Multidrug resistant

Lancet, 2000 Sep 23, 356(9235), 1066 - 71
Tuberculosis control and molecular epidemiology in a South African gold-mining community; Godfrey-Faussett P et al.; BACKGROUND: Gold miners have very high rates of tuberculosis . The contribution of infections imported into mining communities versus transmission within them is not known and has implications for control strategies . METHODS: We did a prospective, population-based molecular and conventional epidemiological study of pulmonary tuberculosis in a group of goldminers . Clusters were defined as groups of patients with Mycobacterium tuberculosis isolates with identical IS6110 DNA fingerprints . We compared the frequency of possible risk factors in the clustered and non-clustered patients whose isolates had fingerprints with more than four bands, and re-interviewed members of 45 clusters . FINDINGS: Of 448 patients, ten were excluded because they had false-positive cultures . Fingerprints were made in 419 of 438, of which 371 had more than four bands . 248 of 371 were categorised into 62 clusters . At least 50% of tuberculosis cases were due to transmission within the community . Patients who had failed treatment at entry to the study were more likely to be in clusters (adjusted odds ratio 3.41 {95% CI 1.25-9.27}) . Patients with multidrug-resistant isolates were more likely to have failed treatment but were less likely to be clustered than those with a sensitive strain (0.27 {0.09-0.83}) . HIV infection was common (177 of 370 tested) but not associated with clustering . INTERPRETATION: Despite a control programme that cures 86% of new cases, most tuberculosis in this mining community is due to ongoing transmission . Persistently infectious individuals who have previously failed treatment may be responsible for one third of tuberculosis cases . WHO targets for cure rates are not sufficient to interrupt transmission of tuberculosis in this setting . Indicators that are more closely linked to the rate of ongoing transmission are needed.

Biochem Pharmacol, 2000 Nov 1, 60(9), 1381 - 90
Phospholipids as multidrug resistance modulators of the transport of epirubicin in human intestinal epithelial Caco-2 cell layers and everted gut sacs of rats; Lo YL; Phospholipids have been increasingly used as carriers for the delivery of a variety of drugs . Studies using cancer chemotherapeutic agents such as epirubicin encapsulated in liposomes, which are made of phospholipids and other ingredients, have generally shown reduced toxicity and enhanced therapeutic efficacy . The recent investigation of the role of P-glycoprotein (P-gp) in phospholipid translocation has opened a new area of research on the possible use of phospholipids as multidrug resistance (MDR) modulators . This study investigated the effects of liposomal encapsulation, empty liposome pretreatment, or free lipid pretreatment on the uptake and transport of epirubicin in the human colon adenocarcinoma cell line Caco-2 and in everted gut sacs of rat jejunum and ileum . Epirubicin uptake experiments, using a flow cytometer, showed that both liposomal encapsulation and empty liposome pretreatment increased the intracellular accumulation of epirubicin in Caco-2 cells significantly . These two treatments substantially increased apical-to-basolateral absorption of epirubicin across Caco-2 monolayers and markedly improved mucosal-to-serosal absorption of epirubicin in rat jejunum and ileum . Enhancement also was observed with both liposome encapsulation and empty liposome pretreatment in the reduction of basolateral-to-apical efflux of epirubicin across Caco-2 monolayers . However, because diffusion of free dipalmitoyl phosphatidylcholine (DPPC) or dipalmitoyl phosphatidylethanolamine (DPPE) lipids across the cell membrane is very slow, these free lipids showed marginal effects on absorption and/or secretion of epirubicin in both Caco-2 cells and rat gut sacs . The study suggests that inhibition of P-gp or other transporter proteins located in the intestines may be partially involved in the reduction of epirubicin efflux . In conclusion, the therapeutic efficacy of epirubicin may be improved by using phospholipids as excipients and MDR modulators in the formulations . Liposomal formulations may have important applications to circumvent drug resistance in cancer chemotherapy.

Biochem Biophys Res Commun, 2000 Sep 16, 276(1), 231 - 7
Equivalent death of P-glycoprotein expressing and nonexpressing cells induced by the protein kinase C inhibitor staurosporine; Tainton KM et al.; P-glycoprotein (P-gp) is an ATP-dependent drug pump that confers multidrug resistance . In addition to its ability to efflux toxins P-gp can also inhibit apoptosis induced by a wide array of cell death stimuli that rely on activation of intracellular caspases for full function . We have previously demonstrated that stimuli including drugs such as hexamethylene bisacetamide (HMBA), the cytotoxic lymphocyte granule protein granzyme B, and pore-forming proteins such as perforin, kill P-gp positive cells in a caspase-independent manner . We therefore hypothesised that drugs that are not effluxed by P-gp and which induce cell death in the absence of caspase activation could induce death of P-gp expressing cells . Staurosporine has been previously shown to kill cells in the absence of caspase activation . Consistent with our hypothesis, we demonstrate here that staurosporine can equivalently kill P-gp(+ve) and P-gp(-ve) tumor cell lines in a caspase-independent manner .

Int J Cancer, 2000 Oct 15, 88(2), 260 - 6
New highly lipophilic camptothecin BNP1350 is an effective drug in experimental human cancer; Van Hattum AH et al.; BNP1350, 7-{(2-trimethylsilyl)ethyl}-20(S)-camptothecin, is a novel semi-synthetic, highly lipophilic, silicon-containing camptothecin and an inhibitor of topoisomerase I . It has been supercomputer engineered for superior oral bioavailability, superior lactone stability, broad anti-tumor activity, increased potency and insensitivity to Pgp/MRP/LRP drug resistance . We determined the efficacy of BNP1350 in experimental human colon cancer and compared its anti-tumor effects with those of CPT-11/SN-38 . We also determined a possible influence of Pgp, MRP and LRP on the efficacy of BNP1350 . The in vitro anti-proliferative capacity of the compounds using various exposure times was assessed in five colon cancer cell lines and indicated that BNP1350 was similarly effective or slightly more potent than SN-38 . Four cell lines of other origin with sublines expressing Pgp, MRP and/or LRP showed that BNP1350 was significantly more effective than SN-38 (p < 0.05) and that the activity of BNP1350 was not reduced in multidrug-resistant cells . For in vivo experiments, BNP1350 was given 1.0 mg/kg i.p . or 1.5 mg/kg p.o . daily x 5 and CPT-11 20 mg/kg i.p . daily x 5 being equitoxic schedules in nude mice bearing s.c . human tumor xenografts . The schedules were studied in colon cancer xenografts COLO320, COLO205 or WiDr as well as in two Pgp-positive xenografts 2780AD and BRO/mdr1.1 and the parental Pgp-negative A2780 ovarian cancer xenografts and BRO melanoma xenografts . Growth inhibition of >50% was obtained for BNP1350 given i.p . in six out of the seven xenografts studied . BNP1350 was similarly effective when given i.p . or p.o . CPT-11 was as effective as BNP1350, except in BRO and BRO/mdr1.1 xenografts . Pgp expression in xenografts in vivo confirmed that there was no negative influence on the efficacy of BNP1350 . In conclusion, BNP1350 shows a broad spectrum of activity in experimental human tumors and is a suitable candidate for oral treatment of cancer .

Am J Physiol Regul Integr Comp Physiol, 2000 Oct, 279(4), R1495 - 503
Expression of members of the multidrug resistance protein family in human term placenta; St-Pierre MV et al.; The placenta serves, in part, as a barrier to exclude noxious substances from the fetus . In humans, a single-layered syncytium of polarized trophoblast cells and the fetal capillary endothelium separate the maternal and fetal circulations . P-glycoprotein is present in the syncytiotrophoblast throughout gestation, consistent with a protective role that limits exposure of the fetus to hydrophobic and cationic xenobiotics . We have examined whether members of the multidrug resistance protein (MRP) family are expressed in term placenta . After screening a placenta cDNA library, partial clones of MRP1, MRP2, and MRP3 were identified . Immunofluorescence and immunoblotting studies demonstrated that MRP2 was localized to the apical syncytiotrophoblast membrane . MRP1 and MRP3 were predominantly expressed in blood vessel endothelia with some evidence for expression in the apical syncytiotrophoblast . ATP-dependent transport of the anionic substrates dinitrophenyl-glutathione and estradiol-17-beta-glucuronide was also demonstrated in apical syncytiotrophoblast membranes . Given the cellular distribution of these transporters, we hypothesize that MRP isoforms serve to protect fetal blood from entry of organic anions and to promote the excretion of glutathione/glucuronide metabolites in the maternal circulation.

Eur J Cancer, 2000 Oct, 36(15), 1974 - 83
Cross-resistance in the 2',2'-difluorodeoxycytidine (gemcitabine)-resistant human ovarian cancer cell line AG6000 to standard and investigational drugs; Bergman AM et al.; Gemcitabine (2'-2'-difluorodeoxycytidine; dFdC) is a deoxycytidine analogue which is effective against solid tumours, including lung cancer and ovarian cancer . dFdC requires phosphorylation by deoxycytidine kinase (dCK) for activation . In the human ovarian cancer cell line A2780 and its 30,000-fold dFdC-resistant variant AG6000 (P<0.001), we investigated the cross-resistance profile to several drugs . AG6000, which has a complete dCK deficiency, was approximately 1000-10,000-fold resistant to other deoxynucleoside analogues such as 1-beta-D-arabinofuranosyl cytosine, 2-chloro-deoxyadenosine, aza-deoxycytidine and 2', 2'-difluorodeoxyguanosine (dFdG) (P<0.001) . dFdG can be activated by dCK and deoxyguanosine kinase (dGK), but the latter enzyme was not altered in AG6000 cells . Thus dFdG resistance was only due to dCK deficiency . AG6000 was 1.6- and 46.7-fold resistant to 5-fluorouracil (5-FU) and ZD1694, respectively (the latter was significant; P<0.01), which may be due to the 1.7-fold higher thymidylate synthase (TS) activity, but AG6000 cells were also 2 . 7-fold resistant to the lipophilic TS inhibitor AG337 (P<0.05) . Remarkably, AG6000 cells were 2.5-fold more sensitive to methotrexate (MTX) (P<0.01) than A2780 cells, but 1.6-fold more resistant to trimetrexate (TMQ) (P<0.10) . However, no differences in reduced folate carrier activity, folylpolyglutamate synthetase (FPGS) activity and polyglutamation of MTX were found between the cell lines . AG6000 cells were approximately 2 to 7.5-fold more resistant to doxorubicin (DOX), daunorubicin (DAU), epirubicin and vincristine (VCR) (the latter was significant; P<0.02) and approximately 4-fold more resistant to the microtubule inhibitors paclitaxel and docetaxel (P<0.001) . Fluorescent activated cell sorter (FACS) analysis revealed no P-glycoprotein (Pgp) or multidrug resistance-associated protein (MRP) expression, but less fluorescence of intercalated DAU in AG6000 cells . An approximately 2-fold resistance to the topoisomerase I and II inhibitors etoposide, CPT-11 and SN38 was found in AG6000 cells . Topoisomerase I and IIalpha RNA expression was decreased in AG6000 cells . AG6000 was 2.4, 2.4, 2.3 and 3.7-fold more resistant to EO9 (P<0.02), mitomycin-C (MMC) (P<0.05), cisplatin (CDDP) (P<0.10) and maphosphamide (MAPH), respectively . DT-diaphorase (DTD), which activates EO9, was 2.2-fold lower in AG6000 cells . CDDP resistance might be related to a reduced retention of DNA adducts in AG6000 . However, glutathione levels were equal in A2780 and AG6000 cells . A 24 h exposure to DOX, VCR and paclitaxel at equimolar and equitoxic concentrations, resulted in more double-strand breaks (1.5- to 2-fold) in A2780 than in AG6000 cells . MAPH at 1120 nM and 17 nM of EO9 did not cause DNA damage in either cell line . In conclusion, AG6000 is a cell line highly cross-resistant to a wide variety of drugs . This cross-resistance might be related to altered enzyme activities and/or increased DNA repair.

Clin Cancer Res, 2000 Sep, 6(9), 3417 - 23
Clinical relevance of the lung resistance protein in diffuse large B-cell lymphomas; Filipits M et al.; Drug resistance of non-Hodgkin's lymphomas may involve mechanisms of the multidrug resistance phenotype including the lung resistance protein (LRP) and the multidrug resistance protein (MRP1) . To determine the clinical relevance of these multidrug resistance factors in previously untreated diffuse large B-cell lymphomas (n = 48), we studied LRP and MRP1 expression in lymphoma cells and their impact on clinical outcome . LRP and MRP1 expression were immunohistochemically assessed by means of the monoclonal antibodies LRP-56 and MRPr1, respectively . LRP was positive in 23% and MRP1 in 44% of the samples . LRP expression was associated with higher tumor stage (P = 0.03), elevated serum lactate dehydrogenase levels (P = 0.01), and the International Prognostic Index (P = 0.0001) . LRP-positive patients had a lower complete response rate to polychemotherapy than LRP-negative patients (18 versus 65%; P = 0.006) . Patients with LRP expression had a shorter overall survival than those without LRP expression (median of 0.9 years versus median not reached; P = 0.001) . MRP1 expression was independent of clinical and laboratory parameters and had no impact on the outcome of chemotherapy or survival of the patients . These data suggest that LRP expression but not MRP1 expression is an important mechanism of drug resistance associated with worse clinical outcome in previously untreated diffuse large B-cell lymphomas . Thus, the reversal of LRP-mediated drug resistance may improve clinical outcome in diffuse large B-cell lymphoma in the future.

Emerg Infect Dis, 2000 Sep-Oct, 6(5), 548 - 51
Double infection with a resistant and a multidrug-resistant strain of Mycobacterium tuberculosis; Niemann S et al.; An immunocompetent patient was dually infected with a resistant and a multidrug-resistant strain of Mycobacterium tuberculosis (TB) . The multidrug-resistant strain, which belongs to the W- strain/Beijing family, was first isolated after 3 months of therapy . Inappropriate treatment led to further drug resistance and unsuccessful therapy . Thus, additional infections with resistant M . tuberculosis strains should be considered when tuberculosis therapy fails.

Br J Haematol, 2000 Sep, 110(3), 591 - 8
Expression of the multidrug resistance-associated protein in myelodysplastic syndromes; Poulain S et al.; In the myelodysplastic syndromes (MDS), P-glycoprotein (P-gp) expression is clinically associated with drug resistance, whereas the clinical significance of multidrug resistance-associated protein (MRP1) is uncertain . Bone marrow from 56 patients with MDS, including six with refractory anaemia (RA)/RA with ringed sideroblasts (RARS), 23 cases of RA with excess blasts/in transformation (RAEB/T), four patients with chronic myelomonocytic leukaemia (CMML) and 23 cases of MDS having progressed to acute myeloid leukaemia (MDS-AML), were studied . MRP1 expression was investigated by immunocytochemistry (ICC) and by flow cytometry using MRPm6 monoclonal antibody . The efflux test using calcein-AM (CAM) +/- probenecid to evaluate MRP1 activity was performed in ten of the 56 patients . Twenty-eight of the 56 cases (50%) expressed MRP1 . MRP1 expression was more frequent in MDS-AML than in MDS (70% vs . 36%) . The efflux test using CAM was positive in three out of the ten patients tested . The results were in agreement with expression of MRP1 in six cases, and were discordant in four cases (1 MRP-/CAM+, 3 MRP+/CAM-) . No correlation was observed between MRP1 expression and P-gp, lung resistance-associated protein (LRP) or CD34 expression, although there was a trend for more frequent MRP1 expression in P-gp-positive cases in MDS-AML (P = 0.08) . Ten of the 26 patients treated with intensive chemotherapy achieved complete remission including six out of 16 MRP1+ and four out of ten MRP1- cases (P = NS) . In conclusion, MRP1 expression was correlated with disease stage in MDS in our study . As for P-gp, discordant expression/function of MRP1 could be found in some cases, suggesting the existence of non-functional transport proteins in MDS . MRP1 expression did not seem to be a prognostic factor in MDS in our experience.

J Pharm Pharm Sci, 2000 May-Aug, 3(2), 268 - 80
Regulation of the multidrug resistance genes by stress signals; Sukhai M et al.; Transporters in the body play a large role in the distribution and elimination of many clinically important therapeutic substances . Of these, perhaps the one that has been best studied is P-Glycoprotein (PGP), a 170 kDa membrane-bound protein which has been implicated as a primary cause of multidrug-resistance in tumors . An understanding of the physiological regulation of these transporters is key to designing strategies for the improvement of therapeutic efficacy of drugs which are their substrates . To that end, we examine herein the current state of understanding of the molecular regulation of PGP by a variety of endogenous and environmental stimuli which evoke stress responses including cytotoxic agents, heat shock, irradiation, genotoxic stress, inflammation, inflammatory mediators, cytokines and growth factors.

J Pharmacol Exp Ther, 2000 Oct, 295(1), 360 - 6
Secretory mechanisms of grepafloxacin and levofloxacin in the human intestinal cell line caco-2; Yamaguchi H et al.; Grepafloxacin and levofloxacin transport by Caco-2 cell monolayers was examined to characterize the intestinal behavior of these quinolones . The levels of transcellular transport of {(14)C}grepafloxacin and {(14)C}levofloxacin from the basolateral to the apical side were greater than those in the opposite direction . The unidirectional transport was inhibited by the presence of excess unlabeled quinolones, accompanied by increased accumulation . The inhibitory effects of cyclosporin A plus grepafloxacin on basolateral-to-apical transcellular transport and cellular accumulation of {(14)C}grepafloxacin were comparable to those of cyclosporin A alone, indicating that the transport of grepafloxacin across the apical membrane was mainly mediated by P-glycoprotein . On the other hand, basolateral-to-apical transcellular transport of {(14)C}levofloxacin in the presence of cyclosporin A was decreased by unlabeled levofloxacin, grepafloxacin, and enoxacin, accompanied by significantly increased cellular accumulation . The organic cation cimetidine, organic anion p-aminohippurate, and the multidrug resistance-related protein (MRP) modulator probenecid did not affect the transcellular transport of {(14)C}grepafloxacin or {(14)C}levofloxacin in the presence of cyclosporin A . The basolateral-to-apical transcellular transport of levofloxacin in the presence of cyclosporin A showed concentration-dependent saturation with an apparent Michaelis constant of 5.6 mM . In conclusion, these results suggested that basolateral-to-apical flux of quinolones was mediated by P-glycoprotein and a specific transport system distinct from organic cation and anion transporters and MRP.

Antimicrob Agents Chemother, 2000 Oct, 44(10), 2905 - 7
The EmrR protein represses the Escherichia coli emrRAB multidrug resistance operon by directly binding to its promoter region; Xiong A et al.; EmrR negatively regulates the transcription of the multidrug resistance pump-encoding operon, emrRAB, by binding to its regulatory region . The binding site spans the promoter and the downstream sequence up to the transcriptional start site of the operon . Structurally unrelated drugs that induce the pump interfere with this binding.

Pharm Res, 2000 Jul, 17(7), 803 - 10
Creation of polarized cells coexpressing CYP3A4, NADPH cytochrome P450 reductase and MDR1/P-glycoprotein; Brimer C et al.; PURPOSE: To develop model polarized cell systems expressing cytochrome P4503A4 . NADPH P450 reductase, and P-glycoprotein (Pgp) . METHODS: LLC-PK1 and derivative L-MDR1 cells stably expressing Pgp, the product of the multidrug resistance gene (MDR1), were transfected stably using either a mammalian neomycin selectable expression vector (CYP3A4-Neo) or an episomal vector based on Epstein-Barr virus (CYP3A4-Hygro) . These CYP3A4 expressing cells were compared with LLC-PK1, L-MDR1, or Caco-2 cells transduced with Adenovirus-3A4 vector (Ad3A4) with or without simultaneous Adenovirus-P450 Reductase (AdRed) transduction . Cells were characterized for expression of CYP3A4 protein and CYP3A4 mediated metabolism towards midazolam and testosterone . Analysis of membrane integrity and drug transport assays were performed to determine whether infection with recombinant Ad3A4 +/- AdRed affected Pgp function . RESULTS: The rank order of optimal CYP3A4 expression and activities in LLC-PKI and L-MDR1 cells from highest to lowest was cells cotransduced with Ad3A4 plus AdRed >> Ad3A4 >>> CYP3A4-Hygro > CYP3A4-Neo . Similarly, coexpression of Ad3A4 plus AdRed led to enhanced CYP3A4 mediated metabolism in Caco-2 cells over cells with Ad3A4 alone . Incubation of transwell cultured cells expressing Ad3A4/AdRed with midazolam led to readily detectable metabolite in the medium . In microsomes from Caco-2 and LLC-PK1 cells, each co-transduced with Ad3A4/AdRed, Vmax values for testosterone 6beta-hydroxylase activity ranged from 414 to 1350 pmoles/min/mg, respectively . For either Caco-2 or LLC-MDR1 cells, TEER values and the rate of apical to basal and basal to apical transport of vinblastine or digoxin were similar in cells with and without Ad3A4/Red transduction . CONCLUSIONS: Polarized cellular systems coexpressing Ad3A4, AdRed, and the MDR1/Pgp transporter were developed and characterized . The results document the utility of these polarized model systems for simultaneous drug transport/drug metabolism studies . Since the experimental approach can be adapted to study the interplay of multiple enzyme/ transporting systems, it may find significant application as a screening tool for the pharmaceutical industry and as a more basic research tool to study the kinetics of intestinal drug bioavailability.

Eur J Pharmacol, 2000 Jul 21, 400(2-3), 195 - 8
Expression and immunolocalization of multidrug resistance protein 2 in rabbit small intestine; Van Aubel RA et al.; Multidrug resistance protein 2 (MRP2) is an ATP-dependent transporter of anionic drugs and conjugates . It functions as an efflux pump in the apical membranes of liver and kidney cells, but its membrane localization in small intestine has not yet been defined . The present study demonstrates exclusive localization of Mrp2 to the brush-border (apical) membrane of villi, decreasing in intensity from the villus tip to the crypts . In immunoblot analysis of crude membranes of various rabbit tissues, Mrp2 was only found in small intestine, kidney and liver . These results are in-line with the supposed function of Mrp2 in drug excretion.

Cancer Res, 2000 Sep 1, 60(17), 4779 - 84
Transport of amphipathic anions by human multidrug resistance protein 3; Zeng H et al.; The multidrug resistance-associated protein 1 (MRP1) and the canalicular multispecific organic anion transporter (cMOAT or MRP2) are ATP-binding cassette transporters that confer resistance to some anticancer drugs and efflux glutathione and glucuronate conjugates from the cell . The MRP subfamily of ABC transporters, however, contains at least four other members of which MRP3 (MOAT-D) bears the closest structural resemblance to MRP1 . Although transfection studies have established that human MRP3 confers increased resistance to several anticancer agents, neither the substrate selectivity nor physiological functions of this transporter have been determined . Here we report the results of investigations of the in vitro transport properties of cloned human MRP3 using membrane vesicles prepared from MRP3-transfected HEK293 cells . It is shown that the expression of MRP3 is specifically associated with enhancement of the MgATP-dependent transport into membrane vesicles of the glucuronide estradiol 17-beta-D-glucuronide (E(2)17betaG), the glutathione conjugates 2,4-dinitrophenyl S-glutathione (DNP-SG) and leukotriene C4 (LTC4), the antimetabolite methotrexate, and the bile acid glycocholate . DNP-SG, LTC4, and E(2)17betaG are transported at moderate affinity and low capacity with Km and Vmax values of 5.7 +/- 1.7 microM and 3.8 +/- 0.1 pmol/mg/min, 5.3 +/- 2.6 microM and 20.2 +/- 5.9 pmol/mg/min, and 25.6 +/- 5.4 microM and 75.6 +/- 5.9 pmol/mg/min, respectively . Methotrexate and glycocholate are transported at low affinity and high capacity with Km and Vmax values of 776 +/- 319 microM and 288 +/- 54 pmol/mg/min and 248 +/- 113 microM and 183 +/- 34 pmol/mg/min, respectively . On the basis of these findings, the osmotic dependence of the transport measured and its inability to transport taurocholate, MRP3, like MRP1 and cMOAT, is concluded to be competent in the transport of glutathione S-conjugates, glucuronides, and methotrexate, albeit at low to moderate affinity . In contrast to MRP1, cMOAT, and all other characterized mammalian ABC transporters, however, MRP3 is active in the transport of the monoanionic human bile constituent glycocholate.

Cancer Res, 2000 Sep 1, 60(17), 4761 - 6
Transactivation of the multidrug resistance 1 gene by T-cell factor 4/beta-catenin complex in early colorectal carcinogenesis; Yamada T et al.; The mutational inactivation of a tumor suppressor gene, adenomatous polyposis coli (APC), results in the accumulation of cytoplasmic beta-catenin protein and the activation of T-cell factor (TCF)/lymphoid enhancer factor transcriptional factors . A colorectal carcinoma cell line, DLD-1, was engineered to suppress transactivation by the TCF4/beta-catenin complex in a dominant-negative manner under the strict control of the tetracycline regulatory system . A large-scale comparison of the expression profiles, using two-color fluorescence hybridization of cDNA microarray, led to the identification of MDR1 as a target gene of the TCF4/beta-catenin complex . Luciferase reporter and gel retardation assays revealed the TCF4/beta-catenin responsive elements in the promoter of the human MDR1 gene . Corresponding to the accumulation of beta-catenin, expression of the MDR1 gene product was steadily up-regulated in adenomas and adenocarcinomas of 10 patients with familial adenomatous polyposis . In combination with cell proliferative activities of c-myc and cyclin D1, MDR1 may initiate colorectal tumorigenesis by suppressing cell death pathways programmed in intestinal epithelial cells.

J Clin Oncol, 2000 Sep 15, 18(18), 3211 - 20
Soft tissue leiomyosarcomas and malignant gastrointestinal stromal tumors: differences in clinical outcome and expression of multidrug resistance proteins; Plaat BE et al.; PURPOSE: Several studies have reported clinical behavior and chemotherapy resistance in leiomyosarcomas, but these studies did not differentiate between soft tissue leiomyosarcomas (LMS) and malignant gastrointestinal stromal tumors (GIST) . Multidrug resistance (MDR) has been associated with the expression of P-glycoprotein (P-gp), multidrug resistance protein (MRP(1)), and lung resistance protein (LRP) . The aim of the present study was to compare LMS and GIST with respect to clinical outcome and MDR parameters . PATIENTS AND METHODS: Clinical outcome was evaluated in 29 patients with a primary deep-seated LMS and 26 patients with a primary malignant GIST . Paraffin-embedded material, available for 26 patients with LMS and 25 with GIST, was used for immunohistochemical detection of P-gp, MRP(1), LRP, and c-kit . RESULTS: Mean overall survival (OS) was 72 months for LMS patients and 31 months for GIST patients (P: <.05) . Metastases occurred in 16 (59%) of 27 assessable LMS patients and in 10 (56%) of 18 assessable GIST patients . LMS predominantly metastasized to the lungs (14 of 16 patients), whereas GIST tended to spread to the liver (five of 10 patients) and the abdominal cavity (three of 10 patients; P: <.001) . P-gp and MRP(1) expression was more pronounced in GIST than in LMS (P: <.05): the mean percentage of P-gp expressing cells was 13.4% in patients with LMS and 38.4% in patients with GIST, and the mean percentage MRP(1) expressing cells was 13.3% in patients with LMS and 35.4% in patients with GIST . LRP expression did not differ between LMS and GIST . c-kit was expressed in 5% of the LMS patients and in 68% of the GIST patients . CONCLUSION: LMS patients have a better survival than GIST patients, and the metastatic pattern is different . Expression of MDR proteins in LMS is less pronounced than in GIST.

Int J Tuberc Lung Dis, 2000 Sep, 4(9), 853 - 9
A one-year prospective study (1994-1995) for a first evaluation of tuberculosis transmission in French prisons; Hanau-Bercot B et al.; SETTING: Ten correctional facilities in Paris, including suburbs . OBJECTIVE: To prospectively determine the incidence of tuberculosis (TB) in prisons during a one-year period and to trace the transmission of tuberculosis by restriction fragment length polymorphism (RFLP) analysis of Mycobacterium tuberculosis strains from inmates . RESULTS: Of 93 cases of tuberculosis observed, 50 were culture-confirmed . The incidence of tuberculosis in correctional facilities was 215 cases per 100,000 inmates . A high turnover of inmates was observed . All patients were male, and a quarter had been homeless . Seventy-two per cent were diagnosed with pulmonary tuberculosis . Several severe cases of TB were observed, including three of tuberculous meningitis . No multidrug-resistant strains were noted . RFLP analysis (n = 24) revealed 22 distinct patterns which made up two clusters . Epidemiological investigation did not show direct tuberculosis transmission, which was, however, probable for one cluster . CONCLUSION: Independently of incarceration, prison inmates run a higher risk of developing active tuberculosis than the general population, which might be the main reason for the high incidence of tuberculosis observed in prisons . However, some cases of transmission may occur inside prisons.

