accumulation of the cells in the G2/M-phase of the cell cycle, and a depletion of microtubules . In vitro, disorazol A1 inhibited the polymerization of tubulin in a concentration-dependent manner and independently of microtubule-associated proteins . Correspondingly it induced a complete depolymerization of microtubules prepared in vitro . Formation of defined degradation structures was not observed . Disorazol is a novel, highly effective antimitotic agent . Efforts are going on to develop it as an anticancer drug.

Mol Pharmacol, 2004 May, 65(5), 1208 - 16
Flavonoids are inhibitors of breast cancer resistance protein (ABCG2)-mediated transport; Zhang S et al.; Breast cancer resistance protein (BCRP) is a newly identified ATP-binding cassette transporter, shown to confer multidrug resistance (MDR) to a number of important anticancer agents and play an important function in governing drug disposition . Flavonoids are a class of polyphenolic compounds widely present in foods and herbal products . The interactions of flavonoids with P-glycoprotein and multidrug resistance-associated protein 1 have been reported; however, their interaction with BCRP is unknown . Our objective was to evaluate the effects of 20 naturally occurring flavonoids on the cellular accumulation and cytotoxicity of mitoxantrone in both BCRP-overexpressing and BCRP-negative human cell lines . BCRP-overexpressing and BCRP-negative human breast cancer cells (MCF-7) and large cell lung carcinoma cells (NCI-H460) were used in these studies . Many of the tested flavonoids (50 microM) increased mitoxantrone accumulation in BCRP-overexpressing cells, completely reversing mitoxantrone resistance, with no effect on the corresponding BCRP-negative cells, indicating that these flavonoids are BCRP inhibitors . The effects of these flavonoids on the cellular accumulation and cytotoxicity of mitoxantrone were flavonoid concentration dependent, and significant changes were produced at concentrations lower than 10 microM for most of the flavonoids . Chrysin and biochanin A were the most potent BCRP inhibitors, producing significant increases in mitoxantrone accumulation at concentrations of 0.5 or 1.0 microM and in mitoxantrone cytotoxicity at a concentration of 2.5 microM . Flavonoid glycosides had no effects on the BCRP-mediated transport of mitoxantrone . The results obtained in this study could be clinically relevant in terms of both MDR reversal in cancer treatment and drug-flavonoid pharmacokinetic interactions.

J Clin Pharmacol, 2004 May, 44(5), 481 - 6
MDR1 mRNA expressions in peripheral blood mononuclear cells of patients with ulcerative colitis in relation to glucocorticoid administration; Hirano T et al.; Overexpression of multidrug resistance (MDR) protein, P-glycoprotein (P-gp), on lymphocytes has been suggested to be implicated in the failure of glucocorticoid (GC) therapy in patients with ulcerative colitis (UC) . However, whether the overexpression of P-gp in a class of patients with inflammatory bowel disease (IBD) is intrinsic or related to the administration of GC is unknown . Relative amounts of MDR1 mRNA expressed in peripheral blood mononuclear cells (PBMCs) were measured using the reverse-transcriptase polymerase chain reaction (RT-PCR) technique in 25 UC patients having no history of GC administration, 25 UC patients having experienced GC therapy, 19 patients with Crohn's disease (CD) with no history of GC therapy, and 27 healthy subjects . Relative amounts of MDR1 mRNA expressed in PBMCs were compared among the groups . The relationship between the amounts of MDR1 mRNA expressed, as well as the total dose of GC administered or the period of GC therapy in UC patients, was examined . The relative amounts of MDR1 mRNA expressed in PBMCs were not significantly different between the healthy subjects and CD patients or UC patients having no history of GC therapy . However, the mean MDR1 mRNA amount in PBMCs of UC patients having experienced GC therapy was significantly greater than that in PBMCs of UC patients with no history of GC administration (p = 0.0375) . The amounts of MDR1 mRNA in PBMCs of UC patients having experienced GC therapy significantly correlated with the total dose of GCs administered (p = 0.0175) . Overexpression of MDR1 mRNA in PBMCs of IBD patients is not intrinsic . However, high-dose administration of GCs for the treatment of UC may result in an increased expression of MDR1 mRNA, which may impair successful GC therapy in these patients.

Infect Immun, 2004 May, 72(5), 2976 - 88
Interleukin-12 therapy reduces the number of immune cells and pathology in lungs of mice infected with Mycobacterium tuberculosis; Nolt D et al.; Alternate modalities for the treatment of Mycobacterium tuberculosis are needed due to the rise in numbers of immunosuppressed individuals at risk for serious disease and the increasing prevalence of multidrug-resistant isolates . Interleukin-12 (IL-12) has been shown to improve immune responses against M . tuberculosis infection in both humans and mice . Previous studies using high-dose IL-12 in various disease models reported a paradoxical immunosuppression . We demonstrate here that exogenous administration of IL-12 for 8 weeks after an aerosolized low dose of M . tuberculosis results in increased survival and decreased pulmonary bacterial loads for CD4-T-cell-deficient mice, most likely due to an early increase in gamma interferon . IL-12 treatment did not impair or enhance the ability of the wild-type mice to control infection, as measured by bacterial numbers . Two novel findings are reported here regarding exogenous IL-12 therapy for M . tuberculosis infections: (i) . IL-12 treatment resulted in decreased numbers of immune cells and reduced frequencies of lymphocytes (CD8(+), CD4(+), and NK cells) in the lungs of infected mice and (ii) . IL-12 therapy reduced the pathology of M . tuberculosis-infected lungs, as granulomas were smaller and less numerous . These studies support an immunoregulatory role for IL-12 in tuberculosis.

Epilepsia, 2004 May, 45(5), 441 - 51
Expression and cellular distribution of multidrug resistance-related proteins in the hippocampus of patients with mesial temporal lobe epilepsy; Aronica E et al.; PURPOSE: This study investigated the cellular distribution of different multidrug resistance (MDR)-related proteins such as P-glycoprotein (P-gp), the multidrug resistance-associated proteins (MRP) 1 and 2, and the major vault protein (MVP) in normal and sclerotic hippocampus of patients with medically refractory mesial temporal lobe epilepsy (MTLE) . METHODS: Single- and double-label immunocytochemistry was used on brain sections of control hippocampus and of hippocampus of refractory MTLE patients . RESULTS: In TLE cases with hippocampal sclerosis (HS), all four MDR proteins examined that had low or no expression in control tissue were upregulated, albeit with different cellular distribution patterns . P-gp immunoreactivity (IR) was observed in astrocytes in regions with diffuse reactive gliosis . In 75% of HS cases, strong P-gp IR was detected in blood vessels, with prominent endothelial labeling . Reactive astrocytes displayed low MRP1 IR . However, glial MRP1 expression was noted in glial endfoot processes around blood vessels . Neuronal MRP1 expression was observed in hypertrophic hilar neurons and in a few residual neurons of the CA1 region . Hippocampal MRP2 expression was observed in the large majority of HS cases in blood vessels . Hypertrophic hilar neurons and blood vessels within the sclerotic hippocampus expressed major vault protein (MVP) . CONCLUSIONS: These findings indicate that MDR proteins are upregulated in concert in the hippocampus of patients with refractory MTLE, supporting their role in the mechanisms underlying drug resistance . The specific cell-distribution patterns within the sclerotic hippocampus suggest different cellular functions, not necessarily linked only to clinical drug resistance.

Genet Mol Res, 2004 Mar 31, 3(1), 134 - 47
Drug resistance in Chromobacterium violaceum; Fantinatti-Garboggini F et al.; Chromobacterium violaceum is a free-living bacterium commonly found in aquatic habitats of tropical and subtropical regions of the world . This bacterium is able to produce a large variety of products of biotechnological and pharmacological use . Although C . violaceum is considered to be non-pathogenic, some cases of severe infections in humans and other animals have been reported . Genomic data on the type strain ATCC 12472(T) has provided a comprehensive basis for detailed studies of pathogenicity, virulence and drug resistance genes . A large number of open reading frames associated with various mechanisms of drug resistance were found, comprising a remarkable feature of this organism . Amongst these, beta-lactam (penicillin and cephalosporin) and multidrug resistance genes (drug efflux pumps) were the most numerous . In addition, genes associated with bacitracin, bicyclomycin, chloramphenicol, kasugamycin, and methylenomycin were also found . It is postulated that these genes contribute to the ability of C . violaceum to compete with other bacteria in the environment, and also may help to explain the common drug resistance phenotypes observed in infections caused by this bacterium.

J Comput Assist Tomogr, 2004 May-Jun, 28(3), 366 - 71
Multidrug-resistant tuberculosis versus drug-sensitive tuberculosis in human immunodeficiency virus-negative patients: computed tomography features; Kim HC et al.; OBJECTIVES: To compare the computed tomography (CT) features of patients with multidrug-resistant tuberculosis with those of patients with drug-sensitive tuberculosis in a country not associated with the human immunodeficiency virus (HIV) epidemic . METHODS: The CT images of 47 patients with multidrug-resistant tuberculosis were compared with those of 47 patients with drug-sensitive tuberculosis as a control group . Each multidrug-resistant tuberculosis patient was age (decade) and gender matched to a drug-sensitive tuberculosis patient . All patients were seronegative to HIV . This study evaluated the presence of centrilobular nodules, consolidation, emphysema, bronchiectasis, lung destruction, calcified granuloma, cavitation, pleural effusion, and lymphadenopathy . A statistical comparison was performed by using the Fisher exact test for univariate analysis and a multiple logistic regression method for multivariate analysis . RESULTS: In univariate analysis, bronchiectasis, lung destruction, a calcified granuloma, and cavitation were more frequently observed in multidrug-resistant tuberculosis than in drug-sensitive tuberculosis . Multivariate analysis showed that cavity formation was the only significant difference between multidrug-resistant tuberculosis and drug-sensitive tuberculosis . In patients with cavitary tuberculosis, multiple cavities (>3 cavities) were observed only in patients with multidrug-resistant tuberculosis . CONCLUSIONS: Most patients with multidrug-resistant tuberculosis had cavity formation on CT . Although the presence of a cavity does not mean multidrug resistance, multiple cavities suggest the possibility of multidrug-resistant tuberculosis.

J Pharmacol Exp Ther, 2004 Sep, 310(3), 1199 - 207 Epub 2004 Apr 20.
Multidrug resistance gene G1199A polymorphism alters efflux transport activity of P-glycoprotein; Woodahl EL et al.; The significance of the human multidrug resistance gene (MDR1) G1199A polymorphism, resulting in a Ser400Asn modification in P-glycoprotein (P-gp), remains unclear . We have developed stable recombinant LLC-PK1 epithelial cells expressing either MDR1wt or MDR11199 to evaluate functional consequences of G1199A {N-(4-{2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl}-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide} . P-gp activity observed in MDR1wt and MDR11199 cells was completely inhibited in the presence of the specific P-gp inhibitor GF120918 . Comparable expression of mRNA and protein in the MDR1-expressed cells and correct localization of P-gp in the apical membrane of recombinant cells was verified . Mean intracellular rhodamine-123 (R123) accumulation, measured by flow cytometry, was approximately 4.75-fold higher in MDR11199 recombinant cells than MDR1wt cells . Cytotoxicity studies have shown that MDR1wt and MDR11199 cells exhibited similar resistance, as measured by EC50 values, to doxorubicin (155 +/- 68 versus 120 +/- 32 nM); however, MDR11199 cells were more resistant to vinblastine (1.41 +/- 0.51 versus 15.7 +/- 4.0 nM; p < 0.001) and vincristine (1.18 +/- 0.56 versus 3.41 +/- 1.47 nM; p < 0.05) . The apparent transepithelial permeability ratios of R123 in MDR1wt and MDR11199 cells were 3.54 +/- 0.94 and 2.02 +/- 0.51 (p < 0.05), respectively . Therefore, the G1199A polymorphism alters the efflux and transepithelial permeability of a fluorescent substrate and sensitivity to select cytotoxic agents, which may influence drug disposition and therapeutic efficacy of some P-gp substrates.

Drug Metab Dispos, 2004 May, 32(5), 519 - 24
Influx and efflux transport of H1-antagonist epinastine across the blood-brain barrier; Ishiguro N et al.; We investigated influx and efflux transporters involved in blood-brain barrier transport of the nonsedative H1-antagonist epinastine . The basal-to-apical transport of {14C}epinastine was markedly higher than that in the opposite direction in LLC-GA5-COL150 cells stably transfected with human multidrug resistance (MDR)1 gene . The brain-to-plasma concentration ratio of {14C}epinastine in mdr1a/b(-/-) mice was 3.2 times higher than that in wild-type mice . The uptake of both {3H}mepyramine and {14C}epinastine into immortalized rat brain capillary endothelial cells (RBEC)1 showed temperature and concentration dependence . The kinetic parameters, K(m), V(max), and uptake clearance (V(max)/K(m)), of the initial uptake of {3H}mepyramine and {14C}epinastine by RBEC1 were 150 microM, 41.8 nmol/min/mg protein, and 279 microl/min/mg protein for mepyramine and 10.0 mM, 339 nmol/min/mg protein, and 33.9 microl/min/mg protein for epinastine, respectively . The uptake of {3H}mepyramine and {14C}epinastine by RBEC1 was inhibited by organic cations such as quinidine, amantadine, and verapamil, but not by other organic cations, tetraethyl ammonium, guanidine, and carnitine . Organic anions such as benzoic acid, estrone-3-sulfate, taurocholate, and neutral digoxin were not inhibitory . Furthermore, some cationic H1 antagonists (chlorpheniramine, cyproheptadine, ketotifen, and desloratadine) inhibited the {3H}mepyramine and {14C}epinastine uptake into RBEC1 . In conclusion, the present study demonstrated that the combination of efficient efflux transport by P-glycoprotein and poor uptake by the influx transporter, which is identical with that responsible for the uptake of mepyramine, account for the low brain distribution of epinastine.

Nucleic Acids Res . 2004 Apr 19;32(7):e64.
Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture; Jacobsen N et al.; LNA oligonucleotides constitute a class of bicyclic RNA analogues having an exceptionally high affinity for their complementary DNA and RNA target molecules . We here report a novel method for highly efficient isolation of intact poly(A)+ RNA using an LNA-substituted oligo(dT) affinity ligand, based on the increased affinity of LNA-T for complementary poly(A) tracts . Poly(A)+ RNA was isolated directly from 4 M guanidine thiocyanate-lysed Caenorhabditis elegans worm extracts as well as from lysed human K562 and vincristine-resistant K562/VCR leukemia cells using LNA_2.T oligonucleotide as an affinity probe, in which every second thymidine was substituted by LNA thymidine . In accordance with the significantly increased stability of the LNA_2.T-A duplexes in 4 M GuSCN, we obtained a 30- to 50-fold mRNA yield increase using the LNA-substituted oligo(T) affinity probe compared with DNA-oligo(dT)-selected mRNA samples . The LNA_2.T affinity probe was, furthermore, highly efficient in isolation of poly(A)+ RNA in a low salt concentration range of 50-100 mM NaCl in poly(A) binding buffer, as validated by selecting the mRNA pools from total RNA samples extracted from different Saccharomyces cerevisiae strains, followed by northern blot analysis . Finally, we demonstrated the utility of the LNA-oligo(T)-selected mRNA in quantitative real-time PCR by analysing the relative expression levels of the human mdr1 multidrug resistance gene in the two K562 cell lines employing pre-validated Taqman assays . Successful use of the NH2-modified LNA_2.T probe in isolation of human mRNA implies that the LNA-oligo(T) method could be automated for streamlined, high throughput expression profiling by real-time PCR by covalently coupling the LNA affinity probe to solid, pre-activated surfaces, such as microtiter plate wells or magnetic particles.

Crit Rev Oncol Hematol, 2004 Apr, 50(1), 39 - 49
Changing picture of cellular drug resistance in human leukemia; Norgaard JM et al.; A relatively well documented and seemingly firm overall picture of mechanisms involved in leukemia-cell drug resistance has evolved since the 1970s, where mechanisms involved in multidrug resistance towards anti-leukemia chemotherapeutic compounds were first described . At that time, based on available data, resistance associated with overexpression of the cell-surface transmembrane ATPase P-glycoprotein (P-170, P-gp, the product of the MDR1 gene) was described as "the" cause of multidrug resistance in cancer cells . However, during the 1980s and later on other mechanisms were described as candidate causes of multidrug resistance in human leukemia . Moreover, research of the past decade has provided us with an enormous increase in the amount of data and knowledge on the cell-biological and--to an even higher extent--the molecular-genetic processes governing cell survival and death in cancer cells . This, in turn, has improved the possibilities of designing and developing better drugs and drug combinations in leukemia . Along this line, based on rational drug design, imatinib, a 2-phenylaminopyrimidine derivative, has very recently been introduced and found to be an efficient inhibitor of the altered tyrosine kinase, which arises as a product of the BCR-ABL fusion transcript in Philadelphia chromosome positive (Ph+) cases of CML . This new compound appears to be the first of a (hopefully) large family of small organic molecules with a more specific inhibiting activity against the pathogenetic defects in leukemia as well as cancer . With this novel compound, as with all other known individual drugs and classes of chemotherapeutic drugs, drug resistance is seen . To what extent drug resistance towards this novel compound (and its successors) will follow patterns of drug resistance that are already known or entirely new mechanisms of drug resistance is yet to be seen.

Di Yi Jun Yi Da Xue Xue Bao, 2004 Apr, 24(4), 392 - 6
{Morphological observation on NK/LAK cell-mediated lysis of human oral carcinoma cells}; Zheng YJ et al.; OBJECTIVE: To understand the relation between cytotoxic activity of immunologic effector cells and multidrug resistance of the tumor cells . METHODS: Continuous observation of the morphological changes and MTT colorimetry were employed to evaluate the cytotoxic activity of lymphokine-activated killer (LAK) cells and natural killer (NK) cells against multidrug-resistant (MDR) human oral carcinoma cell line-KBV200 (before and after reversal of MDR) and parental drug-sensitive cell line KB . The morphologic changes of LAK cells and the 3 target cell lines were observed continuously under inverted microscope 3 h after co-culture of LAK cells with one of three target cell lines respectively . The lysis rates of three tumor cell lines in response to co-culture with LAK or NK cells were determined using MTT colorimetry . RESULTS: In comparison with the parental drug-sensitive cell line KB, both KBV200 and its reserved cell line by verapamil (KBVV) showed earlier adherence and greater number of cells lysed by LAK . In MTT colorimetry assay, the cytotoxicity of both LAK and NK cells against the 3 cell lines was associated with the effector-to-target (E/T) cell ratio; the lysis rates of KBV200 and reversed KBV200 cells by verapamil in response to LAK and NK cells were higher than that of KB cells (P<0.05), but KBV200 and KBVV did not significantly differ (P>0.05) . At the same E/T ratio, LAK cells possessed stronger cytotoxicity than NK cells against all the tumor cell lines (P<0.05) . CONCLUSIONS: Immunologic effector cells possess strong cytotoxic activity against multidrug-resistant cell line KBV200 . Modulation of MDR does not decrease the cytotoxic activity of the immunologic effector cells . The results of this study suggest that adoptive cell immunotherapy with immunologic effector cells may be of value in controlling the progress of MDR tumors.

BMC Cancer . 2004 Apr 17;4(1):13.
Modulation of human multidrug-resistance MDR-1 gene by natural curcuminoids; Limtrakul P et al.; BACKGROUND: Multidrug resistance (MDR) is a phenomenon that is often associated with decreased intracellular drug accumulation in patient's tumor cells resulting from enhanced drug efflux . It is related to the overexpression of a membrane protein, P-glycoprotein (Pgp-170), thereby reducing drug cytotoxicity . A variety of studies have tried to find MDR modulators which increase drug accumulation in cancer cells . METHODS: In this study, natural curcuminoids, pure curcumin, demethoxycurcumin and bisdemethoxycurcumin, isolated from turmeric (Curcuma longa Linn), were compared for their potential ability to modulate the human MDR-1 gene expression in multidrug resistant human cervical carcinoma cell line, KB-V1 by Western blot analysis and RT-PCR . RESULTS: Western blot analysis and RT-PCR showed that all the three curcuminoids inhibited MDR-1 gene expression, and bisdemethoxycurcumin produced maximum effect . In additional studies we found that commercial grade curcuminoid (approximately 77% curcumin, 17% demethoxycurcumin and 3% bisdemthoxycurcumin) decreased MDR-1 gene expression in a dose dependent manner and had about the same potent inhibitory effect on MDR-1 gene expression as our natural curcuminoid mixtures . CONCLUSION: These results indicate that bisdemethoxycurcumin is the most active of the curcuminoids present in turmeric for modulation of MDR-1 gene . Treatment of drug resistant KB-V1 cells with curcumin increased their sensitivity to vinblastine, which was consistent with a decreased MDR-1 gene product, a P-glycoprotein, on the cell plasma membrane . Although many drugs that prevent the P-glycoprotein function have been reported, this report describes the inhibition of MDR-1 expression by a phytochemical . The modulation of MDR-1 expression may be an attractive target for new chemosensitizing agents.

Ai Zheng, 2004 Apr, 23(4), 401 - 5
{MDR-reversing effect of two components of catechin on human hepatocellular carcinoma BEL-7404/Adr in vitro}; Liang G et al.; BACKGROUND & OBJECTIVE: Catechin is composed of a variety of components,some of which have anti-cancer activity . There was no report on effect of catechin on the multidrug resistance of hepatocellular carcinoma so far . The aim of this study was to investigate the MDR-reversing effect of two components of catechin on human hepatocellular carcinoma BEL-7404/Adr in vitro and its potential mechanism . METHODS: MTT was used to test the toxicity of the two components of catechin . The concentration of the drugs inside the cells was determined by fluorospectro-photometry and the expression of MDR1 was measured by RT-PCR . RESULTS: The inhibition rates of BEL-7404/Adr caused by two components of catechin were less than 10% under the dose of 100 mg/L . Epicatechin gallate (ECG) in 60 mg/L or epigallocatechin gallate (EGCG) in 14 mg/L has slight cytotoxicity, but they could decrease the IC(50) of adriamycin (ADM) for BEL-7404/Adr from 36 mg/L to 2.3 mg/L or 1.9 mg/L, respectively, and the reversing folds were 15.8 and 19.2, respectively . The combined administration could increase the concentration of ADM in BEL-7404/Adr cells from 0.76 microg/10(8) cells-2.55 microg/10(8) cells to 2.04 microg/10(8) cells -9.28 microg/10(8) cells (P< 0.01) . The expression of MDR1 was down-regulated by 27.6% and 41.3%, respectively . Furthermore, EGCG is the stronger one . CONCLUSION: ECG and EGCG can reverse the multi-drug resistance of human hepatocellular carcinoma in vitro . The possible mechanism is related to down-regulating the expression of MDR1 and raising the concentration of drugs inside the cells.

Ai Zheng, 2004 Apr, 23(4), 396 - 400
{Effect of PKC inhibitor on P-gp expression and drug-resistance in MGC803 cells}; Chen B et al.; BACKGROUND & OBJECTIVE: It is showed that there is close relationship between multidrug resistance (MDR) and protein kinase C signal transduction system, but the mechanism remains unclarified . The aim of this study was to investigate the correlation between protein kinase C (PKC) signal transduction system and mdr1 gene in human gastric cancer cell line through studying the effect of vincristine (VCR) and a selective inhibitor of PKC, myr-psiPKC on MGC803 cells . METHODS: Western blot analysis was used to analyze the expression of P-glycoprotein (P-gp), which was encoded by mdr1, in transient VCR induced MGC803 cells, which were treated with or without myr-psiPKC . Cell cycle analysis was performed using flow cytometry and MTT assay was used to investigate the drug susceptibility of MGC803 cells which were exposed to VCR with or without myr-psiPKC . RESULTS: High level of P-gp expression was detected in the MGC803 cells after transient exposure to VCR, and its expression was down-regulated when the same VCR induced MGC803 cell line was incubated with myr-psiPKC . FCM results indicated that more MGC803 cells showed significantly higher level of apoptotic phenotype when treated with VCR and myr-psiPKC (ratio 31.23%), than those treated with only VCR (ratio 18.42%) . The IC(50) (284.0+/-13.2 ng/ml) to VCR of MGC803 cells pretreated with VCR exhibited 2.24-fold of negative control group (127.0+/-17.6 ng/ml) and 1.33-fold of the group (212.0+/-30.4 ng/ml) treated with myr-psiPKC . CONCLUSION: The expression of P-gp can be induced by transient exposure to VCR and this induction can be inhibited by myr-psiPKC, which blocks the activity of PKCalpha and beta . PKC signal transduction system may play certain roles in modulating mdr1 expression in gastric cancer.

Ai Zheng, 2004 Apr, 23(4), 391 - 5
{Establishment of human multidrug-resistant hepatocellular carcinoma cell line (HepG2/Adm) and biological characteristics evaluation}; Zhai BJ et al.; BACKGROUND & OBJECTIVE: Multidrug resistance (MDR) is considered to be the major obstacle for chemotherapy . In order to reverse tumor MDR in vitro, we designed this study to establish human multidrug-resistant hepatocellular carcinoma cell line (HepG2/Adm) and to investigate its biological characteristics . METHODS: An adriamycin-resistant human hepatocellular carcinoma cell subline (HepG2/Adm) was established in vitro using gradually increased concentration of adriamycin (ADM) in culture . Cell growth was measured and multidrug resistance to multianticancer agents was evaluated by MTT assay . Flow cytometry (FCM) was performed to determine cell cycle,and to assess the expression of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP),lung-related protein (LRP),and glutathione S-transferase (GST), respectively . MDR mRNA expression related to these proteins were determined with reverse transcription polymerase chain reaction (RT-PCR) . RESULTS: Compared to HepG2, HepG2/Adm had a prolonged doubling time of 30.1 hours . The number of cells in S-phase was significantly decreased (5.6+/-0.03)% in HepG2/Adm while those in G1 and G2-phase increased {(4.2+/-0.09)% and (1.5+/-0.08)%, respectively} . Furthermore, HepG2/Adm was resistant to many anti-tumor agents, and its IC(50) of ADM was 26 times higher than that of parent cell line HepG2 . Significant overexpression of P-gp, MRP, and GST were detected . RT-PCR showed that mRNA expression of P-gp, MRP, LRP, and GST were significantly increased in HepG2/Adm . CONCLUSION: HepG2/Adm is human multidrug-resistant, and its resistance may be closely related to the overexpression of P-gp, MRP, and GST.

Pharmacogenetics, 2004 Apr, 14(4), 213 - 23
Identification and functional characterization of the natural variant MRP3-Arg1297His of human multidrug resistance protein 3 (MRP3/ABCC3); Lee YM et al.; The human multidrug resistance protein 3 (MRP3, symbol ABCC3) is an ATP-binding cassette transporter that mediates the efflux of organic anions, including lipophilic substances conjugated with glucuronate, sulphate or glutathione, across the basolateral membrane of polarized cells (e.g . hepatocytes) into blood . Genetic variants of MRP3 may affect the transport of these substances out of cells . The aims of this study were: (i) to identify MRP3 polymorphisms; (ii) to functionally characterize one relatively frequent MRP3 polymorphism; and (iii) to establish whether MRP3 transports bilirubin glucuronosides . Exonic nucleotide variants in the ABCC3 gene were identified by single-strand conformation polymorphism analysis . The 3890G>A mutation, resulting in MRP3-ArgHis, was introduced into the ABCC3 cDNA which was stably transfected into MDCKII cells . For the functional characterization of MRP3-ArgHis in comparison with MRP3, ATP-dependent transport was analysed in isolated membrane vesicles . Two non-synonymous MRP3 variants were identified with an allele frequency of 0.003 for 1643T>A (MRP3-LeuGln) and 0.08 for 3890G>A (MRP3-ArgHis) . Because of the high frequency of the 3890G>A mutation, and because of the close proximity of Arg to the second nucleotide-binding domain, we pursued the functional characterization of the MRP3-ArgHis polymorphic variant . MRP3-ArgHis was correctly localized to the basolateral membrane of polarized MDCKII cells . We identified monoglucuronosyl bilirubin, bisglucuronosyl bilirubin and leukotriene C4 as substrates for both MRP3 and MRP3-ArgHis . Dehydroepiandrosterone-3-sulphate and 17beta-glucuronosyl oestradiol were transported with similar kinetics by MRP3 and MRP3-ArgHis . This experimental setup provides a useful tool to analyse the functional consequences of polymorphic variants of MRP3.

J Korean Med Sci, 2004 Apr, 19(2), 167 - 71
Six-month therapy with aerosolized interferon-gamma for refractory multidrug-resistant pulmonary tuberculosis; Koh WJ et al.; The aim of this study was to investigate the adjuvant effects of interferon-gamma (IFN-gamma) inhalation therapy for six months in the treatment of refractory multidrug-resistant pulmonary tuberculosis (MDR-TB) . Aerosolized IFN-gamma was given to six MDRTB patients with persistent positive smears and cultures despite long-term medical treatment . The patients received aerosolized two million international units of IFN-gamma three times a week for 6 months while they continued on identical antituberculous chemotherapy . Before IFN-gamma inhalation therapy, the patients received a median of 6.5 (range, 4 to 7) antituberculous drugs for median duration of 29 months (range,7 to 76) . After IFN-gamma inhalation therapy, sputum smears remained persistently positive in all patients throughout the study period . Sputum cultures were transiently negative at the 4th month in two patients, but became positive again at the end of 6 months of IFN-gamma therapy . Five patients had radiological improvement including three patients who showed a decrease in the size of the cavitary lesions . Resectional surgery could be performed in one patient in whom substantial clinical and radiological improvement was noted after IFN-gamma inhalation therapy . These results suggest that IFN-gamma inhalation therapy may be effective for some cases of refractory MDR-TB who are otherwise not responding to conventional therapy.

Am J Cardiol, 2004 Apr 15, 93(8), 1046 - 50
Polymorphisms in the multidrug resistance-1 (MDR1) gene influence the response to atorvastatin treatment in a gender-specific manner; Kajinami K et al.; To test the hypothesis that variations in the multidrug resistance-1 gene influence the response to statin treatment, 2 prevalent polymorphisms (G2677T/A and C3435T) were examined in 344 hypercholesterolemic patients treated with atorvastatin (10 mg) . The C3435T polymorphism was significantly and independently associated with a smaller reduction in low-density lipoprotein cholesterol and with a larger increase in high-density lipoprotein cholesterol, relative to variant allele carriers, in a gender-specific manner . Also, haplotype determination combined with these polymorphisms identified a subgroup that showed a striking response to treatment, which was not defined by a single polymorphism.

Scand J Rheumatol, 2003, 32(6), 325 - 36
Multidrug resistance proteins in rheumatoid arthritis, role in disease-modifying antirheumatic drug efficacy and inflammatory processes: an overview; Jansen G et al.; Drug resistance is generally accepted as an important cause of treatment failure for patients with neoplastic or infectious diseases . Molecular mechanisms underlying drug resistance include the action of drug efflux pumps belonging to the super-family of ATP binding cassette (ABC) proteins, which mediate the cellular extrusion of a large variety of therapeutic drugs, a phenotype that is referred to as multidrug resistance (MDR) . Unlike neoplastic and infectious diseases, chronic inflammatory diseases have received little attention . The potential role of ABC transporters in determining the efficacy of anti-rheumatic drugs, notably disease modifying anti-rheumatic drugs (DMARDs), in patients with rheumatoid arthritis is unclear . Based on knowledge from the field of oncology and immunology, this review concentrates on the pharmacological role of MDR proteins in the (clinical) efficacy of several DMARDs, as well as the physiological role of MDR proteins in transporting signalling molecules important in inflammatory processes.

Hematol J, 2004, 5 Suppl 1, S62 - 7
Use of fludarabine in the treatment of acute myeloid leukemia; Jackson GH; Acute myeloid leukemia (AML) is a disease, which when left untreated, is invariably fatal . The disease is more common in elderly people, who also fare worse than younger patients with AML due to a higher rate of unfavorable prognostic factors, such as poor performance status, multiple comorbidities, reduced tolerance to treatment, 'unfavorable' chromosomal abnormalities and multidrug resistant protein-1 expression . While many patients achieve a complete remission, the rate of relapse is high and prognosis after relapse very poor . Promising results have been published in recent years using fludarabine-containing combination therapy for AML, most commonly fludarabine +cytarabine + granulocyte colony-stimulating factor (G-CSF) {FLAG}, FLAG + mitoxantrone (FLANG), or FLAG + idarubicin (FLAG-Ida) . Such combinations maximize favorable cytotoxic interactions between cytarabine and G-CSF, and between cytarabine and fludarabine . In small studies, such combinations used as second-line therapy have resulted in complete response (CR) rates of 36-59% . Early retrospective analyses suggested higher CR rates in patients with refractory AML than in those with relapsed AML, but this observation has not been confirmed in recent prospective trials . Fludarabine-containing combinations have also been evaluated as first-line therapy in high-risk patients and resulted in CR rates of 34-70%, with median survival from 7 to 16 months . The current large MRC randomized high-risk study will provide further data on the use of fludarabine-containing regimens in patients with poor prognosis AML . Further studies are investigating the use of fludarabine in combination with other agents, such as gemtuzumab ozogamicin and gemcitabine, in patients with AML.

Medicina (Kaunas), 2004, 40 Suppl 1, 131 - 3
{Multidrug-resistance: the modern problem of surgical treatment of pulmonary tuberculosis}; Vencevicius V; The problem of multidrug-resistance is urgent because the part of these patients undergoes treatment failure after chemotherapy . In such cases the operations are performed . In 1998-2002 in the Department of Thoracic Surgery of Republic Santariskes Tuberculosis and Lung Disease Hospital 739 thoracic operations were performed for the patients with pulmonary tuberculosis . Because of multidrug-resistant pulmonary tuberculosis 355 patients (48%) were operated on, radical operations were performed to 190 patients (53.5%) and for 165 (46.5%) - palliative . Positive outcome was documented for 324 patients (91.3%); 31 patients (8.7%) died . Various postoperative complications were confirmed in 76 cases (21.4%) when multidrug-resistance was determined . CONCLUSIONS . When multidrug-resistance is developed, clinic or radiological dynamics of tuberculosis process is not positive, after 3-4 months is it necessary to discuss the question of the operation . In case of monolateral process the radical operations with the type of resection are performed, but in case of widespread process and various complications palliative operations are performed . The treatment with antituberculotic drugs when patient is operated on because multidrug-resistance is continued for 22-24 months.

Oncogene, 2004 Apr 12, 23(16), 2934 - 49
Apoptosis defects and chemotherapy resistance: molecular interaction maps and networks; Pommier Y et al.; Intrinsic (innate) and acquired (adaptive) resistance to chemotherapy critically limits the outcome of cancer treatments . For many years, it was assumed that the interaction of a drug with its molecular target would yield a lethal lesion, and that determinants of intrinsic drug resistance should therefore be sought either at the target level (quantitative changes or/and mutations) or upstream of this interaction, in drug metabolism or drug transport mechanisms . It is now apparent that independent of the factors above, cellular responses to a molecular lesion can determine the outcome of therapy . This review will focus on programmed cell death (apoptosis) and on survival pathways (Bcl-2, Apaf-1, AKT, NF-kappaB) involved in multidrug resistance . We will present our molecular interaction mapping conventions to summarize the AKT and IkappaB/NF-kappaB networks . They complement the p53, Chk2 and c-Abl maps published recently . We will also introduce the 'permissive apoptosis-resistance' model for the selection of multidrug-resistant cells.

Pharmacogenetics, 2004 Feb, 14(2), 91 - 102
Sequence analysis of bile salt export pump (ABCB11) and multidrug resistance p-glycoprotein 3 (ABCB4, MDR3) in patients with intrahepatic cholestasis of pregnancy; Pauli-Magnus C et al.; Intrahepatic cholestasis of pregnancy (ICP) is a liver disorder associated with increased risk of intrauterine fetal death and prematurity . There is increasing evidence that genetically determined dysfunction in the canalicular ABC transporters bile salt export pump (BSEP, ABCB11) and multidrug resistance protein 3 (MDR3, ABCB4) might be risk factors for ICP development . This study aimed to (i) . describe the extent of genetic variability in BSEP and MDR3 in ICP and (ii) . identify new disease-causing mutations . Twenty-one women with ICP and 40 women with uneventful pregnancies were recruited between April 2001 and April 2003 . Sequencing of BSEP and MDR3 spanned 8-10 kb per gene and comprised the promoter region and 100-350 bp of the flanking intronic region around each exon . DNA sequencing of polymerase chain reaction fragments was performed on an ABI3700 capillary sequencer . MDR3 promoter activity of promoter constructs carrying different ICP-specific mutations was studied using reporter assays . A total of 37 and 51 variant sites were detected in BSEP and MDR3, respectively . Three non-synonymous sites in codons for evolutionarily conserved amino acids were specific for the ICP collective (BSEP, N591S; MDR3, S320F and G762E) . Furthermore, four ICP-specific splicing mutations were detected in MDR3 {intron 21, G(+1)A; intron 25, G(+5)C and C(-3)G; and intron 26, T(+2)A} . Activity of the mutated MDR3 promoter was similar to that observed for the wild-type promoter . Our data further support an involvement of MDR3 genetic variation in the pathogenesis of ICP, whereas analysis of BSEP sequence variation indicates that this gene is probably less important for the development of pregnancy-associated cholestasis.

AIDS, 2004 Jan 23, 18(2), 217 - 26
Benefit of treatment interruption in HIV-infected patients with multiple therapeutic failures: a randomized controlled trial (ANRS 097); Katlama C et al.; BACKGROUND: Both highly potent antiretroviral drug rescue therapy and treatment interruption have been suggested to be effective in patients with multiple treatment failure . OBJECTIVE: To assess both the benefits and risks of an 8-week treatment interruption associated with a six to nine-drug rescue regimen in patients with multiple treatment failures . DESIGN: A randomized comparative controlled trial in 19 university hospitals in France . PATIENTS: Sixty-eight HIV-infected patients with multiple previous treatment failures and CD4 cell counts less than 200 x 10(6) cells/l and plasma HIV-1-RNA levels of 50,000 copies/ml or greater . MEASUREMENTS: The primary efficacy outcome was the proportion of patients with at least a 1 log10 decrease (copies/ml) in the plasma HIV-1-RNA level after 12 weeks of therapy . RESULTS: Treatment interruption followed by multidrug salvage therapy led to a greater proportion of patients achieving virological success (i.e . 1 log10 decrease) at 12 weeks compared with patients receiving multidrug therapy alone (62 versus 26%, intent-to-treat analysis; P = 0.007) . The median decrease in the HIV-1-RNA level was -1.91 and -0.37 log10 copies/ml (P = 0.008), respectively . Treatment interruption led to an increase in the number of sensitive drugs of the multidrug regimen (71 versus 35% of regimen with at least two sensitive drugs; P = 0.004) . Factors associated with virological success were treatment interruption, the reversion of at least one mutation to wild type, adequate plasma drug concentration, and the use of lopinavir . CONCLUSION: Treatment interruption was beneficial for treatment-experienced HIV-infected patients with advanced HIV disease and multidrug-resistant virus.

Urology, 2004 Apr, 63(4), 694 - 8
Expression of lung resistance-related protein in transitional cell carcinoma of bladder; Zhu Y et al.; OBJECTIVES: To determine the role of lung resistance-related protein (LRP) in intrinsic multidrug-resistance (MDR) of bladder cancer . METHODS: The study group consisted of 66 patients with newly diagnosed primary bladder cancer . No patient had been treated preoperatively with either radiotherapy or chemotherapy . Reverse transcriptase-polymerase chain reaction was performed to measure LRP, multidrug resistance-associated protein 1 (MDR1), and MRP1 mRNA expression . The expression of LRP, p53 proteins, and p63 proteins was examined by immunohistochemistry . We analyzed the correlation of LRP with the above indexes and the clinical pathologic parameters . RESULTS: The expression rate of LRP mRNA (63.6%) was the greatest among the three MDR markers in primary bladder cancer without exposure to chemotherapy . The LRP mRNA level was significantly greater in normal bladder tissue than in transitional cell carcinoma bladder tissue (P <0.01) and in superficial cancer than in invasive cancer (P = 0.013) . LRP mRNA expression showed no association with either MDR1 or MRP1, but close correlation with the LRP level (P = 0.001) . LRP was associated with low-grade (P <0.01) and low-stage (P <0.05) cancer but had no association with tumor suppressor p53 or p63 . CONCLUSIONS: The grade and stage-related expression pattern of LRP indicates that it may be a predictive index for intrinsic MDR in early bladder cancer . Anticancer drugs out of the MDR spectrum of LRP may be more effective for patients with early bladder cancer.