Int J Tuberc Lung Dis, 2000 Sep, 4(9), 832 - 8
Primary and acquired drug resistance in Polish tuberculosis patients: results of a study of the national drug resistance surveillance programme; Zwolska Z et al.; OBJECTIVE: To determine the prevalence and patterns of primary and acquired drug resistance among Mycobacterium tuberculosis isolates recovered from tuberculosis patients in Poland . DESIGN: In a prospective survey, M . tuberculosis strains were collected from 3970 tuberculosis patients (2976 newly diagnosed cases and 994 previously treated patients) bacteriologically confirmed by culture between November 1996 and October 1997 . METHODS: Drug susceptibility testing to isoniazid (INH), streptomycin, ethambutol and rifampicin (RMP) was performed on Lowenstein-Jensen medium according to the proportion method and/or using the radiometric Bactec 460 TB system . RESULTS AND CONCLUSION: The male to female ratio was 2.61:1 . The patients were aged between 6 and 82 years, with 86% of males and 77% of females aged over 35 years . Primary resistance to any drug was found in 3.6% of new patients; any INH resistance was 2.6%, any RMP resistance was 0.7%, and multidrug resistance (to INH and RMP {MDR}) was 0.6% . In previously treated cases, resistance to any drug was 17.0%, any INH resistance 14.1%, any RMP resistance 7.8%, and MDR 7.0% . Drug-resistant tuberculosis does not present a big problem in Poland; primary drug resistance has been monitored since 1960 with decreasing frequency, and rates remain at the same level as 20 years ago . Studies such as this should be conducted regularly to monitor drug resistance in Poland in order to effectively manage national tuberculosis control efforts.

Biomaterials, 2000 Nov, 21(21), 2203 - 10
Chronic exposure of human ovarian carcinoma cells to free or HPMA copolymer-bound mesochlorin e6 does not induce P-glycoprotein-mediated multidrug resistance; Tijerina M et al.; The acquisition of multidrug resistance in human ovarian carcinoma A2780 cells was investigated after chronic exposure to free mesochlorin e6 monoethylenediamine (Mce6) and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound Mce6 (P(GG)-Mce6) . The dose that inhibits growth by 50% (IC50) was determined for free Mce6 (2.09 +/- 0.32 microM) and P(GG)-Mce6 (204.15 +/- 28.97 microM) to utilize similar effective doses of drug . A total of 14 drug exposures were performed over a period of 78 days . Cells were characterized by IC50 dose, MDR1 gene expression and anti-human P-glycoprotein (P-gp) antibody binding after each drug exposure . At the conclusion of the experiment, neither the A2780 cells habitually exposed to free Mce6 or P(GG)-Mce6 were significantly different than the control A2780 cells indicating cells did not acquire a MDR phenotype . The doxorubicin (DOX)-resistant A2780/AD cells served as a positive control.

Neoplasma, 2000, 47(2), 100 - 6
Cytotoxic activity of several unrelated drugs on L1210 mouse leukemic cell sublines with P-glycoprotein (PGP) mediated multidrug resistance (MDR) phenotype . A QSAR study; Breier A et al.; L1210/VCR-1 and L1210/VCR-2 cell lines are multidrug resistant (MDR) sublines obtained by adaptation of mouse leukemic cell line L1210 to vincristine and, the development of MDR in these cell lines has been found to be associated with an overexpression of P-glycoprotein (PGP) . In the present work we studied the relationship between the structure of 15 cytotoxic active substances (drugs) and their cytotoxicities on L1210/VCR-1 and L1210/VCR-2 resistant cell lines . The resistance of these MDR cells to the respective drugs was expressed as the ratio of IC50 values obtained for resistant and sensitive cells . These values of resistance were correlated with the following physico-chemical constants of the test substances: binding energy, Ebind; total energy of the molecule, Esum; aromaticity, Kpi; molecular weight, Mw; acidobasic constant, pKa; partition coefficient in water/octanol two phase system, log(p) . It has been found that according to the cytotoxic effects the tested drugs may be divided into three groups: (i) drugs with higher cytotoxicity to the resistant cell lines as to sensitive cells (collateral hypersensitivity); (ii) drugs exhibiting approximately similar effects on sensitive and resistant cell lines; (iii) drugs with weaker cytotoxicity to resistant cells than to sensitive cells . No direct correlations with any physico-chemical constant described above could be established for cell resistance to the drug studied . However, resistance values could be fitted by multiple exponential regression with all described physico-chemical constants implied as six independent variables . The latter procedure made us to conclude that the ability of a drug to be a substrate for PGP is connected with its fulfilling the following criteria: (i) flexible structure of its molecule; (ii) molecular weight lower than approximately 1,300 g/mol; (iii) nonprotonized character at pH 7.0.

AIDS, 2000 Aug 18, 14(12), 1767 - 74
Virological and immunological characteristics of HIV treatment failure; Kaufmann D et al.; BACKGROUND: Resistance to antiretroviral treatment is prevalent . There is limited knowledge of the determinants of disease evolution in subjects infected with multidrug-resistant HIV (MDR-HIV) . METHODS: Infectivity, replication, chemokine receptor usage, and env, gag, protease and reverse transcriptase sequence analysis was performed for MDR-HIV isolates from 14 HIV-infected individuals and compared to wild-type HIV isolates from individuals naive to antiretroviral treatment . Expression of CD45RO/RA, Ki67 and interferon-gamma and CD4 proliferative response to various antigens was determined for individuals infected with MDR-HIV and compared to that in individuals with optimal suppression of viral replication . RESULTS: Infectivity and replication are diminished for various MDR-HIV isolates, usually in the context of an increase in CD4 and CD4+CD45RA+ T-cell counts . However, a number of MDR-HIV isolates are associated with high in vivo viraemia and pronounced immunosuppression, and display in vitro levels of infectivity and replication comparable to those of wild-type strains . No specific genetic sequence or chemokine receptor usage predicted the fitness of an MDR isolate . CONCLUSIONS: Despite the biological diversity of resistant viruses and the range of host responses observed, our descriptive analysis indicates that viral factors play a role in determining the degree of immune damage observed in the context of MDR-HIV infection.

Acta Med Okayama, 2000 Aug, 54(4), 139 - 45
Factors influencing response to treatment of pulmonary tuberculosis; Hiyama J et al.; We analyzed 150 patients with pulmonary tuberculosis from 1990 to 1996 (i) to evaluate the frequency of drug resistance, (ii) to elucidate factors influencing the response to chemotherapy, and (iii) to attempt to improve the therapeutic approach . Multidrug-resistant tuberculosis strains were not found . By univariate analysis, there were 8 factors associated with an increased sputum conversion time: male gender, prior treatment, complications, progressive chest radiographic findings, a high Ziehl-Neelsen stain score, lymphocytopenia, a high erythrocyte sedimentation rate (ESR), and hypoproteinemia . Complications, prior treatment, a high Ziehl-Neelsen stain score, and a high ESR were independent predictive factors in a Cox proportional hazard model . Recursive partitioning and amalgamation (RPA) defined 3 subgroups that responded to treatment . In order to reduce the time to sputum conversion, poor responders according to the RPA should be treated with a 4-drug regimen containing pyrazinamide.

Planta Med, 2000 Aug, 66(6), 531 - 6
Leishmanicidal, antiplasmodial and cytotoxic activity of indole alkaloids from Corynanthe pachyceras; Staerk D et al.; Five indole alkaloids, corynantheidine, corynantheine, dihydrocorynantheine, alpha-yohimbine and corynanthine were isolated from bark of Corynanthe pachyceras K . Schum . (Rubiaceae) . The structures were established by spectroscopic methods, including previously unreported assignment of all 1H-NMR resonances by COSY and NOESY experiments . These and related alkaloids showed pronounced activity against Leishmania major promastigotes (IC50 at the micromolar level) but no significant in vitro antiplasmodial activity (against chloroquine-sensitive Plasmodium falciparum) . Cytotoxicity assessed with drug sensitive KB-3-1 and multidrug-resistant KB-V1 cell lines was low; the alkaloids are apparently not substrates for the P-glycoprotein (P-170) efflux pump.

Epidemiol Infect, 2000 Jun, 124(3), 523 - 8
Drug resistance rates of Mycobacterium tuberculosis strains in Austria between 1995 and 1998 and molecular typing of multidrug-resistant isolates . The Austrian Drug Resistant Tuberculosis Study Group; Stauffer F et al.; In this study the drug resistance pattern of 3559 Mycobacterium tuberculosis strains isolated in Austria between 1995 and 98 was evaluated . Of these strains, 165 (4.6%) were resistant to one or more drugs, 113 (3.2%) to one of the tested drugs and 53 (1.5%) to two or more drugs . Monodrug resistance was observed most often to isoniazid (56 strains), followed by streptomycin (44 strains) . Resistance to rifampicin or ethambutol alone was rarely seen (12 strains and 1 strain, respectively) . Of the 53 strains resistant to 2 or more drugs, 25 were resistant to isoniazid and streptomycin, while 17 were multidrug resistant . Molecular typing revealed a large diversity among the multidrug-resistant strains.

Probl Tuberk, 2000, (4), 24 - 6
{Effectiveness of the surgical treatment of patients with pulmonary tuberculosis and multidrug resistance of its causative agent}; Shaikhaev AIa et al.; In 1996-1998, the Central Institute of Tuberculosis, Russian Academy of Medical Sciences performed more than 600 operations . Of them 90 (15%) patients isolated mycobacteria . The drug resistance of M . tuberculosis was found in 69 (76.7%) patients . In 60.9% of cases, the resistance of M . tuberculosis to isoniazid and rifampicin was concurrently accompanied by that to one (21.4%), two (61.0%), and even 3 (14.8%) tuberculostatics . Removal of the lung or its remnants in the extrapleural layer was most common (67.7%); thoracic cavernomyoplastic operations were made in 17.4% of cases, and partial resections accounted for only 13% . Postoperative complications were more frequently encountered in patients with drug-resistant tuberculosis than in controls (6.7%) . Among them, tuberculosis progression and empyema were prevalent (13%) . The clinical efficiency and duration of surgical treatment were directly related to the rates of progression and to the magnitude of drug-resistance of Mycobacteria.

J Biol Chem, 2000 Dec 1, 275(48), 37347 - 56
Multiple signals from dysfunctional mitochondria activate the pleiotropic drug resistance pathway in Saccharomyces cerevisiae; Hallstrom TC et al.; Multiple or pleiotropic drug resistance most often occurs in Saccharomyces cerevisiae due to substitution mutations within the Cys(6)-Zn(II) transcription factors Pdr1p and Pdr3p . These dominant transcriptional regulatory proteins cause elevated drug resistance and overexpression of the ATP-binding cassette transporter-encoding gene, PDR5 . We have carried out a genetic screen to identify negative regulators of PDR5 expression and found that loss of the mitochondrial genome (rho(o) cells) causes up-regulation of Pdr3p but not Pdr1p function . Additionally, loss of the mitochondrial inner membrane protein Oxa1p generates a signal that results in increased Pdr3p activity . Both of these mitochondrial defects lead to increased expression of the PDR3 structural gene . Importantly, the signaling pathway used to enhance Pdr3p function in rho(o) cells is not the same as in oxa1 cells . Loss of previously described nuclear-mitochondrial signaling genes like RTG1 reduce the level of PDR5 expression and drug resistance seen in rho(o) cells but has no effect on oxa1-induced phenotypes . These data uncover a new regulatory pathway connecting expression of multidrug resistance genes with mitochondrial function.

Int J Hematol, 2000 Jul, 72(1), 20 - 7
Current status and future issues in the treatment of HIV-1 infection; Matsushita S; Over the past 5 years, advances in human immunodeficiency virus type 1 (HIV-1) clinical research and data on the effectiveness of potent combination therapy have substantially influenced the overall perspective of the long-term management of HIV-1 disease . It is now generally accepted that the benefits of mono- and bio-therapy for HIV-1 infection are only transient owing mainly to antiviral-drug resistance . To obtain continued benefit from antiviral therapy, current guidelines recommend at least triple-drug combinations, or so-called highly active antiretroviral therapy (HAART) . In Japan, 13 antiretroviral agents are currently available for combination therapy . Ten of them have been approved for clinical use in the past 3 years . Following the introduction of HAART, marked decreases in AIDS-related morbidity and mortality have been observed . However, in some patients, HAART can be problematic, either because it is difficult for the patient to remain compliant or because previous suboptimum therapies have limited the choice of drugs . For compliant, drug-naive patients, HAART should offer long-term virus suppression, when changing from first- to second- to third-line HAART following drug failure . Long-term treatment might ultimately result in multidrug resistance, leaving few options for salvage therapy . HIV-1 drug resistance testing to enable salvage therapy and the development of new drugs and immunotherapeutic agents to allow new options will therefore remain priorities in HIV-1 research.

Leuk Res, 2000 Sep, 24(9), 769 - 74
Identification of the subcellular localization of daunorubicin in multidrug-resistant K562 cell line; Gong Y et al.; We examined the subcellular distribution of daunorubicin (DNR) in resistant K562 cell line which overexpress the P-glycoprotein by confocal laser scanning microscopy . Three fluorescent probes - Rhodamine123, neutral red, NBD-ceramide, which stain the mitochondria, lysosomes, Golgi apparatus respectively, were used to identify the nature of the subcellular compartment sequestering daunorubicin . In sensitive k562 cell line, nuclear and cytoplasmic DNR fluorescence was intense and diffuse . In contrast, resistant K562 cell line showed a different DNR distribution . A bright fluorescence signal was located in the perinuclear region and peripheral plasma, the nucleus and other cytoplasmic region appear as empty, as suggested by the distribution of fluorescent probe Rhodamine123 specifically for mitochondria . Verapamil, an effective resistance modulator in P-glycoprotein MDR cells, restored the DNR distribution closer to that in the parent cells . Golgic inhibitor brefeldin A and lysosomotropic agent chloroquine had little effect on drug sequestration . Our studies demonstrate that daunorubicin may be sequestered in mitochondrial compartment in the resistant cells and P-glycoprotein plays an important role on mediating DNR transport.

J Biol Chem, 2000 Dec 15, 275(50), 39617 - 24
Identification of basic residues involved in drug export function of human multidrug resistance-associated protein 2; Ryu S et al.; Multidrurg resistance-associated protein 2 (MRP2)/canalicular multispecific organic anion transporter (cMOAT) is involved in the ATP-dependent export of organic anions across the bile canalicular membrane . To identify functional amino acid residues that play essential roles in the substrate transport, each of 13 basic residues around transmembrane regions (TMs) 6-17 were replaced with alanine . Wild type and mutant proteins were expressed in COS-7 cells, and the transport activity was measured as the excretion of glutathione-methylfluorescein . Four mutants, K324A (TM6), K483A (TM9), R1210A (TM16), and R1257A (TM17), showed decreased transport activity, and another mutant, K578A (TM11), showed decreased protein expression . These five mutants were normally delivered to the cell surface similar to the other fully active mutants and wild type MRP2 . The importance of TM6, TM16, and TM17 in the transport function of MRP2 is consistent with the previous observation indicating the importance of the corresponding TM1, TM11, and TM12 on P-glycoprotein (Loo, T . W., and Clarke, D . M . (1999) J . Biol . Chem . 274, 35388-35392) . Another observation that MRP2 inhibitor, cyclosporine A, failed to inhibit R1230A specifically, indicated the existence of its binding site within TM16.

Am J Vet Res, 2000 Sep, 61(9), 1122 - 7
Molecular analysis of multidrug resistance in feline lymphoma cells; Okai Y et al.; OBJECTIVE: To evaluate the mechanism of multidrug resistance in feline lymphoma cell lines . SAMPLE POPULATION: A feline lymphoma cell line (FT-1) and its adriamycin (ADM)-resistant subline (FT-1/ADM) . PROCEDURES: The FT-1 cell line was cultivated in the presence of a gradually increasing concentration of ADM to generate its ADM-resistant subline (FT-1/ADM) . Susceptibility of cells from the parental FT-1 cell line and the FT-1/ADM subline to antineoplastic drugs was determined . From the complementary DNA (cDNA) template of FT-1/ADM cells, feline MDR1 cDNA was amplified by use of polymerase chain reaction (PCR) and sequenced . Reverse transcription (RT)-PCR and Western blot analyses were performed to assess expression of the MDR1 gene and P-glycoprotein (P-gp) in FT-1/ADM cells, compared with that in FT-1 cells . RESULTS: A drug sensitivity assay revealed that FT-1/ADM cells were much more resistant to ADM and vincristine than the parental FT-1 cells . The feline MDR7 cDNA amplified by use of PCR was 3,489 base pairs long, corresponding to approximately 90% of the whole open reading frame of human MDR1 cDNA; its amino acid sequence was 91.5, 87.0, and 79.4% identical to that of human MDR1, mouse mdr1a, and mdr1b cDNA, respectively . By RT-PCR analysis, expression of MDR1 messenger RNA was clearly detected in FT-1/ADM cells but not in the parental FT-1 cells . Western blot analysis also revealed the expression of P-gp encoded by the MDR1 gene in FT-1/ADM cells but not in FT-1 cells . CONCLUSIONS: The basic structure of the feline MDR1 gene was essentially the same as that of multidrug-resistance genes of other species . Expression of P-gp appeared to be one of the mechanisms responsible for the development of multidrug resistance in feline lymphoma cell lines in vitro.

Curr Opin Oncol, 2000 Sep, 12(5), 450 - 8
Multidrug resistance transporters and modulation; Tan B et al.; Multidrug resistance (MDR), whereby tumor cells simultaneously possess intrinsic or acquired cross-resistance to diverse chemotherapeutic agents, hampers the effective treatment of cancer . Molecular investigations in MDR resulted in the isolation and characterization of genes coding for several proteins associated with MDR, including P-glycoprotein (P-gp), the multidrug resistance associated protein (MRP1), the lung resistance protein (LRP), and, more recently, the breast cancer resistance protein (BCRP) . These transmembrane proteins cause MDR either by decreasing the total intracellular retention of drugs or redistributing intracellular accumulation of drugs away from target organelles . These proteins are expressed at varying degrees in different neoplasms, including the AIDS-associated non-Hodgkin lymphoma and Kaposi sarcoma and are generally associated with poor prognosis . Several MDR-reversing agents are in various stages of clinical development . First-generation modulators such as verapamil, quinidine, and cyclosporin required high doses of drugs to reverse MDR and were associated with unacceptable toxicities . Second- and third-generation MDR inhibitors include PSC 833, GF120918, VX-710, and LY335979, among others . Limitations to the use of these modulators include multiple and redundant cellular mechanisms of resistance, alterations in pharmacokinetics of cytotoxic agents, and clinical toxicities . Studies to validate the role of MDR reversal in the treatment of various malignancies are underway . A potential use of these agents may be to enhance intestinal drug absorption and increase drug penetration to biologically important protective barriers, such as the blood-brain, blood-cerebrospinal fluid, and the maternal-fetal barriers . The use of MDR modulators with drugs such as the antiviral protease inhibitors and cytotoxics may enhance drug accumulation in sanctuary sites that are traditionally impenetrable to these agents.

Trans R Soc Trop Med Hyg, 2000 May-Jun, 94(3), 271 - 5
RFLP patterns and risk factors for recent tuberculosis transmission among hospitalized tuberculosis patients in Rio de Janeiro, Brazil; Fandinho FC et al.; Isolates of Mycobacterium tuberculosis from 120 tuberculosis patients seen in the 12 months ending September 1994 at 2 tertiary-care centres in Rio de Janeiro were characterized by IS6110 restriction fragment length polymorphism (RFLP) analysis . Ninety-seven patients (81%) had isolates with unique RFLP patterns, while 23 patients (19%) had isolates that belonged to 11 different RFLP cluster patterns . The strains from the latter patients were distributed among 1 group of 3 patients and 10 groups of 2 patients each . The cluster-pattern strains were not associated with gender, age, HIV infection, type of residence, living in shelter, homelessness or previous history of tuberculosis . However, clustering was strongly associated with multidrug resistance (P = 0.006) . These data suggest that recent exogenous transmission may be important for the development of new cases of multidrug-resistant disease in patients attending tertiary-care centres in Rio de Janeiro, Brazil.

Cesk Patol, 2000 Jul, 36(3), 111 - 5
{Biological characteristics of multiple myeloma}; Dusek J et al.; On a series of thirty trephine bone marrow biopsies from patients with multiple myeloma, the authors evaluated expression of markers of cell proliferation or of its blockade (Ki-67, PCNA, topoisomerase IIa, cyclin D-1, AgNOR, and p27kip1) and markers indicating multidrug resistance (P-170 and Bcl-2) . Expression of Ki-67 and of topoisomerase IIa was unfrequent . Marked positivity of PCNA was expressed in about one third of cases, negative staining was exceptional . No expression of cyclin D-1 was noted . Positivity of p27kip1 was frequent . P-170 was demonstrated in a small number of cases, Bcl-2 was strongly positive in most cases . The results characterise multiple myeloma as a tumour with low proliferation rate and, simultaneously, with high resistance to apoptosis.

Acta Cient Venez, 2000, 51(1), 45 - 52
{Multidrug or pleiotropic resistance}; Arvelo F et al.; The resistance to cytotoxic drugs represents a major obstacle to successful cancer therapy . The intrinsic resistance of tumoral cells is one of major causes of treatment failure . The overexpression of a membrane associated glycoprotein, P-glycoprotein, in tumoral cell lines, resistant to a wide range of drugs, permitted the description of a multidrug resistance (MDR) phenotype . This P-glycoprotein, which appears to play a role in drug efflux is encoded by the mdr1 gene in humans . The frequent mdr1 gene overexpression in clinically resistant tumours suggest that this gene may be the cause of treatment failure in human cancer . This review summarizes recent developments in this area, which suggest that both the activity of the pump and its genetic regulation are potential targets for new anticancer therapies.

Cancer Lett, 2000 Oct 16, 159(1), 95 - 101
Establishment and characterization of 5-fluorouracil-resistant gastric cancer cells; Chung YM et al.; Two 5-fluorouracil (5-FU)-resistant cell lines from a Korean gastric cancer cell line were established by incubation of the cells with increasing concentration of 5-FU, and the resultant cell lines showed an over 800-fold increased resistance to 5-FU . To identify the mechanism of 5-FU resistance, the expressions of genes involved in 5-FU metabolism were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) . Expressions of orotate phosphoribosyltransferase (OPRT), thymidine phosphorylase (TP), and uridine phosphorylase (UP) were significantly downregulated in these cell lines, resulting in low incorporation of 5-FU into nucleic acids . In contrast, an increased expression of thymidine kinase (TK) was observed in 5-FU-resistant cells . These results strongly indicate that blocking of 5-FU incorporation into nucleic acids and TK overexpression may play a major role in 5-FU resistance in these cells . Interestingly, these cell lines showed cross-resistance to paclitaxel, cisplatin, and doxorubicin, suggesting that other factors such as HSP27 and Mn-SOD could be also involved in the mechanism of multidrug resistance in these cell lines.

Curr Pharm Des, 2000 Nov, 6(16), 1653 - 68
Monitoring interactions at ATP-dependent drug efflux pumps; Hendrikse NH; Chemotherapeutic treatment of cancer patients is often unsuccessful, due to the involvement of various mechanisms, leading to multidrug resistance (MDR) . In this review, I describe the mechanisms involved in MDR . Furthermore, results obtained by imaging of P-glycoprotein (P-gp) and the multidrug resistance associated protein (MRP) are reviewed . Single photon emission computed tomography (SPECT) and positron emission tomography (PET) are unique techniques to study P-gp- and MRP-mediated transport . The radiopharmaceutical (99m)Tc-sestamibi is a substrate for both P-gp and MRP . This tracer has been used for tumor imaging in clinical studies, and to visualize blockade of P-gp mediated transport after modulation of the P-gp pump . Other (99m)Tc-radiopharmaceuticals such as (99m)Tc- tetrofosmin and several (99m)Tc-Q-complexes are also substrates for P-gp . Until now, for these compounds only results from in vitro and animal studies are available . For quantification of P-gp mediated transport with PET in vivo, several agents, such as {(11)C}colchicine, {(11)C}verapamil and {(11)C}daunorubicin have been evaluated . In vivo results suggest that these radiopharmaceuticals can be used to image P-gp function in tumors . (124)I and (76)Br radiolabeled doxorubicin analogues are also useful to examine P-gp mediated transport . Leukotrienes are specific substrates for MRP . Therefore, N-{(11)C}acetyl-leukotriene E4 provides the opportunity to study MRP function non-invasively . Results obtained with this radiopharmaceutical in MRP(2) mutated GY/TR- rats indicate visualization of MRP-mediated transport . This tracer enables to study MRP transport function abnormalities in vivo such as in Dubin-Johnson patients, who are MRP(2) gene deficient . In conclusion, it is feasible to study the functionality of MDR transporters in vivo, both with SPECT and with PET . Such imaging techniques may become an important factor in the development of novel chemotherapeutic drugs.

Biochem Biophys Res Commun, 2000 Sep 7, 275(3), 795 - 803
Structure-activity studies of verapamil analogs that modulate transport of leukotriene C(4) and reduced glutathione by multidrug resistance protein MRP1; Loe DW et al.; The 190-kDa multidrug resistance protein MRP1 is an ATP-binding cassette protein that confers resistance to multiple antineoplastic agents and actively transports conjugated organic anions . We have previously shown that MRP1-mediated GSH transport is stimulated by verapamil but transport of verapamil in the presence or absence of GSH is not observed . We have now examined 20 sulfur-containing verapamil analogs for their ability to inhibit MRP1-mediated leukotriene C(4) (LTC(4)) transport and stimulate GSH uptake into inside-out membrane vesicles . All of the derivatives were poor inhibitors of LTC(4) uptake . However, the inhibitory potency of the more lipophilic dithiane compounds could be enhanced by coincubation with GSH whereas this was not the case for the more hydrophilic dithiane tetraoxides . The dithiane derivatives stimulated GSH transport whereas, with one exception, the dithiane tetraoxides did not . One pair of dithiane stereoisomers differed significantly in their ability to stimulate GSH transport although their ability to inhibit LTC(4) uptake in the presence of GSH was comparable . Our findings indicate that the GSH transport activity of MRP1 can be dissociated from its conjugated organic anion transport activity .

Brain Res, 2000 Sep 8, 876(1-2), 148 - 53
Expression of various multidrug resistance-associated protein (MRP) homologues in brain microvessel endothelial cells; Zhang Y et al.; Multidrug resistance-associated protein (MRP) actively transports a broad range of anionic compounds out of the cell . To date, six different homologues of MRP (i.e . MRP1-MRP6) have been identified . The current study examines the expression of the various MRP homologues in both primary cultured bovine brain microvessel endothelial cells (BBMEC) and the capillary-enriched fraction from bovine brain homogenates . RT-PCR analysis demonstrated the presence of MRP1, MRP4, MRP5 and MRP6 in both BBMEC and the capillary-enriched fractions of brain homogenates . While low levels of MRP3 were detected in the BBMEC, it was not observed in the capillary-enriched fraction . In addition, RT-PCR and Western blot studies indicated an absence of MRP2 expression in both blood-brain barrier preparations . The presence of several different MRP homologues in the brain microvessel endothelial cells may be important in controlling the permeability of the blood-brain barrier to organic anions.

Br J Clin Pharmacol, 2000 Sep, 50(3), 237 - 46
Effect of P-glycoprotein modulation on the clinical pharmacokinetics and adverse effects of morphine; Drewe J et al.; AIMS: To investigate the effect of acute P-glycoprotein inhibition by the multidrug-resistance (MDR) modulator valspodar (SDZ PSC 833; PSC) on the pharmacokinetics, and potentially adverse pharmacodynamic effects of morphine, and its principal pharmacologically active metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) . METHODS: In a double-blind, three-way crossover study, the pharmacokinetic and potentially adverse pharmacodynamic effects (reaction time, transcutaneous PCO2, blood pressure) of morphine were compared with and without acute inhibition of P-glycoprotein by PSC . The effects of PSC alone were also evaluated . The study was performed in 18 healthy male volunteers and pharmacodynamic effects analysed by measuring the area under the effect (AUE) curve . 150 mg PSC (or its placebo) was given as an i.v . infusion over 2 h . With the expected inhibition of Pgp 1 h after starting PSC infusion, 7.5 morphine HCl (or its placebo) was infused over 2 h . RESULTS: The infusion of PSC resulted in blood concentrations expected to inhibit Pgp mediated transport . While the pharmacokinetics of plasma morphine and M6G . were unaffected there was a small but statistically significant increase in the AUC and Cmax of M3G (11.8 and 8.3%, respectively) . The t(1/2) and tmax were unaffected . The pharmacokinetic parameters of PSC were not affected by coadministration with morphine . PSC did not significantly affect the adverse events of morphine, as assessed by spontaneous reporting . Compared with PSC alone, morphine elicited an increase in reaction time (Emax 48 ms, compared with the predose absolute reaction time of 644 ms), which was not detected by the alertness-drowsiness score, indicating only slight sedation . There was a significant decrease in systolic blood pressure (Emin -9 mm Hg), and a trend for a fall in diastolic blood pressure (Emin -14.5 mm Hg) and respiratory rate (Emin -1.8 breath x min(-1)) . For all these parameters, the effects of PSC/morphine were similar to that of PSC alone, suggesting some attenuation of morphine's effect . In contrast, morphine caused a significant increase in PCO2 (Emax 0.69 kPa) compared to PSC alone, indicating slight respiratory depression . This increase was similar to that of the PSC/morphine combination . CONCLUSIONS: Acute inhibition of P-glycoprotein by PSC in this setting does not affect the pharmacokinetic or safety-related pharmacodynamic profile of morphine in a clinically significant manner.

Br J Cancer, 2000 Oct, 83(7), 921 - 7
Drug resistance features and S-phase fraction as possible determinants for drug response in a panel of human ovarian cancer xenografts; Kolfschoten GM et al.; Multidrug resistance (MDR) and more specifically the expression of P-glycoprotein (Pgp) have been studied extensively in vitro . Unfortunately, it appears that the predictive value of MDR recognized in vitro is mostly an incorrect measure to determine the responsiveness of a particular tumour in the clinic . This misunderstood or overvalued role of MDR might explain the failure of strategies to reverse Pgp function by the use of modulators in solid tumours . To obtain more insight in in vivo drug resistance we investigated a panel of 15 human ovarian cancer xenografts consisting of the most common histological subtypes known in ovarian cancer patients . The response rate to cisplatin, cyclophosphamide and doxorubicin in the xenografts resembled the results of phase II trials with these agents in ovarian cancer patients . This resemblance justifies drug resistance studies in this experimental in vivo human tumour system . We determined the expression levels of MDR 1, MRP 1, LRP and topoisomerase IIalpha mRNA by the RNase protection assay and the presence of MRP1 and LRP proteins by immunohistochemistry . The S-phase fraction was investigated as a separate parameter by flow cytometry . In none of the 15 ovarian cancer xenografts was MDR 1 expression detectable . The expression levels of MRP 1 and LRP were low to moderate and resembled the presence of the MRP1 and LRP proteins . There was a weak, inverse relationship between the expression levels of LRP and sensitivity to cisplatin and cyclophosphamide (r = -0.44 and -0.45), but not to doxorubicin . The levels of topoisomerase IIalpha varied among the xenografts (0.73-2.66) and failed to correlate with doxorubicin resistance (r = 0.14) . The S-phase fraction, however, showed a relation with the sensitivity to cisplatin (r = 0.66) . Among the determinants studied in ovarian cancer in vivo, LRP mRNA and the S-phase fraction were the best predictive factors for drug response and most specifically for the activity of cisplatin .

Br J Cancer, 2000 Oct, 83(7), 892 - 8
MDR 1 activation is the predominant resistance mechanism selected by vinblastine in MES-SA cells; Chen GK et al.; Single-step selection with vinblastine was performed in populations of the human sarcoma cell line MES-SA, to assess cellular mechanisms of resistance to the drug and mutation rates via fluctuation analysis . At a stringent selection with 20 nM vinblastine, resulting in 5-6 logs of cell killing, the mutation rate was 7 x 10(-7)per cell generation . Analysis of variance supported the hypothesis of spontaneous mutations conferring vinblastine resistance, rather than induction of adaptive response elements . Surviving clones displayed a stable multidrug resistance phenotype over a 3-month period . All propagated clones demonstrated high levels of resistance to vinblastine and paclitaxel, and lower cross-resistance to doxorubicin and etoposide . Activation of MDR 1 gene expression and P-glycoprotein function was demonstrable in all clones . No elevation was found in the expression of the mrp gene, the LRP-56 major vault protein and beta-tubulin isotypes (M40, beta4, 5beta, and beta9) in these mutants . We conclude that initial-step resistant mechanism in these vinblastine-selected mutants commonly arises from a stochastic mutation event with activation of the MDR 1 gene .

Cancer Res, 2000 Aug 15, 60(16), 4403 - 11
Human acute myeloid leukemia CD34+/CD38- progenitor cells have decreased sensitivity to chemotherapy and Fas-induced apoptosis, reduced immunogenicity, and impaired dendritic cell transformation capacities; Costello RT et al.; The destruction of cells capable of initiating and maintaining leukemia challenges the treatment of human acute myeloid leukemia . Recently, CD34+/CD38- leukemia progenitors have been defined as new leukemia-initiating cells less mature than colony-forming cells . Here we show that CD34+/CD38- leukemia precursors have reduced in vitro sensitivity to daunorubicin, a major drug used in leukemia treatment, in comparison with the CD34+/CD38+ counterpart, and increased expression of multidrug resistance genes (mrp/lrp) . These precursors show lower expression of Fas/Fas-L and Fas-induced apoptosis than CD34+/CD38+ blasts . Moreover, the CD34+/CD38- leukemic subpopulation induces a weaker mixed leukocyte reaction of responding T-lymphocytes than the CD34+/CD38+ leukemic counterpart, either in a MHC-unmatched or MHC-matched settings . This weaker immunogenicity could be linked to lower expression on CD34+/CD38- leukemia precursors of major immune response molecules (MHC-DR, LFA-3, B7-1, or B7-2) than CD34+/CD38+ leukemic cells . Nonetheless, the susceptibility of the immature CD38- precursors to cytotoxicity was not different from the sensitivity of the CD38+ counterpart . Finally, CD34+/CD38- leukemia precursors, in contrast with CD38+ precursors, failed, under appropriate conditions, to differentiate into dendritic cells, a central step for antigen recognition . This is to our knowledge the first demonstration that the very immature phenotype of CD34+/CD38- leukemic progenitors confers both chemotherapy resistance and decreased capacities to induce an immune response . Because the susceptibility of the immature leukemia cells as cytotoxic targets is maintained, our data underline the importance of improving the initial steps of leukemia recognition, more particularly by defining optimal conditions of dendritic cell transformation of the very immature hematopoietic precursors.

Eur Respir J, 2000 Aug, 16(2), 364 - 71
Standardization of antituberculosis drug resistance surveillance in Europe . Recommendations of a World Health Organization (WHO) and International Union Against Tuberculosis and Lung Disease (IUATLD) Working Group; Schwoebel V et al.; Surveillance of antituberculosis drug resistance is an essential tool for evaluating the quality of tuberculosis control programmes . Consensus-based recommendations on uniform reporting of antituberculosis drug resistance surveillance data in Europe have been developed by a Working Group of the World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Disease (IUATLD) . Laboratories should use standardized methods for testing drug susceptibility with a quality assurance programme including national and international proficiency testing . The proportion of drug resistance, particularly resistance to isoniazid, rifampicin or both (multidrug resistance) among all definite, i.e . culture-positive, tuberculosis cases at the start of treatment is the major indicator of interest . It should be calculated separately among patients treated previously and among those who have never been treated with > or = 1 month of combined antituberculosis drugs . The Working Group recommends that, in countries in which resources allow, laboratories report drug susceptibility test results on all isolates of the Mycobacterium tuberculosis complex . Test results of the specimen at the start of treatment and clinical data from the notification should be linked using a suitable identifier . Results should be presented by calendar year and analysed by age, sex, place of birth, site of disease and sputum smear results . In countries in which a routine system cannot be organized, representative surveys or sentinel systems are possible alternatives . In some countries, the annual prevalence of multidrug-resistant tuberculosis may be estimated through a national laboratory reporting system.

Eur Respir J, 2000 Aug, 16(2), 203 - 8
Results from 8 yrs of susceptibility testing of clinical Mycobacterium tuberculosis isolates in Denmark; Thomsen VO et al.; Increased rates of multidrug-resistant (MDR) tuberculosis (TB) has been reported from countries close to Denmark . This study evaluated the incidence of drug resistance in Denmark in order to determine the magnitude of the problem . Susceptibility testing was performed in isolates from 85.4% of all notified patients during 1991-1998 . Epidemiological information was retrieved from the mandatory notification forms . Total drug resistance remained largely constant, although a minor increase was observed in 1997-1998 . Monoresistance was observed in 7.3%, of the isolates . Among 3.6% polyresistant isolates, resistance to isoniazid and streptomycin accounted for 2.8%, whereas MDR accounted for 0.5% . The MDR strains displayed different restriction fragment length polymorphism (RFLP) patterns, and no matches were identified in the international MDR database . Drug resistance in untreated Danes and foreigners were 5.9% and 14.6%, respectively . Among Danes and foreigners with previous TB, 6.2% and 22.7% had drug resistance, respectively . Increased drug-resistance was found among untreated Danes aged 25-54 yrs mainly due to a single isoniazid and streptomycin-resistant RFLP-cluster . Among all patients with isoniazid and streptomycin-resistance, 77.0% had clustered strains . In conclusion, although drug resistance among untreated Danes was close to the rate estimated in good national programmes, close monitoring is needed in future years, as active transmission of isoniazid- and streptomycin-resistant Mycobacterium tuberculosis was demonstrated.

Life Sci, 2000 Jul 7, 67(7), 759 - 63
Pyronaridine: an effective antimalarial against multidrug-resistant malaria; Dutta GP et al.; Pyronaridine, administered intramuscularly (im) to Swiss mice infected with the lethal multidrug-resistant Plasmodium yoelii nigeriensis, was found to exert high blood schizontocidal activity . The efficacy of doses of pyronaridine ranging from 0.625 to 30 mg (base/kg) was evaluated using a 4 day treatment schedule (drug was administered at 0, 24, 48 and 72 hrs) . It was found that doses of 2.5mg/ kg and higher protected animals completely from the lethal effects of the parasite . The same degree of protection was found when the treatment duration was reduced to 3 days . This study shows that pyronaridine is a potentially useful antimalarial drug that could be exploited for the control of multidrug-resistant malaria infection.

Arch Dermatol Res, 2000 Jul, 292(7), 354 - 61
Expression and activity of P-glycoprotein in transplantable hamster melanomas; Witkowski JM et al.; In the study described here we investigated the possibility of an association between the aggressiveness of melanoma and multidrug resistance phenotype by analyzing the expression and activity of P-glycoprotein (Pgp) in two genetically related transplantable hamster melanomas--a melanotic (Ma) and an amelanotic (Ab) form --which differed in aggressiveness and metastatic potential . Flow cytometric analysis of Pgp activity (using a verapamil-sensitive rhodamine R123 exclusion test) as well as Western blotting of cellular lysates showed its preferential (although not very marked) expression in the Ab melanoma cells . The Ab melanoma cells also exhibited a higher proportion of tumor-infiltrating lymphocytes (TIL), mostly of T cell phenotype, that may have reflected a higher immunogenicity of the tumor . In conclusion, Pgp activity appeared to be associated with less-differentiated more aggressively metastasizing melanoma (the Ab variant) although its role in maintaining this phenotype remains to be established.

Biol Pharm Bull, 2000 Aug, 23(8), 926 - 9
Mithramycin represses MDR1 gene expression in vitro, modulating multidrug resistance; Tagashira M et al.; The effect of an aureolic acid, mithramycin (MTM) on multidrug resistance (MDR) was investigated . At a concentration of 0.02--0.1 mg/ml (about 20--90 microM), MTM repressed MDR1 gene transcription of SBC-3/ADM, a MDR-phenotype subline derived from human small cell lung tumor . Under the same conditions, another aureolic acid, chromomycin A3, showed potent cytotoxicity . FACS analysis revealed that 5 microm MTM depleted the P-glycoprotein (Pgp) and lowered the efflux activity of SBC-3/ADM cells . Furthermore, MTM sensitized the cells against adriamycin . These results suggest that MTM would be a useful modulator of MDR induced by Pgp.

Biochim Biophys Acta, 2000 Aug 31, 1481(1), 63 - 74
Two transport binding sites of P-glycoprotein are unequal yet contingent: initial rate kinetic analysis by ATP hydrolysis demonstrates intersite dependence; Wang EJ et al.; The ATP-dependent transport enzyme known as P-glycoprotein (P-gp) confers multidrug resistance (MDR) against many unrelated drugs and xenobiotics . To understand better the broad substrate specificity of the enzyme as well as the mechanism of substrate transport out of the cell, it is critical to characterize the substrate binding sites . Since approximately 1 ATP is hydrolyzed per transport event, phosphate release rate provides a steady-state kinetics assay . Notably, the substrate H33342 causes a decrease in the baseline hydrolysis of ATP (probably due to competition for transport with an endogenous membrane lipid substrate) providing an excellent tool for a comprehensive graphical kinetic analysis of the interaction of substrate pairs at the transport site(s) allowing the determination of inhibition type and hence characterization of transport binding sites . The substrate H33342 interacted with quinidine, progesterone, and propranolol in a non-competitive manner, indicating that binding of H33342 precludes active transport of these other substrates at a distinct site . Compounds such as TPP+ and verapamil, and perhaps also nicardipine, interacted with H33342 as mixed-type inhibitors . This type of interaction results from a reduced affinity at the opposing active site by a factor of alpha and sometimes a partial activity of a fraction beta . Indeed, H33342 binding caused a roughly four-fold reduced affinity for TPP+ . Using this definitive approach to inhibition kinetics, we were able to establish traits of a second transport site in P-gp . Therefore, the sites are unequal; however, the performance at one site is contingent on the other being unoccupied, and transport is also sometimes mitigated when the other site is occupied.

Cancer Lett, 2000 Oct 1, 158(2), 203 - 10
Transfer of p14ARF gene in drug-resistant human breast cancer MCF-7/Adr cells inhibits proliferation and reduces doxorubicin resistance; Guo-Chang F et al.; The INK4a/ARF locus on human chromosome 9p21 encodes two tumor suppressors, p16INK4a and p14ARF, that restrain cell growth by affecting the functions of the retinoblastoma protein and p53, respectively . Overexpression of ARF results in cell cycle arrest in both G1 and G2 . To elucidate the effect of p14ARF gene on multidrug-resistant tumor cells, we transferred a p14ARF cDNA into p53-mutated MCF-7/Adr human breast cancer cells . In this report we demonstrated for the first time that p14ARF expression was able to greatly inhibit the MCF-7/Adr cell proliferation . Furthermore, p14ARF expression resulted in decrease of MDR-1 mRNA and P-glycoprotein production, which linked to the reducing resistance of MCF-7/Adr cells to doxorubicin . These results imply that drug resistance might be effectively reversed by the wild-type p14ARF expression in human breast cancer cells.

Hepatology, 2000 Sep, 32(3), 556 - 62
Altered gene expression in the liver of gamma-glutamyl transpeptidase-deficient mice; Habib GM et al.; We used mice deficient in gamma-glutamyl transpeptidase (GGT) to analyze the effects of GGT deficiency and altered thiol levels on gene expression in liver . GGT-deficient mice have markedly reduced levels of glutathione (GSH), cysteine, methionine, and cysteinylglycine in liver . Steady-state RNA levels of the catalytic subunit of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme in GSH synthesis, are elevated 4-fold in these mice, while those for glutathione synthetase (GSH syn) are elevated 2-fold . RNA levels of cystathionase (cystathionine gamma-lyase), a key enzyme in the synthesis of cysteine from methionine, are elevated approximately 3.5-fold . In contrast, levels of RNA coding for multidrug resistance protein 2 (MRP2), which transports GSH into bile, are half wild-type values . We found no change in RNA levels of enzymes related to oxidative injury (CuZn and Mn superoxide dismutases {SOD}, catalase, and glutathione peroxidase) . Similarly, RNA levels of glutathione reductase and ribonucleotide reductase were unchanged . Furthermore, in contrast to previous in vitro results, methyl methanesulfonate did not induce stress-activated signal transduction as measured by c-jun phosphorylation in livers of GGT-deficient mice, despite further depletion of GSH by buthionine sulfoximine . Our findings indicate that GGT deficiency itself and/or altered thiol levels regulate expression of genes involved in GSH metabolism, but have no effect on the expression of other antioxidant genes.

Pediatr Infect Dis J, 2000 Aug, 19(8), 695 - 9
Transmission of multidrug-resistant tuberculosis; Schaaf HS et al.; AIM: To compare the Mycobacterium tuberculosis isolates of adult index cases with multidrug-resistant (MDR) tuberculosis to the isolates obtained from their child contacts . PATIENTS AND METHODS: A 4-year prospective study in the Western Cape Province of South Africa . We evaluated 149 child contacts of 80 adult MDR pulmonary tuberculosis cases . This report includes those cases where a culture for M . tuberculosis was obtained from both the adult source case and the child contact . Isolates were compared by drug susceptibility pattern and restriction fragment length polymorphism analysis . RESULTS: Six adult-child pairs with cultures for M . tuberculosis were identified . Two children had contact with more than one adult tuberculosis case . One child received previous isoniazid prophylaxis . Drug susceptibility pattern and restriction fragment length polymorphism analysis were identical for five adult-child pairs . One child, with no other known source case, had a strain different from that of the identified source case, but the MDR M . tuberculosis strain with which he was infected was prevalent in the community in which he resided . All children responded well to treatment . CONCLUSION: This study confirms that most of the childhood contacts of adults with MDR tuberculosis are likely to be infected by these MDR source cases despite their exposure to other drug-susceptible adults with tuberculosis in some instances . Child contacts of adults with MDR tuberculosis should be treated according to the drug susceptibility patterns of the likely source cases' M . tuberculosis strains unless their own strain's susceptibility testing indicates otherwise . Contact tracing remains of fundamental importance in identifying children at risk.

Invest New Drugs, 2000 Aug, 18(3), 205 - 20
Development of multidrug-resistance convertors: sense or nonsense?
van Zuylen L, Nooter K, Sparreboom A, Verweij J.
This review describes the clinical relevance of the two drug transporters P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP) and the in vitro phenomenon which is referred to as multidrug resistance (MDR) . The attempts to try to block these resistance mechanisms are summarized with specific attention for the intentionally designed "second generation" MDR-convertors . Potential explanations of the limited clinical success rate are given and recommendations for the design of future studies provided.

S Afr Med J, 2000 Apr, 90(4), 381 - 6
Drug-resistant pulmonary tuberculosis in a cohort of southern African goldminers with a high prevalence of HIV infection; Murray J et al.; OBJECTIVES: To determine rates of drug resistance to Mycobacterium tuberculosis and associated risk factors, including HIV infection . DESIGN: Prospective cohort study of patients with pulmonary tuberculosis . SETTING: The study population comprised 28,522 men working on four goldmines in Westonaria, Gauteng . Health care is provided at a 240-bed mine hospital, Gold Fields West Hospital, and its primary health care facilities . SUBJECTS: All 425 patients with culture-positive pulmonary tuberculosis identified in 1995 . OUTCOME MEASURES: Tuberculosis drug resistance on enrollment and after 6 months' treatment . RESULTS: There were 292 cases of new tuberculosis, 77 of recurrent disease and 56 prevalent cases in treatment failure . Two hundred and seven patients (48.7%) were HIV infected . Primary resistance to one or more drugs (9%) was similar to the 11% found in a previous study done on goldminers in 1989 . Primary multidrug resistance (0.3%) was also similar (0.8%) . Acquired multidrug resistance was 18.1%: 6.5% for recurrent disease and 33.9% in treatment failure cases . Neither HIV infection nor the degree of immunosuppression as assessed by CD4+ lymphocyte counts was associated with drug resistance at the start or end of treatment . New patterns of drug resistance were present in 9 of 52 patients in treatment failure at 6 months, 1 of whom was HIV-infected . CONCLUSION: Primary and acquired drug resistance rates are stable in this population and are not affected by the high prevalence of HIV infection.

J Org Chem, 2000 Aug 11, 65(16), 4973 - 83
The synthesis and evaluation of a solution phase indexed combinatorial library of non-natural polyenes for reversal of P-glycoprotein mediated multidrug resistance; Andrus MB et al.; A combinatorial library of polyenes, based on (-)-stipiamide, has been constructed and evaluated for the discovery of new multidrug resistance reversal agents . A palladium coupling was used to react each individual vinyl iodide with a mixture of the seven acetylenes at near 1:1 stoichiometry . The coupling was also used to react each individual acetylene with the mixture of six vinyl iodides to create 13 pools indexed in two dimensions for a total of 42 compounds . Individual compounds were detected at equimolar concentration . The vinyl iodides, made initially using a crotylborane addition to generate the anti1,2-hydroxylmethyl products, were now made using a more efficient norephedrine propionate boron enolate aldol reaction . The indexed approach, ideally suited for cellular assays that involve membrane-bound targets, allowed for the rapid identification of reversal agents using assays with drug-resistant human breast cancer MCF7-adrR cells . Intersections of potent pools identified new compounds with promising activity . Aryl dimension pools showed R = ph and naphthyl as the most potent . The acetylene dimension had R' = phenylalaninol and alaninol as the most potent . Isolated individual compounds, both active and nonpotent, were assayed to confirm the library results . The most potent new compound was 4ek (R = naphthyl, R' = phenylaninol) at 1.45 microM . Other nonnatural individual naphthyl-amide compounds showed potent MDR reversal including the morpholino-amide 4ej (1.69 microM) . Synergistic activities attributed to the two ends of the molecule were also identified . Direct interaction with Pgp was established by ATPase and photoaffinity displacement assays . The results indicate that both ends of the polyene reversal agent are involved in Pgp interaction and can be further modified for increased potency.

Int J Cancer, 2000 Sep 15, 87(6), 818 - 23
Human papillomavirus type 16-immortalized endocervical cells selected for resistance to cisplatin are malignantly transformed and have a multidrug resistance phenotype; Ding Z et al.; Cis-diamminedichloroplatinum (II) (cisplatin, CDDP) is a highly effective chemotherapeutic agent against cervical cancer, but drug resistance is a major obstacle in its clinical application . The mechanism of drug resistance in human cervical cancer is not well understood . Here, we established an in vitro endocervical, cisplatin-resistant cell system that mimics the development of cisplatin resistance in the human cervix . Human papillomavirus (HPV) type 16-immortalized human endocervical cells (HEN-16-2) were treated with cisplatin, and the cisplatin-selected cells (HEN-16-2/CDDP) were resistant to cisplatin, paclitaxel, actinomycin D, doxorubicin, etoposide, and 5-fluorouracil, thus demonstrating a multidrug resistance (MDR) phenotype . Furthermore, compared with a similar passage of drug-sensitive HEN-16-2 cells, HEN-16-2/CDDP cells exhibited the general growth characteristics of cancer cell lines: faster growth in medium containing serum and high calcium levels, higher saturation density, anchorage-independent growth, and formation of tumors in nude mice . These results provided the first in vitro evidence that cisplatin selection can transform HPV-immortalized endocervical cells and cause a phenotype of MDR .

Clin Cancer Res, 2000 Aug, 6(8), 3205 - 14
P-glycoprotein and multidrug resistance protein activities in relation to treatment outcome in acute myeloid leukemia; van der Kolk DM et al.; Despite treatment with intensive chemotherapy, a considerable number of patients with acute myeloid leukemia (AML) die from their disease due to the occurrence of resistance . Overexpression of the transporter proteins P-glycoprotein (P-gp) and multidrug resistance protein (MRP) 1 has been identified as a major cause of cross-resistance to functionally and structurally unrelated drugs . In the present study, the functional activity of P-gp and MRP was determined in 104 de novo AML patients with a flow cytometric assay using rhodamine 123 (Rh123) in combination with PSC833 and carboxyfluorescein (CF) in combination with MK-571 . The results were compared with clinical outcome and with known prognostic factors . The functional activity of P-gp and MRP, expressed as Rh123 efflux blocking by PSC833 and CF efflux blocking by MK-571, demonstrated a great variability in the AML patients . A strong negative correlation was observed between Rh123 efflux blocking by PSC833 and Rh123 accumulation (r(s) = -0.69, P < 0.001) and between CF efflux blocking by MK-571 and CF accumulation (r(s) = -0.59, P < 0.001) . A low Rh123 accumulation and a high Rh123 efflux blocking by PSC833 were associated with a low complete remission (CR) rate after the first cycle of chemotherapy (P = 0.008 and P = 0.01, respectively) . Patients with both low Rh123 and CF accumulation (n = 16) had the lowest CR rate (6%), whereas patients with both high Rh123 and CF accumulation (n = 11) had a CR rate of 73% . AML patients with French-American-British classification M1 or M2 showed a lower Rh123 accumulation than patients with French-American-British classification M4 or M5 (P = 0.02) . No association was observed between the multidrug resistance parameters and overall survival of the AML patients . Risk group was the only predictive parameter for overall survival (P = 0.003).

Hum Gene Ther, 2000 Aug 10, 11(12), 1671 - 81
GST-pi gene-transduced hematopoietic progenitor cell transplantation overcomes the bone marrow toxicity of cyclophosphamide in mice; Matsunaga T et al.; Autologous transplantation of bone marrow cells (BMCs) transduced with the multidrug resistance 1 (MDR1) gene or dihydrofolate reductase (DHFR) gene has already been applied in clinical chemoprotection trials . However, anticancer drugs frequently used in high-dose chemotherapy (HDC), such as alkylating agents, are not relevant to MDR1 or DHFR gene products . In this context, we have previously reported that glutathione S-transferase-pi (GST-pi) gene-transduced human CD34(+) cells showed resistance in vitro against 4-hydroperoxicyclophosphamide, an active form of cyclophosphamide (CY) . In the present study, a subsequent attempt was made in a murine model to evaluate the effectiveness of transplantation of GST-pi-transduced BMCs to protect bone marrow against high-dose CY . The gene transfection was carried out retrovirally, employing a recombinant fibronectin fragment . Transfection efficiency into CFU-GM was 30% . After the transplantation, recipient mice (GST-pi mice) received three sequential courses of high-dose CY . As the chemotherapy courses advanced, both shortening of recovery period from WBC nadir and shallowing of WBC nadir were observed . In contrast to the fact that three of seven control mice died, possibly due to chemotoxicity, all seven GST-pi mice were alive after the third course, at which point the vector GST-pi gene was detected in 50% of CFU-GM derived from their BMCs and peripheral blood mononuclear cells . When BMCs obtained from these seven mice were retransplanted into secondary recipient mice, 20% of CFU-GM from BMCs showed positive signals for vector GST-pi DNA after 6 months . These data indicate that the GST-pi gene can confer resistance to bone marrow against CY by being transduced into long-term repopulating cells.

Anticancer Res, 2000 Jul-Aug, 20(4), 2691 - 6
Induction of P-glycoprotein expression on the plasma membrane of human melanoma cells; Molinari A et al.; Melanoma cells exhibit, both in vivo and in vitro, intrinsic drug resistance to various chemotherapeutic agents . Cultured human melanoma cells (M14) intrinsically express significant amounts of multidrug resistance-related protein (MRP1) and P-glycoprotein (P-gp) in the Golgi apparatus, but do not express these drug transporters on the plasma membrane . A panel of multidrug resistant (MDR) melanoma cell lines (M14Dx), showing different degrees of resistance to doxorubicin (DOX), were isolated . In M14Dx lines, the appearance of surface P-gp, but not of MRP1 or lung resistance related protein (LRP), occurred in cells grown in the presence of DOX concentrations higher than 60 nM . Furthermore, P-gp levels appeared to be dose-dependent . Flow cytometry, laser scanning confocal microscopy and cytotoxicity studies demonstrated that the activity of the drug extrusion system was related to both surface P-gp expression and resistance to DOX . In conclusion, P-gp, but not MRP1 or LRP, might play a pivotal role in the pharmacologically-induced MDR phenotype of melanoma cells.

Anticancer Res, 2000 Jul-Aug, 20(4), 2617 - 23
Effects of MDR reversing agent combinations on the 3H-daunomycin accumulation in drug-sensitive and drug-resistant human cancer cells; Moins N et al.; BACKGROUND: As multidrug resistant (MDR) tumour cells generally exhibit a drug accumulation deficit, the effects of three prototype modulators and their combinations were investigated by studying the modulation of 3H-dounomycin cellular accumulation . MATERIALS AND METHODS: Two cell lines derived from a rhino-pharingeal human carcinoma, either sensitive (KB-3-1) or selected as MDR (KB-A1) were used . Verapamil (10mumol.L-1), PSC 833 (lmumol.L-1) and S9788 (5mumol.L-1) were tested alone or in association two by two . The cells were characterized by reverse transcriptase polymerase chain reaction (RT-PCR) in terms of pleiotropic resistance gene expression . RESULTS: A strong mdr1 and a light LRP gene expression were found in KB-A1 resistant cells compared to KB-3-1, whereas MRP expression was found to a similar extent . Relative to the KB-3-1, cells, accumulation of 3H-daunomycin was reduced to 31 +/- 5% in the KB-A1 cells . In these KB-A1 cells, the three agents tested significantly increased the 3H-daunomycin intracellular concentration, S9788 being the most active (311 +/- 37%) and inducing a near complete reversion to the basal level of the sensitive cells . Verapamil and PSC 833 demonstrated an additive effect (252 +/- 69% compared to 188 +/- 33% and 126 +/- 27%, respectively) . On KB-3-1 sensitive cells, S9788 had no effect, while verapamil or PSC 833 moderately increased the 3H-daunomycin accumulation, without additive effect . CONCLUSION: These results show a strong MDR reversing effect of S9788, which appears specific to P-glycoprotein (Pgp) and an additive effect between verapamil and PSC 833, suggesting a better therapeutic efficiency if used in well defined combinations.