Drug Metab Rev, 2004 Feb, 36(1), 57 - 104
Herbal modulation of P-glycoprotein; Zhou S et al.; P-glycoprotein (Pgp) is a 170 kDa phosphorylated glycoprotein encoded by human MDR1 gene . It is responsible for the systemic disposition of numerous structurally and pharmacologically unrelated lipophilic and amphipathic drugs, carcinogens, toxins, and other xenobiotics in many organs, such as the intestine, liver, kidney, and brain . Like cytochrome P450s (CYP3A4), Pgp is vulnerable to inhibition, activation, or induction by herbal constituents . This was demonstrated by using an ATPase assay, purified Pgp protein or intact Pgp-expressing cells, and proper probe substrates and inhibitors . Curcumin, ginsenosides, piperine, some catechins from green tea, and silymarin from milk thistle were found to be inhibitors of Pgp, while some catechins from green tea increased Pgp-mediated drug transport by heterotropic allosteric mechanism, and St . John's wort induced the intestinal expression of Pgp in vitro and in vivo . Some components (e.g., bergamottin and quercetin) from grapefruit juice were reported to modulate Pgp activity . Many of these herbal constituents, in particular flavonoids, were reported to modulate Pgp by directly interacting with the vicinal ATP-binding site, the steroid-binding site, or the substrate-binding site . Some herbal constituents (e.g., hyperforin and kava) were shown to activate pregnane X receptor, an orphan nuclear receptor acting as a key regulator of MDR1 and many other genes . The inhibition of Pgp by herbal constituents may provide a novel approach for reversing multidrug resistance in tumor cells, whereas the stimulation of Pgp expression or activity has implication for chemoprotective enhancement by herbal medicines . Certain natural flavonols (e.g., kaempferol, quercetin, and galangin) are potent stimulators of the Pgp-mediated efflux of 7,12-dimethylbenz(a)-anthracene (a carcinogen) . The modulation of Pgp activity and expression by these herb constituents may result in altered absorption and bioavailability of drugs that are Pgp substrates . This is exemplified by increased oral bioavailability of phenytoin and rifampin by piperine and decreased bioavailability of indinavir, tacrolimus, cyclosporine, digoxin, and fexofenadine by coadministered St . John's wort . However, many of these drugs are also substrates of CYP3A4 . Thus, the modulation of intestinal Pgp and CYP3A4 represents an important mechanism for many clinically important herb-drug interactions . Further studies are needed to explore the relative role of Pgp and CYP3A4 modulation by herbs and the mechanism for the interplay of these two important proteins in herb-drug interactions.

Pharm Res, 2004 Mar, 21(3), 467 - 75
ATP-dependent transport of a novel thromboxane A2 receptor antagonist, {2-(4-chlorophenylsulfonylaminomethyl)indan-5-yl}acetate (Z-335) and its xenobiotic taurine conjugate (Z-335-Tau) by rat bile canalicular membrane vesicles; Kawabata Y et al.; PURPOSE: The characteristics of bile canalicular transport processes for xenobiotic taurine conjugates have not yet been clarified . To elucidate the biliary excretion characteristics of xenobiotic taurine conjugates, we investigated the transport of a novel thromboxane A2 receptor antagonist, Z-335, and its taurine conjugate (Z-335-Tau) across the bile canalicular membrane . METHODS: We examined the uptake of Z-335 and Z-335-Tau by isolated bile canalicular membrane vesicles (CMVs) from Sprague Dawley and Eisai-hyperbilirubinemic rats (EHBRs) which EHBRs have a hereditary defect of canalicular multidrug resistance-associated protein 2 (Mrp2) function . Also, the in vitro and in vivo kinetics of Z-335-Tau uptake and excretion were compared . RESULTS: Z-335 uptake by CMVs from normal rats exhibited marked ATP-dependence, whereas ATP-dependent uptake of Z-335 into CMVs from EHBRs was not observed . In contrast, Z-335-Tau uptake into CMVs from both normal rats and EHBRs was ATP dependent . The initial uptake velocity was concentration-dependent, with an in vitro Michaelis constant for initial uptake of 189 microM, which was similar to the in vivo value . CONCLUSIONS: The biliary excretion of Z-335 involves Mrp2, whereas that of Z-335-Tau involves active transport systems that remain intact in EHBRs and show marked ATP dependence, which ATP-dependent transport is involved in the biliary excretion of Z-335-Tau in vivo.

Pharm Res, 2004 Mar, 21(3), 406 - 12
Chemosensitivity assessed by collagen gel droplet embedded culture drug sensitivity test, and MDR1, MRP1, and MRP2 mRNA expression in human colorectal adenocarcinomas; Nakahara T et al.; PURPOSE: To evaluate chemosensitivity and its correlation with expression levels of the multidrug resistant transporter (MDR1) and the multidrug resistance-associated proteins 1 and 2 (MRP1, MRP2) mRNA in human colorectal adenocarcinomas . METHODS: Colorectal adenocarcinomas were obtained as surgical samples from 25 patients . The chemosensitivity of 12 anticancer drugs was assessed by the collagen gel droplet embedded culture drug sensitivity test (CD-DST) . The expression levels of MDR1, MRP1, and MRP2 mRNA in colorectal adenocarcinomas were also evaluated by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) . RESULTS: The chemosensitivity was successfully evaluated for 16 of 25 patients, and the anticancer drugs were effective against the samples showing a relatively high growth rate . Gemcitabine hydrochloride was found to be more promising than those often prescribed for the treatment of colorectal adenocarcinoma . There was no correlation of the mRNA expression levels of MDR1 and MRP1 with the chemosensitivity of any anticancer drugs tested, but mitomycin C was found to be more effective for the colorectal adenocarcinoma with relatively high expression of MRP2 mRNA.

Oncol Rep, 2004 May, 11(5), 1091 - 5
Effect of short-term morphine exposure on P-glycoprotein expression and activity in cancer cell lines; Pajic M et al.; Multidrug resistance (MDR) is a common problem in various types of cancer . One important factor in the development of MDR is overexpression of P-glycoprotein, encoded by the MDR1 gene . Morphine is the opioid of choice for moderate to severe cancer pain, and is a substrate of P-glycoprotein . Recently, morphine has been shown to induce P-glycoprotein expression in the rat brain . Using Western blot analysis and cytotoxicity assays respectively, we have investigated the effects of short-term (72 h) morphine treatment on P-glycoprotein expression in a panel of human cancer cell lines, and its effects on cellular resistance to the known P-glycoprotein substrates, vinblastine and colchicine . The effect of morphine on P-glycoprotein expression and activity in the mouse fibroblast NIH-3T3 cell was assessed to establish whether morphine effects are species specific . Short-term exposure to morphine did not result in any significant differences in P-glycoprotein expression or activity in any cancer cell lines . Morphine pre-treatment resulted in a moderate but significant increase in sensitivity of NIH-3T3 cells to vinblastine, but not colchicine . This study suggests that morphine effects may be cell-type specific . Importantly, however, it appears that short-term morphine treatment does not affect the MDR phenotype of tumour cells.

Leuk Res, 2004 May, 28(5), 449 - 55
Expression of the neural cell adhesion molecule CD56 is not associated with P-glycoprotein overexpression in core-binding factor acute myeloid leukemia; Suvannasankha A et al.; Acute myeloid leukemia (AML) with rearrangement of the core-binding factor (CBF) alpha or beta subunit gene has a favorable prognosis, but CD56 expression in CBFalpha-AML is associated with short disease-free survival . A proposed mechanism is overexpression of the multidrug resistance (MDR) protein P-glycoprotein (Pgp) . CD56 expression, Pgp expression and function, and expression of the additional MDR proteins multidrug resistance protein-1 (MRP-1), lung resistance protein (LRP) and breast cancer resistance protein (BCRP) were studied in pretreatment blasts from 25 CBF-AML patients . CD56 expression was frequent in CBFalpha but rare in CBFbeta, and Pgp expression and function were frequent in both subtypes . CD56 expression did not correlate with Pgp expression or function, nor with expression of the other MDR proteins . Treatment failure associated with CD56 expression in CBFalpha-AML is not likely attributable to Pgp.

Epilepsy Res, 2004 Jan, 58(1), 67 - 79
Increased expression of the multidrug transporter P-glycoprotein in limbic brain regions after amygdala-kindled seizures in rats; Volk HA et al.; Increased expression of the multidrug transporter P-glycoprotein (Pgp; ABCB1) has previously been found in epileptogenic brain tissue from patients with pharmacoresistant temporal lobe epilepsy (TLE) as well as in the hippocampus and other limbic brain regions in the rat kainate model of TLE . Approaches to the quantification of Pgp expression have mainly been based on subjective visual estimation of the level of Pgp immunoreactivity in brain sections . In the present study, computer-assisted image analysis based on optical density (OD) measurements was used to examine immunohistochemical expression of Pgp in the kindling model of TLE . Sections from kainate-treated rats were used for comparison . Using diaminobenzidine as chromogen, Pgp was exclusively located in brain capillary endothelial cells, which was confirmed by double-labeling with an antibody against the endothelial glucose transporter (GLUT-1) . After kainate-induced seizures, the intensity of endothelial Pgp staining significantly increased by 70-80% in the dentate gyrus . A significant, albeit less marked increase in Pgp expression in this area was also seen after amygdala-kindled seizures . Furthermore, Pgp was upregulated after kindling in the hilus of the dentate gyrus, the CA1 and CA3 sectors of the hippocampus, and the piriform and cerebral cortex . In kindled rats, most Pgp alterations occurred ipsilateral to the electrode in the basolateral amygdala . The data demonstrate that computer-assisted image analysis using OD is an accurate and rapid method to determine the relative amount of Pgp protein in brain sections and the effects of seizures on this multidrug transporter . The fact that Pgp overexpression in brain capillary endothelial cells occurs in two established models of difficult-to-treat TLE substantiates the notion that seizure-induced upregulation of Pgp contributes to multidrug resistance (MDR) in epilepsy.

Epilepsy Res, 2004 Jan, 58(1), 53 - 66
Valproic acid uptake by bovine brain microvessel endothelial cells: role of active efflux transport; Gibbs JP et al.; The basis for low brain permeability of valproic acid (VPA) appears to be the result of efflux transport at the blood-brain barrier (BBB); however, the identity of the putative efflux transporter has not been investigated . The objective of our studies was to determine whether the multidrug resistance-associated protein (MRP) might be involved in efflux transport of VPA . Brain microvessel endothelial cells (BMEC) were isolated from cow brains and grown to confluence . MRP messenger RNA (mRNA) in BMEC were verified by reverse transcriptase-polymerase chain reaction (RT-PCR) . Functional activity was demonstrated using the steady-state retention of calcein and MRP inhibitors, indomethacin (IND) and probenecid (PRB) . Probenecid (0.50 mM) and indomethacin (10 microM) produced a 26 and 13% ( P<0.05 ) elevation in steady-state cellular VPA uptake following a 30-min-incubation with tracer 3H-VPA and 30 microM cold VPA . In contrast, at higher concentrations of probenecid (2 mM) and indomethacin (500 microM), an 11 and 31% reduction in VPA uptake was observed . The biphasic pattern of VPA uptake suggested concurrent inhibition of uptake and efflux transporters by the inhibitor with differing sensitivities, i.e . the efflux transporter being more susceptible to inhibition than the influx transporter . Similar results were obtained in the MRP overexpressing cell line A549 . Overall, the results suggest that MRP(s) is(are) involved in the efflux transport of VPA, but do not preclude the possible contribution(s) of other organic anion transporters . The findings also adds to the growing evidence that up-regulation of active drug efflux transporters at the BBB may contribute to the development of drug resistance to antiepileptic drug therapy.

J Biomed Opt, 2004 Mar-Apr, 9(2), 385 - 94
Mitochondria in tumor cells studied by laser scanning confocal microscopy; Villa AM et al.; We present here a confocal fluorescence microscopy study of mitochondria in sensitive and resistant carcinoma cells by using two potentiometric probes of mitochondria, rhodamine 123 (R123) and dimethylaminostyryl-methylpyridiniumiodine . We have found that active mitochondria in sensitive MCF-7 and multidrug resistant MCF-7/DX carcinoma cells are very different in localization and morphology . In sensitive cells active mitochondria are found in the perinuclear region, whereas in the multidrug resistance (MDR) subline they are confined to the cell periphery . Interestingly, the MDR revertant verapamil has been found to restore in MCF-7/DX cells the same pattern of active mitochondria seen in sensitive cells . We have also studied R123 in human lung carcinoma A549 cells, which display a low responsivity to doxorubicin, and overexpress the lung resistance-related protein . In addition to perinuclear mitochondria, peripheral mitochondria with weaker fluorescence can be seen in this cell line . Interestingly, in the two examined carcinoma lines we have been able to recognize by image analysis a common new star-lobed morphology . Our results indicate that in resistant carcinoma cells two populations of mitochondria coexist with different localization, morphology, and activity . (c) 2004 Society of Photo-Optical Instrumentation Engineers.

J Med Assoc Thai, 2004 Feb, 87(2), 180 - 9
Direct identification of Mycobacterium tuberculosis from sputum on Ziehl-Neelsen acid fast stained slides by use of silica-based filter combined with polymerase chain reaction assay; Tansuphasiri U et al.; This paper describes a method for isolation of deoxyribonucleic acid (DNA) from Ziehl-Neelsen stained sputum smears on glass slides; and isolated DNA was used for the IS6110 polymerase chain reaction (PCR)-based identification of M . tuberculosis . A total of 221 samples from newly diagnosed suspected tuberculosis cases were first examined by microscopic examination . For DNA extraction by silica-based filter, a home-made modified spin column gave the efficacy as did the nucleospin tissue reagent kit and therefore was selected for PCR template preparation . The extracted DNA was amplified by the IS6110 PCR using a primer pair that amplifies a 377-bp target, and the product was analyzed by agarose gel electrophoresis with confirmation by Southern blot hybridization . In comparison with culture, PCR with template prepared by the silica based filter showed overall sensitivity and specificity of 91.7 and 100 per cent, respectively . This study used the over one year and less than one year slides samples to study the effect of storage time . In the more than one year storage group, PCR assay gave a sensitivity and specificity of 83.3 and 100 per cent, respectively . In conclusion, the applicability of the PCR directly to DNA extracted from Ziehl-Neelsen stained smears could become a valuable alternative approach for rapid identification of M . tuberculosis, and could be used to evaluate quality of the control of local laboratories in tuberculosis (TB) screening and solve the problem of specimen transportation . In addition, the method could be used in retrospective studies involving a wide range of PCR-based analyses, such as detection of rifampicin resistant gene in multidrug-resistant tuberculosis (MDR-TB) study.

Nucl Med Commun, 2004 Jan, 25(1), 19 - 27
P-glycoprotein expression is associated with FDG uptake and cell differentiation in patients with untreated lung cancer; Higashi K et al.; In vitro studies demonstrated that the accumulation of 2-deoxy-D-glucose was reduced in multidrug resistant cell lines . In animal study, it has been suggested that 2-{18F}fluoro-2-deoxy-D-glucose (FDG) may be a marker for multidrug resistance (MDR) . The aim of this clinical study was to compare MDR characteristics by immunohistochemical assay with FDG uptake and investigate whether FDG is a marker for MDR in patients with untreated lung cancer . Forty-seven patients with 49 untreated lung cancers, who had undergone both preoperative FDG PET imaging and thoracotomy, were enrolled in this study . Before surgery, FDG PET was performed 40 min after injection, and standardized uptake values (SUVs) were obtained . Patients were classified into low-SUV (< or = 5) and high-SUV (> 5) groups . After surgery, the expression of P-glycoprotein (Pgp) was investigated by immunohistochemistry, and the lung cancer FDG uptake was analysed for possible association with Pgp expression . The strong intensity of Pgp immunoreactivity was seen only in the low-SUV group . The percentage of the Pgp positive area was significantly lower in the high-SUV group (21.7 +/- 13.4%) than in the low-SUV group (44.1 +/- 29.7%) (P = 0.015) . In the high-SUV group, the percentage of Pgp positive area did not exceed 50% . In lung adenocarcinoma, the intensity of Pgp immunoreactivity and the percentage of Pgp positive area increased with degree of cell differentiation, while FDG uptake decreased with degree of cell differentiation . Bronchioloalveolar carcinoma, in particular, showed overexpression of Pgp and modest uptake of FDG . In conclusion, Pgp expression was found to be inversely related to FDG uptake in untreated lung cancer . Pgp expression correlated with the degree of cell differentiation in adenocarcinomas, whilst FDG uptake was inversely related to cell differentiation . FDG may be an in vivo marker for MDR in patients with untreated lung cancer.

Cancer Chemother Pharmacol, 2004 May, 53(5), 363 - 9 Epub 2004 Jan 27.
Broad-spectrum modulation of ATP-binding cassette transport proteins by the taxane derivatives ortataxel (IDN-5109, BAY 59-8862) and tRA96023; Minderman H et al.; PURPOSE: The taxanes paclitaxel and docetaxel are substrates for P-glycoprotein (Pgp), an ATP-binding cassette (ABC) transport protein associated with multidrug resistance (MDR) . In contrast, the synthetic taxane ortataxel (BAY 59-8862, IDN-5109) is effective against Pgp-expressing cells by virtue of modulation of Pgp-mediated transport . The synthetic taxane tRA96023 also modulates Pgp and is noncytotoxic due to removal of the tubulin-binding side chain at the C-13 position of the taxane backbone . We studied the effects of ortataxel and tRA96023 on the other MDR-associated ABC transport proteins, multidrug resistance protein (MRP-1) and breast cancer resistance protein (BCRP, MXR, ABCG2) . METHODS: Modulation of mitoxantrone, daunorubicin and doxorubicin retention and cytotoxicity by ortataxel and tRA96023 was studied in established cell lines overexpressing Pgp, MRP-1 and wild type (BCRP(R482)) and mutant (BCRP(R482T)) BCRP, and was compared with modulation by the established Pgp-, MRP-1- and BCRP-specific modulators PSC-833, probenecid and fumitremorgin C, respectively . RESULTS: Ortataxel effectively modulated drug retention and cytotoxicity in cell lines overexpressing MRP-1 and BCRP(R482), in addition to Pgp . tRA96023 modulated drug retention and cytotoxicity in cell lines overexpressing BCRP(R482) and Pgp, but not those overexpressing MRP-1 . Neither ortataxel nor tRA96023 modulated BCRP(R482T) . CONCLUSIONS: The synthetic taxane derivatives ortataxel and tRA96023 are broad-spectrum ABC protein modulators . Further studies will seek to identify a noncytotoxic synthetic taxane that modulates Pgp, MRP-1 and BCRP.

Clin Pharmacol Ther, 2004 Apr, 75(4), 352 - 61
Tacrolimus therapy according to mucosal MDR1 levels in small-bowel transplant recipients; Masuda S et al.; To clarify the clinical applicability of intestinal absorptive barriers, we have quantified messenger ribonucleic acid (mRNA) expression levels of multidrug resistance 1 (MDR1) protein and cytochrome P450 (CYP) 3A4 in intestinal biopsy specimens from 2 small-bowel transplant recipients . Postoperative immunosuppressive therapy was started with intravenous and oral administrations of tacrolimus and a small amount of steroids . The daily dosage of tacrolimus was modified mainly on the basis of trough levels . After confirmation that the enterocyte MDR1 level was decreasing, tacrolimus was administered via the oral route only . The mRNA levels in the biopsy specimens varied widely throughout the period . With high-dose steroid-pulse treatment, the enterocyte mRNA expression of CYP3A4, but not of MDR1, was markedly enhanced . The mRNA levels of MDR1, but not CYP3A4, correlated well with the concentration/oral dose ratio and the oral dosage of tacrolimus . The good progress after transplantation in both cases suggested that monitoring the change in expression of MDR1 mRNA in the graft intestine might be helpful for understanding the pharmacokinetic profile and determining when to change the route of tacrolimus administration in small-bowel transplant recipients.