Anticancer Res, 2000 Jul-Aug, 20(4), 2449 - 56
Establishment and characterization of a paclitaxel-resistant human non-small cell lung cancer cell line; Chu JJ et al.; We have established a paclitaxel-resistant mutant cell line called H460/TAX which was derived from human non-small cell lung cancer (NSCLC) H460 . A 64-fold greater resistant was shown in our assay as compared with the parental cells . High specificity of drug resistance was also observed since this mutant was not cross-resistant to several other anticancer drugs . Drug accumulation in H460/TAX was significantly less than that in H460 . Many endogenous protein profiles were intact, including the expression level of P-glycoprotein, multidrug resistance-associated protein, the 70 kDa heat shock proteins as well as the phosphorylation of Bcl-2 in H460/TAX cells, except that the total amount of alpha- and beta- tubulins was higher in H460/TAX than in H460 cells . Higher drug concentration and longer treatment for paclitaxel were required in H460/TAX to exert the phosphorylation of keratin 19 which was then accompanied by reorganization of the intermediate filament and the microtubule networks . Since all of the aforementioned factors involved in paclitaxel-resistance in other systems were not found to be significantly altered in H460/TAX, there must be other paclitaxel-resistance mechanisms(s) which remains to be identified in human lung cancers.

Antimicrob Agents Chemother, 2000 Sep, 44(9), 2431 - 4
Nonylphenolethoxylates as malarial chloroquine resistance reversal agents; Crandall I et al.; Malaria-associated morbidity and mortality are increasing because of widespread resistance to one of the safest and least expensive antimalarials, chloroquine . The availability of an inexpensive agent that is capable of reversing chloroquine resistance would have a major impact on malaria treatment worldwide . The interaction of nonylphenolethoxylates (NPEs, commercially available synthetic surfactants) with drug-resistant Plasmodium falciparum was examined to determine if NPEs inhibited the growth of the parasites and if NPEs could sensitize resistant parasites to chloroquine . NPEs inhibited the development of the parasite when present in the low- to mid-micromolar range (5 to 90 microM), indicating that they possess antimalarial activity . Further, the presence of <10 microM concentrations of NPEs caused the 50% inhibitory concentrations for chloroquine-resistant lines to drop to levels (< or =12 nM) observed for sensitive lines and generally considered to be achievable with treatment courses of chloroquine . Long-chain (>30 ethoxylate units) NPEs were found to be most active in P . falciparum, which contrasts with previously observed maximal activity of short-chain ( approximately 9 ethoxylate units) NPEs in multidrug-resistant mammalian cell lines . NPEs may be attractive chloroquine resistance reversal agents since they are inexpensive and may be selectively directed against P . falciparum without inhibiting mammalian tissue P glycoproteins . Antimalarial preparations that include these agents may prolong the effective life span of chloroquine and other antimalarials.

Antimicrob Agents Chemother, 2000 Sep, 44(9), 2395 - 8
Therapeutic responses to quinine and clindamycin in multidrug-resistant falciparum malaria; Pukrittayakamee S et al.; Therapeutic responses to clindamycin in combination with quinine were assessed in adult Thai patients with uncomplicated multidrug-resistant Plasmodium falciparum malaria . In total 204 patients were randomized to receive a 7-day oral treatment regimen of quinine (Q(7)) either alone (n = 68), in combination with clindamycin (Q(7)C(7); n = 68), or in combination with tetracycline (Q(7)T(7); n = 68) . All patients had uncomplicated recoveries with no serious adverse effects . Fever clearance times for both of the two combination regimens (median of 47 h and range of 8 to 120 h for Q(7)C(7) and median of 36 h and range of 8 to 117 h for Q(7)T(7)) were significantly shorter than that for the Q(7)-only regimen (median, 56; range, 4 to 152 h) (P = 0.002) . Parasite clearance times (overall mean +/- standard deviation, 78 +/- 23 h) were not significantly different between the three treatment groups (P = 0 . 98) . The cure rates assessed at 28 days of follow-up were 100% for Q(7)C(7) and 98% for Q(7)T(7), whereas the cure rate was 87% for the Q(7)-only regimen (P < or = 0.04) . Clindamycin in combination with quinine is a safe and effective treatment for multidrug-resistant P . falciparum malaria . This combination may be of particular value in children and pregnant women, in whom tetracyclines are contraindicated.

Eur J Biochem, 2000 Sep, 267(17), 5369 - 77
Evaluating limited specificity of drug pumps reduced relative resistance in human MDR phenotypes; Jongsma AP et al.; In the parallel paper, we developed a property to characterize drug efflux pumps, i.e . the reduced relative resistance (RRR) . Using this RRR, we here investigate whether the observed diversity in human multidrug resistance (MDR) phenotypes might be due to variable levels of P-glycoprotein encoded by MDR1 . We analyzed resistance phenotypes of various human cell lines in which either one, or both, classical human multidrug resistance genes, MDR1 and MDR3, are overexpressed . In addition, RRR values were calculated for MDR phenotypes presented in the literature . The results suggest that more than a single mechanism is required to account for the observed phenotypic diversity of classical multidrug resistance . This diversity is only partly due to differences in plasma membrane permeabilities between cell line families . It is discussed whether the alternative MDR phenotypes might be MDR1 phenotypes modified by other factors that do not themselves cause MDR . The method we here apply may also be useful for other nonspecific enzymes or pumps.

Eur J Biochem, 2000 Sep, 267(17), 5355 - 68
Relating multidrug resistance phenotypes to the kinetic properties of their drug-efflux pumps; Westerhoff HV et al.; The simplest model for pump-mediated multidrug resistance is elaborated quantitatively . The way in which toxicity data should be evaluated to characterize most effectively the drug-efflux pump is then examined . The isotoxic drug dose (D10) depends on too many unrelated properties . The D10 of a cell line taken relative to that of the parental (nonresistant) cell line has been called the relative resistance (RR) . This is inappropriate for characterizing the drug pump, as it depends on the extent of amplification of the latter . The reduced RR (RRR) is newly defined as the ratio of the (RR - 1) for one drug to the (RR - 1) for a different drug . This RRR should be independent of both the drug-target affinity and the extent of amplification of the drug pump in cell lines belonging to a family . The RRR depends on the avidities with which the pump extrudes the drugs relative to the passive membrane permeabilities of the latter . In plots of RRR for one drug combination vs . that for a second drug combination, cell lines that have the same pump amplified should cluster, whereas those with amplification of (functionally) different drug-efflux pumps should segregate . Both a set of new experimental data and literature results are discussed in terms of RRR . RRRs discriminate between human MDR1 and mouse mdr1a and mdr1b, between hamster pgp1 and a mutant thereof, as well as between human MDR1 and a mutant thereof . RRRs are not affected by changes in membrane surface area . Our results indicate that RRR may be used to (a) characterize drug-resistance mechanisms and (b) determine which drug-resistance mechanism is operative . Moreover, our analysis suggests that some of the reported phenotypic diversity among multidrug-resistant cell lines may not be due to diversity in the resistance mechanism.

Eur J Biochem, 2000 Sep, 267(17), 5306 - 12
Nucleotide-binding domain 1 of cystic fibrosis transmembrane conductance regulator production of a suitable protein for structural studies; Duffieux F et al.; Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) . This protein belongs to the large ATP-binding cassette (ABC) family of transporters . Most patients with cystic fibrosis bear a mutation in the nucleotide-binding domain 1 (NBD1) of CFTR, which plays a key role in the activation of the channel function of CFTR . Determination of the three dimensional structure of NBD1 is essential to better understand its structure-function relationship, and relate it to the biological features of CFTR . In this paper, we report the first preparation of recombinant His-tagged NBD1, as a soluble, stable and isolated domain . The method avoids the use of renaturing processes or fusion constructs . ATPase activity assays show that the recombinant domain is functional . Using tryptophan intrinsic fluorescence, we point out that the local conformation, in the region of the most frequent mutation DeltaF508, could differ from that of the nucleotide-binding subunit of histidine permease, the only available ABC structure . We have undertaken three dimensional structure determination of NBD1, and the first two dimensional 15N-1H NMR spectra demonstrate that the domain is folded . The method should be applicable to the structural studies of NBD2 or of other NBDs from different ABC proteins of major biological interest, such as multidrug resistance protein 1 or multidrug resistance associated protein 1.

Eur J Biochem, 2000 Sep, 267(17), 5298 - 305
Symmetry and structure in P-glycoprotein and ABC transporters what goes around comes around; Jones PM et al.; The ABC superfamily of membrane transporters is one of the largest classes of proteins across all species and one of the most intensely researched . ABC proteins are involved in the trafficking of a diverse variety of biological molecules across cell membranes, with some members implicated in medical syndromes such as cystic fibrosis and multidrug resistance to anti-cancer drugs . In the absence of X-ray crystallographic data, structural information has come from spectroscopy, electron microscopy, secondary structure prediction algorithms and residue substitution, epitope labelling and cysteine cross-linking studies . These have generally supported a model for the topology of the transmembrane domains of ABC transporters in which a single aqueous pore is formed by a toroidal ring of 12 alpha helices, deployed in two arcs of six helices each . Although this so-called 6 + 6 helix model can be arranged in either mirror or rotational symmetry configurations, experimental data supports the former . In this review, we put forward arguments against both configurations of this 6 + 6 helix model, based on what is known generally about symmetry relationships in proteins . We relate these arguments to P-glycoprotein, in particular, and discuss alternative models for the structure of ABC transporters in the light of the most recent research.

J Histochem Cytochem, 2000 Sep, 48(9), 1215 - 22
CFTR, MDR1, and MRP1 immunolocalization in normal human nasal respiratory mucosa; Wioland MA et al.; CFTR (cystic fibrosis transmembrane conductance regulator), MDR1 (multidrug resistance), and MRP1 (multidrug resistance-associated protein), members of the ABC transporter superfamily, possess multiple functions, particularly Cl(-), anion, and glutathione conjugate transport and cell detoxification . They are also hypothesized to have a number of complementary functions . It is generally accepted that data obtained from nasal mucosa can be extrapolated to lower airway cell physiology . The aim of the present study was to investigate by immunohistochemistry the differential localization of CFTR, MDR1, and MRP1 in the normal mucosa of 10 human nasal turbinates . In ciliated epithelial cells, CFTR was inconstantly expressed at the apical cell surface, intense membranous labeling was observed for MDR1, and intense cytoplasmic labeling was observed for MRP1 . In the glands, a higher level of expression was observed on serous cells, at the apical surface (for CFTR), on lateral membranes (for MDR1), and with an intracytoplasmic distribution (for MRP1) . In conclusion, CFTR, MDR1 and MRP1 are expressed in the epithelium and glands of the nasal respiratory mucosa, but with different patterns of expression . These results suggest major roles for CFTR, MDR1, and MRP1 in serous glandular cells and a protective function for MDR1 and MRP1 in respiratory ciliated cells . (J Histochem Cytochem 48:1215-1222, 2000)

J Chemother, 2000 Aug, 12(4), 360 - 6
A pilot study of low dose hydroxyurea as a novel resistance modulator in metastatic renal cell cancer; Hao D et al.; Mechanisms of chemoresistance in renal cell carcinoma include P-glycoprotein, overexpression of multidrug resistance-1 (mdr1) gene, and unstable chromosomal aberrations . In vitro exposure of resistant tumor cells to low dose hydroxyurea causes loss of chromosomal aberrations, decrease in the mdr1 gene copies, and increased sensitivity to vinblastine . Patients received continuous hydroxyurea 500 mg every Monday, Wednesday and Friday . Vinblastine 5 mg/m2 was given intravenously on days 1 and 8 every 21 days . Seventeen patients with a median age of 63 (range 40-80) received a median of 3 courses of vinblastine (range 1-14) . Toxicities included: > or = grade 3 non-hematologic toxicity (1) and febrile neutropenia (2) . No treatment related mortality occurred . Three patients (17.6%) had partial responses . The median survival was 38.0 weeks (95% CI = 26.9-49.1 weeks) . The addition of hydroxyurea given at the dose of 500 mg orally three times weekly had no major impact on the expected antitumor effect of vinblastine.

Int J Tuberc Lung Dis, 2000 Aug, 4(8), 758 - 64
Multidrug-resistant tuberculosis: long-term treatment outcome in the Netherlands; Geerligs WA et al.; SETTING: Tuberculosis units (Beatrixoord, Haren; and Dekkerswald, Groesbeek) in the Netherlands . OBJECTIVE: To study the long-term treatment outcome of patients with multidrug-resistant tuberculosis (MDR-TB) . DESIGN: Descriptive analysis of all consecutively admitted patients with MDR-TB between 1 January 1985 and 1 September 1998, with follow-up until 1 August 1999 . RESULTS: Of 44 patients (31 male) enrolled in the study, 33 were foreign born and none were human immunodeficiency virus positive . At diagnosis 38 patients had sputum-smear positive pulmonary TB, and converted culture negative after a mean of 6 weeks, while six converted to negative later (mean 69 weeks) . Most patients had micro-organisms resistant to several antimycobacterial drugs (mean = median: 5), including resistance to isoniazid and rifampin . In-patient treatment lasted a mean of 164 days (range 31-481), and patients were treated with six drugs on average . Side effects were common . Treatment lasted for a mean of 608 days (range 268-1626); five patients are still on treatment . Four patients were operated for TB, and two others were operated for post-TB sequelae . During the follow-up period six patients died, of whom three had active TB; 33 (75%) were considered cured . CONCLUSION: Mortality was only 14% after a mean follow-up period of 53 months . MDR-TB can be successfully treated, but requires much effort from both patients and carers, and the costs may be higher than is affordable in resource-poor countries.

Int J Tuberc Lung Dis, 2000 Aug, 4(8), 752 - 7
Trends in antituberculosis drug resistance in Karonga District, Malawi, 1986-1998; Warndorff DK et al.; SETTING: Karonga District, Malawi . OBJECTIVES: To examine long term trends in initial and acquired resistance to antituberculosis drugs in a rural area of Africa . DESIGN: Monitoring of all patients with culture-confirmed tuberculosis 1986-1998 . RESULTS: Initial drug resistance results were available for 1121 patients . The proportion resistant to any of the first line drugs (streptomycin, isoniazid, rifampicin or ethambutol) was 9.6%, and to isoniazid 7.2% . Initial resistance to at least isoniazid and rifampicin (multidrug resistance) was seen in only six patients . No initial resistance to ethambutol was found . There was no significant change in initial drug resistance over time . Overall, 22/120 (18%) patients with previous treatment were resistant to at least one drug; only one had multidrug resistance . Acquired resistance decreased over the period of the study . There were no associations between age, sex or human immunodeficiency virus (HIV) status and initial or acquired drug resistance . CONCLUSIONS: Changes in acquired resistance may reflect the recent performance of a control programme more quickly than those in initial resistance . It is encouraging that acquired resistance decreased and levels of multidrug resistance were low despite more than a decade of use of rifampicin . The lack of association between HIV and drug resistance confirms findings elsewhere in Africa.

Int J Hyperthermia, 2000 Jul-Aug, 16(4), 291 - 303
Thermosensitivity of multidrug-resistant human gastric and pancreatic carcinoma cells; Lage H et al.; Often, tumour cells acquire drug resistance phenotypes, which include the classical multidrug resistance (MDR) phenomenon accompanied by the synthesis of the P-glycoprotein (Pgp) and atypical MDR phenotypes mediated by different, in part unknown, mechanisms . To investigate the susceptibility of tumour cells exhibiting different kinds of MDR to treatment with heat, the hyperthermic survival of established human gastric and pancreatic carcinoma cell lines were studied and sublines exhibiting a classical and an atypical MDR phenotype were derived, respectively . Arrhenius analysis of this panel of gastrointestinal tumour cells revealed that both the classical and the atypical MDR variants exhibited no breaking points (T*) in contrast to the parent tumour cells . The activation enthalpies E(A) were about 40% lower at T > T* in comparison to the E(A) at lower temperatures . Classical MDR variants of both gastrointestinal tumour cell types exhibited a similar E(A) value, whereas the E(A) of atypical MDR gastric carcinoma cells was 1.6-fold higher than the E(A) of corresponding pancreatic carcinoma cells . In comparison to the parent lines, the drug resistant variants exhibited a 2.1-fold (gastric carcinoma, classical MDR), 2.7-fold (gastric carcinoma, atypical MDR) and 1.4-fold (pancreatic carcinoma, classical MDR) increase of activation enthalpies and a nearby unchanged E(A) in pancreatic carcinoma cells exhibiting an atypical MDR.

Biochem J, 2000 Sep 1, 350 Pt 2, 555 - 61
Multidrug resistance protein MRP1 protects against the toxicity of the major lipid peroxidation product 4-hydroxynonenal; Renes J et al.; 4-Hydroxynonenal (4HNE) is the most prevalent toxic lipid peroxidation product formed during oxidative stress . It exerts its cytotoxicity mainly by the modification of intracellular proteins . The detection of 4HNE-modified proteins in several degenerative disorders suggests a role for 4HNE in the onset of these diseases . Efficient protection mechanisms are required to prevent the intracellular accumulation of 4HNE . The toxicity of 4HNE was tested with the small cell lung cancer cell lines GLC(4) and the multidrug-resistance-protein (MRP1)-overexpressing counterpart GLC(4)/Adr . In the presence of the MRP1 inhibitor MK571 or the GSH-depleting agent buthionine sulphoximine, both cell lines became more sensitive and showed decreased survival . Transport experiments were performed with the (3)H-labelled glutathione S-conjugate of 4HNE ({(3)H}GS-4HNE) with membrane vesicles from GLC(4)-derived cell lines with different expression levels of MRP1 . {(3)H}GS-4HNE was taken up in an ATP-dependent manner and the transport rate was dependent on the amount of MRP1 . The MRP1 inhibitor MK571 decreased {(3)H}GS-4HNE uptake . MRP1-specific {(3)H}GS-4HNE transport was demonstrated with membrane vesicles from High Five insect cells overexpressing recombinant MRP1 . Kinetic experiments showed an apparent K(m) of 1.6+/-0.21 microM (mean+/-S.D.) for MRP1-mediated {(3)H}GS-4HNE transport . In conclusion, MRP1 has a role in the protection against 4HNE toxicity and GS-4HNE is a novel MRP1 substrate . MRP1, together with GSH, is hypothesized to have a role in the defence against oxidative stress.

Biochem J, 2000 Sep 1, 350 Pt 2, 531 - 5
Multidrug resistance protein 1 regulates lipid asymmetry in erythrocyte membranes; Dekkers DW et al.; The role of multidrug resistance protein 1 (MRP1) in the maintenance of transbilayer lipid asymmetry in the erythrocyte membrane was investigated . The transbilayer distribution of endogenous phospholipids and {(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino}hexanoyl (NBD)-labelled lipid analogues was compared in the absence and the presence of inhibitors of MRP1 . At equilibrium the transbilayer distribution of the NBD analogues (in the absence of MRP1 inhibitors) was very similar to that of the endogenous lipids . Inhibition of MRP1 by verapamil or indomethacin resulted in a shift in the amount of probe that was internalized: approx . 50% of NBD-labelled phosphatidylcholine (PtdCho) and 9% of NBD-sphingomyelin (NBD-Spm) were no longer extractable by BSA in cells treated with inhibitor, in comparison with 25% and 3% for control cells respectively . To verify whether inhibition of MRP1 also affected the distribution of the endogenous phospholipids, phospholipase A2 and sphingomyelinase were used to assess the amount of each of the various lipid classes present in the membrane outer leaflet . No shift in phospholipid distribution was observed after 5 h of incubation with verapamil or indomethacin . However, after 48 h of incubation with these inhibitors, significantly smaller amounts of PtdCho and Spm were present in the outer membrane leaflet . No appreciable change was observed in the distribution of phosphatidylethanolamine or phosphatidylserine . Decreased hydrolysis of PtdCho and Spm was not due to endovesicle formation, as revealed by electron microscopy . This is the first report to show that MRP1 has a role in the maintenance of the outwards orientation of endogenous choline-containing phospholipids in the erythrocyte membrane.

Biochem J, 2000 Sep 1, 350 Pt 2, 443 - 51
Physiological oxygen tensions modulate expression of the mdr1b multidrug-resistance gene in primary rat hepatocyte cultures; Hirsch-Ernst KI et al.; P-Glycoprotein transporters encoded by mdr1 (multidrug resistance) genes mediate extrusion of an array of lipophilic xenobiotics from the cell . In rat liver, mdr transcripts have been shown to be expressed mainly in hepatocytes of the periportal region . Since gradients in oxygen tension (pO(2)) may contribute towards zonated gene expression, the influence of arterial and venous pO(2) on mRNA expression of the mdr1b isoform was examined in primary rat hepatocytes cultured for up to 3 days . Maximal mdr1b mRNA levels (100%) were observed under arterial pO(2) after 72 h, whereas less than half-maximal mRNA levels (40%) were attained under venous pO(2) . Accordingly, expression of mdr protein and extrusion of the mdr1 substrate rhodamine 123 were maximal under arterial pO(2) and reduced under venous pO(2) . Oxygen-dependent modulation of mdr1b mRNA expression was prevented by actinomycin D, indicating transcriptional regulation . Inhibition of haem synthesis by 25 microM CoCl(2) blocked mdr1b mRNA expression under both oxygen tensions, whereas 80 microM desferrioxamine abolished modulation by O(2) . Haem (10 microM) increased mdr1b mRNA levels under arterial and venous pO(2) . In hepatocytes treated with 50 microM H(2)O(2), mdr1b mRNA expression was elevated by about 1.6-fold at venous pO(2) and 1.5-fold at arterial pO(2) . These results support the conclusion that haem proteins are crucial for modulation of mdr1b mRNA expression by O(2) in hepatocyte cultures and that reactive oxygen species may participate in O(2)-dependent signal transduction . Furthermore, the present study suggests that oxygen might be a critical modulator for zonated secretion of mdr1 substrates into the bile.

Shock, 2000 Aug, 14(2), 176 - 81
Intraabdominal sepsis down-regulates transcription of sodium taurocholate cotransporter and multidrug resistance-associated protein in rats; Kim PK et al.; Hepatic dysfunction in sepsis is characterized by hyperbilirubinemia and intrahepatic cholestasis . We hypothesize that sepsis causes decreased hepatic transcription of the bile acid transporter sodium taurocholate cotransporter (Ntcp) and the organic anion transporter multidrug resistance-associated protein (Mrp2) and that interleukin (IL)-6 is important in the down-regulation of Ntcp and Mrp2 expression . Male Sprague-Dawley rats underwent induction of mild, nonlethal sepsis by cecal ligation and single puncture (CLP) or fulminant sepsis by cecal ligation and double puncture (2CLP) . Hepatic transcription of Ntcp and Mrp2 rapidly decreased after CLP or 2CLP . Seventy-two hours later, transcription was 60% of baseline in CLP and 14% of baseline in 2CLP . Serum bilirubin was elevated from 24 h onward and cholestasis was observed on fixed liver specimens at 24, 48, and 72 h after 2CLP but not after CLP . Steady-state Ntcp and Mrp2 mRNA was decreased in IL-6-treated cultured hepatocytes and in normal rats given 1 mg/kg intravenous IL-6 . We conclude that 1) Ntcp and Mrp2 transcription is down-regulated transiently after CLP and persistently after 2CLP; 2) 2CLP results in hyperbilirubinemia and cholestasis, in part due to persistently decreased transcription of Ntcp and Mrp2; and 3) altered Ntcp and Mrp2 transcription is mediated in part by IL-6.

J Pharmacol Exp Ther, 2000 Sep, 294(3), 837 - 43
Cellular uptake of dietary flavonoid quercetin 4'-beta-glucoside by sodium-dependent glucose transporter SGLT1; Walgren RA et al.; Although it has been suggested that the intestinal glucose transporter may actively absorb dietary flavonoid glucosides, there is a lack of direct evidence for their transport by this system . In fact, our previous studies with the human Caco-2 cell model of intestinal absorption demonstrated that a major dietary flavonoid, quercetin 4'-beta-glucoside, is effluxed by apically expressed multidrug resistance-associated protein-2, potentially masking evidence for active absorption . The objective of this study was to test the hypothesis that quercetin 4'-beta-glucoside is a substrate for the intestinal sodium-dependent D-glucose cotransporter SGLT1 . Cellular uptake of quercetin 4'-beta-glucoside was examined with Caco-2 cells and SGLT1 stably transfected Chinese hamster ovary cells (G6D3 cells) . Although quercetin 4'-beta-glucoside is not absorbed across Caco-2 cell monolayers, examination of the cells by indirect fluorescent microscopy as well as by HPLC analysis of cellular content revealed cellular accumulation of this glucoside after apical loading . Consistent with previous observations, the accumulation of quercetin 4'-beta-glucoside in both Caco-2 and G6D3 cells was markedly enhanced in the presence of multidrug resistance-associated protein inhibition . Uptake of quercetin 4'-beta-glucoside was greater in SGLT1-transfected cells than in parental Chinese hamster ovary cells . Uptake of the glucoside by Caco-2 and G6D3 cells was sodium-dependent and was inhibited by the monovalent ionophore nystatin . In both Caco-2 and G6D3 cells, quercetin 4'-beta-glucoside uptake was inhibited by 30 mM glucose and 0.5 mM phloridzin . These results demonstrate for the first time that quercetin 4'-beta-glucoside is transported by SGLT1 across the apical membrane of enterocytes.

J Pharmacol Exp Ther, 2000 Sep, 294(3), 830 - 6
Efflux of dietary flavonoid quercetin 4'-beta-glucoside across human intestinal Caco-2 cell monolayers by apical multidrug resistance-associated protein-2; Walgren RA et al.; Although there is strong evidence to suggest that flavonoid consumption is beneficial to human health, the extent to which flavonoids are absorbed and the mechanisms involved are controversial . Contrary to common dogma, we previously demonstrated that quercetin 4'-beta-glucoside, the predominant form of the most abundant dietary flavonoid, quercetin, was not absorbed across Caco-2 cell monolayers . The aim of this study was to test the hypothesis that a specific efflux transporter is responsible for this lack of absorption . Transport of quercetin 4'-beta-glucoside, alone or with inhibitors, was examined with Caco-2 cell monolayers . In addition, subcellular localization of the multidrug resistance-associated proteins MRP1 and MRP2 was examined by immunofluorescent confocal microscopy . Efflux of quercetin 4'-beta-glucoside, a saturable process, was not altered by verapamil, a P-glycoprotein inhibitor, but was competitively inhibited by MK-571, an MRP inhibitor . These data in combination with immunofluorescent localization of MRP2 to the apical membrane support a role for MRP2 in the intestinal transcellular efflux of quercetin 4'-beta-glucoside . These results suggest a role for MRP2 in the transport of a new class of agents, dietary glucosides.

Cancer Res, 2000 Aug 1, 60(15), 4238 - 44
Increased resistance to anticancer therapy of mouse cells lacking the poly(ADP-ribose) polymerase attributable to up-regulation of the multidrug resistance gene product P-glycoprotein; Wurzer G et al.; Mouse embryo fibroblasts lacking poly(ADP-ribose) polymerase (PARP)-1 express a barely detectable level of wild-type (wt) p53 protein . Doxorubicin at concentrations activating wt p53 in normal mouse embryo fibroblasts failed to induce it in mutant cells . wt p53 was only activated in response to a 10-fold higher doxorubicin dose . Treatment with higher doxorubicin concentrations was cytotoxic for normal but not for PARP-1 -/- cells . The latter was also resistant to other anticancer agents . The increased resistance of mutant cells to drugs resembled a unique phenomenon known as multidrug resistance (MDR) . Interestingly, the MDR gene product P-glycoprotein was clearly up-regulated in PARP-1-deficient cells as compared with normal counterparts . Pretreatment with verapamil reversed the MDR phenotype.

J Natl Cancer Inst, 2000 Aug 16, 92(16), 1295 - 302
A family of drug transporters: the multidrug resistance-associated proteins; Borst P et al.; The human multidrug resistance-associated protein (MRP) family currently has seven members . The ability of several of these membrane proteins to transport a wide range of anticancer drugs out of cells and their presence in many tumors make them prime suspects in unexplained cases of drug resistance, although proof that they contribute to clinical drug resistance is still lacking . Recent studies have begun to clarify the function of the MRP family members . MRPs are organic anion transporters; i.e., they transport anionic drugs, exemplified by methotrexate, and neutral drugs conjugated to acidic ligands, such as glutathione (GSH), glucuronate, or sulfate . However, MRP1, MRP2, and MRP3 can also cause resistance to neutral organic drugs that are not known to be conjugated to acidic ligands by transporting these drugs together with free GSH . MRP1 can even confer resistance to arsenite and MRP2 to cisplatin, again probably by transporting these compounds in complexes with GSH . MRP4 overexpression is associated with high-level resistance to the nucleoside analogues 9-(2-phosphonylmethoxyethyl) adenine and azidothymidine, both of which are used as anti-human immunodeficiency virus drugs . MRPs may, therefore, also have a role in resistance against nucleoside analogues used in cancer chemotherapy . Mice without Mrp1, a high-affinity leukotriene C(4) transporter, have an altered response to inflammatory stimuli but are otherwise healthy and fertile . MRP2 is the major transporter responsible for the secretion of bilirubin glucuronides into bile, and humans without MRP2 develop a mild liver disease known as the Dubin-Johnson syndrome . The physiologic functions of the other MRPs are not known . Whether long-term inhibition of MRPs in humans can be tolerated (assuming that suitable inhibitors will be found) remains to be determined.

Proc Natl Acad Sci U S A, 2000 Aug 15, 97(17), 9677 - 82
Ethionamide activation and sensitivity in multidrug-resistant Mycobacterium tuberculosis; DeBarber AE et al.; Ethionamide (ETA) is an important component of second-line therapy for the treatment of multidrug-resistant tuberculosis . Synthesis of radiolabeled ETA and an examination of drug metabolites formed by whole cells of Mycobacterium tuberculosis (MTb) have allowed us to demonstrate that ETA is activated by S-oxidation before interacting with its cellular target . ETA is metabolized by MTb to a 4-pyridylmethanol product remarkably similar in structure to that formed by the activation of isoniazid by the catalase-peroxidase KatG . We have demonstrated that overproduction of Rv3855 (EtaR), a putative regulatory protein from MTb, confers ETA resistance whereas overproduction of an adjacent, clustered monooxygenase (Rv3854c, EtaA) confers ETA hypersensitivity . Production of EtaA appears to be negatively regulated by EtaR and correlates directly with {(14)C}ETA metabolism, suggesting that EtaA is the activating enzyme responsible for thioamide oxidation and subsequent toxicity . Coding sequence mutations in EtaA were found in 11 of 11 multidrug-resistant MTb patient isolates from Cape Town, South Africa . These isolates showed broad cross-resistance to thiocarbonyl containing drugs including ETA, thiacetazone, and thiocarlide.

Cytometry, 2000 Sep 1, 41(1), 62 - 72
Modulation of daunorubicin cellular resistance by combination of P-glycoprotein blockers acting on drug efflux and intracellular drug sequestration in Golgi vesicles; Merlin JL et al.; BACKGROUND: S9788 and PSC833 were developped as P-glycoprotein (Pgp) blockers and found to act additionally on daunorubicin subcellular distribution, involving different putative targets . On this basis, combinations of S9788 and PSC833 were evaluated in Pgp-expressing MCF7(DXR) cells in which we recently demonstrated that daunorubicin was sequestered in Golgi vesicles (Bour-Dill et al.: Cytometry, 39: 16-25, 2000) . METHODS: Combinations of S9788 and PSC833 consisted in complementary fractions of iso-effective concentrations (IEC) leading to 90% (IEC90) and median (IEC50) reversion of daunorubicin resistance . Resistance modulation was assessed using cytotoxicity assays, flow cytometry determination of intracellular daunorubicin, and fluorescence microscopy analysis of daunorubicin subcellular distribution . RESULTS: Individually, both S9788 and PSC833 were found to be very potent with IEC90 of 5 and 15 micromol/l, and IEC50 of 0.1 and 0.2 micromol/l, respectively, for S9788 and PSC833 . When combined, synergistic cytotoxicity was observed for both IEC90 and IEC50 combinations while intracellular daunorubicin fluorescence was only synergistically increased for IEC90 combinations . For IEC50 combinations, no increase in intracellular fluorescence was observed, and fluorescence microscopy examination of the cells suggested that daunorubicin sequestration in Golgi vesicles could be modulated at concentrations that do not significantly increase daunorubicin cellular concentration . Using immunofluorescence and reverse transcription-polymerase chain reaction analyses, multidrug resistance-associated protein, major vault lung-resistance protein, and anthracycline-resistance associated protein were not found to be implicated . CONCLUSIONS: Synergistic combinations of S9788 and PSC833 might offer alternative ways to decrease the toxicity generated by high-dose Pgp-blockers without altering the efficacy of the resistance modulation .

J Biol Chem, 2000 Nov 3, 275(44), 34166 - 72
Functional reconstitution of substrate transport by purified multidrug resistance protein MRP1 (ABCC1) in phospholipid vesicles; Mao Q et al.; The 190-kDa multidrug resistance protein MRP1 (ABCC1) is a polytopic transmembrane protein belonging to the ATP-binding cassette transporter superfamily . In addition to conferring resistance to various antineoplastic agents, MRP1 is a transporter of conjugated organic anions, including the cysteinyl leukotriene C(4) (LTC(4)) . We previously characterized the ATPase activity of reconstituted immunoaffinity-purified native MRP1 and showed it could be stimulated by its organic anion substrates (Mao, Q., Leslie, E . M., Deeley, R . G., and Cole, S . P . C . (1999) Biochim . Biophys . Acta 1461, 69-82) . Here we show that purified reconstituted MRP1 is also capable of active transport of its substrates . Thus LTC(4) uptake by MRP1 proteoliposomes was osmotically sensitive and could be inhibited by two MRP1-specific monoclonal antibodies . LTC(4) uptake was also markedly reduced by the competitive inhibitor, S-decyl-glutathione, as well as by the MRP1 substrates 17 beta-estradiol 17-beta-(d-glucuronide), oxidized glutathione, and vincristine in the presence of reduced glutathione . The K(m) for ATP and LTC(4) were 357 +/- 184 microm and 366 +/- 38 nm, respectively, and 2.14 +/- 0.75 microm for 17 beta-estradiol 17-beta-(d-glucuronide) . Transport of vincristine required the presence of both ATP and GSH . Conversely, GSH transport was stimulated by vincristine and verapamil . Our data represent the first reconstitution of transport competent purified native MRP1 and confirm that MRP1 is an efflux pump, which can transport conjugated organic anions and co-transport vincristine together with GSH.

Leukemia, 2000 Aug, 14(8), 1444 - 50
Increased sensitivity of multidrug-resistant myeloid leukemia cell lines to lovastatin; Maksumova L et al.; Lovastatin, a competitive inhibitor of HMG-CoA reductase, reportedly inhibits proliferation and induces apoptosis of tumor cells with MDR-1 coded P-glycoprotein (Pgp) expression . In this study we investigated the sensitivity to lovastatin of eight myeloid leukemia cell lines: K562, NOMO-1, NB4 and its retinoic acid (RA) resistant subline NB4/RA, and their multidrug-resistant (MDR) sublines: K562/ADR, NOMO-1/ADR, NB4/MDR and NB4/RA/MDR . MTT and apoptosis assays revealed that K562/ADR, NOMO-1/ADR and NB4/RA/MDR were more sensitive to lovastatin than their parental cell lines, while NB4/MDR showed the same level of sensitivity as parental NB4 cells, which already were very sensitive to lovastatin . Significant elevation of transcript levels of HMG-CoA reductase was observed by semiquantitative RT-PCR analysis in more than three lovastatin-sensitive MDR sublines, but not in NB4/MDR compared with the parental cell lines . HMG-CoA reductase mRNA levels were up-regulated more than two-fold by the exposure to lovastatin in all of the parental non-Pgp-expressing cell lines . In NB4/MDR, HMG-CoA reductase mRNA level was elevated to a similar extent as in parental NB4, whereas in three other MDR sublines which showed preferential sensitivity to lovastatin, their HMG-CoA reductase mRNA levels were not significantly elevated after 24- and 48-h treatment with lovastatin . These results indicate a connection between drug resistance and regulation of the mevalonate pathway, and further strengthen the clinical possibility that drug resistant leukemias would be susceptible to treatment with lovastatin.

Leukemia, 2000 Aug, 14(8), 1436 - 43
Calicheamicin-conjugated humanized anti-CD33 monoclonal antibody (gemtuzumab zogamicin, CMA-676) shows cytocidal effect on CD33-positive leukemia cell lines, but is inactive on P-glycoprotein-expressing sublines; Naito K et al.; Calicheamicin-conjugated humanized anti-CD33 mouse monoclonal antibody, CMA-676, has recently been introduced to clinics as a promising drug to treat patients with acute myeloid leukemia (AML) in relapse . However, the mechanism of action of CMA-676 has not been well elucidated . The cytotoxic effect of CMA-676 on HL60, NOMO-1, NB4, NKM-1, K562, Daudi, and the multidrug-resistant sublines, NOMO-1/ADR and NB4/MDR, was investigated by cell cycle distribution and morphology . These studies were done by a video-microscopic system, DNA fragmentation, dye exclusion and 3H-thymidine uptake after analysis of CD33, CD34, P-glycoprotein (P-gp), multidrug resistance (MDR)-associated protein and lung-related protein on these cells . A dose-dependent, selective cytotoxic effect of CMA-676 was observed in cell lines that expressed CD33, and was dependent on the amount of CD33 and the proliferative speed of the cells . Sensitive cells were temporally arrested at the G2/M phase before undergoing morphological changes . CMA-676 is not effective on P-gp-expressing multidrug-resistant sublines compared with parental cell lines . MDR modifiers, MS209 and PSC833, restored the cytotoxic effect of CMA-676 in P-gp-expressing sublines . CMA-676 is a promising agent in the treatment of patients with AML that expresses CD33 . The combined use of CMA-676 and MDR modifiers may increase the selective cytotoxic effect in multidrug-resistant AML.

Head Neck, 2000 Sep, 22(6), 599 - 608
Prognostic value of c-erbB2 and other markers in patients treated with chemotherapy for recurrent head and neck cancer; Shiga H et al.; BACKGROUND: Chemotherapy is widely used in patients with recurrent head and neck cancer, but no clear markers are available that can predict response to treatment or survival in these patients . METHODS: Twenty-nine patients evaluated in this study had recurrent head and neck squamous carcinomas, previously treated with surgery and/or radiotherapy . Patients received either cisplatin and 5-fluorouracil (5-FU) (n = 15) or cisplatin and paclitaxel (Taxol) (n = 14) . Expression of c-erbB2, p53, glutathione S-transferase pi, multidrug resistance-associated protein, thymidylate synthase, and glutathione synthetase were evaluated in biopsy tissues . RESULTS: Response to chemotherapy was significantly correlated with improved survival (progression-free survival, p =.0005; overall survival, p = . 007) . Of the factors examined, expression of c-erbB2 was associated with significantly decreased progression-free survival (p =.023) and overall survival (p =.029) . This was seen in patients treated with cisplatin/taxol but not in patients treated with cisplatin/5-FU . CONCLUSION: Expression of c-erbB2 may be a clinically useful predictor of survival in this group of patients.

Acta Haematol, 2000, 103(3), 144 - 51
Down-regulation of CD98 in melphalan-resistant myeloma cells with reduced drug uptake; Harada N et al.; Although melphalan has been used as a therapeutic reagent for multiple myeloma, many patients become refractory . To elucidate the mechanism of resistance to melphalan, we generated a melphalan-resistant myeloma cell line, KHM-11(EMS), by treating a parental line, KHM-11, with a mutagen, ethylmethanesulfonate . KHM-11(EMS) is 55 times more resistant to melphalan . gamma-Glutamylcysteine synthetase, P-glycoprotein, multidrug-resistance-associated protein, lung-resistance-related protein and the Bcl-2 family of proteins were not responsible for the drug resistance in KHM-11(EMS) . Intracellular incorporation of melphalan to myeloma cells was determined by using {(14)C}-labeled melphalan . Accumulation of melphalan in KHM-11(EMS) was 43% of KHM-11, while the efflux rates were comparable in both cell lines . The uptake of melphalan was inhibited by the addition of L-phenylalanine, indicating that melphalan is incorporated through the L-phenylalanine transporter as reported previously . Expression of CD98, which was recently cloned as an L-phenylalanine transporter, was 6-fold decreased in KHM-11(EMS), suggesting that CD98 may be correlated with the incorporation of melphalan . CD98 expression and incorporation of melphalan were analyzed in fresh purified myeloma cells from 5 patients . All myeloma cells from 4 cases expressed CD98 at a high level and incorporated melphalan . However, tumor cells from 1 case expressed CD98 at low levels and did not incorporate melphalan . Taken together, reduced melphalan uptake could be responsible for the drug resistance in KHM-11(EMS), and down-regulation of CD98 may be related to this phenomenon . Further investigation of the correlation between impaired drug uptake and down-regulation of CD98 in myeloma cells should be important to understand the mechanism of resistance to melphalan .

Int J Oncol, 2000 Sep, 17(3), 579 - 86
Altered activity of MDR-reversing agents on KB3-1 cells transfected with Gly(185)-->Val human P-glycoprotein; Watanabe T et al.; P-glycoprotein (P-gp) is a transmembrane glycoprotein that confers multidrug resistance (MDR) . It has been demonstrated that the Gly185 residue within the cytoplasmic loop between predicted transmembrane portions 2 and 3 plays an important role in substrate specificity of human P-gp . Derivatives of cyclosporin interact with and reverse the ability of P-gp to act as a drug efflux pump . To determine if the Gly185 residue of human P-gp is also important for the interaction of P-gp with closely related cyclosporin derivatives, we examined the effect of PSC-833 and CsA on P-gp in KB3-1 cells transfected with human wild-type P-gp (GSV-2) or with the mutant P-gp (VSV-1) that habored the Gly185-->Val substitution . While the ability of CsA to sensitize VSV-1 cells to anticancer agents was enhanced, no changes in the potency of PSC-833 against cells transfected with either the wild-type or mutant P-gp were observed . In addition, VSV-1 transfected cells were more sensitive to CsA inhibition of verapamil-stimulated ATPase activity than cells transfected with wild-type P-gp . Furthermore, the intracellular accumulation of CsA was low in GSV-2 P-gp-expressing cells, compared with its accumulation in VSV-1 cells and it was found to be as high as in non-P-gp expressing KB3-1 cells . These results indicated an enhanced sensitivity of Val185-P-gp expressing cells to CsA that correlated with increased intracellular accumulation in these cells . In contrast, no significant difference in the accumulation of PSC-833 was observed among the parental, wild-type or resistant cells . Since PSC-833 was found to be more potent than CsA, these studies provided insight into the effects of the structure of MDR modulators in mediating sensitivity to anticancer drugs.

J Orthop Res, 2000 May, 18(3), 449 - 55
Multidrug resistance-1 gene expression does not increase during tumor progression in the MGH-OGS murine osteosarcoma tumor model; Trammell RA et al.; In addition to its possible role in drug resistance, expression of the multidrug resistance-1 gene may also be associated with a more malignant phenotype and tumor progression . This study evaluated its expression during tumor progression in the MGH-OGS transplantable murine osteosarcoma tumor model . Three variables of tumor progression were analyzed: tumor size, local recurrence, and metastasis . With a highly sensitive reverse transcription-polymerase chain reaction method, mRNA levels of multidrug resistance-1 were compared in primary tumors of different sizes . In addition, the levels were compared in primary, locally recurrent, and metastatic tumors isolated from individual mice . No significant difference was found in the levels of expression with increasing primary tumor size . In addition, the levels in primary, locally recurrent, and metastatic tumors were not significantly different . Our results indicate that--at least in the MGH-OGS tumor model, which is analogous to the majority of spontaneously occurring human osteosarcomas in that it has low levels of multidrug resistance-1/P-glycoprotein and is sensitive to doxorubicin--there is no evidence of upregulation of multidrug resistance-1 expression during tumor progression.

Turk J Pediatr, 2000 Apr-Jun, 42(2), 145 - 7
Drug-resistant tuberculosis in Turkish children; Dilber E et al.; The purpose of this study was to determine the prevalence of anti-tuberculosis drug resistance in children followed at Hacettepe University Ihsan Dogramaci Children's Hospital . Sixty cases with tuberculosis for whom susceptibility testing was available were searched retrospectively . Teh overall drug resistance was 26.7 percent . Resistance to streptomycin (sm) was the most frequent (18.3%), followed by isoniazid (6.7%), rifampicin (6.5%), and ethambutol (4.2%) . Strain resistant to more than one drug was present in two cases (3.3%) . In summary, excluding SM, both single and multidrug resistance were relatively low in our pediatric patients.

Mol Biotechnol, 1999 Nov, 13(1), 21 - 8
Therapeutic genes for cancer gene therapy; Walther W et al.; Cancer still represents a disease of high incidence and is therefore one major target for gene therapy approaches . Gene therapy for cancer implies that ideally selective tumor cell killing or inhibition of tumor cell growth can be achieved using nucleic acids (DNA and RNA) as the therapeutic agent . Therefore, the majority of cancer gene therapy strategies introduce foreign genes into tumor cells which aim at the immunological recognition and destruction, the direct killing of the target cells or the interference with tumor growth . To achieve this goal for gene therapy of cancer, a broad variety of therapeutic genes are currently under investigation in preclinical and in clinical studies . These genes are of very different origin and of different mechanisms of action, such as human cytokine genes, genes coding for immunostimulatory molecules/antigens, genes encoding bacterial or viral prodrug-activating enzymes (suicide genes), tumor suppressor genes, or multidrug resistance genes.

Bone Marrow Transplant, 2000 May, 25 Suppl 2, S118 - 24
Human multidrug resistance-1 gene transfer to long-term repopulating human mobilized peripheral blood progenitor cells; Schiedlmeier B et al.; Mobilized peripheral blood progenitor cells (PBPC) are an attractive target for the retrovirus-mediated transfer of cytostatic drug resistance genes . We analyzed NOD/SCID mouse repopulating CD34+ PBPC from cancer patients following retroviral Transwell transduction in various cytokine combinations with the FMEV-based (Friend-mink cell focus forming/murine embryonic stem cell virus) hybrid vector SF-MDR carrying the human multidrug resistance-1 (MDR1) gene . Five to 10 weeks following transplantation of 2.0 x 10(6) CD34+ PBPC into NOD/SCID mice we observed medium to high levels of human cell engraftment with up to 33% . The extent of vector-marked human cells was assessed by a quantitative real-time polymerase chain reaction (PCR) . SF-MDR gene transfer into long-term in vivo repopulating human hematopoietic cells was optimal in the presence of either IL-3/IL-6/SCF/FL or FL/TPO/SCF resulting in three-fold (12.4% +/- 1.7%) or four-fold (16.5% +/- 6.8%) higher average proportions of gene-marked human cells in NOD/SCID mice as compared to IL-3 alone (P < 0.01) . In conclusion, we could optimize the engraftment capacity and the retroviral gene transfer to CD34+ PBPC using cocktails of early acting cytokines in combination with the recombinant fibronectin fragment CH-296 . Our data suggest that the NOD/SCID model provides a valid assay to estimate the gene transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation settings.

Bone Marrow Transplant, 2000 May, 25 Suppl 2, S114 - 7
Clinical scale production of an improved retroviral vector expressing the human multidrug resistance 1 gene (MDR1); Eckert HG et al.; Retroviral vectors are currently the most important and best characterized tools for ex vivo genetic modification of hematopoietic progenitor/stem cells . As a prerequisite for clinical applications, large volumes of high-titer vector supernatants have to be generated in compliance with 'GMP' guidelines . This goal can be reached using a carefully selected producer cell clone and a conventional large-scale cell culture system . The retroviral vector SF1m provides efficient expression of the human multidrug resistance 1 (MDR1) gene in hematopoietic progenitor/stem cells in vitro and in NOD/SCID mouse repopulating human cells in vivo . Currently, a clinical phase I/II study is in preparation to test whether intensified consolidation chemotherapy is enabled by autologous transplantation of peripheral blood progenitor/stem cells that have been genetically modified with SF1m . Using multi-tray cell factories >19 l of serum-free vector containing supernatant were generated from cells of a previously established SF1m-producer clone, based on the PG13 packaging cell line . Testing of the final samples revealed sufficient quality (>1.5 x 10(6) infectious particles/ml) for clinical scale transduction of CD34+ cells . Results from the production runs and the applied biosafety concept are described.

Bone Marrow Transplant, 2000 May, 25 Suppl 2, S71 - 4
Genetic modification of haematopoietic cells for combined resistance to podophyllotoxins, other agents covered by MDR1-mediated efflux activity and nitrosoureas; Baum C et al.; Genetic transfer and expression of drug-resistance functions into haematopoietic stem and progenitor cells is a promising means to overcome both the acute and longterm side-effects of cytotoxic drugs in bone marrow . Here, we describe a functional analysis of a retroviral vector that co-expresses human cDNAs for multidrug resistance 1/P-glycoprotein (MDR1) and a double mutant of O(6)-alkylguanine-alkyltransferase (hATPA/GA) to high levels . The hATPA/GA protein contains two amino acid substitutions that render it resistant to compounds such as O(6)-benzylguanine that inhibit the wild-type protein which is often overexpressed in resistant tumour cells . Evidence for simultaneous drug resistance of genetically modified primary murine progenitor cells to colchicine or the podophyllotoxin etoposide, both covered by MDR1-mediated efflux activity, and the nitrosourea BCNU, which is counteracted by hATPA/GA, is presented using in vitro colony assays.

Neoplasia, 1999 Jun, 1(2), 118 - 22
Cyclosporin A reverses chemoresistance in patients with gynecologic malignancies; Sood AK et al.; Multidrug resistance is a major obstacle in successful systemic therapy of gynecologic malignancies . The objectives of this study are to evaluate the activity of cyclosporin A used to overcome drug resistance in a variety of gynecologic malignancies . Forty women (29 with ovarian cancer, 7 with uterine cancer, 3 with cervical cancer, and 1 with choriocarcinoma) were treated with cyclosporin A, 4 mg/kg intravenously, 6 hours before and 18 hours after the specific chemotherapeutic agent, to which the tumor had developed drug resistance . All patients had shown resistance to the chemotherapy agent used in combination with cyclosporin A . All patients had been heavily pretreated (mean, 2.8 previous chemotherapy regimens) . Overall, among 38 available patients with gynecologic malignancies, a 29% objective response rate was observed . Twenty-six (65%) of all patients received three or more cycles of cyclosporin A . There was a 25% response rate for patients with ovarian cancer patients and 50% for those with uterine cancer . There were no responses among the three patients with cervical cancer, and the patient with choriocarcinoma had a complete response . All patients were evaluable for toxicity . Leukopenia and nausea were the most common toxic reactions, but in most cases they were transient, and only three patients required a treatment delay . The most common grade 3 or 4 toxicity was thrombocytopenia, which was observed in 22% of the patients . Cyclosporin A is well tolerated and has significant potential for reversal of chemoresistance in heavily pretreated patients with ovarian and uterine malignancies.

Ann Biol Clin (Paris), 2000 Jul-Aug, 58(4), 439 - 44
{The role of integrons in dissemination of antibiotic resistance}; Ploy MC et al.; Bacteria can transfer genetic information to get protection against most antibiotics . The acquisition of resistance genes involves genetic mobile elements such as plasmids and transposons . Another genetic structures, named integrons, have been described and contain one or more gene cassettes located at a specific site . Integrons contain an intI gene encoding a site-specific recombinase belonging to the integrase family and a recombination site attI . A gene cassette includes an open reading frame and, at the 3'-end, a recombination site attC . Integration or excision of cassettes occurs by a site-specific recombination mechanism catalyzed by the integrase . However, insertion can rarely occur, at non-specific sites leading to a stable situation for the cassette . Cassettes are transcribed from a common promoter located in the 5'-conserved segment and expression of distal genes is reduced by the presence of upstream cassettes . Most gene cassettes encode antibiotic resistant determinants but antiseptic resistant genes have also been described . Integrons seem to have a major role in the spread of multidrug resistance in Gram-negative bacteria but integrons in Gram-positive bacteria have been recently described . Moreover, the finding of super-integrons with gene cassettes coding for other determinants (biochemical functions, virulence factors) in different Gram negative bacteria suggests that integrons are probably implied in bacterial genome evolution.

Br J Haematol, 2000 Jul, 110(1), 154 - 60
Constitutive expression levels of CD95 and Bcl-2 as well as CD95 function and spontaneous apoptosis in vitro do not predict the response to induction chemotherapy and relapse rate in childhood acute lymphoblastic leukaemia; Wuchter C et al.; CD95 (Fas/APO-1) expression and function and Bcl-2 expression, as well as spontaneous apoptosis in vitro, have been shown to be predictive markers for the in vivo response to chemotherapy in acute myeloid leukaemia (AML) . To determine the clinical significance of apoptosis-regulating factors in acute lymphoblastic leukaemia (ALL), we investigated cell samples of children with ALL who had been included in the German ALL Berlin-Frankfurt-Munster (BFM) study using flow cytometry for constitutive expression levels of CD95 (n = 110) and Bcl-2 (n = 110) . Furthermore, we determined the extent of spontaneous apoptosis in vitro (n = 102) and susceptibility to anti-CD95-induced apoptosis (CD95-sensitivity) (n = 97) . We correlated these findings with the functional activity of the multidrug resistance (MDR)-associated P-glycoprotein (P-gp), as detected by the rhodamine123 efflux test, immunophenotype, cytogenetics and clinical data of the patients examined . Good responders to initial prednisone therapy ('prednisone response') revealed significantly higher Bcl-2 expression levels {5.4 +/- 3.4 relative fluorescence intensity (RFI), n = 68} than poor responders (3.7 +/- 2.6 RFI, n = 42; P = 0.002) . There was no significant correlation between the other investigated parameters and prednisone response . Moreover, neither the CD95 and Bcl-2 expression levels nor the extent of spontaneous apoptosis in vitro, CD95 sensitivity or P-gp function were correlated with the response to induction chemotherapy or relapse rate, either for B-cell precursor ALL or T-cell ALL . No consistent pattern of change in CD95 (n = 10) and Bcl-2 expression (n = 9) was noted in cases studied at both initial diagnosis and relapse . In conclusion, our findings underline the different cell biological features of primary AML and ALL cells.

Biochem Pharmacol, 2000 Sep 15, 60(6), 831 - 7
BCRP/MXR/ABCP expression in topotecan-resistant human breast carcinoma cells; Yang CH et al.; We have previously described a mitoxantrone-resistant MCF7 cell line that is cross-resistant to topotecan, 7-ethyl-10-{4-(1-piperidino)-1-piperidino}carbonyloxy-camptothecin (CPT-11), and 9-aminocamptothecin, but not to camptothecin . A novel mechanism that resulted in decreased topotecan accumulation in MCF7/MX cells was proposed (Yang et al . Cancer Res 55: 4004-4009, 1995) . We now have developed a topotecan-resistant cancer cell line from wild-type MCF7 cells . MCF7/TPT300 cells were 68.9-fold resistant to topotecan, 68.3-fold to 10-hydroxy-7-ethylcamptothecin (SN-38), and 116-fold to mitoxantrone, but only 4.1-fold to camptothecin . Topotecan efflux was increased in MCF7/TPT300 cells compared with MCF7/WT cells, and this increase was reversed upon ATP depletion by sodium azide, suggesting an energy-dependent drug efflux mechanism . However, MCF7/TPT300 cells did not overexpress P-glycoprotein or the multidrug resistance-associated protein (MRP1) . In contrast, overexpression of the breast cancer resistance protein (BCRP/MXR/ABCP) was observed in MCF7/TPT300 cells as well as DNA topoisomerase I down-regulation . Our data suggest that enhanced topotecan efflux contributes partly to topotecan resistance in MCF7/TPT300 cells, possibly mediated by BCRP/MXR/ABCP.

Arch Dermatol Res, 2000 Jun, 292(6), 292 - 300
Expression of lung resistance protein in epithelioid sarcoma in vitro and in vivo; Kusakabe H et al.; The incidence of epithelioid sarcoma among patients with malignant soft tissue tumors is small, but the rates of recurrence and metastasis of this type of sarcoma are high . To date, effective chemotherapy for advanced epithelioid sarcoma has not been established and, furthermore, epithelioid sarcoma is known to exhibit multidrug resistance (MDR) . The chemosensitivities to anticancer agents of two cell lines established from epithelioid sarcoma were examined in this study . The results showed that the ES-OMC-MN and SFT-8606 cell lines were resistant to vincristine (IC50 1190 nM and 872 nM, respectively) and Adriamycin (IC50 921 nM and 650 nM, respectively), but sensitive to actinomycin D (IC50 < 10 nM) . P-glycoprotein (p-Gp) and MDR-associated protein (MRP) were not expressed in these cell lines, but a high expression level of lung resistance protein (LRP) was observed . The original tumor tissues from which the two cell lines were established were also found to be LRP-positive but not to express p-Gp or MRP . Their chemosensitivities to Adriamycin were not significantly altered in the presence of 2.5 microg/ml anti-LRP antibody (LRP-56), but the IC50 of vincristine was much less (IC50 128 nM and 27 nM, respectively) than that for an untreated cell line . It is thus suggested that the vincristine resistance in the two cell lines is LRP-mediated . Since cyclosporin A, known to be a modifier of p-Gp, also induced reversal of vincristine resistance in the ES-OMC-MN and SFT-8606 cell lines (IC50 6.2 nM and 17 nM, respectively), it is suggested that cyclosporin A acts as a modifier of MDR mediated by LRP.