Zhonghua Yi Xue Za Zhi, 2004 Feb 17, 84(4), 323 - 8
{Mechanisms of the drug resistance of a 2', 2-difluorodeoxycytide (gemcitabine)-resistant variant of the human lung adenocarcinoma cell line}; Dong M et al.; OBJECTIVE: To investigate the mechanisms of the drug resistance of gemcitabine resistance variant of the human lung adenocarcinoma cell line A549-Gem.METHOD: Immunohistochemistry and RT-PCR were used to tested the expression of P-glycoprotein and transcription of mRNA of multidrug resistance gene and deoxycytidine kinase . Using a cDNA microarray compared the expression profiles between the resistant A549-Gem and the parent cell line A549 . RESULTS: The A549-Gem shown slight positive expression of P-glycoprotein compared with positive control by immunohistochemistry, but A549 was negative for P-gp . RT-PCR amplified mRNA, using specific dCK and multidrug resistant gene 1 (mdr1) primers, demonstrated that A549-Gem express amplicon of mdr1 but no products of mdr1 was detected in A549 . In the parent cell line, dCK mRNA amplication product could be detected, but did not found in resistant cells . About 18.8% of the total DNA elements had substantially altered level of expression in resistant A549-Gem compared with A549 by cDNA microarray . The biochemical functions of the different expressed genes are diverse and include oncogene and tumor suppressor gene, cell cycle regulator, heat shock protein, apoptotic and antiapoptotic factors, DNA transcription factors, DNA repair and recombination factors, signal transduction genes, protein translation genes, as well as a large number of metabolic genes . Several cluster genes overexpressed in resistant cell line, including ubiquitin-proteasome system, zinc finger protein, glutaredoxin and heat shock protein, may be related to the mechanisms of gemcitabine resistance.CONCLUSION: The mechanisms of resistance to gemcitabine are multifactorial, may include multidrug resistance and dCK deficiency . Differential expression genes monitored by cDNA microarray between A549-Gem and A549 may be related to the mechanisms of gemcitabine resistance in human lung cancer and potentially suggest the prevalence of expression of these genes in drug resistance . The use of cDNA microarray has provided a global view of the response of lung cancer cells to gemcitabine at the genomic level, it should be suitable for examining the development of drug resistance in cancer.

Hepatology, 2004 Apr, 39(4), 1099 - 109
Kupffer cells alter organic anion transport through multidrug resistance protein 2 in the post-cold ischemic rat liver; Kudo A et al.; Although Kupffer cells (KCs) may play a crucial role in post-cold ischemic hepatocellular injury, their role in nonnecrotic graft dysfunction remains unknown . This study examined reveal the role of KC in post-cold ischemic liver grafts . Rat livers treated with or without liposome-encapsulated dichloromethylene diphosphonate, a KC-depleting reagent, were stored in University of Wisconsin (UW) solution at 4 degrees C for 8 to 24 hours and reperfused while monitoring biliary output and constituents . The ability of hepatocytes to excrete bile was assessed through laser-confocal microfluorography in situ . Cold ischemia-reperfused grafts decreased their bile output significantly at 8 hours without any notable cell injury . This event coincided with impaired excretion of glutathione and bilirubin-IXalpha (BR-IXalpha), suggesting delayed transport of these organic anions . We examined whether intracellular relocalization of multidrug resistance protein-2 (Mrp2) occurred . Kinetic analyses for biliary excretion of carboxyfluorescein, a fluoroprobe excreted through this transporter, revealed significant delay of dye excretion from hepatocytes into bile canaliculi . The KC-depleting treatment significantly attenuated this decline in biliary anion transport mediated through Mrp2 in the 8-hour cold ischemic grafts via redistribution of Mrp2 from the cytoplasm to the canalicular membrane . Furthermore, thromboxane A(2) (TXA(2)) synthase in KC appeared involved as blocking this enzyme improved 5-carboxyfluorescein excretion while cytoplasmic internalization of Mrp2 disappeared in the KC-depleting grafts . In conclusion, KC activation is an important determinant of nonnecrotic hepatocellular dysfunction, jeopardizing homeostasis of the detoxification capacity and organic anion metabolism of the post-cold ischemic grafts.

Gastroenterology, 2004 Apr, 126(4), 1044 - 53
Consequences of bile duct obstruction on intestinal expression and function of multidrug resistance-associated protein 2; Dietrich CG et al.; BACKGROUND & AIMS: Multidrug resistance-associated protein 2 (MRP2), a transporter of organic anions in hepatocytes, renal epithelial cells, and enterocytes, is differentially regulated in liver and kidney during cholestasis, but little is known about its regulation in the intestine . METHODS: We investigated duodenal protein expression of MRP2 in male Sprague-Dawley rats with bile duct ligation (BDL) or biliary diversion as well as in 20 cholestatic patients with biliary obstruction . RESULTS: In biliary obstruction, but not biliary depletion, intestinal Mrp2 protein mass was reduced to 9.3% +/- 5.5% of controls and mRNA to 40.5% +/- 20.8% of controls after 7 days . Binding of RXR alpha:RAR alpha heterodimers to the Mrp2 promoter element was significantly reduced in BDL rats . Cytokine blockade identified IL-1 beta as the responsible inducer of Mrp2 down-regulation . In humans with obstructive cholestasis, intestinal MRP2 protein expression was reduced to 27.3% +/- 20.3% of control patients; this reduction correlated with the duration of cholestasis and was reversible after reconstitution of bile flow by stenting of the common bile duct . However, no significant differences in MRP2 mRNA levels were detected by RT-PCR in humans . Intestinal protein expression of P-glycoprotein, breast cancer resistance protein (BCRP), and MRP3 was unchanged . In BDL rats, oral bioavailability of the Mrp2 substrate and food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) was elevated 2.5 times compared with sham-operated rats . CONCLUSIONS: Cholestasis promotes down-regulation of MRP2 expression in the duodenum of rats and humans . Selective down-regulation during cholestasis might be the consequence of species-specific transcriptional and posttranscriptional mechanisms and contributes to higher bioavailability of a food-derived carcinogen.

Mem Inst Oswaldo Cruz, 2004 Feb, 99(1), 111 - 3 Epub 2004 Mar 31.
Drug susceptibility testing of Mycobacterium tuberculosis by the broth microdilution method with 7H9 broth; Coban AY et al.; In this study, we have evaluated the broth microdilution method (BMM) for susceptibility testing of Mycobacterium tuberculosis . A total of 43 clinical isolates of M . tuberculosis and H37Rv as a control strain were studied . All isolates were tested by the proportion method and the BMM for isoniazid (INH), rifampicin (RIF), streptomycin (STR), and ethambutol (ETM) . The proportion method was carried out according to the National Committee for Clinical Laboratory Standards (NCCLS) on Lowenstein-Jensen (LJ) medium . The BMM was carried out using 7H9 broth with 96 well-plates . All strains were tested at 3.2-0.05 micro g/ml, 16-0.25 micro g/ml, 32-0.5 micro g/ml, and 32-0.5 micro g/ml concentrations for INH, RIF, STR, and ETM, respectively . When the BMM was compared with the proportion method, sensitivity was 100, 100, 96.9, and 90.2%, while specificity was 100, 85.7, 90.9, and 100% for INH, RIF, STR, and ETM, respectively . The plates were examined 7, 10, 14, and 21 days after incubation . The majority of the result were obtained at 14th days after incubation, while the proportion method result were ended in 21-28 days . According to our results, it may be suggested that the BMM is suitable for early determining of multidrug-resistance-M . tuberculosis strains in developed or developing countries.

Anticancer Drugs, 2004 Apr, 15(4), 303 - 9
Carvedilol: a new candidate for reversal of MDR1/P-glycoprotein-mediated multidrug resistance; Takara K et al.; In 1983, carvedilol {1-{carbazolyl-(4)-oxy}-3-{(2-methoxyphenoxyethyl)amino}-2-propanol} was designed and developed as a beta-adrenoceptor antagonist with vasodilating activity for efficacious and safe treatment of hypertension and coronary artery disease . Carvedilol belongs to the 'third generation' of beta-adrenoceptor antagonists and shows selectivity for the beta1- rather than beta2-adrenoceptor . Carvedilol is also an alpha1-blocking agents, with around 2- to 3-fold more selectivity for beta1- than alpha1-adrenoceptors . This degree of alpha1-blockade is responsible for the moderate vasodilator properties of carvedilol, being different from other beta-adrenoceptor antagonists . In addition, carvedilol is a potent antioxidant, with a 10-fold greater activity than vitamin E . Some carvedilol metabolites found in human plasma also exhibit antioxidative activity approximately 50- to 100-fold greater than carvedilol and other antioxidants . These unique properties of carvedilol, i.e . adrenergic (beta1, beta2 and alpha1) blockade and antioxidative activity, may be important in preventing progressive deterioration of left ventricular dysfunction and chronic heart failure . Recently, carvedilol has been demonstrated to reverse multidrug resistance (MDR) to anticancer drugs in tumor cells in vitro and its reversal effects were comparable with verapamil, which has been used in the first clinical trial for the reversal of MDR . This review introduces the reversal activity and usefulness against MDR, as well as an overview of the pharmacological and pharmacokinetic properties, of carvedilol.

Cell Biochem Biophys, 2004, 40(2), 185 - 206
Plasmalemmal Vacuolar-Type H+-ATPase in Cancer Biology; Sennoune SR et al.; Vacuolar-type H+-adenosine triphosphatase (V-ATPase) is one of the most fundamental enzymes in nature . V-ATPases are responsible for the regulation of proton concentration in the intracellular acidic compartments . It has similar structure with the mitochondrial F0F1-ATP synthase (F-ATPase) . dagger The V-ATPases are composed of multiple subunits and have various physiological functions, including membrane and organelle protein sorting, neurotransmitter uptake, cellular degradative processes, and cytosolic pH regulation . The V-ATPases have been involved in multidrug resistance . Recently, plasma membrane V-ATPases have been involved in regulation of extracellular acidity, essential for cellular invasiveness and proliferation in tumor metastasis . The current knowledge regarding the structure and function of V-ATPase and its role in cancer biology is discussed.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2004 Feb, 26(1), 43 - 6
{Rat model for the multidrug resistant glioma cell line}; Zhang J et al.; OBJECTIVE: To evaluate the animal model of the multidrug resistant glioma cell line C6/adr for further in vivo studies . METHODS: The rat glioma cells C6 and multidrug resistance cells C6/adr were cultured in vitro and implanted into the brain of S-D rats . After implantation, all these animals were examined continually with magnetic resonance imaging (MRI) and histological examination . The growth procedure of intracranial implanted glioma and the survival span of the animal model were evaluated . The statistical analysis was made between the survival data of the two cell lines . RESULTS: The symptoms of intracranial hypertension did not occur until 4 weeks after inoculation . The MRI findings of the implanted glioma in the rat brain were much earlier than the abnormal behavior observed . Pathological results after inoculation demonstrated the MRI findings . The two cell lines had similar growth characteristics and no significant differences in survival times . CONCLUSION: These results suggest that by means of MRI and histology the growth procedure of the implanted glioma in vivo be successfully observed . All these data will proved to be a useful basis for study of glioma in vivo.

Biochem Cell Biol, 2004 Feb, 82(1), 156 - 69
Genetic analysis of intracellular aminoglycerophospholipid traffic; Voelker DR; Inter- and intramembrane phospholipid transport processes are central features of membrane biogenesis and homeostasis . Relatively recent successes in the molecular genetic analysis of aminoglycerophospholipid transport processes in both yeast and mammalian cells are now providing important new information defining specific protein and lipid components that participate in these reactions . Studies focused on phosphatidylserine (PtdSer) transport to the mitochondria reveal that the process is regulated by ubiquitination . In addition, a specific mutation disrupts PtdSer transport between mitochondrial membranes . Analysis of PtdSer transport from the endoplasmic reticulum to the locus of PtdSer decarboxylase 2 demonstrates the requirement for a phosphatidylinositol-4-kinase, a phosphatidylinositol-binding protein, and the C2 domain of the decarboxylase . Examination of NBD-phosphatidylcholine transport demonstrates the involvement of the prevacuolar compartment and a requirement for multiple genes involved in regulating vacuolar protein sorting for transport of the lipid to the vacuole . In intramembrane transport, multiple genes are now identified including those encoding multidrug resistant protein family members, DNF family members, ATP binding cassette transporters, and pleiotropic drug resistance family members . The scramblase family constitutes a collection of putative transmembrane transporters that function in an ATP-independent manner . The genetic analysis of lipid traffic is uncovering new molecules involved in all aspects of the regulation and execution of the transport steps and also providing essential tools to critically test the involvement of numerous candidate molecules.

Pathobiology, 2004, 71(3), 123 - 8
Modulation of multidrug-resistance-associated P-glycoprotein in human U-87 MG and HUV-ECC cells with antisense oligodeoxynucleotides to MDR1 mRNA; Rittierodt M et al.; OBJECTIVE: Glioblastoma is the highest dedifferentiated form of astrocytic brain tumors, and it is refractory to chemotherapy in most cases . To improve the clinical outcome of such tumors, new therapeutic strategies are needed . While malignancy is mainly associated with a nonfunctional apoptotic pathway, the lack of chemotherapeutic success correlates with overexpression of the multidrug resistance 1 (MDR1) gene product P-glycoprotein (P-gp) . Previous investigations have shown that not only glioblastoma cells but also endothelial cells are important in the response to chemotherapy . The aim of the present investigations was to reduce the expression of P-gp in the human glioblastoma cell line U-87 MG and in the human endothelial cell line HUV-ECC . METHODS: Therefore, these cells were treated with antisense oligodeoxynucleotides (asn-ODN) directed against the P-gp mRNA in order to increase the intracellular retention of doxorubicin (DOX) which had been given previously . RESULTS: Flow cytometry revealed about 4-fold increased intracellular retention of DOX in both asn-ODN-treated cell lines as compared to asn-ODN non-treated cell lines . CONCLUSION: These results suggest that asn-ODN-mediated inhibition of P-gp expression is an efficient way to increase intracellular retention of DOX .

J Org Chem, 2004 Apr 2, 69(7), 2417 - 22
Indium-mediated atom-transfer and reductive radical cyclizations of iodoalkynes: synthesis and biological evaluation of HIV-protease inhibitors; Yanada R et al.; Novel indium-mediated radical cyclization reactions of aliphatic iodoalkynes have been studied . Treatment of iodoalkynes with a catalytic amount of In (0.1 equiv) and I(2) (0.05 equiv) promotes atom-transfer 5-exo cyclization to give five-membered alkenyl iodides . In contrast, reaction with In (2 equiv) and I(2) (1 equiv) yields reductive 5-exo cyclization products via the same 5-exo cyclization . Both processes are most likely initiated by low-valent indium species . To demonstrate versatility of these reactions, optically active HIV protease inhibitors were synthesized by this reductive cyclization method . Among them, several products, which contain a hydroxyethylamine dipeptide isostere as a transition state-mimicking substructure, proved to possess potent activity (IC(50) = 5-39 nM) against a wide spectrum of HIV strains, including multidrug-resistant variants.

Cell Mol Neurobiol, 2004 Feb, 24(1), 101 - 7
Transient expression of MDR-1/P-glycoprotein in a model of partial cortical devascularization; Ramos AJ et al.; 1 . MDR-1 gene product confer to expressing cells the multidrug resistance phenotype to a broad range of drugs and xenobiotics . 2 . It is known that different stress signals are able to induce MDR-1 expression through different promoters . 3 . In a rat model of ischemia by partial cortical devascularization we studied the expression profile and the cellular localization of MDR-1 after 1, 3, 7, 14 and 28 days post lesion (DPL) . 4 . Using two different antibody clones we found that MDR-1 is expressed in cortical and striatal neurons ipsilateral to the devascularizing lesion, starting at 1DPL, showing a maximum at 7DPL to be thereafter reduced until undetectable levels by 28DPL . 5 . MDR-1 expression may be defining a neuronal subset with a particular pharmacological profile.

Cell Mol Neurobiol, 2004 Feb, 24(1), 77 - 85
Neuronal and glial expression of the multidrug resistance gene product in an experimental epilepsy model; Lazarowski A et al.; 1 . Failure of anticonvulsive drugs to prevent seizures is a common complication of epilepsy treatment known as drug-refractory epilepsy but their causes are not well understood . It is hypothesized that the multidrug resistance P-glycoprotein (Pgp-170), the product of the MDR-1 gene that is normally expressed in several excretory tissues including the blood brain barrier, may be participating in the refractory epilepsy . 2 . Using two monoclonal antibodies against Pgp-170, we investigated the expression and cellular distribution of this protein in the rat brain during experimentally induced epilepsy . Repeated seizures were induced in male Wistar rats by daily administration of 3-mercaptopropionic acid (MP) 45 mg/kg i.p . for either 4 days (MP-4) or 7 days (MP-7) . Control rats received an equivalent volume of vehicle . One day after the last injection, rats were sacrificed and brains were processed for immunohistochemistry for Pgp-170 . As it was previously described, Pgp-170 immunostaining was observed in some brain capillary endothelial cells of animals from control group . 3 . Increased Pgp-170 immunoreactivity was detected in MP-treated animals . Besides the Pgp-170 expressed in blood vessels, neuronal, and glial immunostaining was detected in hippocampus, striatum, and cerebral cortex of MP-treated rats . Pgp-170 immunolabeled neurons and glial cells were observed in a nonhomogeneous distribution . MP-4 animals presented a very prominent Pgp-170 immunostaining in the capillary endothelium, surrounding astrocytes and some neighboring neurons while MP-7 group showed increased neuronal labeling . 4 . Our results demonstrate a selective increase in Pgp-170 immunoreactivity in the brain capillary endothelial cells, astrocytes, and neurons during repetitive MP-induced seizures . 5 . The role for this Pgp-170 overexpression in endothelium and astrocytes as a clearance mechanism in the refractory epilepsy, and the consequences of neuronal Pgp-170 expression remain to be disclosed.

Thorax, 2004 Apr, 59(4), 279 - 85
Outbreak of isoniazid resistant tuberculosis in north London; Ruddy MC et al.; BACKGROUND: A description is given of a major outbreak of isoniazid monoresistant tuberculosis (TB) chiefly in north London, including prisons . The earliest case was diagnosed in 1995 with most cases appearing after 1999 . METHODS: Confirmation of a local cluster of cases was confirmed by restriction fragment length polymorphism (RFLP IS6110) typing or "rapid epidemiological typing" (RAPET) . Further cases were found by retrospective analysis of existing databases, prospective screening of new isolates, and targeted epidemiological case detection including questionnaire analysis . RESULTS: By the end of 2001, 70 confirmed cases in London had been linked with a further 13 clinical cases in contacts and nine epidemiologically linked cases outside London . The epidemic curve suggests that the peak of the outbreak has not yet been reached . Cases in the outbreak largely belong to a social group of young adults of mixed ethnic backgrounds including several individuals from professional/business backgrounds . Compared with other cases of TB reported to the enhanced surveillance scheme in London during 1999-2001, the cases are more likely to be of white (26/70 (37%) v 1308/7666 (17%)) or black Caribbean ethnicity (17/70 (24%) v 312/7666 (4%)), born in the UK (41/70 (59%) v 1335/7666 (17%)), and male (52/70 (74%) v 4195/7666 (55%)) . Drug misuse and/or prison detention are factors common to many cases . CONCLUSIONS: The investigation of the outbreak revealed significant problems on an individual patient and population based level including difficulties with contact tracing, compliance, and the risk of developing multidrug resistance . This incident has demonstrated the value of molecular strain typing in investigating an extensive outbreak of TB . This is the first documented outbreak involving a UK prison.

J Biol Chem, 2004 Jun 11, 279(24), 25527 - 34 Epub 2004 Mar 26.
Folate deprivation results in the loss of breast cancer resistance protein (BCRP/ABCG2) expression . A role for BCRP in cellular folate homeostasis; Ifergan I et al.; Breast cancer resistance protein (BCRP/ABCG2) is currently the only ABC transporter that exports mono- and polyglutamates of folates and methotrexate (MTX) . Here we explored the relationship between cellular folate status and BCRP expression . Toward this end, MCF-7 breast cancer cells, with low BCRP and moderate multidrug resistance protein 1 (MRP1/ABCC1) levels, and their mitoxantrone (MR)-resistant MCF-7/MR subline, with BCRP overexpression and low MRP1 levels, were gradually deprived of folic acid from 2.3 microm to 3 nm resulting in the sublines MCF-7/LF and MCF-7/MR-LF . These cell lines expressed only residual BCRP mRNA and protein levels and retained a poor MRP2 (ABCC2) through MRP5 (ABCC5) expression . Furthermore, MCF-7/MR-LF cells also displayed 5-fold decreased MRP1 levels relative to MCF-7/MR cells . In contrast, BCRP overexpression was largely retained in MCF-7/MR cells grown in MR-free medium containing 2.3 microm folic acid . Loss of BCRP expression in MCF-7/LF and MCF-7/MR-LF cells resulted in the following: (a) a prominent decrease in the efflux of Hoechst 33342, a BCRP substrate; (b) an approximately 2-fold increase in MR accumulation as revealed by flow cytometry; this was accompanied by a 2.5- and approximately 84-fold increased MR sensitivity in these cell lines, respectively . Consistently, Ko143, a specific BCRP inhibitor, rendered MCF-7 and MCF-7/MR cells 2.1- and approximately 16.4-fold more sensitive to MR, respectively . Loss of BCRP expression also resulted in the following: (c) an identical MTX sensitivity in these cell lines thereby losing the approximately 28-fold MTX resistance of the MCF-7/MR cells; (d) an approximately 2-fold increase in the 4- and 24-h accumulation of {(3)H}folic acid . Furthermore, MCF-7/MR-LF cells displayed a significant increase in folylpoly-gamma-glutamate synthetase activity . Hence, consistent with the mono- and polyglutamate folate exporter function of BCRP, down-regulation of BCRP and increased folylpoly-gamma-glutamate synthetase activity appear to be crucial components of cellular adaptation to folate deficiency conditions . This is the first evidence for the possible role of BCRP in the maintenance of cellular folate homeostasis.

J Biol Chem, 2004 Jun 4, 279(23), 24569 - 77 Epub 2004 Mar 25.
Molecular determinants of multi-nucleoside analogue resistance in HIV-1 reverse transcriptases containing a dipeptide insertion in the fingers subdomain: effect of mutations D67N and T215Y on removal of thymidine nucleotide analogues from blocked DNA primers; Matamoros T et al.; Human immunodeficiency virus type 1 isolates having dipeptide insertions in the fingers subdomain of the reverse transcriptase (RT) show high level resistance to 3 '-azido-3 '-deoxythymidine (AZT) and other nucleoside analogues . Insertions are usually associated with thymidine analogue resistance mutations, such as T215Y . The resistance phenotype correlates with increased ATP-dependent phosphorolytic activity, which facilitates removal of thymidine analogues from inhibitor-terminated primers . In this report, we show that substituting Thr, Ser, or Asn for Tyr-215 in a multidrug-resistant RT, bearing a Ser-Ser insertion between codons 69 and 70, leads to AZT and stavudine resensitization through the loss of the ATP-mediated removal activity . The mutation D67N, which is rarely found in insertion-containing strains, had no effect on excision and a minor influence on resistance . Substituting Tyr-215 had a larger effect than deleting the dipeptide insertion . The presence of both the insertion and mutation T215Y in the wild-type BH10 RT conferred significant ATP-mediated removal activity and moderate resistance to AZT . However, resistance levels and unblocking activities were lower than those observed with the multidrug-resistant enzyme . Removal reactions can be inhibited by the next complementary dNTP . Both Tyr-215 and the dipeptide insertion affect RT-DNA.DNA-dNTP ternary complex formation, an effect that was not detected in the presence of foscarnet . Based on crystal structures of binary and ternary complexes of HIV-1 RT, we propose that Tyr-215 exerts its action by facilitating a proper orientation of the pyrophosphate donor molecule, whereas the effects on dNTP binding are indirect and could be related to significant conformational changes occurring during polymerization.

Gynecol Oncol, 2004 Apr, 93(1), 144 - 8
Phase II trial of the combination of bryostatin-1 and cisplatin in advanced or recurrent carcinoma of the cervix: a New York Gynecologic Oncology Group study; Nezhat F et al.; OBJECTIVES: Bryostatin-1 is a macrocyclic lactone that has been shown to regulate protein kinase C (PKC) activity and thereby potentially inhibit tumor invasion, angiogenesis, cell adhesion, and multidrug resistance . In preclinical experiments, bryostatin-1 induces tumor growth inhibition and enhances cytotoxicity when combined with other agents including cisplatin in cervical cancer cells . It was therefore anticipated that combination bryostatin-1-cisplatin therapy would be effective in patients with cervical cancer . The current study was conducted to evaluate this therapeutic approach in patients with recurrent or advanced-stage cervical carcinoma . METHODS: An IRB-approved New York Gynecologic Oncology Group (NYGOG) trial was activated for patients with a histological diagnosis of metastatic cervical cancer or in patients with recurrent disease not eligible for surgery or radiation . Enrolled patients received bryostatin-1 (50-65 microg/m(2)) as a 1-h infusion followed by cisplatin (50 mg/m(2)) . The combined treatment was administered every 21 days . RESULTS: Fourteen patients were enrolled . The majority of patients had squamous cell carcinoma . Ten out of fourteen patients had recurrent disease . Fifty percent of the patients received bryostatin at 50 microg/m(2) and 50% received bryostatin at 65 microg/m(2) . Seventy-one percent completed two cycles of treatment . The most common grade II-III toxicities were myalgia, anemia, and nausea or vomiting . One patient developed a hypersensitivity reaction and one developed grade III nephrotoxicity . Seventy-one percent (10/14) of patients were evaluated for tumor response . Eight out of ten (80%) of patients had progressive disease and 2/10 (20%) had stable disease . There were no treatment responses . CONCLUSIONS: Despite promising preclinical data, this clinical trial indicates that the combination of cisplatin and bryostatin-1 at the doses and schedule used is not effective in patients with advanced-stage or recurrent cervical cancer . There is even the possibility of therapeutic antagonism . The development of a serum assay for bryostatin-1 and additional mechanistic studies would be useful for future bryostatin clinical trials.

Gynecol Oncol, 2004 Apr, 93(1), 98 - 106
Expression of multidrug resistance-1 protein inversely correlates with paclitaxel response and survival in ovarian cancer patients: a study in serial samples; Penson RT et al.; OBJECTIVES: The role of MDR1 in clinical paclitaxel resistance remains poorly characterized . This study sought to identify the incidence and significance of P-glycoprotein (P-gp) over-expression on survival, tumor response to paclitaxel and the effect of prior cytotoxic exposure on P-gp expression in patients with paired primary and recurrent ovarian cancer samples . METHODS: Retrospective survival analysis . P-gp expression was evaluated immunohistochemically with antibodies c494 and c219 . RESULTS: Thirty-two patients were identified from the tumor registry . Median interval between primary and secondary surgery was 17.9 (5.7-40.9) months . Only five primary tumors (16%) demonstrated +++ staining for P-gp . First-line treatment contained paclitaxel in 17 patients (53%) and 26 patients (81%) had been exposed to P-gp exportable chemotherapy before second surgery . Only seven of the recurrent tumors (22%) were +++ . Only one of seven (14% (95% CI 0-46%)) recurrent tumors with ++ or +++ staining responded to subsequent paclitaxel, while 8 of 10 (80% (CI 46-100%)) recurrent tumors with 0/+ staining responded (P = 0.025) . In multivariate analysis of outcome following second surgery, response to paclitaxel (P = 0.004) and P-gp over-expression (P < 0.001) were significant predictors of survival . CONCLUSIONS: De novo strong P-gp over-expression is uncommon, appears to change little over time or with prior exposure to chemotherapy . However, P-gp over-expression is a significant prognostic factor, and at the time of disease, relapse is inversely correlated with tumor response to paclitaxel.

Biochem Biophys Res Commun, 2004 Apr 23, 317(1), 235 - 43
SP-transcription factors are involved in basal MVP promoter activity and its stimulation by HDAC inhibitors; Steiner E et al.; The major vault protein (MVP) has been implicated in multidrug resistance, cellular transport, and malignant transformation . In this study we aimed to identify crucial MVP promoter elements that regulate MVP expression . By mutation as well as deletion analysis a conserved proximal GC-box element was demonstrated to be essential for basal human MVP promoter transactivation . Binding of Sp-family transcription factors but not AP2 to this element in vitro and in vivo was shown by EMSA and ChIP assays, respectively . Inhibition of GC-box binding by a dominant-negative Sp1-variant and by mithramycin A distinctly attenuated MVP promoter activity . In Sp-null Drosophila cells, the silent human MVP promoter was transactivated by several human Sp-family members . In human cells the MVP promoter was potently stimulated by the histone deacetylase (HDAC) inhibitors butyrate (NaB) and trichostatin A (TSA), resulting in enhanced MVP expression . This stimulation was substantially decreased by mutation of the single GC-box and by application of mithramycin A . Treatment with HDAC inhibitors led to a distinct decrease of Sp1 but increase of Sp3 binding in vivo to the respective promoter sequence as demonstrated by ChIP assays . Summarising, this study identifies variations in Sp-transcription factor binding to a single proximal GC-box element as critical for basal MVP promoter activation and its stimulation by HDAC inhibitors.

Biochem Biophys Res Commun, 2004 Apr 23, 317(1), 46 - 53
Rat multidrug resistance protein 4 (Mrp4, Abcc4): molecular cloning, organ distribution, postnatal renal expression, and chemical inducibility; Chen C et al.; In the present study, we report cloning of the rat Mrp4 cDNA . The cDNA is 4526 bp, containing a 3975 bp open reading frame . The deduced polypeptide has 1325 amino acids and is 83% and 91% identical to human MRP4 and mouse Mrp4, respectively . Phylogenetic analysis revealed that the cloned rat cDNA is closely related to human MRP4 and mouse Mrp4 . Additionally, an alternatively spliced variant, 111 bp shorter than the full-length form, was cloned . Rat Mrp4 mRNA was detectable in 11 tissues examined, with levels being highest in kidney, and lowest in liver . Mrp4 mRNA levels in kidney were higher in males than females, and at birth were about half of adult levels . Mrp4 expression in liver and kidney of rats treated with six classes of microsomal enzyme inducers was examined . Mrp4 mRNA in liver was induced by two electrophile response element activators, namely ethoxyquin and oltipraz.

Oncology (Huntingt), 2002 Mar, 16(3), 343 - 52, 355-6; discussion 357, 362, 365-6
Treatment of acute myelogenous leukemia; Estey EH; The treatment of patients with acute myelogenous leukemia (AML) ranges from palliative care only, to standard therapy, to investigational approaches . Acute myelogenous leukemia is, in fact, several different diseases, and the percentage of clinical responses varies with disease and prognostic subsets . A high proportion of AML patients remain refractory to primary treatment or relapse later . This article aims to identify those subsets of patients who may benefit from standard treatment and those who may be candidates for investigational therapies . It describes standard therapy and reviews new agents and new modes of therapy targeted at CD33, multidrug resistance, hypermethylated (suppressor) genes, and apoptotic pathways . The evolution of new therapies for acute promyelocytic leukemia also is discussed, and the article provides a perspective on the changing role of hematopoietic stem cell transplantation for treatment of AML.

Cancer Biol Ther, 2004 Jun, 3(6), 561 - 5 Epub 2004 Jun 15.
Reversal of Multidrug Resistance of Gastric Cancer Cells by Downregulation of TSG101 with TSG101siRNA; Shen H et al.; We have previously identified tumor susceptibility gene101 (TSG101), overexpressed in vincristine-resistant human gastric adenocarcinoma cell line SGC7901/VCR using a modified subtractive hybridization method and Western blot . In the present study, we constructed two siRNA eukaryotic expression vectors of TSG101 and transfected them into SGC7901/VCR cells to examine whether the down-regulation of TSG101 increased cell sensitivity towards chemotherapeutic drugs . After transfection, the expression of TSG101 was dramatically decreased in TSG101 siRNA transfectants compared with that in parental cells and empty vector control cells . The down-regulation of TSG101 could significantly enhance the sensitivity of SGC7901/VCR cells to vincristine and adriamycin . The capacity of cells to efflux adriamycin markedly decreased in TSG101 siRNA transfectants . These observations suggested that the siRNA constructs of TSG101 we made could effectively down-regulate the expression of TSG101 and reverse the resistant phenotype of gastric cancer cells . The preliminary study suggested that TSG101 might play an important role in multidrug resistance of gastric carcinoma.

Mol Pharmacol, 2004 Apr, 65(4), 897 - 905
Glutathione S-transferase M1 and multidrug resistance protein 1 act in synergy to protect melanoma cells from vincristine effects; Depeille P et al.; Previous studies have shown that glutathione S-transferases (GSTs) can operate in synergy with efflux transporters, multi-drug resistance proteins (MRPs), to confer resistance to several carcinogens, mutagens and anticancer drugs . To address the poorly documented role of the GSTM1 in cancer chemoresistance, we used CAL1 human melanoma cells expressing no endogenous GSTM1 and a high level of MRP1 . Cells were transfected with an expression vector containing the GSTM1 cDNA, and different clones were selected expressing different levels of GSTM1 (RT-PCR, Western blot, and enzyme activity) . Cells overexpressing GSTM1 displayed a 3- to 4-fold increase in resistance to anticancer drugs vincristine (VCR) and chlorambucil (CHB) in proliferation, cytotoxic, and clonogenic survival assays . Inhibitors of MRP1 (sulfinpyrazone, verapamil) and GST (dicumarol, curcumin) completely reversed the GSTM1-associated resistance to VCR, indicating that a MRP efflux function is necessary to potentiate GSTM1-mediated resistance to VCR . Conversely, MRP1 inhibitors had no effect on the sensitivity to CHB . Using immunofluorescence assay, GSTM1 was also shown to protect microtubule network integrity from VCR-induced inhibition of microtubule polymerization . In conclusion, these results show that GSTM1 alone is involved in melanoma resistance to CHB, whereas it can act in synergy with MRP1 to protect cells from toxic effects of VCR.

Biochem Biophys Res Commun, 2004 Apr 16, 316(4), 1173 - 7
Multidrug-resistant tumor cells with decreased malignancy: a role for integrin alphavbeta3; Kozlova NI et al.; We studied whether acquisition of multidrug resistance (MDR) by tumor cells can alter their integrin profile and malignant behavior . Hamster fibroblast cell line HET-SR-2SC-LNM was selected for MDR, yielding the 2SC/20 subline . Compared with the parental cells, the 2SC/20 subline weakly adhered to denatured collagen (dCol) which correlated with decreased expression of alphavbeta3, a dCol receptor . Importantly, 2SC/20 subline demonstrated significantly decreased activity of collagenase MMP-2, lower ability to invade Matrigel, and attenuated metastasis in syngeneic animals . We provide evidence for the first time that selection for MDR can be associated with down-regulation of alphavbeta3 integrin, supporting our recent proof of the pro-apoptotic role of this integrin (Oncogene 20 (2001) 4710) . Lack of alphavbeta3 expression may link cell survival under toxic conditions with decreased malignancy of the resulting drug resistant tumor.

Nucleosides Nucleotides Nucleic Acids, 2004, 23(1-2), 385 - 99
Synthesis and evaluation of a novel synthetic phosphocholine lipid-AZT conjugate that double-targets wild-type and drug resistant variants of HIV; Kucera LS et al.; INK-20, a synthetic phosphocholine lipid-AZT conjugate, was evaluated for antiviral activity against wild-type HIV-1, a matched pair of pre-AZT and post-AZT and multidrug resistant clinical isolates . In addition, it was tested for activity against viruses resistant to nucleoside (AZT, 3TC) and nonnucleoside (nevirapine) reverse transcriptase and protease (saquinavir) inhibitors using the syncytial plaque reduction assay for infectious virus multiplication . The EC50 values were 0.004, and 0.005 microM against wild-type HIV-1 for INK-20 and AZT, respectively . INK-20 showed little or no cytotoxicity when assayed in CEM-SS cells and four other cell types including PBMC . This resulted in a selective index of > 25,000 and > 20,000 for INK-20 and AZT, respectively . When tested against a matched pair of pre-AZT and post-AZT clinical isolates, the EC50 values were 0.01 and 0.03 microM for INK-20 and 0.0005 and 0.33 microM for AZT, respectively . INK-20 had moderate to good activity against two other AZT resistant variants and very good activity against a multi-drug resistant clinical isolate compared to marked resistance of these viruses to AZT alone . INK-20 retained significant activity against viruses resistant to 3TC, nevirapine, and saquinavir . The synthetic phosphocholine lipid-AZT conjugate INK-20 represents a novel class of anti-HIV compounds, which may provide new strategies for the treatment of HIV drug-resistant variants.

Biochem Pharmacol, 2004 Apr 15, 67(8), 1541 - 8
Folate concentration dependent transport activity of the Multidrug Resistance Protein 1 (ABCC1); Hooijberg JH et al.; The Multidrug Resistance Protein MRP1 (ABCC1) can confer resistance to a variety of therapeutic drugs . In addition, MRP1/ABCC1 mediates cellular export of natural folates, such as folic acid and l-leucovorin . In this study we determined whether cellular folate status affected the functional activity of MRP1/ABCC1 mediated efflux of an established substrate, the anthracycline daunorubicin (DNR) . As a model system we used the human ovarian carcinoma cell line 2008wt, and its MRP1/ABCC1 transfected subline 2008/MRP1 . Both types of these moderate- and high-MRP1/ABCC1 expressing cells displayed efflux of DNR when maintained in standard culture media (2.3microM folic acid) . The initial total cellular DNR efflux rate in 2008/MRP1 cells was approximately 2-fold higher compared to 2008wt cells . This efflux consisted of MRP1/ABCC1 mediated transport, possibly non-MRP1 mediated transport, as well as passive diffusion . Benzbromarone, a specific MRP1 inhibitor, decreased the initial efflux rate in 2008/MRP1 cells (4-fold) and in 2008wt cells (2-fold) . When 2008/MRP1 cells were challenged for 2 days in folate-free medium, total cellular DNR efflux was decreased to 43% of the initial efflux rate under folate-rich conditions . In 2008wt cells DNR efflux was decreased to 84% of the folate-rich conditions . Benzbromarone did not inhibit DNR efflux after the folate-free period in both cell lines . Repletion of folate by a 2-24hr exposure to 2.5microM l-leucovorin or folic acid resulted in a complete restoration of DNR efflux . In contrast, expression of MRP1/ABCC1 protein was not changed significantly during the folate-free period or the repletion-period, nor were cellular ATP or ADP pools . In conclusion, this study demonstrates that the cellular folate status can influence the transport activity of MRP1/ABCC1 . These results have potentially important implications in the understanding of the (patho-)physiological roles of MRP1/ABCC1, and possibly other ABC transporter proteins in cellular folate homeostasis and drug resistance.

Antivir Ther, 2004 Feb, 9(1), 77 - 84
Intracellular and plasma pharmacokinetics of nelfinavir and M8 in HIV-infected patients: relationship with P-glycoprotein expression; Ford J et al.; One of the targets of antiretroviral therapy is within cells infected with HIV . In order to improve therapeutic efficacy, it is therefore important that the intracellular pharmacokinetics of drugs, such as nelfinavir mesylate and its active metabolite M8, are studied in addition to plasma pharmacokinetics . Previously, the intracellular accumulation of protease inhibitors has been reported in vivo, displaying the following hierarchy: nelfinavir > saquinavir > ritonavir > indinavir . Multidrug resistance transporters, such as P-glycoprotein (P-gp), may result in a lower intracellular concentration of drug via an efflux mechanism, thus contributing to sanctuary site formation . The objective of this study was to determine concentrations of nelfinavir and M8 in plasma and peripheral blood mononuclear cells from HIV-infected patients, and to ascertain the relationship between intracellular accumulation and lymphocyte P-gp expression . Venous blood samples from 12 HIV-infected patients (viral load <50 copies/ml) receiving nelfinavir (1250 mg twice daily) and dual nucleoside reverse transcriptase inhibitor therapy were collected over a full dosage interval (0, 2, 4, 8 and 12 h) . Plasma and intracellular (cell-associated) drug concentrations were measured by HPLC-MS/MS . Drug exposure in plasma and cells was expressed as the area under the concentration-time curve (AUC(0-12h)), derived from non-compartmental modelling . The ratio of intracellular AUC(0-12h)/total plasma AUC(0-12h) was calculated to determine cellular drug accumulation . P-gp expression on lymphocytes was determined by flow cytometry . The median (range) AUC(0-12h) of nelfinavir in plasma and cellular compartments was 21.8 mg x h x l(-1) (5.64-50.8) and 104.6 mg x h x l(-1) (23.1-265.7), respectively . Corresponding values for M8 in plasma and cells were 6.60 mg x h x l(-1) (2.16-17.3) and 19.6 mg x h x l(-1) (5.14-60.8) . A ratio of plasma M8/plasma nelfinavir (AUC(0-12h)) and intracellular M8/intracellular nelfinavir (AUC(0-12h)) gave median values of 0.32 and 0.17, respectively . The cellular accumulations {median; (range)} of nelfinavir and M8 were 5.30 (2.28-16.2) and 2.32 (1.01-10.7), respectively . A significant correlation between plasma and intracellular nelfinavir minimum concentration (Cmin) (r2=0.34; P=0.049), but not between plasma and intracellular M8 Cmin was observed . C(0h) concentrations were higher than C(12h) for both nelfinavir and M8 . No relationship was observed between nelfinavir or M8 accumulation and lymphocyte cell surface expression of P-gp . This study illustrates that intracellular concentrations were higher than plasma concentrations for both nelfinavir and M8, suggesting lymphocyte accumulation . The mechanism of differential intracellular accumulation of nelfinavir and M8 remains to be elucidated . It may be that affinities for influx transporters or fundamental drug characteristics play a major role in the greater accumulation of nelfinavir than M8.