Radiologe, 2000 Jun, 40(6), 507 - 17
{Pulmonary tuberculosis--the current radiological diagnosis of an old disease}; Hlawatsch A et al.; INCIDENCE: Decreasing numbers of tuberculosis cases in the western countries have led to diminished attention towards this disease . But worldwide, tuberculosis still is the leading cause of mortality due to any one single infectious agent . In the industrialized countries, immigration, growth of low-income groups and increasing numbers of immunocompromised patients, mainly due to the HIV epidemic, supply a reservoir for tuberculosis . DIAGNOSIS: Because of the option of a specific therapy, early diagnosis of tuberculosis is crucial for the course of the disease . In cases of multidrug resistant strains, further spread has to be prevented . Radiology with chest films and computed tomography has a central role in diagnosing tuberculosis . FINDINGS: However, as the disease produces a broad spectrum of radiographic findings, there are often difficulties in determining the underlying diagnosis . Additionally, there have been reports of atypical presentations of tuberculosis in immunocompromised as well as immunocompetent patients . This article reviews the current state of radiological diagnosis of pulmonary tuberculosis.

Anticancer Res, 2000 May-Jun, 20(3B), 1921 - 5
A case of pulmonary adenocarcinoma with overexpression of multidrug resistance-associated protein and p53 aberration; Nakamura M et al.; A 66-year-old female patient underwent left upper lobectomy and dissection of the mediastinal lymph nodes . The pathological diagnosis was well-differentiated papillary adenocarcinoma of the lung with metastasis to the mediastinal lymph nodes, p-T2N2MO, stage IIIA . After the operation, she was treated by chemotherapy including lipophilic anticancer compounds (carboplatin and VP-16) . The patient unexpectedly showed long survival for 6 years and 2 months without obvious recurrence or metastasis of the cancer . The anticancer compounds were not effective on the recurrent lesions and then she died due to respiratory failure 8 months after recurrence . The autopsy revealed pleural dissemination and intrapulmonary metastasis . Immunohistochemical analysis showed increased multidrug resistance-associated protein (MRP)-positive tumor cells in the recurrent and metastatic lesions, while few MRP-positive cells were apparent in the primary lesion . The MRP-positive cells were accompanied by p53 nuclear accumulation in the carcinoma . This was a case of pulmonary adenocarcinoma with acquired multidrug resistance caused by MRP overexpression and aberrant p53 after chemotherapy.

Anticancer Res, 2000 May-Jun, 20(3A), 1819 - 23
Hyperthermia enhanced chemosensitivity of human malignant glioma cells; Hermisson M et al.; In an effort to overcome chemoresistance of human malignant glioma cells, the modulation of drug-induced cell death by hyperthermia was assessed in 4 human malignant glioma cells lines, LN-18, LN-229, T98G and U87MG . Compared to normothermic conditions, pulsed 24 h drug exposure enhanced the sensitivity of glioma cells most strikingly with teniposide, treosulfan, topotecan and cisplatin, moderately with vincristine, CCNU and doxorubicin, but not with gemcitabine . Susceptibility to hyperthermia-mediated drug sensitization, varied significantly with T98G and LN-229 being strongly sensitized and U87MG being most resistant to the effects of hyperthermia . Hyperthermia did not significantly modulate drug-induced changes in cell cycle distribution . The degree of sensitization was independent of p53 status and of multidrug resistance (mdr) activity . Hyperthermia may thus be a useful approach to overcome, chemoresistance of human malignant glioma cells.

Anticancer Res, 2000 May-Jun, 20(3A), 1467 - 70
Technetium-99m tetrofosmin mammoscintigraphy findings related to the expression of P-glycoprotein mediated multidrug resistance; Sun SS et al.; We studied 30 patients with infiltrating ductal breast carcinomas to evaluate the relationship between the degree of accumulation of technetium-99m tetrofosmin (Tc-TETRO) and p-glycoprotein (Pgp) expression in breast tumor tissues . All of the 30 patients underwent Tc-TETRO mammoscintigraphy to calculate breast tumor uptake of Tc-TETRO to background (T/B) ratios before surgery or biopsy . Immunohistochemical analysis was performed to determine Pgp expression in the pathological specimens of the 30 breast tumors . The T/B ratios were significantly lower for tumors in 12 patients with positive Pgp expression (Group A) than for those in 18 patients with negative expression (Group B) (1.20 +/- 0.12 and 1.94 +/- 0.30, p < 0.05) . Our results supported the opinion that Tc-TETRO mammoscintigraphy is helpful for determining in vivo the presence of multidrug resistance due to Pgp expression in breast carcinoma patients.

Br J Cancer, 2000 Aug, 83(3), 375 - 83
Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export; Evers R et al.; The multidrug resistance proteins MRP1 and MRP2 are members of the same subfamily of ATP-binding cassette transporters . Besides organic molecules conjugated to negatively charged ligands, these proteins also transport cytotoxic drugs for which no negatively charged conjugates are known to exist . In polarized MDCKII cells, MRP1 routes to the lateral plasma membrane, and MRP2 to the apical plasma membrane . In these cells MRP1 transports daunorubicin, and MRP2 vinblastine; both transporters export reduced glutathione (GSH) into the medium . We demonstrate that glutathione transport in MDCKII-MRP1 cells is inhibited by the inhibitors of organic anion transporters sulfinpyrazone, indomethacin, probenecid and benzbromarone . In MDCKII-MRP2 cells, GSH export is stimulated by low concentrations of sulfinpyrazone or indomethacin, whereas export is inhibited down to control levels at high concentrations . We find that unmodified sulfinpyrazone is a substrate for MRP2, also at concentrations where GSH export is inhibited . We also show that GSH export in MDCKII-MRP2 cells increases in the presence of vinblastine, and that the stoichiometry between drug and GSH exported is between two and three . Our data indicate that transport of sulfinpyrazone and vinblastine is associated with GSH export . However, at high sulfinpyrazone concentrations this compound is transported without GSH . Models of MRP action are discussed that could explain these results.

Br J Cancer, 2000 Aug, 83(3), 366 - 74
Inhibitory effect of the reversal agents V-104, GF120918 and Pluronic L61 on MDR1 Pgp-, MRP1- and MRP2-mediated transport; Evers R et al.; The human multidrug transporter MDR1 P-glycoprotein and the multidrug resistance proteins MRP1 and MRP2 transport a range of cytotoxic drugs, resulting in multidrug resistance in tumour cells . To overcome this form of drug resistance in patients, several inhibitors (reversal agents) of these transporters have been isolated . Using polarized cell lines stably expressing human MDR1, MRP1 or MRP2cDNA, and 2008 ovarian carcinoma cells stably expressing MRP1 cDNA, we have investigated in this study the specificity of the reversal agents V-104 (a pipecolinate derivative), GF120918 (an acridone carboxamide derivative also known as GG918), and Pluronic L61 (a (poly)oxypropethylene and (poly)oxypropylene block copolymer) . Transport experiments with cytotoxic drugs with polarized cell lines indicate that all three compounds efficiently inhibit MDR1 Pgp . Furthermore, V-104 partially inhibits daunorubicin transport by MRP1 but not vinblastine transport by MRP2 . V-104 reverses etoposide resistance of 2008/MRP1 cells, whereas GF120918 does not reverse resistance due to MRP1 . V-104 partially inhibits the export of the organic anion dinitrophenyl S-glutathione by MDCKII-MRP1 but not by MDCKII-MRP2 cells . Unexpectedly, export of the organic anion calcein by MDCKII-MRP1 and MDCKII-MRP2 cells is stimulated by Pluronic L61, probably because it relieves the block on entry of calcein AM into the cell by endogenous MDR1 Pgp.

Br J Cancer, 2000 Aug, 83(3), 338 - 45
Characterization and modulation of drug resistance of human paediatric rhabdomyosarcoma cell lines; Cocker HA et al.; The role of multidrug resistance (MDR) and p53 functional status in the treatment of paediatric rhabdomyosarcoma is unclear . We have characterized a panel of seven human rhabdomyosarcoma cell lines for MDR and p53 phenotype . None of the cell lines had P-glycoprotein (P-gp) or multidrug resistance-related protein (MRP) detectable by Western blotting, whereas immunohistochemistry suggested that very low levels of MDR proteins may be present in some of the lines . RT-PCR studies indicated that mdr-1, mrp-1 and Irp mRNA was present in 5/7, 7/7 and 5/7 lines respectively . The function of p53 is compromised in six of the lines, either through mutation of the p53 gene or by overexpression of mdm-2 . The sensitivity of many of the cell lines to vincristine could be modulated above 2-fold and as high as 16-fold using two modulating agents, PSC833 and VX710 (with VX710 being a significantly more potent modulator of the rhabdomyosarcoma lines) . PSC833 also increased vincristine accumulation in all of the lines from 1.2- to 2.2-fold . These results suggest that some of these cell lines have low levels of multidrug resistance . The level of MDR proteins is very low and therefore difficult to detect, but may be sufficient to confer low-level, but clinically relevant, resistance to some cytotoxic agents, especially vincristine . These cell lines will therefore provide a suitable model to test new strategies in treatment and for further understanding relationships between protein expression and drug resistance.

Int J Cancer, 2000 Sep 1, 87(5), 615 - 28
Intracellular P-glycoprotein expression is associated with the intrinsic multidrug resistance phenotype in human colon adenocarcinoma cells; Meschini S et al.; The 2 clones, LoVo 5 and LoVo 7, derived from untreated LoVo WT human colon adenocarcinoma cells and exhibiting different sensitivity to doxorubicin (DOX), were compared in order to identify possible determinants of intrinsic drug resistance . A multidrug resistant variant cell line, selected from LoVo WT cells by continuous exposure to DOX (LoVo DX), was also included in the study . Analysis of the expression and organization of cytoskeletal elements by flow cytometry and fluorescence microscopy evidenced a positive correlation between vimentin expression and DOX resistance in LoVo 7 and LoVo DX cells, whereas differences in actin, tubulin or cytokeratin did not seem to relate to drug response . The expression and localization of different drug transporters commonly implicated in drug resistance, i.e., the MDR1 gene product P-glycoprotein (P-gp), the multidrug resistance-related protein MRP and the lung resistance-related protein LRP were also investigated by means of flow cytometry and fluorescence microscopy, following labeling with specific monoclonal antibodies . Surface expression of P-gp was only detected in LoVo DX cells, which also exhibited increased MRP and LRP protein levels . However, significant amounts of P-gp were found at intracellular sites in the intrinsically resistant LoVo 7 clone . Modulation of P-gp function by cyclosporin A was found to alter DOX accumulation and efflux in LoVo 7 cells, indicating that intracellular P-gp plays a functional role in drug trafficking and suggesting possible implications in determining the intrinsic resistance displayed by this clone .

Biochemistry, 2000 Aug 8, 39(31), 9327 - 34
Novel function of human RLIP76: ATP-dependent transport of glutathione conjugates and doxorubicin; Awasthi S et al.; Active transport of conjugated and unconjugated electrophiles out of cells is essential for cellular homeostasis . We have previously identified in human tissues a transporter, DNP-SG {S-(2, 4-dinitrophenyl)glutathione} ATPase, capable of carrying out this function {Awasthi et al . (1998) Biochemistry 37, 5231-5238, 5239-5248} . We now report the cloning of DNP-SG ATPase . The sequence of the cDNA clone was identical to that of human RLIP76, a known Ral-binding protein . RLIP76 expressed in E . coli was purified by DNP-SG affinity chromatography . Purified recombinant RLIP76: (1) had ATPase activity stimulated by DNP-SG or doxorubicin (DOX), and the K(m) values of RLIP76 for ATP, DOX, and DNP-SG were similar to those reported for DNP-SG ATPase; (2) upon reconstitution with asolectin as well as with defined lipids, catalyzed ATP-dependent transport of DNP-SG and DOX with kinetic parameters similar to those of DNP-SG ATPase; (3) when transfected into K562 cells, resulted in increased resistance to DOX, and increased ATP-dependent transport of DNP-SG and DOX by inside-out membrane vesicles from transfected cells; (4) direct uptake of purified RLIP76 protein into mammalian cells from donor proteoliposomes confers DOX resistance . These results indicate that RLIP76, in addition to its role in signal transduction, can catalyze transport of glutathione conjugates and xenobiotics, and may contribute to the multidrug resistance phenomenon.

Farmaco, 2000 Mar, 55(3), 206 - 8
Novel approaches in the rational design of antifungal agents of low toxicity; Borowski E; This paper presents an overview of studies on novel strategies for the rational design of antifungal agents of low toxicity and overcoming the multidrug resistance (MDR) of fungi . This goal was achieved both due to the introduction of a novel target, glucosamine-6-phosphate synthase, as well as to the recognition of molecular basis of selectivity of action of amphotericin B derivatives.

Arzneimittelforschung, 2000 Jun, 50(6), 576 - 9
Effect of polyoxyl 35 castor oil and Polysorbate 80 on the intestinal absorption of digoxin in vitro; Cornaire G et al.; Surfactants are classically used to improve the solubilization of lipophilic drugs such as digoxin . Polysorbate 80 and Cremophor EL (polyoxyl 35 castor oil) are such surfactants but they may also modulate the action of P-glycoprotein, an energy-dependent "counter-transport" system implicated in the phenomenon of multidrug resistance in cancer cells . P-glycoprotein is also present in the intestine on the apical membrane of mature enterocytes and can potentially reduce the absorption of a wide range of drugs . In this study, using the improved everted gut sac method, the effects of Polysorbate 80, Cremophor EL and cyclosporin on the absorption of digoxin were studied . An increase in the uptake of digoxin in the presence of these three products could be shown with our in vitro model . Cremophor EL and Polysorbate 80 had no toxic effects at the concentrations used . These results suggest that surfactants such as Cremophor EL and Polysorbate 80 should not only support solubilization but can also modulate the P-glycoprotein system to improve the bioavailability of poorly absorbed drugs.

J Biol Chem, 2000 Aug 4, 275(31), 23530 - 9
MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drugs as judged by interference with nucleotide trapping; Smith AJ et al.; The human MDR3 gene is a member of the multidrug resistance (MDR) gene family . The MDR3 P-glycoprotein is a transmembrane protein that translocates phosphatidylcholine . The MDR1 P-glycoprotein related transports cytotoxic drugs . Its overexpression can make cells resistant to a variety of drugs . Attempts to show that MDR3 P-glycoprotein can cause MDR have been unsuccessful thus far . Here, we report an increased directional transport of several MDR1 P-glycoprotein substrates, such as digoxin, paclitaxel, and vinblastine, through polarized monolayers of MDR3-transfected cells . Transport of other good MDR1 P-glycoprotein substrates, including cyclosporin A and dexamethasone, was not detectably increased . MDR3 P-glycoprotein-dependent transport of a short-chain phosphatidylcholine analog and drugs was inhibited by several MDR reversal agents and other drugs, indicating an interaction between these compounds and MDR3 P-gp . Insect cell membranes from Sf9 cells overexpressing MDR3 showed specific MgATP binding and a vanadate-dependent, N-ethylmaleimide-sensitive nucleotide trapping activity, visualized by covalent binding with {alpha-(32)P}8-azido-ATP . Nucleotide trapping was (nearly) abolished by paclitaxel, vinblastine, and the MDR reversal agents verapamil, cyclosporin A, and PSC 833 . We conclude that MDR3 P-glycoprotein can bind and transport a subset of MDR1 P-glycoprotein substrates . The rate of MDR3 P-glycoprotein-mediated transport is low for most drugs, explaining why this protein is not detectably involved in multidrug resistance . It remains possible, however, that drug binding to MDR3 P-glycoprotein could adversely affect phospholipid or toxin secretion under conditions of stress (e.g . in pregnant heterozygotes with one MDR3 null allele).

Gene Ther, 2000 Jul, 7(14), 1224 - 33
Modulation of the typical multidrug resistance phenotype by targeting the MED-1 region of human MDR1 promoter; Marthinet E et al.; Multidrug resistance of cancer (MDR) is the major cause of failure of chemotherapy . The typical MDR phenotype is due to the overexpression of membrane proteins among which the main representative is P-glycoprotein (Pgp) encoded by the MDR1 gene . Many attempts to modulate MDR by chemosensitizers have been unsuccessful in human therapy due to their intrinsic toxic effects . In an effort to modulate the MDR phenotype efficiently we designed an antisense and a transcriptional decoy strategy targeting the TATA-less human MDR1 gene promoter . The choice of the start point of transcription in a multiple start site window is related to an upstream MED-1 cis-element, the sequence and configuration of which are specific to human MDR1 gene expressed in Pgp-overproducing cancer cells . A 12mer antisense oligodeoxynucleotide (ODN) and a 12mer double-stranded ODN, both containing the MED-1 sequence, were designed and efficiently vectorized into the nucleus with the chimerical MPG peptide . A synthetic cellular model (NIH-EGFP) and highly resistant human CEM/VLB0.45 leukemia cells, significantly responded to transfection with the ODN/MPG complex . The level of EGFP fluorescence in NIH-EGFP cells decreased, and thus its production, and viability of CEM/VLB0.45 cells decreased by 63% in the presence of vinblastine, revealing that their resistance to the anticancer drug was reversed . These results open new insights into transcriptional decoy and anti-gene therapies of MDR cancers that overproduce Pgp . Gene Therapy (2000) 7, 1224-1233.

Rev Esp Quimioter, 2000 Jun, 13(2), 167 - 70
{Current state of multidrug resistance in tuberculosis}; Casal M et al.; Given the current concern about multiresistance to drugs in tuberculosis, the data from a large study in two different centers are presented . At the Hospital Universitario Reina Sofia de Cordoba 71,359 clinical isolates of Mycobacterium tuberculosis were processed and at the Centro de Referencia de Micobacterias 2,350 cultures were studied . The percentage of total multiresistance at the Hospital Universitario was 6% from 1996-1998, and 6.7% at the Centro de Referencia in 1998 . Although these figures are high, they are lower than in previous years and could indicate the beginning of a decreasing trend, following years of increasing in the 1990s.

J Clin Microbiol, 2000 Aug, 38(8), 3119 - 22
Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis isolates from Brazil; Valim AR et al.; Mutations in a 69-bp region of the rpoB gene associated with rifampin resistance (Rif(r)) in 100 isolates (82 Rif(r)) from three states of Brazil were studied . Twenty-one different kinds of mutations were identified in the Rif(r) isolates, and six new alleles are described.

Zhonghua Xue Ye Xue Za Zhi, 1998 Feb, 19(2), 67 - 9
{The correlation between p170, p26 and multidrug resistance in refractory/relapse acute leukemia}; Gao X et al.; OBJECTIVE: To explore the role of two membrane proteins, p170 and p26, in clinical drug resistance and their correlation . METHODS: The expression of p170 and p26 in bone marrow cells from 39 acute leukemia patients were detected by flow cytometry . RESULTS: The expression ratios of p170 and p26 were 15.89% +/- 4.41% and 17.32% +/- 10.20%, respectively, in previously untreated group, and 31.02% +/- 14.33% and 33.78% +/- 15.97%, respectively, in refractory/relapse group . The difference between the two groups was significant, while there were no correlation between p170 and p26 (r = -0.1578, P > 0.5) . CONCLUSION: Both p170 and p26 were related to clinical multidrug resistance, but the mechanisms were different.

Zhonghua Xue Ye Xue Za Zhi, 1998 Feb, 19(2), 63 - 6
{The clinical significance of multidrug resistance associated protein(MRP) gene expression in acute leukemia}; Wang F et al.; OBJECTIVE: To investigate the relationship between the expression of the multidrug resistance-associated protein(MRP) gene and clinical drug resistance in acute leukemia(AL) . METHODS: Semi-quantitative reverse transcriptase polymerase chain reaction was used to examine the expression of MRP gene in 65 bone marrow aspirates of AL patients and 15 normal volunteer's peripheral blood mononuclear cells . The expression level of MRP were expressed as ratio of MRP/beta 2M(beta 2-microglobulin) and the ratio of MRP/beta 2M > or = 0.3 was defined as MRP positive . RESULTS: The level of MRP mRNA and the positive percentage of MRP in relapsed-refractory group were significantly higher than that in normal control group . In newly diagnosed group, the first complete remission rate of MRP negative patients(84%) was significantly higher than that of MRP positive(25%) patients . MRP expression level was different in leukemia sub-type . MRP and mdr-1 genes were examined simultaneously in 65 AL patients, the mean expression level and positive percentage of MRP and mdr-1 in clinical resistance group were significantly higher than those in non-resistance group . No significant correlation was found between the MRP and mdr-1 expression(rs = 0.1683) . CONCLUSION: High expression of MRP leads to drug resistance and it is an unfavorable factor to prognosis.

Zhonghua Xue Ye Xue Za Zhi, 1998 Jan, 19(1), 20 - 2
{Synergistic reversal effect of quinine in combination with modulators on multidrug resistant cell line K562/HHT}; Luo M et al.; OBJECTIVE: To develop effective combination of drug resistance modulators . METHODS: The reversal effects of quinine (Quin) in combination with cyclosporin A (CsA), dipyridamole (DPM) or tamoxifen (Tam), respectively on the drug resistance of K562/HHT were studied by MTT, flow cytometry and median-effect principle . RESULTS: The reversal effectiveness of the modulator combinations was 2-3 times as much as that of each modulator alone . Synergistic interaction between Quin and DPM or Tam was greater than that between Quin and CsA . Quin combined with CsA increased intracellular DNR accumulation significantly as compared with either of them alone . CONCLUSION: There was synergistic interactions between Quin and CSA, DPM or Tam, and combination of modulators with different operating mechanisms had a greater synergistic interaction.

Zhonghua Zhong Liu Za Zhi, 1998 Jan, 20(1), 40 - 2
{Molecular biological evidence for the genetic stability of in vivo passaged doxorubicin resistant cell line S-180R}; Zheng G et al.; OBJECTIVE: To assess the genetic stability of doxorubicin resistant sarcoma 180 cell line(S-180R) after in vivo passages . METHODS: Flow cytometry, Southern blot, Northern blot and RT-PCR were used to examine genes and molecules related to drug resistance . RESULTS: The drug-efflux of S-180R was nearly 100-fold as high as that of the parental cells . The ratio of half peak width to peak height was 0.23 as compared to 0.56 measured two years before when the S-180R cell line was initially established . The mdr-1 gene was significantly amplified and transcribed while the transcription of topoisomerase II alpha gene was decreased . However there was no increase in mRNA expression of the multidrug resistance associated protein(MRP) . CONCLUSION: Compared with the initially established S-180R, its resistance to doxorubicin is not only maintained but in fact has been increased since in vivo passage for 2 years . The major mechanism is amplification and over-expression of mdr-1 gene, but decreased topoisomerase II alpha also contributes . S-180R is an ideal experimental model for the study of doxorubicin resistance and its reversion.

Schweiz Med Wochenschr, 2000 May 20, 130(20), 727 - 31
{Clinical pharmacology: proteins for the transport of drugs}; Drewe J et al.; Most drugs have to pass cellular barriers in order to reach their site of action . This can be accomplished passively by diffusion, but more often it is an energy-consuming process using specific carrier proteins . Two groups of such proteins which are well characterised, the organic cation transporters and the multidrug resistance proteins, are discussed in detail in the present review . The clinical significance of these proteins is due not only to their role in drug distribution and elimination, but also to possible drug interactions when different drugs and/or endogenous substrates compete with the same carrier protein . Inhibition of multidrug resistance proteins could be of therapeutic value in impairing transport of drugs from their site of action and this could be particularly beneficial in the treatment of malignant diseases.

Hepatology, 2000 Aug, 32(2), 341 - 7
Regulation of multidrug resistance 2 P-glycoprotein expression by bile salts in rats and in primary cultures of rat hepatocytes; Gupta S et al.; Biliary phospholipid secretion is tightly coupled to the secretion of free cholesterol and bile salts . The secretion of phospholipids across the canalicular membrane of hepatocytes occurs via the multidrug resistance 2 (mdr2) P-glycoprotein (Pgp) . The mechanism underlying the coupling of bile salt and phospholipid secretion has not been elucidated . The aims of this study were to determine the effects of bile acid structure on the expression of mdr2 in vitro and in vivo . Under optimal culture conditions, taurine-conjugated bile acids (50 micromol/L) increased mdr2 messenger RNA (mRNA) levels in the following order: taurocholate (TCA) (288 +/- 36%, P < . 005) = taurodeoxycholate (TDCA) (276 +/- 36%, P <.025) > taurochenodeoxycholate (TCDCA) (216 +/- 34%, P <.025) > tauroursodeoxycholate (TUDCA) (175 +/- 28%, P <.05) of control levels . The increase in mdr2 mRNA levels by TCA was both time and concentration dependent . Cholate feeding to rats with intact enterohepatic circulation increased mdr2 transcriptional activity by 4-fold and protein mass by 1.9-fold . Chronic biliary diversion (CBD) decreased mdr2 mRNA levels to 66 +/- 9% (P <.025) of sham-operated controls . Intraduodenal infusion of TCA for 48 hours in CBD rats caused a significant increase in mdr2 mRNA levels (224%) as compared with CBD controls . A diet high in cholesterol (4%) decreased mdr2 mRNA levels to 57% +/- 2 (P <.001) of pair-fed controls . Squalestatin (1 micromol/L), an inhibitor of cholesterol biosynthesis, increased mdr2 mRNA levels by 8.8-fold (P <.005) in hepatocyte cultures after 24 hours . In conclusion, in the rat, bile acids up-regulated mdr2 transcriptional activity whereas cholesterol decreased mdr2 mRNA both in vitro and in vivo.

Am J Physiol Gastrointest Liver Physiol, 2000 Aug, 279(2), G417 - 25
ATP-dependent GSH and glutathione S-conjugate transport in skate liver: role of an Mrp functional homologue; Rebbeor JF et al.; Multidrug resistance-associated proteins 1 and 2 (Mrp1 and Mrp2) are thought to mediate low-affinity ATP-dependent transport of reduced glutathione (GSH), but there is as yet no direct evidence for this hypothesis . The present study examined whether livers from the little skate (Raja erinacea) express an Mrp2 homologue and whether skate liver membrane vesicles exhibit ATP-dependent GSH transport activity . Antibodies directed against mammalian Mrp2-specific epitopes labeled a 180-kDa protein band in skate liver plasma membranes and stained canaliculi by immunofluorescence, indicating that skate livers express a homologous protein . Functional assays of Mrp transport activity were carried out using (3)H-labeled S-dinitrophenyl-glutathione (DNP-SG) . DNP-SG was accumulated in skate liver membrane vesicles by both ATP-dependent and ATP-independent mechanisms . ATP-dependent DNP-SG uptake was of relatively high affinity {Michaelis-Menten constant (K(m)) = 32 +/- 9 microM} and was cis-inhibited by known substrates of Mrp2 and by GSH . Interestingly, ATP-dependent transport of (3)H-labeled S-ethylglutathione and (3)H-labeled GSH was also detected in the vesicles . ATP-dependent GSH transport was mediated by a low-affinity pathway (K(m) = 12 +/- 2 mM) that was cis-inhibited by substrates of the Mrp2 transporter but was not affected by membrane potential or pH gradient uncouplers . These results provide the first direct evidence for ATP-dependent transport of GSH in liver membrane vesicles and support the hypothesis that GSH efflux from mammalian cells is mediated by members of the Mrp family of proteins.

Bioorg Med Chem Lett, 2000 Jul 17, 10(14), 1605 - 8
Michael adducts of ascorbic acid as inhibitors of protein phosphatase 2A and inducers of apoptosis; Fathi AR et al.; Michael adducts of ascorbic acid with alpha,beta-unsaturated carbonyl compounds have been shown to be potent inhibitors of protein phosphatase 1 (PP1) without affecting cell viability at the respective concentrations . Here we were able to show that higher concentrations can partially inhibit PP2A activity and concomitantly induce apoptotic cell death . A nitrostyrene adduct of ascorbic acid proved to be a more potent and effective inhibitor of PP2A as well as a stronger inducer of apoptosis . These adducts only slightly lost their cytotoxic potential in multidrug resistant cells that were 10-fold less sensitive to apoptosis induction by okadaic acid and vinblastine.