World J Gastroenterol, 2004 Mar 15, 10(6), 795 - 9
Effect of mitogen-activated protein kinase signal transduction pathway on multidrug resistance induced by vincristine in gastric cancer cell line MGC803; Chen B et al.; AIM: To investigate the correlation between mitogen-activated protein kinase (MAPK) signal transduction pathway and multidrug resistance (MDR) in MGC803 cells . METHODS: Western blot was used to analyze the expression of MDR associated gene in transient vincristine (VCR) induced MGC803 cells, which were treated with or without the specific inhibitor of MAPK, PD098059 . Morphologic analysis of the cells treated by VCR with or without PD098059 was determined by Wright-Giemsa staining . The cell cycle analysis was performed by using flow cytometric assay and the drug sensitivity of MGC803 cells which were exposed to VCR with or without PD098059 was tested by using MTT assay . RESULTS: Transient exposure to VCR induced P-gp but not MRP1 or GST-pi expression in MGC803 cells and the expression of P-gp was inhibited by PD098059 . Apoptotic bodies were found in the cells treated with VCR or VCR+PD098059 . FCM results indicated that more MGC803 cells showed apoptotic phenotype when treated by VCR and PD098059 (rate: 31.23%) than treated by VCR only (rate: 18.42%) (P<0.05) . The IC(50) (284+/-13.2 mug/L) of MGC803 cells pretreated with VCR was 2.24-fold as that of negative control group (127+/-17.6 mug/L) and 1.48-fold as that of the group treated with PD098059 (191+/-27.9 mug/L) . CONCLUSION: This study shows that the expression of P-gp can be induced by transient exposure to VCR and this induction can be prevented by PD098059, which can block the activity of MAPK . MAPK signal transduction pathway may play some roles in modulating MDR1 expression in gastric cancer.

J Cell Physiol, 2004 May, 199(2), 252 - 61
Bile canalicular formation in hepatic organoid reconstructed by rat small hepatocytes and nonparenchymal cells; Sudo R et al.; The morphogenesis and movement of bile canaliculi (BC) are not well understood . This is because culture of hepatocytes that maintain polarity of cell membranes and possess highly differentiated functions has never been successful . We found that small hepatocytes (SHs), which are known to be hepatic progenitor cells, could proliferate and differentiate into mature hepatocytes and that BC-like structures developed between rising/piled-up cells . We investigated how BC-like structures developed with maturation of SHs and whether the structures were functionally active as BC . Hepatic cells, including SHs, were isolated from an adult rat liver and cultured . Immunocytochemistry and immunoblotting for BC proteins, such as ectoATPase, 5'-nucleotidase, dipeptidylpeptidase IV, and multidrug-resistance associated protein 2, were examined and time-lapse microscopy was used for the observation of BC contractions . Secretion of bilirubin into the reconstructed BC was also observed . The results of immunocytochemistry, immunoblots, and immunoelectron micrographs revealed that BC proteins were localized in the intercellular space that coincided with BC-like structures reconstructed between rising/piled-up cells . Tight junction-associated protein ZO-1 was also expressed along the BC-like structures . Bilirubin added to the medium were secreted into BC-like structure and accumulated without leakage . Time-lapse microscopy showed continuous contractions of reconstructed BC . In conclusion, BC-like structures reconstructed by SHs may be functional with membrane polarity, secretory ability, and motility . These results show that this culture system may suitable for investigating the mechanism of the formation of BC and their functions.

Biochem Pharmacol, 2004 Feb 1, 67(3), 523 - 37
Synthetic 1,4-anthracenedione analogs induce cytochrome c release, caspase-9, -3, and -8 activities, poly(ADP-ribose) polymerase-1 cleavage and internucleosomal DNA fragmentation in HL-60 cells by a mechanism which involves caspase-2 activation but not Fas signaling; Perchellet EM et al.; Synthetic analogs of 1,4-anthraquinone (AQ code number), a compound that mimics the antiproliferative effects of daunorubicin (daunomycin) in the nanomolar range in vitro but has the advantage of blocking nucleoside transport and retaining its efficacy in multidrug-resistant tumor cells, were tested for their ability to induce apoptosis in the HL-60 cell system . AQ10 and, especially, the new lead antiproliferative compounds AQ8 and AQ9 reduce the growth and integrity of wild-type, drug-sensitive, HL-60-S cells more effectively than AQ1, suggesting that various methyl group substituents at C6 may enhance the bioactivity of the parent compound . Internucleosomal DNA fragmentation, a late marker of apoptosis, is similarly induced in a biphasic manner by increasing concentrations of AQ8 and AQ9 at 24 hr . Poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, an early event required for cells committed to apoptosis, is detected within 3-6 hr in HL-60-S cells treated with AQ9 . In accord with the fact that the caspases 9 and 3 cascade is responsible for PARP-1 cleavage, the activities of initiator caspase-9 and effector caspase-3 are induced by AQ9 in the same time- and concentration-dependent manners and to the same maximal degrees in both the HL-60-S and multidrug-resistant HL-60-RV cell lines . Interestingly, a 1-hr pulse treatment is sufficient for AQ8 and AQ9 to maximally induce caspase-9 and -3 activities at 6 hr . The release of mitochondrial cytochrome c (Cyt c) is also detected within 3-6hr in HL-60-S cells treated with AQ9, a finding consistent with the fact that Cyt c is the apoptotic trigger that activates caspase-9 . Moreover, AQ analogs induce Cyt c release, caspase-9 and -3 activities and PARP-1 cleavage in relation with their abilities to decrease tumor cell growth and integrity, AQ8 and AQ9 being consistently the most effective . Since apical caspases 2 and 8 may both act upstream of mitochondria to promote Cyt c release, it is significant to show that AQ9 maximally induces caspase-2 and -8 activities at 6 and 9 hr, respectively . During AQ8 treatment, the caspase-2 inhibitor benzyloxycarbonyl (z)-Val-Asp-Val-Ala-Asp (VDVAD)-fluoromethyl ketone (fmk) totally blocks caspase-9, -3, and -8 activations, whereas the caspase-8 inhibitor z-Ile-Glu-Thr-Asp-(IETD)-fmk does not prevent caspase-2, -9, and -3 activations, suggesting that AQ-induced caspase-2 activity is an upstream event critical for the activation of the downstream caspases 9 and 3 cascade, including the mitochondrial amplification loop through caspase-8 . However, these caspase-2 and -8 inhibitors fail to alter AQ8-induced Cyt c release, suggesting that AQs might also target mitochondria independently from caspase activation . Furthermore, the antagonistic anti-Fas DX2 and ZB4 monoclonal antibodies (mAbs), which block the induction of Cyt c release and caspase-2, -8, and -9 activities by the agonistic anti-Fas CH11 mAb, and the neutralizing anti-Fas ligand (FasL) NOK-1 mAb all fail to inhibit AQ9-induced Cyt c release and caspase-2, -8, and -9 activities, suggesting that the FasL/Fas signaling pathway is not involved in the mechanism by which antiproliferative AQ analogs trigger apoptosis in HL-60 cells.

Biochemistry, 2004 Mar 30, 43(12), 3696 - 703
Ionophoric activity of pluronic block copolymers; Krylova OO et al.; Pluronic block copolymers (triblock copolymers of poly(ethylene oxide) and poly(propylene oxide)) exhibit a chemosensitizing effect on multidrug resistant cell lines . Changes in membrane permeability are hypothesized to be responsible because inhibition of drug transport mediated by both the multidrug-resistance-associated protein and the P-glycoprotein drug efflux system has been observed . To test this hypothesis, we now have studied the ion conductivity mediated by Pluronic L61 . Besides a detergent-like action, the copolymer was able to form regular channels and to exhibit carrier activity . Long living ion channels were formed by polymer oligomerization . Aggregate equilibrium was shifted toward L61 monomers and dimers, which operated as mobile carriers . Copolymer-induced membrane permeability for potassium ions (1 M KCl) was less than 10(-8) cm s(-1), whereas the permeability for uncharged doxorubicin molecules (1 mM) was equal to 5 x 10(-4) cm s(-1) . The results are consistent with reports about an increased doxorubicin accumulation in cells (Venne, Li, S., Mandeville, R., Kabanov, A., and Alakhov, V . Y . (1996) Cancer Res . 56, 3626-3629) . However, the increased permeability contrasts with the polymer-mediated decrease of drug efflux from cells . Preferential polymer binding to membrane proteins may mask the unspecific effect of L61 observed on lipid bilayers.

Ann N Y Acad Sci, 2003 Dec, 1010, 311 - 5
Antiproliferative and apoptosis-inducing effects of hemin in hepatoma cells; Diaconu CC et al.; Hemin is an extremely versatile molecule that may have cytotoxic or cytoprotective effects on certain cells . We investigated the effect of hemin on the growth of hepatoma cells, including the multidrug-resistant ones . Searching for new tools that interfere with the growth of hepatomas is an important area of clinical research . Cell viability and proliferation of drug-sensitive and multidrug-resistant hepatoma cell lines was determined using the trypan-blue exclusion test XTT/PMS and colony-forming ability assays . Apoptosis was assessed by confocal microscopy and DNA ladder assay . Hemin inhibited the proliferation and induced apoptosis in both drug-sensitive and multidrug-resistant hepatoma cells overexpressing functional P-glycoprotein . zVAD-fmk inhibited the hemin-induced decrease in cell viability, pointing to a role of caspases in hemin-induced apoptosis . The antiproliferative and apoptosis-inducing effects of hemin might be considered in the design of treatment for patients with hepatoma.

Am J Trop Med Hyg, 2004 Mar, 70(3), 256 - 9
Widespread occurrence of the Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene haplotype SVMNT in P . falciparum malaria in India; Vathsala PG et al.; The Plasmodium falciparum chloroquine resistance transporter (Pfcrt) K76T mutation and haplotype (amino acids 72-76) and the P . falciparum multidrug resistance 1 (Pfmdr1) mutation (N86Y) were analyzed as markers of chloroquine resistance in the DNAs of 73 blood samples from patients with P . falciparum malaria in India . Seventy of the 73 DNAs had the Pfcrt K76T mutation . Of these, 66 had the SVMNT haplotype and four had CVIET, the African/Southeast Asian haplotype . Only 20 of 69 DNAs had the Pfmdr1 N86Y mutation . It is surprising that the Pfcrt haplotype in India is predominantly SVMNT, rather than that seen in Southeast Asia . The widespread prevalence of the Pfcrt K76T mutation is a cause for concern.

Am J Trop Med Hyg, 2004 Mar, 70(3), 251 - 5
Molecular analysis of Plasmodium falciparum from drug treatment failure patients in Papua New Guinea; Casey GJ et al.; A study was conducted in Papua New Guinea to analyze Plasmodium falciparum drug resistance polymorphisms in patients presenting with resistant malaria . One hundred ninety-nine P . falciparum-positive patients were recruited at two sites, Madang and Maprik . Exposure to the 4-aminoquinolines chloroquine and amodiaquine was uniformly high, at 84% overall . However, 59% of these were taken in various combinations of sulfadoxine/pyrimethamine and/or primaquine and/or quinine . Two markers for 4-aminoquinoline resistance, P . falciparum chloroquine resistance transporter 76T and P . falciparum multidrug resistance 1, were fixed in the population and two markers for pyrimethamine resistance, dihydrofolate reductase (dhps) 59R and 108N, were found at moderate to high levels, overall 60% and 75%, respectively . No polymorphisms in dhps associated with sulfadoxine resistance were present . Differences between the two sites are analyzed . The study period encompasses a change in standard malaria treatment policy . These findings stress the need for regular monitoring of the effects of standard drug treatment of uncomplicated malaria in Papua New Guinea.

Mol Cancer Ther, 2004 Mar, 3(3), 345 - 52
Retinoic acid-induced CD38 antigen as a target for immunotoxin-mediated killing of leukemia cells; Mehta K et al.; A major obstacle in the successful delivery of antibody-based therapeutics to tumor cells is the heterogeneity of target antigen expression . We reported previously that retinoic acid (RA) is a potent and selective inducer of the cell-surface antigen CD38 in myeloid leukemia cells . The purpose of this study was to determine whether the RA-induced CD38 antigen could be a target for an anti-CD38-based immunotoxin to induce selective killing of leukemia cells . The combination of RA and the anti-CD38 gelonin immunotoxin induced a synergistic killing of leukemia cells . Thus, coculture of myeloid leukemia cells and cell lines with as little as 1 nM RA in the presence of immunotoxin induced substantial killing (>90%) of leukemia cell clones . More importantly, the blasts of myeloid leukemia patients, irrespective of their morphological and phenotypic features, also responded to the RA and immunotoxin combination when cultured ex vivo . A similar synergistic effect between RA and immunotoxin was observed against a multidrug-resistant variant subline of HL-60 cells . However, another variant of HL-60 cells, HL-60R, in which the retinoid receptor function has been abrogated by a trans-dominant-negative mutation, exhibited complete resistance to the immunotoxin-induced killing effect in the presence or absence of RA . Our results suggest that RA combined with anti-CD38-based therapeutic agent may offer exciting opportunities for the treatment of myeloid leukemias despite their multiplicity of genetic and clinical varieties.

Clin Chim Acta, 2004 Apr, 342(1-2), 115 - 26
A competitive nucleic acid sequence-based amplification assay for the quantification of human MDR1 transcript in leukemia cells; Hayashi T et al.; BACKGROUND: Because clinical drug resistance is caused by low-grade expression of a responsible gene, highly sensitive methods are desirable for its detection in clinical settings . We developed a quantitative nucleic acid sequence-based amplification (NASBA) assay for multidrug resistance-1 (MDR1) transcripts, and applied it to clinical samples . METHOD: MDR1 transcripts were amplified using the NASBA technique combined with sandwich hybridization of amplified MDR1 mRNA followed by chemiluminescence detection on an automated analyzer . Quantification of MDR1 mRNA was achieved through competitive coamplification of in vitro-generated RNA, which acts as an internal control . RESULTS: The competitive NASBA assay exhibited higher sensitivity (reliable detection limit was 100 copies of MDR1 mRNA) and linearity over a broader dynamic range (7 logarithmic orders) than the competitive reverse transcription-polymerase chain reaction assay . All 33 clinical samples obtained from patients with leukemia were successfully assayed, demonstrating its feasibility . MDR1 expression-compensated with beta-actin expression-ranged from 1.4 x 10(2) to 2.5 x 10(6) (median 4.8 x 10(5)) copies/microg RNA, while the range of MDR1 expression in peripheral blood samples from 15 healthy adults was from 8.9 x 10(4) to 5.2 x 10(5) (median 2.2 x 10(5)) copies/microg RNA . MDR1 expression in 8 of 33 clinical samples exceeded the median of healthy adult samples . CONCLUSIONS: The competitive NASBA assay is applicable to MDR1 mRNA quantification in clinical samples and would contribute to detection of clinical multidrug resistance.

Antiviral Res, 2004 Apr, 62(1), 1 - 7
The potential impact of drug transporters on nucleoside-analog-based antiviral chemotherapy; Borst P et al.; Several ATP-binding cassette (ABC) transporters can transport drugs out of cells against steep concentration gradients resulting in resistance to the drugs transported . Recent work has shown that at least three members of the family of human Multidrug Resistance-associated Proteins (MRPs), MRP4, 5 and 8, are able to transport some nucleoside-monophosphate analogs . This can result in resistance to the base, nucleoside or nucleotide precursors of these results, at least in cell lines with high levels of transporter . The affinity of these transporters for the nucleotide analogs studied thus far is relatively low (millimolar rather than micromolar), and this limits their potential impact on the resistance . We briefly review how ABC transporters in general, and MRPs in particular, could affect the disposition and cellular accumulation of antiviral compounds.

Mol Genet Genomics, 2004 Apr, 271(3), 325 - 38 Epub 2004 Mar 13.
Predicted ATP-binding cassette systems in the phytopathogenic mollicute Spiroplasma kunkelii; Zhao Y et al.; Spiroplasma kunkelii is a cell wall-free, helical, and motile mycoplasma-like organism that causes corn stunt disease in maize . The bacterium has a compact genome with a gene set approaching the minimal complement necessary for cellular life and pathogenesis . A set of 21 ATP-binding cassette (ABC) domains was identified during the annotation of a draft S . kunkelii genome sequence . These 21 ABC domains are present in 18 predicted proteins, and are components of 16 functional systems, which account for 5% of the protein coding capacity of the S . kunkelii genome . Of the 16 systems, 11 are membrane-bound transporters, and two are cytosolic systems involved in DNA repair and the oxidative stress response; the genes for the remaining three hypothetical systems harbor nonsense and/or frameshift mutations, so their functional status is doubtful . Assembly of the 11 multicomponent transporters, and comparisons with other known systems permitted functional predictions for the S . kunkelii ABC transporter systems . These transporters convey a wide variety of substrates, and are critical for nutrient uptake, multidrug resistance, and perhaps virulence . Our findings provide a framework for functional characterization of the ABC systems in S . kunkelii.

Cancer, 2004 Mar 15, 100(6), 1199 - 207
Pegylated liposomal doxorubicin-efficacy in patients with recurrent high-grade glioma; Hau P et al.; BACKGROUND: Doxorubicin exhibits high efficacy in malignant glioma cell cultures . Nonetheless, as a standard formulation, doxorubicin has not been used clinically, due to poor penetration of the blood-brain barrier . Furthermore, doxorubicin is known to induce tumor resistance genes . To address both of these issues, the authors investigated the use of pegylated liposomal doxorubicin (Caelyx; Essex Pharma, Munich, Germany) alone (Trial 1) and in combination with tamoxifen (Trial 2) in two sequentially performed nonrandomized prospective Phase II trials involving patients with recurrent high-grade glioma . METHODS: Twenty patients were included in each trial . Progression-free survival at 6 months (PFS-6) and toxicity were the primary endpoints . Expression of the tumor resistance proteins multidrug resistance protein 1 (MDR-1) and multiple resistance protein (MRP) was evaluated by immunohistochemical methods and by sestamibi-single-photon emission computed tomography (SPECT) . RESULTS: The overall response rate (including cases of disease stabilization) was 40% in both Trial 1 and Trial 2 . PFS-6 was 15%, and the median time to disease progression was 17 weeks . It is noteworthy that 40% of patients with Grade III tumors had long-term responses, which lasted for up to 3 years . There was no significant difference between Trial 1 and Trial 2 in terms of efficacy . Both regimens were well tolerated, with the main side effect being palmoplantar erythrodysesthesia . The authors found no correlation between clinical response and expression of tumor resistance genes or between clinical response and SPECT data . CONCLUSIONS: Pegylated liposomal doxorubicin administered alone or in combination with tamoxifen is safe and moderately effective in patients with recurrent high-grade glioma . None of the putative predictors for response that were evaluated proved to be significant in this setting .