Clin Cancer Res, 2000 Jul, 6(7), 2751 - 8
p53 but not bcl-2 immunostaining is predictive of poor clinical complete response to primary chemotherapy in breast cancer patients; Bottini A et al.; Preoperative chemotherapy administered to breast cancer (BC) patients is a model for studying in vivo the interaction between cytotoxic treatment and clinical and biological parameters . Apoptosis induced by anticancer agents is a mechanism of treatment activity; therefore, overexpression of genes inhibiting the apoptotic pathway could produce drug resistant tumors . In the present study, the two most studied inhibitors of apoptosis, the bcl-2 gene and the mutant p53, have been evaluated to assess whether they may play a role in modulating response of BC to primary chemotherapy . From August 1990 to January 1997, 143 patients bearing T(2-4)N(0-1)M0 primary BC were submitted to two different chemotherapeutic regimens before surgery . The first 64 received the cyclophosphamide, methotrexate, 5-fluorouracil (CMF) regimen (on days 1 and 8 and every 28 days thereafter) associated with tamoxifen (30 mg daily) in case of estrogen receptor (ER)-positive BC, and the remaining 79 were submitted to single agent epirubicin (120 mg/m2 every 21 days) . The expression of p53, bcl-2, Ki67, ER, progesterone receptor, c-erbB2, and the multidrug resistance P-glycoprotein (gp-170) was evaluated in BC specimens obtained at diagnosis by incision biopsy and at postchemotherapy surgery . At the end of chemotherapy administration (median, 3 cycles; range, 2-6), the clinical complete response (cCR) rate was superimposable in the patient subgroups with bcl-2-positive or -negative primary tumors; conversely, p53 expression, at a cutoff of 10% positive cells, was significantly associated with a lower cCR rate (9.4 versus 27.0%; P < 0.04) . p53 was a significant predictor for poor cCR in the subset submitted to epirubicin (3.6 versus 25.5%; P < 0.02; in patients with p53+ and p53- BC, respectively); by contrast, only a trend toward lower cCR has been observed in patients with p53+ tumors receiving CMF +/- tamoxifen with respect to p53- ones . The distribution of cCR according to the gp-170-positive or -negative tumors was 8 versus 22% in patients submitted to epirubicin and 29 versus 30% in those receiving CMF +/- tamoxifen, respectively . In a multivariate regression analysis, after adjusting for treatment administered (epirubicin versus CMF +/- tamoxifen), menopausal status, tumor and node status, histology grade, ER, progesterone receptor, c-erbB2, Ki67, bcl-2, and gp-170 expression, the p53 status maintained an independent predictive role for cCR . Most of the tumors undergoing change in percentage of p53 expression after both treatments originally harbored mutant protein, and only four BC specimens that were p53 negative before chemotherapy became positive afterward . These data confirm in vivo the concept that the responsiveness of tumors to chemotherapy in part derives from the capability of BC cells to undergo apoptosis . The role of mutated p53 in preventing response is more evident in patients submitted to epirubicin, and this may be caused by the up-regulation of multidrug resistance gene expression by p53 inactivation . p53 is a stable phenotype and is not inducible by at least three or four chemotherapy cycles.

J Toxicol Environ Health A, 2000 Jun, 60(4), 275 - 89
Dieldrin induces cytosolic {3H}7, 12-dimethylbenz{a}anthracene binding but not multidrug resistance proteins in rainbow trout liver; Curtis LR et al.; Previously it was demonstrated that biliary excretion of a single dose of {14C}dieldrin or {3H}7, 12-dimethylbenz/alanthracene (DMBA) was stimulated up to 700% and 300%, respectively, in rainbow trout fed 0.3-0.4 mg dieldrin/kg/d for 9-12 wk . This was not explained by increased activities of hepatic microsomal xenobiotic-metabolizing enzymes or increased amounts of any of six cytochrome P-450 isozymes quantitated by Western blots . It was hypothesized that stimulated excretion was explained by induction of (1) cytosolic binding proteins that facilitated intracellular trafficking of DMBA to sites of metabolism, or (2) ATP-dependent proteins that transport xenobiotic metabolites from liver to bile . Binding of 15 and 60 nmol {3H}DMBA/mg protein increased about 200% in hepatic cytosol from dieldrin-fed fish . A 50-fold molar excess of unlabeled DMBA reduced binding of 15 nmol {3H}DMBA/mg protein (nonspecific binding) by the same amount in cytosol from control and dieldrin-fed fish, indicating that dieldrin induced specific binding . Liver sections from control and dieldrin-fed fish were treated with multidrug resistance (MDR) protein monoclonal antibodies C494, C219, and JSB-1, and polyclonal antibody MDR Ab-1 . There were no marked differences in optical densities of immunohistochemical staining near bile canaliculi of control and dieldrin-fed fish . Induction of xenobiotic binding capacity in cytosol of dieldrin-fed rainbow trout at least partially explained altered DMBA disposition in fish pretreated with this cyclodiene insecticide.

Leukemia, 2000 Jul, 14(7), 1266 - 75
Study of apoptosis-related responses of leukemic blast cells to in vitro anthracycline treatment; Belaud-Rotureau MA et al.; Anthracyclines trigger an apoptotic cell death but their molecular targets are not totally explored . We investigated the apoptotic response of blast cells and lymphocytes from medullary samples of 31 de novo acute leukemia . Mononuclear cells were treated in vitro by therapeutic concentrations of either daunorubicin (DNR) or idarubicin (IDA) for 1 h, washed and cultured for 18 h . A multivariate analysis using flow cytometry and a CD45 gating on lymphocytes and blast cells was performed . DNR and IDA induced a Fas enhancement on both leukemic and normal cells . In blast cells the DEVDases were activated and the caspase 3 was cleaved in relation to phosphatidyl serine exposure, showing a caspase-dependent pathway in anthracycline-induced apoptosis . Apoptotic percentages were always higher for blast cells than for lymphocytes, confirming that anthracycline toxicity mainly affected tumor cells . Moreover, drug-induced apoptosis was not related to spontaneous apoptosis, suggesting that variations in response intensities were due to individual variations of sensitivity rather than to programmed life span time . The apoptotic response of P-glycoprotein-expressing blast cells was not significant, giving biological argument for the poor prognosis of multidrug resistance leukemia . Finally, Fas induction and anthracycline-induced apoptosis on blast cells were significantly higher when a complete remission was achieved, thus shedding light on potential new prognostic factors in acute leukemia.

Anticancer Drugs, 2000 Jun, 11(5), 377 - 84
Toxicity and DNA binding of dextran-doxorubicin conjugates in multidrug-resistant KB-V1 cells: optimization of dextran size; Lam W et al.; We previously showed that conjugating doxorubicin to very large 70-500 kDa dextran decreased its removal rate from P-glycoprotein (P-gp) over-expressing, multidrug-resistant KB-V1 cells . Furthermore these conjugates could act synergistically with other cancer drugs . In the drug-sensitive 3-1 clone, but not in the V1 subclone which was 300-fold more resistant to free doxorubicin, conjugation led to a size-related decrease in toxicity . Here we identified the optimal size of dextran for avoiding P-gp-mediated efflux and yet preserving as much as possible doxorubicin toxicity . Chemically reduced, intracellularly stable 3.4-10 kDa conjugates were prepared . Confocal microscopy and fluorescence quenching experiments showed that these conjugates entered nuclei and interacted with DNA . In 3-1 cells, but not in V1 cells, cytotoxicity of conjugates decreased 14- to 45-fold linearly related to log size of the carrier (r=0.95) . In V1 cells toxicity of the 10 kDa conjugate exceeded that of free doxorubicin . After conjugation the equilibrium binding constant of the DNA-drug complex (KA) decreased only by up to 3-fold . In 3-1 cells, but not in VI cells, DNA binding kinetics was an important factor and toxicity could be linearly correlated to 1/KA of conjugate (r=0.94) . Drug accumulation decreased with an increase in dextran size but drug removal was decreased only in V1 cells . It appeared that drug uptake was also sensitive to dextran conjugation . In Vl cells drug removal was sensitive to the P-gp inhibitor verapamil or energy starvation . Ratios of V1/3-1 toxicity, drug accumulation and drug removal correlated linearly with log dextran size . When these ratios equaled 1, dextran sizes were estimated to be 32, 103 and 21 kDa, respectively.

Anticancer Drugs, 2000 Jun, 11(5), 339 - 52
1,4-Anthraquinone: an anticancer drug that blocks nucleoside transport, inhibits macromolecule synthesis, induces DNA fragmentation, and decreases the growth and viability of L1210 leukemic cells in the same nanomolar range as daunorubicin in vitro; Perchellet EM et al.; 1,4-Anthraquinone (AQ) was synthesized and shown to prevent L1210 leukemic cells from synthesizing macromolecules and growing in vitro . In contrast, its dihydroxy-9,10anthraquinone precursor, quinizarin, was inactive . The antitumor activity of AQ was compared to that of daunorubicin (DAU), which is structurally different from AQ but also contains a quinone moiety . AQ is equipotent to DAU against L1210 tumor cell proliferation (IC50: 25 nM at day 2 and 9 nM at day 4) and viability (IC50: 100 nM at day 2 and 25 nM at day 4), suggesting that its cytostatic and cytotoxic activities are a combination of drug concentration and duration of drug exposure . Since AQ does not increase but rather decreases the mitotic index of L1210 cells at 24 h, it is not an antitubulin drug but might arrest early stages of cell cycle progression . Like DAU, a 1.5-3 h pretreatment with AQ is sufficient to inhibit the rates of DNA, RNA and protein syntheses (IC50: 2 microM) determined over 30-60 min periods of pulse-labeling in L1210 cells in vitro . In contrast to DAU, which is inactive, a 15 min pretreatment with AQ has the advantage of also inhibiting the cellular transport of both purine and pyrimidine nucleosides (IC50: 2.5 microM) over a 30 s period in vitro . Hence, AQ may prevent the incorporation {3H}thymidine into DNA because it rapidly blocks the uptake of these nucleosides by the tumor cells . After 24 h, AQ induces as much DNA cleavage as camptothecin and DAU, two anticancer drugs producing DNA strand breaks and known to, respectively, inhibit topoisomerase I and II activities . However, the concentration-dependent induction of DNA cleavage by AQ, which peaks at 1.6-4 microM and disappears at 10-25 microM, resembles that of DAU . The mechanism by which AQ induces DNA cleavage is inhibited by actinomycin D, cycloheximide and aurintricarboxylic acid, suggesting that AQ activates endonucleases and triggers apoptosis . The abilities of AQ to block nucleoside transport, inhibit DNA synthesis and induce DNA fragmentation are irreversible upon drug removal, suggesting that this compound may rapidly interact with various molecular targets in cell membranes and nuclei to disrupt the functions of nucleoside transporters and nucleic acids, and trigger long-lasting antitumor effects which persist after cessation of drug treatment . Because of its potency and dual effects on nucleoside transport and DNA cleavage, the use of bifunctional AQ with antileukemic activity in the nM range in vitro might provide a considerable advantage in polychemotherapy to potentiate the action of antimetabolites and sensitize multidrug-resistant tumor cells.

Cancer Chemother Pharmacol, 2000, 46(1), 27 - 34
Evaluation of toremifene for reversal of multidrug resistance in renal cell cancer patients treated with vinblastine; Braybrooke JP et al.; PURPOSE: Expression of P-glycoprotein (Pgp), which confers the multidrug resistance (MDR) phenotype, is thought to contribute to the insensitivity of renal cell cancer (RCC) to chemotherapy . The development of Pgp inhibitors for clinical application has been hampered by unacceptable toxicity at doses required to achieve adequate cellular concentration . Toremifene is able to reverse MDR and sensitise RCC to vinblastine in vitro . However, in vivo toremifene is tightly bound to serum proteins, in particular the acute phase protein alpha1-acid glycoprotein (AAG), which may limit tissue availability . In this phase I-II study we assessed the tolerability of short courses of high dose toremifene in combination with vinblastine and evaluated the key determinants of MDR reversal in vivo . METHODS: Twenty-seven patients with metastatic RCC received escalating doses of oral toremifene for 3 days every 2 weeks in combination with vinblastine 6 mg/m2 i.v . on day 3 of each cycle . The serum concentration of toremifene, its metabolites and AAG were measured and the effect of patients' serum on inhibition of Pgp in vitro was determined . RESULTS: Twenty-six patients were evaluable for response . Eight patients (31%) had stable disease and 18 patients (69%) progressive disease . The mean serum concentration of toremifene at 780 mg daily for 3 days was 7.82 microM {standard deviation (SD) 2.48, range 2.50 to 14.70}, which exceeds that known to reverse MDR in vitro . The serum concentration of the major metabolite of toremifene, N-demethyltoremifene, which also reverses MDR, was 5.13 microM (SD 1.78, range 1.80 to 9.00) . In 60% of patients the pre-treatment AAG concentration was above that known to block the effects of toremifene in vitro . However, addition of serum from patients on toremifene to MCF-7 adr cells in vitro inhibited Pgp-mediated efflux of rhodamine 123 . CONCLUSIONS: We have shown that short course, high-dose toremifene in combination with vinblastine is generally well tolerated and that the concentration of toremifene required to reverse MDR in vitro is achievable in vivo.

Blood, 2000 Aug 1, 96(3), 1070 - 9
Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance; Mahon FX et al.; Targeting the tyrosine kinase activity of Bcr-Abl with STI571 is an attractive therapeutic strategy in chronic myelogenous leukemia (CML) . A few CML cell lines and primary progenitors are, however, resistant to this compound . We investigated the mechanism of this resistance in clones of the murine BaF/3 cells transfected with BCR-ABL and in 4 human cell lines from which sensitive (s) and resistant (r) clones were generated by various methods . Although the resistant cells were able to survive in the presence of STI571, their proliferation was approximately 30% lower than that of their sensitive counterparts in the absence of the compound . The concentration of STI571 needed for a 50% reduction in viable cells after a 3-day exposure was on average 10 times higher in the resistant (2-3 micromol/L) than in the sensitive (0.2-0.25 micromol/L) clones . The mechanism of resistance to STI571 varied among the cell lines . Thus, in Baf/BCR-ABL-r, LAMA84-r, and AR230-r, there was up-regulation of the Bcr-Abl protein associated with amplification of the BCR-ABL gene . In K562-r, there was no Bcr-Abl overexpression, but the IC(50) for the inhibition of Bcr-Abl autophosphorylation was increased in the resistant clones . Sequencing of the Abl kinase domain revealed no mutations . The multidrug resistance P-glycoprotein (Pgp) was overexpressed in LAMA84-r, indicating that at least 2 mechanisms of resistance operate in this cell line . KCL22-r showed neither Bcr-Abl up-regulation nor a higher threshold for tyrosine kinase inhibition by STI571 . We conclude that BCR-ABL-positive cells can evade the inhibitory effect of STI571 by different mechanisms, such as Bcr-Abl overexpression, reduced intake mediated by Pgp, and, possibly, acquisition of compensatory mutations in genes other than BCR-ABL.

Blood, 2000 Aug 1, 96(3), 902 - 9
Enforced P-glycoprotein pump function in murine bone marrow cells results in expansion of side population stem cells in vitro and repopulating cells in vivo; Bunting KD et al.; The human multidrug resistance-1 (MDR1) gene product, P-glycoprotein (P-gp), is well known for its ability to confer drug resistance; however, recent evidence suggests that P-gp expression can have more general effects on cellular development . In support of this idea, it was previously shown that retroviral-mediated MDR1 expression in murine bone marrow cells resulted in the expansion of stem cells in culture and in the development of a myeloproliferative syndrome in transplanted mice . It is now reported that MDR1-mediated stem cell expansion is associated with an increase in side population (SP) stem cells, defined by Hoechst dye staining . Transduction of murine bone marrow cells with an MDR1 retroviral vector resulted in an almost 2 log increase in SP cell numbers over 12 days in culture, whereas there was a rapid loss of SP cells from control cultures . Stem cell amplification was not limited to ex vivo expansion cultures but was also evident when MDR1-transduced cells were directly transplanted into irradiated mice . In these cases, stem cell expansion was associated with relatively high vector copy numbers in stem cell clones . As previously reported, some cases were associated with a characteristic myeloproliferative syndrome . A functionally inactive MDR1 mutant cDNA was used to show that P-gp pump function was required both for amplification of phenotypically defined SP cells and functionally defined repopulating cells . These studies further support the concept that ABC transporter function can have important effects on hematopoietic stem cell development.

Cancer Res, 2000 Jul 1, 60(13), 3514 - 21
Methotrexate cross-resistance in a mitoxantrone-selected multidrug-resistant MCF7 breast cancer cell line is attributable to enhanced energy-dependent drug efflux; Volk EL et al.; Cellular resistance to the antifolate methotrexate (MTX) is often caused by target amplification, uptake defects, or alterations in polyglutamylation . Here we have examined MTX cross-resistance in a human breast carcinoma cell line (MCF7/MX) selected in the presence of mitoxantrone, an anticancer agent associated with the multidrug resistance (MDR) phenotype . Examination of protein expression and enzyme activities showed that MCF7/MX cells displayed none of the classical mechanisms of MTX resistance . They did, however, exhibit an ATP-sensitive accumulation defect accompanied by reduced polyglutamylation . Although the kinetics of drug uptake was similar between parental and resistant cells, the resistant cells exhibited increased energy-dependent drug efflux . This suggested the involvement of an ATP-binding cassette (ABC) transporter . However, cells transfected with the breast cancer resistance protein (BCRP)-the ABC transporter known to be highly overexpressed in MCF7/MX cells and to confer mitoxantrone resistance (D . D . Ross et al., J . Natl . Cancer Inst . 91: 429-433, 1999)-were not MTX resistant, which suggested that this transporter is not involved in MTX cross-resistance . Moreover, members of the MRP protein family of transport proteins, which had previously been implicated in MTX resistance, were not found to be overexpressed in the MCF7/MX cells . Thus, our data suggest that a novel MTX-specific efflux pump may be involved in this unusual cross-resistance phenotype.

Int J Tuberc Lung Dis, 2000 Jul, 4(7), 673 - 83
Clinical and programmatic mismanagement rather than community outbreak as the cause of chronic, drug-resistant tuberculosis in Buenaventura, Colombia, 1998; Laserson KF et al.; SETTING: Buenaventura, Colombia . OBJECTIVE: To assess whether antituberculosis drug resistance was generated by poor management or community transmission . DESIGN: Treatment-failure and new tuberculosis (TB) patients identified between May 1997 and June 1998 were interviewed and their treatment histories reviewed . Bacteriologic testing, including drug susceptibility profiles (DSP) and DNA fingerprinting by restriction fragment length polymorphism (RFLP), was performed and human immunodeficiency virus (HIV) testing was offered . RESULTS: DSP and RFLP fingerprints were obtained for isolates from 34 of 64 treatment-failure patients; 25 (74%) were resistant to > or = one drug . Fifteen of the 25 patients consented to HIV testing; none were positive . An average of 2.8 major treatment errors per patient was identified . RFLP from the treatment-failure patients revealed 20 unique isolates and six clusters (isolates with identical RFLP); 4/6 clusters contained isolates with different DSP . Analysis of the RFLP from both treatment-failure and new patients revealed that 44/111 (40%) isolates formed 18 clusters . Four of 47 (9%) new patients had multidrug-resistant TB (MDR-TB) . Eleven isolates belonged to the Beijing family, related to the MDR strain W . CONCLUSION: Drug resistance in Buenaventura results from both poor management and community transmission . Dependence on DSP to identify TB transmission is inadequate when programmatic mismanagement is common.

Int J Tuberc Lung Dis, 2000 Jul, 4(7), 665 - 72
Surveillance of Mycobacterium tuberculosis drug resistance in France, 1995-1997 . AZAY Mycobacteria Study Group; Robert J et al.; OBJECTIVE: To measure the rate of primary and secondary drug resistance of Mycobacterium tuberculosis on an ongoing basis . DESIGN: Data on all culture-positive tuberculosis were collected prospectively from 1995 through 1997 from a microbiological laboratory network of 19 university hospitals throughout France, and submitted quarterly to the National Reference Centre for Surveillance of Mycobacterial Diseases . RESULTS: A total of 2998 patients were included in the study . Among the 2333 (78%) previously untreated patients, 8.6% had isolates resistant to any drug, 4.8% to streptomycin (SM) alone, 1.2% to isoniazid (INH) alone, 1.8% to SM + INH, and 0.3% to INH + rifampicin (RMP) or multidrug resistance (MDR) . Foreign birth was independently associated with a higher risk of primary resistance to any drug (odds ratio {OR} 1.5) . Among the 268 (9%) previously treated patients, 20.9% had isolates resistant to any drug, 6.3% to SM alone, 3.4% to INH alone, 4.1% to SM + INH, and 3.7% to INH + RMP . Foreign birth (OR = 2.3), and human immunodeficiency virus positive status (OR = 4.4) were independently associated with a higher risk of secondary resistance to any drug . CONCLUSION: During the last 30 years there has been no increase in resistance to any drug among previously untreated patients . As expected, secondary resistance was highly associated with foreign birth . MDR-TB remains a rare event in France.

Lancet, 2000 Jun 17, 355(9221), 2147 - 52
Hit HIV-1 hard, but only when necessary; Harrington M et al.; Randomised, controlled trial data show that combination antiretroviral therapy for HIV-1 infection benefits people with CD4-cell counts less than 350 cells/microL . Based on currently known risks and benefits, we believe that if CD4-cell counts and viral load are monitored carefully, and highly active antiretroviral therapy (HAART) is started commonly when the CD4-cell count drops below 350 cells/microL, then clinically relevant immune-system damage and progression to AIDS and death can be greatly delayed or prevented . This approach is dictated by three features of HIV-1 infection that are not typical of infectious diseases: no available regimen can eradicate HIV-1; all currently effective regimens may cause undesirable, sometimes life-threatening, toxic effects; and, unless regimens are strictly adhered to, multidrug resistance can develop, limiting future treatment options . If therapy is started too early, cumulative side-effects of the drugs used and the development of multidrug resistance may outweigh the net benefits of the lengthening of life . If therapy is started too late, increases in disease progression and mortality outweigh the risk of adverse events . A patients' activist (MH) and a clinician (CCJC) discuss data that justify this balanced approach and the feasibility of randomised controlled trials to provide clearer answers about when to start treatment.

Drug Metab Dispos, 2000 Aug, 28(8), 951 - 8
Glutathione S-transferase metabolism of the antineoplastic pentafluorophenylsulfonamide in tissue culture and mice; Frankmoelle WP et al.; The microtubule disrupting agent 2-fluoro-1-methoxy-4-pentafluorophenylsu lfonamidobenzene (T138067) binds covalently and selectively to beta-tubulin and has been shown to evade drug-efflux pumps that confer multidrug resistance to other antimitotic drugs that are used in cancer chemotherapy (Shan et al., 1999) . In addition to these resistance mechanisms, eukaryotic cells have developed other protection mechanisms that involve enzymes that modify electrophilic xenobiotics . To determine whether T138067 is a substrate for such enzymatic detoxification pathways, a metabolism study was initiated . GSH conjugation was shown to play a major role in T138067 metabolism . T138067-GSH conjugates were isolated from the culture media of T138067-treated cells and the bile of mice treated i.v . with T138067 . The major T138067-GSH degradation products were also isolated from these sources . 19F NMR studies of the metabolites showed that metabolic conversions occurred through substitution of the para fluorine atom in the pentafluorophenyl ring of T138067 . The T138067-GSH conjugate was also isolated from T138067 incubation buffer that had been exposed to mouse, rat, dog, or human liver slices, suggesting that this mechanism is not species-specific . All three human glutathione S-transferases (alpha, mu, and pi), which are expressed in a wide variety of tissues including human tumors, were shown to metabolize T138067 effectively in vitro . The combined data show that T138067 is being metabolized, in vitro and in vivo, predominantly via a glutathione S-transferase-mediated metabolic pathway.

J Bone Joint Surg Am, 2000 Jul, 82-A(7), 963 - 9
Intrinsic resistance to chemotherapeutic agents in murine osteosarcoma cells; Takeshita H et al.; BACKGROUND: There are two general categories of drug resistance: acquired and intrinsic . The mechanisms involved in acquired drug resistance have been extensively studied, and several mechanisms have been described . However, the mechanisms responsible for intrinsic drug resistance have not been elucidated, to our knowledge . The purpose of the present study was to investigate the cytological and biochemical differences between acquired and intrinsic drug resistance in osteosarcoma cells . METHODS: We previously isolated a clonal cell line (MOS/ADR1) to study acquired resistance in osteosarcoma by exposure of parental murine osteosarcoma cells (MOS) to doxorubicin . In the present study, we cloned a new, intrinsically resistant cell line (MOS/IR1) by single-cell culture of MOS cells and we investigated the differences in cell phenotype and the mechanisms of resistance in both of these resistant clones . RESULTS: The MOS/ADR1 and MOS/IR1 cells were sevenfold and fivefold more resistant to doxorubicin than the parental murine osteosarcoma cells . Morphologically, the MOS/ADR1 cell line was composed of polygonal cells, whereas the MOS/IR1 cell line consisted of plump spindle cells with long cytoplasmic processes . The MOS/IR1 cells showed a much lower level of alkaline phosphatase activity than did the MOS/ ADR1 and MOS cells . There were no substantial differences in the cellular DNA content or the doubling time among these three lines . Overexpression of the P-glycoprotein involved in the function of an energy-dependent drug-efflux pump was detected in the MOS/ADR1 cells but not in the MOS/ IR1 cells . After the cells were incubated with doxorubicin for one hour, the two resistant lines had less accumulation of the drug than did the parent line (p < 0.05) . The addition of a P-glycoprotein antagonist, verapamil, or the depletion of cellular adenosine triphosphate resulted in a marked increase in the accumulation of doxorubicin in the MOS/ADR1 cells (p < 0.05) but not in the MOS/ IR1 cells . The MOS/ADR1 cells were found to exhibit cross-resistance only to substrates for P-glycoprotein (such as doxorubicin, vincristine, and etoposide), whereas the MOS/IR1 cells were resistant to all of the drugs studied (including cisplatin and methotrexate) . The degree of drug resistance in the MOS/IR1 cells was found to be associated with the molecular weight of the drugs (p < 0.05) . Permeabilization of the plasma membrane by saponin increased both the accumulation of doxorubicin (p < 0.05) and the cytotoxic activity of this drug in all lines, but the effects were most pronounced in the MOS/IR1 cells . CONCLUSIONS: Taken together, this data suggests that reduced drug accumulation in the MOS/IR1 cells may be due to the effect of decreased permeability of the plasma membrane on the transport of drugs from the extracellular environment into the cytosol of the cell and that this may be the mechanism responsible for intrinsic resistance to multiple drugs in the MOS/IR1 cell line . CLINICAL RELEVANCE: Current drug treatment for human osteosarcoma may include multiple chemotherapeutic agents, such as doxorubicin, cisplatin, and methotrexate . These drugs exhibit different cytotoxic actions and, thus, the mechanisms of resistance to individual drugs vary . Clinical resistance to multidrug chemotherapy may be observed in tumors that recur after repetitive chemotherapy and in previously untreated tumors . In the former group, a tumor cell may express multidrug resistance by combining several different mechanisms due to its exposure to various drugs . In the latter group, however, this is not likely . Decreased intracellular drug accumulation due to reduced permeability of the plasma membrane, found in the MOS/IR1 cells, is one possible mechanism and may explain the intrinsic resistance to multidrug chemotherapy for the treatment of osteosarcoma . Further study regarding the resistance mechanism in the MOS/IR1 cells may help to overcome the intrinsic drug resistance in oste

Probl Tuberk, 2000, (3), 9 - 11
{Treatment of multidrug resistant tuberculosis in Santakiskes tuberculosis hospital}; Mishkinis K et al.; The authors studies 98 patients (82 males and 16 females) in 1994-1996 . The patients were found sputum Mycobacteria tuberculosis (MT) resistant to two essential antituberculosous drugs: isoniazid and rifampicin . In 67 (68.4%) cases MT resistance was verified in other laboratories of the country . The examinees were 13 new cases and 17 had relapses . Sixty eight patients were diagnosed as having chronic tuberculosis . After multidrug resistant strains were identified, the patients were treated by an individual regimens by choosing adequate drugs from different groups . Twenty patients were operated on . Sputum conversion occurred in only 24 (24.5%) patients . In 14 of them clinical and X-ray lesion disappeared . In 74 (75%) good treatment outcomes were not achieved and MT remained in the sputum . There were statistically significant differences in the treatment outcomes among new, relapsing, and chronic cases (poor treatment outcomes were in 38.5, 64.7, and 85.3%, respectively) . The surgical outcomes proved to be no better than those in drug-treated patients due to the incorrect definition of indications for surgery or advanced disease . The findings show that the outcomes were poor in chronic MT multidrug-resistant patients and fair results could be achieved in new cases of the disease diagnosed in time.