J Pharmacol Exp Ther, 2004 Jul, 310(1), 376 - 85 Epub 2004 Mar 12.
Disposition mechanisms of raloxifene in the human intestinal Caco-2 model; Jeong EJ et al.; The purpose of this study was to determine the mechanisms responsible for transport of raloxifene and its hydrophilic conjugates . Human intestinal Caco-2 cell culture model and Caco-2 cell lysate were used for the studies . The results indicated that absorptive permeability (PAB) of raloxifene was lower than its secretory permeability (PAB) . As the concentration increased, the efflux ratio (PBA/PAB) decreased, but PBA increased . PAB was also increased in the presence of verapamil and cyclosporine A, two P-glycoprotein inhibitors . Raloxifene was extensively metabolized into sulfated and glucuronidated conjugates . The extent of metabolism or clearance was decreased as the concentration increased from 3.4 (96%) to 30 (22%) microM . Multidrug resistance-related protein inhibitors MK-571 (C26H26ClN2O3S2) and leukotriene C4 significantly decreased (maximal 80%) apical efflux of both conjugates . They also significantly decreased (maximal 85%) basolateral efflux of glucuronides but not sulfates . On the other hand, organic anion transporter (OAT) inhibitor estrone sulfate and estrone glucuronide significantly decreased (maximal 50%) the efflux of sulfate from both sides but had variable effects on glucuronide efflux . Inhibition of conjugate efflux with the OAT inhibitor estrone sulfate was concentration dependent, which resulted in increased transport of intact raloxifene (maximal 90%) . This increase in raloxifene transport was also observed in the presence of another OAT inhibitor estrone glucuronide (70%) . In conclusion, this is the first report that inhibition of an efflux transporter responsible for the transport of metabolites can result in increase in the transport of the intact compound . It also provides additional explanation why raloxifene has low bioavailability but a long half-life.

J Clin Oncol, 2004 Mar 15, 22(6), 1078 - 86
Mitoxantrone, etoposide, and cytarabine with or without valspodar in patients with relapsed or refractory acute myeloid leukemia and high-risk myelodysplastic syndrome: a phase III trial (E2995); Greenberg PL et al.; PURPOSE: To determine whether adding the multidrug resistance gene-1 (MDR-1) modulator valspodar (PSC 833; Novartis Pharmaceuticals, Hanover, NJ) to chemotherapy provided clinical benefit to patients with poor-risk acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS) . PATIENTS AND METHODS: A phase III randomized study was performed using valspodar plus mitoxantrone, etoposide, and cytarabine (PSC-MEC; n=66) versus MEC (n=63) to treat patients with relapsed or refractory AML and high-risk MDS . RESULTS: For the PSC-MEC versus MEC arms, complete response (CR) was achieved in 17% versus 25% of patients, respectively (P=not significant) . For patients who had not received prior intensive chemotherapy (ie, with secondary AML or high-risk MDS), the CR rate was increased--35% versus 15% for the remaining patients (P=.018); CR rates did not differ between treatment arms . The median disease-free survival in those achieving CR was similar in the two arms (10 versus 9.3 months) as was the patients' overall survival (4.6 versus 5.4 months) . The CR rates in MDR+ (69% of patients) versus MDR- patients were similar for those receiving either chemotherapy regimen (16% versus 24%) . The CR rate for unfavorable cytogenetic patients (45% of patients) was 13% compared to the remainder, 28% (P=.09) . Population pharmacokinetic analysis demonstrated that the clearances of mitoxantrone and etoposide were decreased by 59% and 50%, respectively, supporting the empiric dose reductions in the PSC-MEC arm designed in anticipation of drug interactions between valspodar and the chemotherapeutic agents . CONCLUSION: CR rates and overall survival were not improved by using PSC-MEC compared to MEC chemotherapy alone in patients with poor-risk AML or high-risk MDS.

Toxicol Lett, 2004 Mar 14, 148(1-2), 41 - 51
The role of mdr2 in manganese-bilirubin induced cholestasis in mice; Akoume MY et al.; The pathogenesis of manganese-bilirubin (Mn-BR) induced cholestasis has only been studied in rats and is associated with alteration in the hepatic homeostasis of cholesterol and phospholipids . Multidrug resistance-2 (mdr2) transporter, which mediates excretion of these lipids, is suggested to be involved in this phenomenon . The present study was undertaken to examine if Mn-BR induced cholestasis is reproducible in mice, then to clarify the role of mdr2 in its pathogenesis, using mice with disrupted mdr2 gene (mdr2 (-/-)) . Results showed that Mn-BR combination decreased bile flow in mice . This reduction in bile flow was similar in mdr2 (-/-) and the wild type mdr2 (+/+) . Furthermore, the change in biliary lipid excretion was comparable in both genotypes . These data indicate that Mn-BR induced cholestasis is reproducible in mice and provide evidence that mdr2 alteration is not a primary event in this form of cholestasis.

Hum Gene Ther, 2004 Mar, 15(3), 251 - 62
Anti-human immunodeficiency virus hematopoietic progenitor cell-delivered ribozyme in a phase I study: myeloid and lymphoid reconstitution in human immunodeficiency virus type-1-infected patients; Amado RG et al.; A phase I gene transfer clinical study was undertaken to examine the ability to introduce a potential anti-human immunodeficiency virus (HIV) gene therapeutic into hematopoietic progenitor cells (HPC), thereby contributing to multilineage engraftment . The potential therapeutic effect of genetically modifying HPC with protective genes in HIV-infected adults depends in part on the presence of adult thymic activity and myeloid capacity in the setting of HIV replication . Herein we report the presence and expression of a retroviral vector encoding an anti-HIV-1 ribozyme in mature hematopoietic cells of different lineages, and de novo T-lymphocyte development ensuing from genetically engineered CD34(+) HPC . Sustained output of vector-containing mature myeloid and T-lymphoid cells was detected even in patients with multidrug-resistant infection . In addition, the study showed that the degree of persistence of gene-containing cells was dependent on transduced HPC dose . These novel findings support the concept of gene therapy as a modality to effect immune reconstitution with cells engineered to inhibit HIV replication and this report represents the first demonstration of long-term maintenance of a potential therapeutic transgene in HIV disease.

Dermatol Clin, 2004 Jan, 22(1), 51 - 61
Skin infections in the elderly; Weinberg JM et al.; The diagnosis and management of viral diseases of the skin are significant issues in the elderly population . With advances in these areas, there are new tools to combat these diseases and limit morbidity . It is important for clinicians to monitor and treat these diseases aggressively in the elderly because of the potential for immunosuppression in this population . Further advances in antiviral therapy and the potential for the development of antiviral vaccines will aid in the therapy of these diseases . Onychomycosis is found more frequently in the elderly . In this population, the most common clinical presentations are distal and lateral subungual onychomycosis (which usually affects the great or first toe) and white superficial onychomycosis (which generally involves the third or fourth toes) . Over the past several years, new treatments for this disorder have emerged that offer shorter courses of therapy and greater efficacy than previous therapies . The treatment of bacterial skin and skin structure infections in the elderly is an important issue . There has been an alarming increase in the incidence of gram-positive infections, including resistant bacteria, such as MRSA and drug-resistant pneumococci . Although vancomycin has been considered the drug of last defense against gram-positive multidrug-resistant bacteria, the late 1980s saw a rise in vancomycin-resistant bacteria, including VRE . With treatment options limited, it has become critical to identify antibiotics with novel mechanisms of activity . Several new drugs have emerged as possible therapeutic alternatives, including linezolid and quinupristin-dalfopristin.

Hua Xi Kou Qiang Yi Xue Za Zhi, 2004 Feb, 22(1), 23 - 5
{Expression and implication of Pgp, MRP, LRP, GST-pi, Topo II alpha in tongue squamous cell carcinoma}; Leng WD et al.; OBJECTIVE: To explore the correlation of chemotherapy efficacy in tongue squamous cell carcinoma(SCC) with expression level of P-glycoprotein(Pgp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathiones-tranferase (GST-pi), DNA topo-isomerase II alpha (Topo II alpha) . METHODS: The expression patterns of Pgp, MRP, LRP, GST-pi and Topo II alpha in 40 patients (pre and post-chemotherapy, respectively) with tongue SCC were examined by immunohistochemically labelled streptavidin bioein method (LsAB) . RESULTS: The expression ratios of Pgp, MRP, LRP, GST-pi and Topo II alpha in pre-chemotherapy cases were 47.5%, 50%, 35%, 45%, 82.5%, respectively . No relations between expression of Pgp, MRP, LRP, GST-pi, Topo II alpha and clinic indexes were established (P > 0.05) . Expression ratios of Pgp, MRP in post-chemotherapy cases were higher than that in pre-chemotherapy cases (P < 0.05) . Expression of Pgp and MRP showed relevance with drug resistance (P < 0.05) . The co-expression was common, the ratios of co-expression of Pgp, MRP, GST-pi and MRP, GST-pi in chemotherapy non-responders were 40% and 50%, respectively, but 0 in responders . CONCLUSION: The intrinsic multidrug resistance of tongue SCC is relevant to the effects of Pgp, MRP, GST-pi.

J Neurooncol, 2004 Jan, 66(1-2), 65 - 70
Multidrug resistance-associated protein MRP1 expression in human gliomas: chemosensitization to vincristine and etoposide by indomethacin in human glioma cell lines overexpressing MRP1; Benyahia B et al.; The 190 kDa multidrug resistance protein MRP1 is likely to be involved in the multidrug resistance phenotype of human gliomas . MRP1 expression was evaluated in surgical tumor samples from 17 patients with gliomas . In addition, the impact of the MRP's inhibitor, indomethacin, on the chemosensitivity to etoposide (VP16) and vincristine (VCR) of two glioblastoma cell lines expressing MRP1 (GL15 and 8MG) was investigated . When evaluated in tumor samples, MRP1 expression was observed in all of them with more than 90% of stained tumor cells in 14/15 high-grade gliomas . MRP1 was also strongly expressed at the membrane of the vascular endothelial cells in the same 14 tumor samples, suggesting that the permeability to anticancer drugs could be also limited across brain tumor vessels . At concentrations comprised between 5 and 50 microM, indomethacin significantly increased the cytotoxic effect of etoposide in both cell lines but it was more efficient in increasing the cytotoxicity of VCR on GL15 cells, as compared with 8MG cells . These results suggest that the association of indomethacin to VCR or etoposide could be of interest in the clinical management of gliomas.

Anticancer Res, 2004 Jan-Feb, 24(1), 325 - 31
The impact of stromal cell contamination on chemosensitivity testing of head and neck carcinoma; Dollner R et al.; BACKGROUND: Reliable chemosensitivity testing of head and neck squamous cell carcinoma (HNSCC) still faces methodical limitations . Since stromal cell contamination has been found to preclude reliable radiosensitivity testing of HNSCC as well as chemosensitivity testing of lung tumors, the present study investigates the impact of stromal cell contamination on chemosensitivity testing of HNSCC . PATIENTS AND METHODS: Seventeen biopsies from HNSCC were analyzed . The specimens were investigated using an ex vivo colony formation assay which allows for the quantitative and separate determination of the overall, as well as the epithelial, and stromal response to carboplatin, 5-fluorouracil and docetaxel . RESULTS: The overall chemoresponse was dominated by stromal cell multidrug resistance . However, by selective evaluation of the epithelial chemoresponse, individual chemosensitivity patterns could be identified . CONCLUSION: Multidrug-resistant stromal cells preclude the reliable assessment of the chemoresponse of HNSCC specimens . Carefiul correction for stromal cell effects is a prerequisite for the generation of therapeutically useful information.

Anticancer Res, 2004 Jan-Feb, 24(1), 291 - 5
Multidrug resistance proteins in primary and metastatic soft-tissue sarcomas: down-regulation of P-glycoprotein during metastatic progression; Komdeur R et al.; BACKGROUND: Chemotherapy sensitivity of soft-tissue sarcomas (STS) is limited, which may be due to multidrug resistance (MDR) . MDR is associated with expression of P-glycoprotein (P-gp), Multidrug Resistance-associated Protein 1 (MRP1) and Lung Resistance-related Protein (LRP) . It is unknown whether in STS metastasis is more resistant than the primary counterpart . MATERIALS AND METHODS: In 35 chemonaive STS and their metastases (86% chemonaive), MDR proteins were immunohistochemically assessed . Eleven metastases presented synchronously, 24 metachronously . Expression was scored positive (>5% positive tumour cells) or negative . RESULTS: P-gp was positive in 31/34 primaries (91%), versus 22/32 metastases (69%) (p=0.005) . This difference was significant for metachronous metastases (p=0.008) . MRP1 was positive in 18/32 primaries (56%) and 22/33 metastases (67%) . MRP1 was more expressed in synchronous metastases than primaries (p=0.047), but for the overall group this significance disappeared . LRP expression did not differ: 27/34 primaries (80%), versus 28/34 metastases (82%) . CONCLUSION: P-gp, MRP1, LRP expression in the primary tumours was high . Metastatic progression did not coincide with MDR-protein up-regulation.

Clin Cancer Res, 2004 Mar 1, 10(5), 1826 - 34
VX-710 (biricodar) increases drug retention and enhances chemosensitivity in resistant cells overexpressing P-glycoprotein, multidrug resistance protein, and breast cancer resistance protein; Minderman H et al.; PURPOSE: The pipecolinate derivative VX-710 (biricodar; Incel) is a clinically applicable modulator of P-glycoprotein (Pgp) and multidrug resistance protein (MRP-1); we studied its activity against the third multidrug resistance (MDR)-associated drug efflux protein, breast cancer resistance protein (BCRP) . EXPERIMENTAL DESIGN: VX-710 modulation of uptake, retention, and cytotoxicity of mitoxantrone, daunorubicin, doxorubicin, topotecan, and SN38 was studied in cell lines overexpressing Pgp, MRP-1 and wild-type (BCRP(R482)) and mutant (BCRP(R482T)) BCRP . RESULTS: In 8226/Dox6 cells (Pgp), VX-710 increased mitoxantrone and daunorubicin uptake by 55 and 100%, respectively, increased their retention by 100 and 60%, respectively, and increased their cytotoxicity 3.1- and 6.9-fold, respectively . In HL60/Adr cells (MRP-1), VX-710 increased mitoxantrone and daunorubicin uptake by 43 and 130%, increased their retention by 90 and 60%, and increased their cytotoxicity 2.4- and 3.3-fold . In 8226/MR20 cells (BCRP(R482)), VX-710 increased mitoxantrone uptake and retention by 60 and 40%, respectively, and increased cytotoxicity 2.4-fold . VX-710 increased daunorubicin uptake and retention by only 10% in 8226/MR20 cells, consistent with the fact that daunorubicin is not a substrate for BCRP(R482), but, nevertheless, it increased daunorubicin cytotoxicity 3.6-fold, and this increase was not associated with intracellular drug redistribution . VX-710 had little effect on uptake, retention, or cytotoxicity of mitoxantrone, daunorubicin, doxorubicin, topotecan, or SN38 in MCF7 AdVP3000 cells (BCRP(R482T)) . CONCLUSIONS: VX-710 modulates Pgp, MRP-1, and BCRP(R482), and has potential as a clinical broad-spectrum MDR modulator in malignancies such as the acute leukemias in which these proteins are expressed.

Clin Cancer Res, 2004 Mar 1, 10(5), 1691 - 7
Breast cancer resistance protein impacts clinical outcome in platinum-based chemotherapy for advanced non-small cell lung cancer; Yoh K et al.; PURPOSE: The purpose of this study was to investigate the relationship between the level of expression of ATP-binding cassette (ABC) transporter proteins, and response to chemotherapy and prognosis in advanced non-small cell lung cancer (NSCLC) . EXPERIMENTAL DESIGN: Expression of ABC transporter proteins, including P-glycoprotein, multidrug resistance protein (MRP) 1, MRP2, MRP3, and breast cancer resistance protein (BCRP), was examined immunohistochemically in 72 formalin-fixed tumor samples from untreated stage IIIB or IV NSCLC patients . All of the patients received platinum-based chemotherapy . Response to chemotherapy, progression-free survival (PFS), and overall survival were compared in relation to expression of each of the ABC transporter proteins and clinicopathological factors . RESULTS: Expression of P-glycoprotein, MRP1, and MRP3 was not significantly associated with response to chemotherapy or survival . MRP2 expression was associated with overall survival (P = 0.002) but not with response to chemotherapy and PFS . By contrast, the response rate to chemotherapy of patients with BCRP-negative tumors was 44%, as opposed to 24% in patients with BCRP-positive tumors . Response rate was lower in BCRP-positive tumors, although this difference was not statistically significant (P = 0.08) . BCRP-positive patients had also shorter PFS (P = 0.0003) and overall survival (P = 0.004) than BCRP-negative patients . Multivariate analysis confirmed BCRP status as an independent variable related to PFS (P = 0.001) . CONCLUSIONS: Positive immunostaining for BCRP appears to be a predictor of survival in patients with advanced NSCLC . These findings indicate that BCRP may serve as a molecular target for reducing drug resistance to chemotherapy in advanced NSCLC patients.

FEBS Lett, 2004 Mar 12, 561(1-3), 207 - 12
Overexpression of LaMDR2, a novel multidrug resistance ATP-binding cassette transporter, causes 5-fluorouracil resistance in Leishmania amazonensis; Katakura K et al.; The ATP-binding cassette (ABC) proteins play an important role in drug resistance and detoxification in various organisms . Here we isolated LaMDR2, a new member of the multidrug resistance (MDR) subfamily of ABC proteins in Leishmania amazonensis . LaMDR2 exhibited 47% amino acid identity to its most closely related protein, LaMDR1, which was previously isolated from the same species . Promastigotes that overexpressed LaMDR2 showed significant resistance to 5-fluorouracil (5-FU), but not to LaMDR1 substrates . Expression of LaMDR2 in the transfectants was relatively higher in the log phase than the stationary phase, and a lower accumulation of {(3)H}5-FU was observed in the log-phase cells . These results suggest that LaMDR2 is involved in extrusion of xenobiotics, but functionally different from LaMDR1.

Bioorg Med Chem Lett, 2004 Feb 23, 14(4), 881 - 5
Inhibitors of multidrug resistance (MDR) have affinity for MDR substrates; Zloh M et al.; Multidrug-resistance (MDR) occurs in many bacterial species and tumour cells . MDR functions by membrane proteins which export drugs from cells, resulting in a low ineffective concentration of the drug . We have shown by molecular modelling that inhibitors of MDR have affinity for substrates of MDR transporters . This affinity may facilitate drug entry into cells and a large inhibitor-drug complex may be a poorer substrate for the MDR mechanism . This complex would effectively 'cloak' the drug rendering it unavailable for efflux.

J Gastroenterol Hepatol, 2004 Apr, 19(4), 413 - 7
Biliary excretion of azelnidipine, a calcium antagonist, in rats; Hanawa N et al.; BACKGROUND AND AIMS: Azelnidipine (CS-905) is a novel dihydropyridine calcium antagonist that is known to be excreted in feces . To examine the mechanism of biliary excretion of azelnidipine, the authors studied its biliary excretion in Eisai hyperbilirubinuria rats (EHBR), multidrug resistance protein (Mrp)2-deficient rats, and the effect of cholephilic compounds on the biliary excretion of azelnidipine in rats . METHODS: Radiolabeled azelnidipine was intravenously administered to EHBR and control rats, and the biliary excretion of radiolabeled metabolites was studied . Furthermore, the effect of sulfobromophthalein, taurocholate and vinblastine on the biliary excretion of azelnidipine metabolites was also studied in control rats . RESULTS: The biliary excretion of azelnidipine metabolites was delayed in EHBR . The biliary excretion of azelnidipine metabolites was inhibited by sulfobromophthalein and vinblastine, but was not inhibited by taurocholate or phenothiazine pretreatment . CONCLUSION: These findings suggest that the metabolites of azelnidipine are excreted into the bile partly by Mrp2 and P-glycoprotein.

Nephrology (Carlton), 2003 Oct, 8 Suppl, S10 - 5
Development of bioartificial kidneys; Saito A; Bioartificial kidneys, which consist of continuous haemofiltration and bioartificial tubules using tubular epithelial cells, have been studied since 1987 . The bioartificial tubules consist of hollow fibre modules and tubular epithelial cells grown on the hollow fibre membranes after coating with extracellular matrices . The kinds of tubular epithelial cells, extracellular matrices and artificial membranes therefore have been investigated and then the most appropriate cell and materials have been selected on the basis of the development of bioartificial kidneys . Successful seeding to form confluent monolayers on the surfaces of hollow fibers is not easy, but this method has already been established . Renal assist devices using human renal proximal tubular epithelial cells have been used in the treatment of acute renal failure patients with endotoxemia by Humes et al., and successful treatment of acute renal failure patients with these devices was reported in 2001 and 2002, in which the improved mortality rate of those patients was shown . A bioartificial kidney, in which cDNA of multidrug resistance protein-1 was transfected into tubular epithelial cells that were then grown on the outer surfaces of hollow fibers, was used in the experimental treatment of digoxin-intoxicated dogs . Rapidly reduced digoxin levels were noted in the plasma of the dogs after treatment . Bioartificial kidneys, however, have never been used in the long-term treatment of a maintenance dialysis patient, although patients need those kidneys . In order to establish long-term treatment with a bioartificial kidney, each haemofilter has to function for more than one week without systemic anticoagulation and a bioartificial tubule must function for 3-4 weeks.

Mol Microbiol, 2004 Mar, 51(6), 1563 - 75
Localization and activity of multidrug resistance protein 1 in the secretory pathway of Leishmania parasites; Dodge MA et al.; Upregulation of the multidrug resistance protein 1 (LeMDR1) in the protozoan parasite, Leishmania enriettii, confers resistance to hydrophobic drugs such as vinblastine, but increases the sensitivity of these parasites to the mitochondrial drug, rhodamine 123 . In order to investigate the mechanism of action of LeMDR1, the subcellular localization of green fluorescent protein (GFP)-tagged versions of LeMDR1 and the fate of the traceable-fluorescent LeMDR1 substrate calcein AM were examined in both Leishmania mexicana and L . enriettii LeMDR1 -/- and overexpressing cell lines . The LeMDR1-GFP chimera was localized by fluorescence microscopy to a number of secretory and endocytic compartments, including the Golgi apparatus, endoplasmic reticulum (ER) and a multivesicular tubule (MVT)-lysosome . Pulse-chase labelling experiments with calcein AM suggested that the Golgi and ER pools, but not the MVT-lysosome pool, of LeMDR1 were active in pumping calcein AM out of the cell . Cells labelled with calcein AM under conditions that slow vesicular transport (low temperature and stationary growth) inhibited export and resulted in the accumulation of fluorescent calcein in both the Golgi and the mitochondria . We propose that LeMDR1 substrates are pumped into secretory compartments and exported from the parasite by exocytosis . Accumulation of MDR substrates in the ER can result in alternative transport to the mitochondrion, explaining the reciprocal sensitivity of drug-resistant Leishmania to vinblastine and rhodamine 123.

Chest, 2004 Mar, 125(3), 974 - 80
Hypokalemia among patients receiving treatment for multidrug-resistant tuberculosis; Shin S et al.; INTRODUCTION: Between January 1999 and December 2000, 125 patients in Lima, Peru were enrolled in individualized treatment for multidrug-resistant tuberculosis (MDR-TB) . Hypokalemia was observed to be an important adverse effect encountered in this cohort . OBJECTIVE: To identify risk factors associated with the development and persistence of hypokalemia during MDR-TB therapy, and to review the incidence and management of hypokalemia in patients receiving MDR-TB therapy . METHODS: A retrospective case series of 125 patients who received individualized therapy for MDR-TB between January 1, 1999, and December 31, 2000 . RESULTS: Among 115 patients who were screened for electrolyte abnormalities, 31.3% had hypokalemia, defined as a potassium level of < 3.5 mEq/L . Mean serum potassium at time of diagnosis was 2.85 mEq/L . Diagnosis of low serum potassium occurred, on average, after 5.1 months of individualized therapy . Multivariate analysis of risk factors for this adverse reaction identified two causes: administration of capreomycin, and low initial body weight . Normalization of potassium levels was achieved in 86% of patients . CONCLUSIONS: Electrolyte disturbance was frequently encountered in our cohort of patients with MDR-TB . Successful screening and management of hypokalemia was facilitated by training the health-care team in the use of a standardized algorithm . Morbidity from hypokalemia can be significant; however, effective management of this side effect is possible without sacrificing MDR-TB treatment efficacy.

Biochem Pharmacol, 2004 Mar 15, 67(6), 1195 - 202
Mediation of annexin 1 secretion by a probenecid-sensitive ABC-transporter in rat inflamed mucosa; Wein S et al.; Annexin 1 is secreted by mammalian cells but lacks a leader signal sequence necessary to lead it to the classical secretory pathway via the endoplasmic reticulum . The mechanisms involved in the secretion of leaderless proteins remain uncertain . It has been suggested to involve membrane translocation via an ABC-transporter (ATP binding cassette) . Using cultured inflamed mucosa from rectocolitis induced in rats, we studied if annexin 1 secretion followed the two main characteristics of ABC-transporter substrates: dependency on ATP hydrolysis and competitive inhibition by several other ABC-transporter substrates . Annexin 1 secretion is inhibited in a dose-dependent manner by two ATPase inhibitors . The inhibition reached 63.2+/-3.2%, 66.1+/-3.73% and 88.6+/-1.4% in the presence of 2mM vanadate, 0.5 and 1mM pervanadate, respectively . The efflux of calcein, a known ABC-transporter substrate, is similarly inhibited by 69.4+/-2.8% in the presence of 1mM pervanadate . Probenecid, an inhibitor of several ABC-transporters of the subfamilly ABCC or MRP (multidrug resistant associated protein), also inhibited annexin 1 secretion in a dose-dependent manner . As compared to control, 10mM probenecid reduced annexin 1 secretion by 72+/-20% and 20mM by 95.0+/-9% . By contrast, annexin 1 secretion is not blocked by other inhibitors of MRP1 (indomethacin, MK571), MRP2 (ochratoxin A1 or MK571), MRP5 (trequinsin or sulfinpyrazone) or by verapamil, cyclosporin A or glyburide . Taken together, our results show that annexin 1 secretion appears to share the efflux properties of ABC-transporter substrates.

Biochem Pharmacol, 2004 Mar 15, 67(6), 1111 - 21
Evaluation of the binding of the tricyclic isoxazole photoaffinity label LY475776 to multidrug resistance associated protein 1 (MRP1) orthologs and several ATP- binding cassette (ABC) drug transporters; Dantzig AH et al.; Several of the ATP-binding cassette (ABC) transporters confer resistance to anticancer agents and/or antiviral agents when overexpressed in drug-sensitive cells . Recently a MRP1 (ABCC1) tricyclic isoxazole inhibitor, LY475776 was shown to be a glutathione-dependent photoaffinity label of human MRP1 and showed poor labeling of murine mrp1, an ortholog that does not confer anthracycline resistance . In the present study, the specificity of LY475776 was examined for its ability to modulate or photolabel orthologs of MRP1 and several other drug efflux transporters of the ABC transporter family . LY475776 modulated MRP1 and Pgp-mediated resistance (MDR, ABCB1) in, respectively, HeLa-T5 and CEM/VLB(100) cells to both vincristine and doxorubicin . LY475776 photolabeled 170kDa Pgp and was inhibited by the potent Pgp inhibitor LY335979 (Zosuquidar.3HCl) . The labeling of the 190kDa MRP1 protein in membranes of HeLa-T5 cells was inhibited by substrates of MRP1 such as leukotriene C(4), vincrisine, and doxorubicin and by the inhibitor, MK571 . LY475776 did not photolabel human MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCC5) or breast cancer resistance protein (ABCG2) . Because LY475776 photolabels murine mrp1 less well than human MRP1 and binds to a region believed important for anthracycline binding, studies were conducted with monkey and canine MRP1 which also show a reduced ability to confer resistance to anthracyclines . Unlike murine mrp1, both orthologs were photolabeled well by LY475776 . These studies indicate that the specificity of LY475776 is fairly limited to Pgp and MRP1 and further studies will help to define the binding regions.

Antibiot Khimioter, 2003, 48(10), 11 - 5
{Inhibition of ABC-transporter(s)' function in non-small cell lung cancer cells by platinum drugs}; Bogush TA et al.; With an account of the literature data that platinum drugs react with many cellular targets, including ATP and proteins, the authors suggested that disturbance of the function of energy-dependent ABC-transporters (markers of multidrug resistance, MDR) under the effect of platinum drugs could be a cause of increased efficacy of MDR agents (agents, MDR to which is developed by the classical mechanism) when used in combination with platinum drugs even in the treatment of multidrug resistant lung cancer . The cisplatin and carboplatin effect on accumulation of MDR doxorubicin in cells of non-small cell cancer was studied by flow cytometry with the use of biopsy specimens . The MDR phenotype of the tumors was determined by a change in doxorubicin intracellular accumulation under the action of the ABC-transporter(s)' inhibitors: verapamil and genistein (specific inhibitors of Pgp and MRP respectively) and sodium azide (an inhibitor of all energy-dependent ABC-transporters) . The MDR phenotypes, i.e . Pgp-MRP+ or Pgp+MRP+, were detected in all the tumors investigated . Two types of changes in doxorubicin intracellular accumulation under the action of the inhibitors and the platinum drugs were shown: (a) an increase in doxorubicin cytoplasmic accumulation and (b) a change in subcellular distribution of the anthracycline (increased accumulation of doxorubicin in the cell nucleus and its higher binding to DNA) . Cisplatin and carboplatin had an inhibitory effect on ABC-transporter(s) in all the tumors investigated but the effect of carboplatin was less pronounced . It was concluded that cisplatin and carboplatin stimulation of doxorubicin intracellular accumulation, as well as a change in subcellular distribution of the anthracycline under the action of the platinum drugs (increased doxorubicin accumulation in the cell nucleus) in multidrug resistant lung tumors could be at least partly explained by inhibition of the MDR transporter(s)' function . The results could provide a basis for the use of the sequential combination cisplatin (or carboplatin)-->doxorubicin in the treatment of multidrug resistant lung cancer.

J Immunol, 2004 Mar 15, 172(6), 3604 - 11
Glycoprotein 170 induces platelet-activating factor receptor membrane expression and confers tumor cell hypersensitivity to NK-dependent cell lysis; Geromin D et al.; Multidrug resistance (MDR) confers resistance to anticancer drugs and reduces therapeutic efficiency . It is often characterized by the expression of the MDR1 gene product P-glycoprotein (or gp170) at the membrane of tumor cells . To further propose a potential complementary tool in cancer treatment, the sensitivity of gp170 tumor cells to NK-dependent lysis was investigated . Two kinds of cells were generated from wild-type K562 erythroleukemic cells: the first were derived from Taxol-selected cells and cloned, whereas the second were retrovirally transduced by the cDNA of the MDR1 gene . The last process was also applied to the human embryonal carcinoma cells called Tera-2 cells . First, both cloned and MDR-1 K562 cells appeared highly susceptible to naive NK cell killing . Interestingly, in addition, Tera-2 cells that were not sensitive to NK lysis could be killed when they expressed gp170 at their membranes . In previous data, we demonstrated that NK cell release of bimolecular complexes composed of perforin and platelet-activating factor (PAF) interacting with the PAF-R, which has to be expressed on the target cell membranes, were components of NK tumor cell killing . In the present study, we show that gp170 has the capacity to drive constitutive PAF-R expression on tumor cells, which could be responsible for hypersensitivity to NK lysis and accelerated cell death.

Anal Biochem, 2004 Mar 15, 326(2), 262 - 6
Microanalysis for MDR1 ATPase by high-performance liquid chromatography with a titanium dioxide column; Kimura Y et al.; MDR1 is clinically important because it is involved in multidrug resistance of cancer cells and affects the pharmacokinetics of various drugs . Because MDR1 harnesses adenosine 5'-triphosphate (ATP) hydrolysis for transporting drugs, examining the effect on ATPase activity is imperative for understanding the interactions between drugs and MDR1 . However, conventional assay systems for ATPase activity are not sensitive enough for screening drugs using purified MDR1 . Here we report a novel method to measure ATPase activity of MDR1 using high-performance liquid chromatography equipped with a titanium dioxide column . The amount of adenosine 5'-diphosphate (ADP) produced by the ATPase reaction was determined within 2 min with a titanium dioxide column (4.6 mm ID x 100 mm) . The relationship between ADP amount and chromatogram peak area was linear from 5 pmol to 10 nmol . This method made it possible to reduce the amount of purified MDR1 required for a reaction to 0.5 ng, about 1/20th of the conventional colorimetric inorganic phosphate detection assay . This method is sensitive enough to detect any subtle changes in ATPase activity of MDR1 induced by drugs and can be applied to measure ATPase activity of any protein.

Biochem Biophys Res Commun, 2004 Mar 26, 316(1), 93 - 9
Caspase 3 activation is controlled by a sequence located in the N-terminus of its large subunit; Pelletier M et al.; We report that the induction and completion of the apoptotic program is delayed in a doxorubicin-resistant cell line (HL60/ADR) . This hindrance to cell death occurred downstream of the multidrug-resistant protein (mrp), a transmembrane transporter . In vitro studies showed that these cells were incapable of correctly activating procaspase 3 (pC3), the main executioner of apoptosis . Sequencing of HL60/ADR pC3 revealed point mutations in a sequence located in the N-terminal region of the large subunit of caspase 3 (C3, amino acids 31-37; i.e., immediately after the propeptide) . We called this particular form of C3, the C3 N-terminal modified (C3-NTM), and show that it is partially active when transfected into MCF-7 cells shown to have little or no endogenous pC3 . As a deletion of the amino acids 31-37 in wild-type C3 leads to the same phenotype, we conclude that this sequence is involved in C3 activation during apoptosis.

Int J Neuropsychopharmacol, 2004 Sep, 7(3), 341 - 9 Epub 2004 Mar 05.
Ethyl-eicosapentaenoate and dexamethasone resistance in therapy-refractory depression; Murck H et al.; Preliminary evidence shows that ethyl-eicosapentaenoate (E-EPA) has a marked clinical effect when used as an adjunct in therapy-refractory depression . EPA belongs to the class of polyunsaturated omega-3 fatty acids . The mechanism of its action in depression is not fully understood . There are two related fields where the pathophysiology of refractory depression meets the effect of EPA . First, a general immunosuppressive effect of EPA meets a general immunoactivation in severe depression, especially an increase in CD4/CD8 ratio, neutrophilia, and an increase in interleukins (IL)-6 and IL-12 and of prostaglandin E2 (PGE2) . Secondly, a resistance to dexamethasone (Dex) suppression of the HPA axis meets the effects of EPA on multidrug resistance reversing and HPA axis suppression . The effects of EPA on the immune system, the HPA axis, and multidrug resistance are connected through the action of a transport protein called p-glycoprotein (p-gp) . Physiological and synthetic steroids such as cortisol and Dex are substrates of p-gp, and so Dex resistance in depression may be related to dysfunction of this protein . In addition, expression of p-gp is induced by PGE2, and EPA inhibits the synthesis of PGE2 . The reversal of drug resistance by EPA may be mediated via this immunological mechanism and lead to its antidepressive efficacy . In addition, antidepressants such as amitriptyline, which have special efficacy in severe depression, decrease p-gp function . EPA may, furthermore, enhance the action of antidepressants, like many SSRIs that are p-gp substrates, which are actively transported out of the intracerebral space at the level of the blood-brain barrier.

Genome Biol . 2004;5(3):R15 . Epub 2004 Feb 11.
The ABC transporter gene family of Caenorhabditis elegans has implications for the evolutionary dynamics of multidrug resistance in eukaryotes; Sheps JA et al.; BACKGROUND: Many drugs of natural origin are hydrophobic and can pass through cell membranes . Hydrophobic molecules must be susceptible to active efflux systems if they are to be maintained at lower concentrations in cells than in their environment . Multi-drug resistance (MDR), often mediated by intrinsic membrane proteins that couple energy to drug efflux, provides this function . All eukaryotic genomes encode several gene families capable of encoding MDR functions, among which the ABC transporters are the largest . The number of candidate MDR genes means that study of the drug-resistance properties of an organism cannot be effectively carried out without taking a genomic perspective . RESULTS: We have annotated sequences for all 60 ABC transporters from the Caenorhabditis elegans genome, and performed a phylogenetic analysis of these along with the 49 human, 30 yeast, and 57 fly ABC transporters currently available in GenBank . Classification according to a unified nomenclature is presented . Comparison between genomes reveals much gene duplication and loss, and surprisingly little orthology among analogous genes . Proteins capable of conferring MDR are found in several distinct subfamilies and are likely to have arisen independently multiple times . CONCLUSIONS: ABC transporter evolution fits a pattern expected from a process termed 'dynamic-coherence' . This is an unusual result for such a highly conserved gene family as this one, present in all domains of cellular life . Mechanistically, this may result from the broad substrate specificity of some ABC proteins, which both reduces selection against gene loss, and leads to the facile sorting of functions among paralogs following gene duplication.

Parasitology, 2004 Jan, 128(Pt 1), 43 - 52
Excretion of fluorescent substrates of mammalian multidrug resistance-associated protein (MRP) in the Schistosoma mansoni excretory system; Sato H et al.; The protonephridium of platyhelminths including Schistosoma mansoni plays a pivotal role in their survival by excretion of metabolic wastes as well as xenobiotics, and can be revealed in the living adult parasite by certain fluorescent compounds which are concentrated in excretory tubules and collecting ducts . To determine the presence of the multidrug resistance-associated protein (MRP) as a possible transporter in protonephridial epithelium, adult schistosomes were exposed to a fluorescent Ca2+ indicator, fluo-3 acetyloxymethyl ester, which is a potential substrate of mammalian MRP . Specific fluorescence related to fluo-3/Ca2+ chelate delineated the whole length of the protonephridial system . Simultaneously, a fluorescent substance was accumulated in the posterior part of collecting ducts and the excretory bladder . Similarly, when other fluorogenic substrates for mammalian MRP such as monoclorobimane, fluorescein diacetate, and 5(6)-carboxyfluorescein diacetate were applied to adult schistosomes, these fluorescent markers were observed in the excretory tubules through to the excretory bladder . The excretory system of mechanically-transformed schistosomula was not labelled with any of these 4 fluorescent markers . These findings suggest that the protonephridial epithelium of adult schistosomes, but not schistosomula, might express the homologue of the mammalian MRP transporting organic anionic conjugates with glutathione, glucuronate or sulphate as well as unconjugated amphiphilic organic anions.

J Biol Chem, 2004 May 7, 279(19), 19781 - 9 Epub 2004 Mar 04.
Characterization of oligomeric human half-ABC transporter ATP-binding cassette G2; Xu J et al.; Human ATP-binding cassette G2 (ABCG2, also known as mitoxantrone resistance protein, breast cancer-resistance protein, ABC placenta) is a member of the superfamily of ATP-binding cassette (ABC) transporters that have a wide variety of substrates . Overexpression of human ABCG2 in model cancer cell lines causes multidrug resistance by actively effluxing anticancer drugs . Unlike most of the other ABC transporters which usually have two nucleotide-binding domains and two transmembrane domains, ABCG2 consists of only one nucleotide-binding domain followed by one transmembrane domain . Thus, ABCG2 has been thought to be a half-transporter that may function as a homodimer . In this study, we characterized the oligomeric feature of human ABCG2 using non-denaturing detergent perfluoro-octanoic acid and Triton X-100 in combination with gel filtration, sucrose density gradient sedimentation, and gel electrophoresis . Unexpectedly, we found that human ABCG2 exists mainly as a tetramer, with a possibility of a higher form of oligomerization . Monomeric and dimeric ABCG2 did not appear to be the major form of the protein . Further immunoprecipitation analysis showed that the oligomeric ABCG2 did not contain any other proteins . Taken together, we conclude that human ABCG2 likely exists and functions as a homotetramer.

Blood, 2004 Jul 1, 104(1), 178 - 83 Epub 2004 Mar 04.
An anti-CD19 antibody inhibits the interaction between P-glycoprotein (P-gp) and CD19, causes P-gp to translocate out of lipid rafts, and chemosensitizes a multidrug-resistant (MDR) lymphoma cell line; Ghetie MA et al.; We have previously demonstrated that an anti-CD19 monoclonal antibody (MAb; HD37) inhibits the function of the P-glycoprotein (P-gp) pump in a multidrug-resistant (MDR) B-lymphoma cell line, Namalwa/MDR1, and that this effect is not due to the recognition of a cross-reactive epitope on P-gp . In this study, we have used the same cell line to define the mechanisms responsible for the effect of HD37 on the P-gp pump . Using fluorescence resonance energy transfer (FRET), we show that CD19 and P-gp are constitutively associated in cells . In the absence of treatment with anti-CD19, 40% of P-gp molecules expressed by Namalwa/MDR1 cells reside in the low-density lipid (ie, cholesterol-rich) microdomains (lipid rafts) . Following treatment of the cells with HD37 and disruption of the interactions between P-gp and CD19, P-gp translocated out of lipid rafts and CD19 translocated into lipid rafts . The effect of chemosensitization on Namalwa/MDR1 cells was specific for CD19; an anti-CD22 MAb had no such effect, although the cells express CD22 . These results suggest that anti-CD19 might chemosensitize P-gp(+) cells by interfering with interactions between CD19 and P-gp, rapidly resulting in the translocation of P-gp into a compartment on the plasma membrane where it is no longer active.

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