J Pharmacol Exp Ther, 2000 Aug, 294(2), 480 - 7
The influence of coordinate overexpression of glutathione phase II detoxification gene products on drug resistance; O'Brien M et al.; Glutathione (GSH), glutathione S-transferases (GSTs), and the multidrug resistance-associated protein 1 (MRP1) have been independently studied for their contributions to drug resistance . Single cDNA transfection experiments have provided inconsistent and disparate conclusions with respect to the importance of GSH and GST in conferring a resistant phenotype . Because these three proteins can act as a concerted coordinated pathway, we reasoned that equivalent increases may be required for enhanced resistance to be expressed . We have assembled these proteins together, or in various combinations, to determine whether they show cooperativity in determining drug response . Increased expression through single cDNA transfection of GSTpi, gamma-glutamylcysteine synthetase (gamma-GCS) (regulatory plus catalytic subunits), or MRP1 enhanced resistance to a number of anticancer drugs . Cotransfection of GSTpi and GCS, gave higher resistance to doxorubicin, etoposide, and vincristine than with either alone . Resistance toward chlorambucil and ethacrynic acid was similar in cells overexpressing either component or overexpressing GST alone . Coexpression of GSTpi with MRP1 conferred significant resistance above that seen for MRP1 alone to chlorambucil, etoposide, ethacrynic acid, and vincristine . The combination of GCS and MRP1 did not afford additional resistance above MRP1 alone . When all three were transfected, significantly higher levels of resistance were found for doxorubicin and etoposide . These results support the concept that coordinate enhancement of focal thiol elements of detoxification pathways provides a more efficient protective phenotype than do single components alone.

Bioconjug Chem, 2000 Jul-Aug, 11(4), 564 - 8
In vitro interleukin-3 binding to leukemia cells predicts cytotoxicity of a diphtheria toxin/IL-3 fusion protein; Alexander RL et al.; Patients with acute myeloid leukemia frequently develop chemotherapy resistant blasts . To overcome multidrug resistance, a diphtheria toxin fusion protein (DTIL3) was engineered by fusing the catalytic and translocation domains of diphtheria toxin (DT) to human interleukin-3 (IL-3) . However, when blasts were isolated from patients and tested for colony growth inhibition by DTIL3, only a third of the samples showed sensitivity to the fusion protein . Prior to clinical development, we need to be able to identify which patients are likely to respond to therapy with DTIL3 . In this report, we compared the inhibition of thymidine incorporation in human leukemia cell lines by DTIL3 to the IL-3 receptor number and affinity . We found DTIL3 was cytotoxic to four of the eight cell lines tested with half-maximal inhibition of thymidine incorporation (IC(50)) from 1 to 50 pM . The IL-3 receptor density for these cell lines ranged from 0 to 2635 receptors per cell . The dissociation constant for an IL-3 high-affinity receptor agonist was 0.5 nM for cell lines with receptors . We found a correlation for the cell lines between the presence of high-affinity IL-3 receptors and sensitivity to DTIL3 (p = 0.03) . These results suggest the variability in sensitivity of patient leukemic progenitors to DTIL3 may be due in part to the presence or absence of high-affinity IL-3 receptors.

Haematologica, 2000 Jul, 85(7), 711 - 21
Clinical significance of P-glycoprotein expression and function for response to induction chemotherapy, relapse rate and overall survival in acute leukemia; Wuchter C et al.; BACKGROUND AND OBJECTIVES: A multidrug-resistance (MDR) phenotype mediated by P-glycoprotein (P-gp) contributes to chemotherapy failure in acute leukemia . However, the exact prognostic significance of this resistance mechanism is still unclear, mostly due to methodologic problems in P-gp detection . We therefore investigated, whether P-gp expression levels or functional P-gp activity better predict response to induction chemotherapy, relapse rate and overall survival in acute leukemia . DESIGN AND METHODS: We examined cell samples of 121 adults with de novo acute myeloid leukemia (AML) and 102 children with newly diagnosed acute lymphoblastic leukemia (ALL) for P-gp expression and functional P-gp activity by flow cytometry . P-gp function was determined by the rhodamine 123 (rh123)-efflux test (AML n=121, ALL n=102) and P-gp expression levels using the P-gp specific monoclonal antibodies (moabs) MRK-16 (AML n=51, ALL n=31), 4.E3 (AML n=35, ALL n=32), or UIC-2 (AML n=68, ALL n=50) . We correlated our findings with the immunophenotype, FAB morphology, cytogenetics and clinical data of the examined patients . RESULTS: P-gp expression levels as detected by MRK-16 and 4.E3 were very low and did not differ between AML and ALL as estimated using relative fluorescence intensity (RFI) values and D-values by Kolmogorow-Smirnov (KS) statistics . For moab UIC-2, P-gp expression levels were higher in AML than in ALL . Within AML, moab UIC-2 mainly reacted with myelomonocytic-differentiated leukemic cells of the FAB M4/5 subtypes . No correlation between P-gp expression levels as detected by MRK-16, 4.E3 or UIC-2 and the response to induction chemotherapy or relapse rate, both in AML and ALL, was observed . However, a prognostic impact of P-gp expression levels on overall survival in AML was seen for moab MRK-16 . Moreover, within AML, P-gp function was higher in immature blast cells as defined by immunophenotype and FAB morphology and correlated with response to induction chemotherapy, relapse rate, overall survival as well as cytogenetic risk groups . In ALL, the overall functional P-gp activity was lower than in AML and did not correlate with immunophenotypical subgroups, response to induction chemotherapy, relapse rate or overall survival . INTERPRETATION AND CONCLUSIONS: Our data demonstrate a prognostic impact of P-gp in AML but not ALL and indicate that the functional rh123-efflux assay should be preferred for flow-cytometric P-gp evaluation in acute leukemia compared with P-gp expression analysis by monoclonal antibodies.

J Clin Oncol, 2000 Jul, 18(14), 2685 - 94
MDR1 gene expression and outcome in osteosarcoma: a prospective, multicenter study; Wunder JS et al.; PURPOSE: Increased expression of the multidrug resistance gene (MDR1) has been implicated in osteosarcoma prognosis . This study represents the first prospective assessment of the prognostic value of MDR1 mRNA expression in patients with newly diagnosed extremity osteosarcoma . PATIENTS AND METHODS: A series of patients with high-grade, nonmetastatic extremity osteosarcoma were enrolled from six tertiary care institutions and observed prospectively for tumor recurrence (median follow-up duration, 30 months) . All patients were treated with (neo)adjuvant chemotherapy and surgery . Tumors from 123 patients were analyzed for MDR1 mRNA expression . The association of the level of MDR1 expression with the risk of systemic recurrence was examined using survival analyses with traditional and histologic markers as prognostic factors . RESULTS: Using the highest MDR1 value for each patient, a dose-response relationship was not identified between the level of MDR1 expression and systemic relapse (relative risk, 1.15; P =.44) . Analyses based on biopsy or resection values alone gave similar results (P =.11 and.41, respectively, log rank test) . In multivariate analysis, large tumor size (> 9 cm) was the only significant independent predictor of systemic outcome (relative risk, 2.8; P =.002) . CONCLUSION: We did not identify any correlation between MDR1 mRNA expression and disease progression in patients with osteosarcoma . It is likely that alterations in other genes are involved in resistance to chemotherapy in osteosarcoma and that they play a more critical role than MDR1 in this disease.

J Neurooncol, 2000, 46(2), 157 - 71
Preliminary individual adjuvant therapy for gliomas based on the results of molecular biological analyses for drug-resistance genes; Tanaka S et al.; New adjuvant therapy individualized by the results of reverse transcription-polymerase chain reaction (RT-PCR) for drug-resistance genes has been used to treat malignant gliomas . Protocol studies for malignant gliomas were not so encouraging in their therapeutic results because of heterogeneity and the various drug-sensitivities of the tumors . Individualization of glioma therapy is recommended . Drug-resistance genes messenger ribonucleic acid (mRNA) expressions were investigated in drug-resistant human glioma cell lines derived from U87MG and 46 frozen samples of retrospectively examined neuroepithelial tumors (12 low grade neuroepithelial tumors, 16 Grade III gliomas, 11 glioblastomas, and 7 other malignant neuroepithelial tumors such as medulloblastomas and primitive neuroectodermal tumors) by RT-PCR with the specific primers for O6-methylguanine DNA methyltransferase (MGMT), multidrug-resistance gene 1 (MDR1), multidrug-resistance-associated protein (MRP), and glutathione-S-transferase-pi (GST-pi) . Thirty-seven preliminary individual adjuvant therapies (IAT) based on RT-PCR results, mainly in MGMT expression, were performed on 30 consecutive patients with neuroepithelial tumors . In the retrospectively examined series, the initial response to 1-(4-amino-2-methyl-5-pyrimidynyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) was correlated most significantly to the MGMT mRNA expression among 11 independent prognostic factors (p = 0.0037) in multivariate logistic regression analysis . In the preliminary IAT, 17 of 32 evaluable therapies had a partial or complete response (53.1% response rate) . Our IAT based on RT-PCR seemed to be more effective than conventional therapies for malignant gliomas.

J Biol Chem, 2000 Sep 29, 275(39), 30069 - 74
The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides; Jedlitschky G et al.; Cellular export of cyclic nucleotides has been observed in various tissues and may represent an elimination pathway for these signaling molecules, in addition to degradation by phosphodiesterases . In the present study we provide evidence that this export is mediated by the multidrug resistance protein isoform MRP5 (gene symbol ABCC5) . The transport function of MRP5 was studied in V79 hamster lung fibroblasts transfected with a human MRP5 cDNA . An MRP5-specific antibody detected an overexpression of the glycoprotein of 185 +/- 15 kDa in membranes from MRP5-transfected cells and a low basal expression of hamster Mrp5 in control membranes . ATP-dependent transport of 3',5'-cyclic GMP at a substrate concentration of 1 micrometer was 4-fold higher in membrane vesicles from MRP5-transfected cells than in control membranes . This transport was saturable with a K(m) value of 2.1 micrometer . MRP5-mediated transport was also detected for 3',5'-cyclic AMP at a lower affinity, with a K(m) value of 379 micrometer . A potent inhibition of MRP5-mediated transport was observed by several compounds, known as phosphodiesterase modulators, including trequinsin, with a K(i) of 240 nm, and sildenafil, with a K(i) value of 267 nm . Thus, cyclic nucleotides are physiological substrates for MRP5; moreover, MRP5 may represent a novel pharmacological target for the enhancement of tissue levels of cGMP.

Lancet, 2000 Jul 1, 356(9223), 22 - 5
Classification of drug-resistant tuberculosis in an epidemic area; Van Rie A et al.; BACKGROUND: Traditionally, patients with drug-resistant tuberculosis are classified as having acquired drug-resistant or primary drug-resistant disease on the basis of a history of previous tuberculosis treatment . Only cases of primary drug resistance are assumed to be due to transmission of drug-resistant strains . METHODS: This descriptive study of 63 patients with drug-resistant tuberculosis assessed the relative contribution of transmission of drug-resistant strains in a high-incidence community of Cape Town, South Africa, by restriction-fragment length polymorphism (RFLP) . The RFLP results were compared with the results obtained by traditional classification methods . FINDINGS: According to RFLP definitions, 52% (33 cases) of drug-resistant tuberculosis was caused by transmission of a drug-resistant strain . The proportion of cases due to transmission was higher for multidrug-resistant (64%; 29 cases) than for single-drug-resistant (no cases) tuberculosis . By the clinical classification, only 18 (29%) patients were classified as having primary drug-resistant tuberculosis (implying transmission) . The clinical classification was thus misleading in 25 patients . INTERPRETATION: The term acquired drug resistance includes patients infected with strains that truly acquired drug resistance during treatment and patients who were initially infected with or reinfected with a drug-resistant strain . This definition could lead to misinterpretation of surveillance studies, incorrect evaluation of tuberculosis programmes, and delayed diagnosis and treatment of patients with multidrug-resistant disease . The clinical term acquired drug resistance should be replaced with the term "drug resistance in previously treated cases", which includes cases with drug resistance due to true acquisition as well as that due to transmitted drug-resistant strains.

Biochem Biophys Res Commun, 2000 Jul 14, 273(3), 913 - 9
Expression and characterization of the N- and C-terminal ATP-binding domains of MRP1; Kern A et al.; The His(6)-tagged N- and C-terminal nucleotide binding (ATP Binding Cassette, ABC) domains of the human multidrug resistance associated protein, MRP1, were expressed in bacteria in fusion to the bacterial maltose binding protein and a two-step affinity purification was utilized . Binding of a fluorescent ATP-analogue occurred with micromolar dissociation constants, MgATP was able to inhibit the ATP-analogue binding with 70 and 200 micromolar apparent inhibition constants, while AMP was nearly ineffective . Both MRP1 nucleotide binding domains showed ATPase activities (V(max) values between 5-10 nmoles/mg protein/min), which is fifty to hundred times lower than that of parent transporter . The K(M) value of the ATP hydrolysis by the nucleotide binding domains were 1.5 mM and 1.8 mM, which is similar to the K(M) value of the native or the purified and reconstituted transporter, N-ethylmaleinimide and A1F(4) inhibited the ATPase activity of both nucleotide binding domains .

Proc Natl Acad Sci U S A, 2000 Jul 18, 97(15), 8490 - 4
Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts; Watanabe T et al.; Several groups have attempted to develop gene therapy strategies to treat cancer via introduction of the wild-type (wt) p53 cDNA into cancer cells . Unfortunately, these approaches do not result in regulated expression of the p53 gene and do not reduce expression of the mutant p53 that is overexpressed in cancerous cells . These shortcomings may greatly limit the utility of this gene replacement approach . We describe an alternative strategy with trans-splicing ribozymes that can simultaneously reduce mutant p53 expression and restore wt p53 activity in various human cancers . The ribozyme accomplished such conversion by repairing defective p53 mRNAs with high fidelity and specificity . The corrected transcripts were translated to produce functional p53 that can transactivate p53-responsive promoters and down-modulate expression of the multidrug resistance (MDR1) gene promoter . The level of wt p53 activity generated was significant, resulting in a 23-fold induction of a p53-responsive promoter and a 3-fold reduction in MDR1 promoter expression in transfected cancer cells . Once efficient delivery systems are developed, this strategy should prove useful for making human cancers more responsive to p53 activity and more sensitive to chemotherapeutic agents.

J Med Chem, 2000 Jun 29, 43(13), 2547 - 56
Aureobasidins: structure-activity relationships for the inhibition of the human MDR1 P-glycoprotein ABC-transporter; Tiberghien F et al.; Cyclic depsipeptide cyclo-{D-Hmp(1)-L-MeVal(2)-L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle+ ++(6)-L-MeVal(7)-L-Leu(8)-L-betaHOMeVal(9)}, the antifungal antibiotic aureobasidin A (AbA), was reported to interfere with ATP-binding cassette (ABC) transporters in yeast and mammalian cells, particularly the MDR1 P-glycoprotein (Pgp), a transmembrane phospholipid flippase or "hydrophobic vacuum cleaner" that mediates multidrug resistance (MDR) of cancer cells . In a standardized assay that measures Pgp function by the Pgp-mediated efflux of the calcein-AM Pgp substrate and uses human lymphoblastoid MDR-CEM (VBL(100)) cells as highly resistant Pgp-expressing cells and the cyclic undecapeptide cyclosporin A (CsA) as a reference MDR-reversing agent (IC(50) of 3.4 microM), AbA was found to be a more active Pgp inhibitor (IC(50) of 2.3 microM) . Out of seven natural analogues and 18 chemical derivatives of AbA, several were shown to display even more potent Pgp-inhibitory activity . The Pgp-inhibitory activity was increased about 2-fold by some minor modifications such as those found in the naturally occurring aureobasidins AbB ({D-Hiv(1)}-AbA), AbC ({Val(6)}-AbA), and AbD {gammaHOMeVal(9)}-AbA) . The replacement of the {Phe(3)-MePhe(4)-Pro(5)} tripeptide by an 8-aminocaprylic acid or the N(7)()-desmethylation of MeVal(7) led to only a 3.3-fold decreased capacity to inhibit Pgp function, suggesting that the Pgp inhibitory potential of aureobasidins, though favored by the establishment of an antiparallel beta-sheet between the {D-Hmp(1)-L-MeVal(2)-L-Phe(3)} and {L-aIle(6)-L-MeVal(7)-L-Leu(8)-} tripeptides, does not critically depend on the occurrence of the {L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle(6)} type II' beta-turn secondary structure . In contrast, the most potent Pgp inhibitors were found among AbA analogues with {betaHO-MeVal(9)} residue alterations, with some data suggesting a negative impact of the {L-Leu(8)-L-betaHOMeVal(9)-D-Hmp(1)} gamma-turn secondary structure on Pgp inhibitory potential . The {2,3-dehydro-MeVal(9)}-AbA was the most potent Pgp inhibitory aureobasidin, being 13-fold more potent than AbA and 19-fold more potent (on a molar basis) than CsA . Finally, there was no correlation between the SAR for the human MDR1 Pgp inhibition and the SAR for Saccharomyces cerevisiae antifungal activity, which is mediated by an inositol phosphoceramide synthase activity.

JAMA, 2000 Jul 12, 284(2), 223 - 8
Immune restoration with antiretroviral therapies: implications for clinical management; Lederman MM et al.; Recent dramatic decreases in acquired immunodeficiency syndrome-related mortality are largely due to the introduction of highly active antiretroviral therapy (HAART) . Although immune restoration due to suppression of human immunodeficiency virus (HIV) replication is a critical determinant of these trends, the magnitude of immune restoration seen after treatment with HAART varies substantially among treated persons and is generally incomplete . Nonetheless, even partial immune restoration is sufficient to provide protection from most major opportunistic infections; these risks can be largely predicted by the number of circulating CD4 cells . Limited data suggest that treatment earlier during the course of HIV infection may result in greater preservation of immune function, though this has not been studied in great detail . Preliminary studies performed among persons with multidrug-resistant virus whose treatment regimens are failing suggest that there is likely a benefit to continuation of therapy that may be related to diminished pathogenicity of drug-resistant virus . As deaths related to opportunistic infections diminish, the spectrum of causes of mortality in HIV infection is changing . Except for Kaposi sarcoma, there is insufficient information to conclude that the risks of non-Hodgkin lymphoma and other malignancies are diminishing among persons with HIV infection . How much immune restoration will be enough to ensure long-term survival in persons with HIV infection remains an open question . JAMA . 2000;284:223-228

Curr Opin Oncol, 2000 Jul, 12(4), 330 - 6
Osteosarcomas and other cancers of bone; Bramwell VH; The results of several studies suggest that alterations in various cell cycle regulatory genes are involved in the pathogenesis of osteosarcomas . Experiments in animal models provide preliminary data on the feasibility of gene therapy in osteosarcoma and chondrosarcoma . Prediction of response to chemotherapy remains a major focus of imaging research . Several clinicopathologic studies have explored the mechanisms underlying multidrug resistance in osteosarcoma patients receiving neoadjuvant chemotherapy . HER2/erbB2 expression, linked to poor prognosis, has been proposed as a therapeutic target in osteosarcoma . In clinical studies of osteosarcoma, further data confirm the activity of ifosfamide and carboplatin but provide little support for the use of immunotherapy . A retrospective analysis showed no value for dose intensification of doxorubicin/cisplatin, but the results of a prospective trial should be more informative . Recent evidence confirms that secondary osteosarcomas and malignant fibrous histiocytomas of bone should be treated with aggressive chemotherapy regimens, similar to those used for osteosarcomas.

Pharm Res, 2000 May, 17(5), 505 - 14
The influence of cytotoxicity of macromolecules and of VEGF gene modulated vascular permeability on the enhanced permeability and retention effect in resistant solid tumors; Minko T et al.; PURPOSE: To study the influence of cytotoxicity of macromolecules, VEGF gene expression, and vascular permeability on the enhanced permeability and retention (EPR) effect . METHODS: Mice bearing xenografts of A2780 multidrug resistant human ovarian carcinoma were treated by free doxorubicin (DOX) and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound DOX (P(GFLG)-DOX), Texas Red (P-TR), and FITC (P-FITC) . Antitumor activity, drug distribution in tumor, vascular permeability, VEGF gene expression, and DNA fragmentation were studied . RESULTS: The accumulation of free DOX led to the VEGF gene overexpression and increased the vascular permeability, which in turn enhanced the drug accumulation in the same location . This positive feedback loop led to a highly inhomogeneous distribution of the drug within the tumor . In contrast, P(GFLG)-DOX down-regulated the VEGF gene and decreased vascular permeability . This negative feedback seemed to prevent additional drug accumulation in dead necrotic tissue, resulting in a more uniform drug distribution and enhanced the antitumor activity P(GFLG)-DOX . CONCLUSIONS: The EPR effect significantly differed for macromolecules containing DOX when compared to macromolecules without drug . The cytotoxicity of P(GFLG)-DOX amplified the EPR effect, led to a more homogenous distribution of the drug, increased the average drug concentration in tumor and augmented its efficacy.

Jpn J Pharmacol, 2000 Mar, 82(3), 265 - 8
Reversal of multidrug resistance in human leukemia K562 by tamolarizine, a novel calcium antagonist; Miyake N et al.; A new type of organic Ca2+ channel blocker, tamolarizine, was examined for its reversing effect on multidrug-resistant tumor cells . Tamolarizine synergistically potentiated the cytotoxicity of doxorubicin for doxorubicin-resistant K562 cells (K562/DXR) at a concentration of 0.1-10 microM, but had hardly any synergistic effects in the parental cell line (K562) at the same concentration . Moreover, tamolarizine inhibits the P-glycoprotein pump-efflux activity in a dose-related manner and reduces the expression of the immunoreactive P-glycoprotein in K562/DXR cells as evaluated by cytofluorimetric assay . These results indicate that tamolarizine reverses the multidrug-resistance phenotype through direct interaction with P-glycoprotein.

Anticancer Drug Des, 2000 Feb, 15(1), 17 - 28
PKC412--a protein kinase inhibitor with a broad therapeutic potential; Fabbro D et al.; The staurosporine derivative PKC412 was originally identified as an inhibitor of protein kinase C (PKC) and subsequently shown to inhibit other kinases including the kinase insert domain receptor (KDR) (vascular endothelial growth factor receptor, VEGF-R2), the receptor of platelet-derived growth factor, and the receptor for the stem cell factor, c-kit . PKC412 showed a broad antiproliferative activity against various tumor and normal cell lines in vitro, and was able to reverse the Pgp-mediated multidrug resistance of tumor cells in vitro . Exposure of cells to PKC412 resulted in a dose-dependent increase in the G2/M phase of the cell cycle concomitant with increased polyploidy, apoptosis and enhanced sensitivity to ionizing radiation . PKC412 displayed a potent antitumor activity as single agent and was able to potentiate the antitumor activity of some of the clinically used cytotoxins (Taxol and doxorubicin) in vivo . The combined treatment of PKC412 with loco-regional ionizing irradiation showed significant antitumor activity against tumors which are resistant to both ionizing radiation and chemotherapeutic agents (dysfunctional p53) . The finding that PKC412 is an inhibitor of the VEGF-mediated cellular signaling via inhibition of KDR and PKC in vitro is consistent with the in vivo inhibition of VEGF-dependent angiogenesis in a growth factor implant model . Orally administered PKC412 also strongly inhibited retinal neovascularization as well as laser-induced choroidal neovascularization in murine models . In summary, PKC412 may suppress tumor growth by inhibiting tumor angiogenesis in addition to directly-inhibiting tumor cell proliferation via its effects on PKC and/or other protein kinases . PKC412 is currently in Phase I clinical trials for treatment of advanced cancer as well as for the treatment of ischemic retinopathy.

J Invest Dermatol, 2000 Jul, 115(1), 19 - 23
Neonatal murine epidermal cells express a functional multidrug-resistant pump; Sleeman MA et al.; Phospho-glycoproteins are members of the ABC transporter family encoded by the multidrug-resistant genes . These proteins are highly expressed in many tumor cells derived from patients undergoing treatment with anti-cancer drugs . Phospho-glycoproteins are large 12 transmembrane spanning molecules of 170 kDa, involved in adenosine-5'-triphosphate-dependent efflux of molecules out of the cell, known currently as multidrug-resistant pumps . Expression analysis of phospho-glycoproteins in mice and humans indicates widespread distribution in a number of organs, such as brain and testis . We have analyzed skin, and more particularly keratinocytes, to determine whether they express phospho-glycoproteins and express the multidrug-resistant phenotype . Immunofluorescent staining of skin showed that keratinocytes located in the basal layer of the epidermis preferentially expressed phospho-glycoproteins, as did the outer root sheath cells of hair follicles . Phospho-glycoprotein expression on the basal cells was restricted to the cell surface . Polymerase chain reaction analysis of first strand cDNA from keratinocytes identified the phospho-glycoproteins to be mdr1b . Using beta1 integrin expression and density gradient centrifugation we were able to enrich and identify the basal cell compartment by flow cytometric analysis and assay this subset of cells for phospho-glycoprotein activity . Basal cells loaded with rhodamine 123, a substrate for multidrug-resistant pumps, effluxed the molecule from the cells in a time-dependent manner . This study shows that basal layer keratinocytes express functional phospho-glycoproteins . We speculate that phospho-glycoproteins may play a role in regulating the level of environmental toxins and differentiation factors, as has been suggested for other progenitor cell compartments.

Proc Natl Acad Sci U S A, 2000 Jul 5, 97(14), 7681 - 6
Production of resistant HIV mutants during antiretroviral therapy; Ribeiro RM et al.; HIV drug therapy often fails because of the appearance of multidrug-resistant virus . There are two possible scenarios for the outgrowth of multidrug-resistant virus in response to therapy . Resistant virus may preexist at low frequencies in drug-naive patients and is rapidly selected in the presence of drugs . Alternatively, resistant virus is absent at the start of therapy but is generated by residual viral replication during therapy . Currently available experimental methods are generally too insensitive to distinguish between these two scenarios . Here we use deterministic and stochastic models to investigate the origin of multidrug resistance . We quantify the probabilities that resistant mutants preexist, and that resistant mutants are generated during therapy . The models suggest that under a wide range of conditions, treatment failure is most likely caused by the preexistence of resistant mutants.

Anticancer Drugs, 1999 Jul, 10(6), 511 - 8
Effect of cyclosporin A on protein binding of teniposide in cancer patients; Toffoli G et al.; We investigated the effect of cyclosporin A (CSA) on protein binding of teniposide (VM26) in 16 patients with metastatic renal cell carcinoma receiving i.v . VM26 alone over 24 h (total dose, 200 mg/m2) and in association with CSA (5 mg/kg/2 h followed by 30 mg/kg/48 h i.v.) . CSA was used in an attempt to overcome multidrug resistance . The unbound fraction (%fu) of VM26 was significantly (p=0.04) higher in the cycles with CSA (median 0.8; range 0.4-1.9) than in the cycles with VM26 alone (median 0.5; range 0.1-1.6) . Both total VM26 area under curve concentration (AUC0-infinity) and free VM26 AUC0-infinity increased after treatment with CSA, but the median increase in free AUC0-infinity was higher (2.7-fold) than total AUC0-infinity (1.5-fold) (p = 0.04) . Bilirubin was significantly (p<0.01) increased after CSA but no association was observed between bilirubin level and %fu of VM26 . Albumin was in the normal range after both VM26 alone and VM26 plus CSA . The nadir of absolute neutrophil count (ANC) after VM26 plus CSA (median 700/microl, range <100-2860/microl) was lower than after VM26 alone (median 1900/microl, range 200-6000/microl) (p = 0.0007) . The median percentage of ANC compared to the pretreatment value (ANC nadir/ANC pretreatment x 100) was 39.0% (range 3.1-98.8%) in the cycles with VM26 alone and 16.9% (range 1.4-97.9%) (p = 0.007) after VM26 plus CSA . Percentage change of neutrophils significantly correlated with free AUC0-infinity VM26 in the cycles with VM26 alone and VM26 plus CSA (p = 0.04, r = -0.53 and p = 0.04, r = -0.52, respectively) . Only a trend which failed to reach significance was observed between total AUC0-infinity VM26 and percentage change of neutrophils in the cycles with VM26 alone and in association with CSA (p = NS, r = -0.33 and p = 0.055, r = -0.49, respectively) . In conclusion, patients treated with CSA had higher systemic exposure to unbound VM26.

Infection, 1999, 27 Suppl 2, S55 - 8
Drug resistance in Plasmodium falciparum malaria; Warhurst DC; Four classes of drugs are reviewed: blood schizontocides acting only on the hemoglobin-digesting blood stages, the antifolates which attack tetrahydrofolate synthesis in all the growing stages, antimitochondrials affecting synthesis and electron transport, and 8-aminoquinolines which interfere with redox processes . Drug efflux via a multidrug resistance membrane protein, and the production of a protein competing with the drug for the target hemin are thought to be responsible for resistance to blood schizontocides . Structural changes in target enzymes are responsible for easily-developed resistance to antifolates and antimitochondrials . The judicious use of drug combinations can help to avoid development of resistance and combat resistant infections, but new drugs are urgently needed.

Naunyn Schmiedebergs Arch Pharmacol, 2000 Jun, 361(6), 654 - 64
Acidic dopamine metabolites are actively extruded from PC12 cells by a novel sulfonylurea-sensitive transporter; Lamensdorf I et al.; Incubation of PC 12 cells with the sulfonylurea drug, glipizide (1-100 microM), increased intracellular levels of the acidic metabolites of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) . The levels of these acids in the medium were decreased, indicating the presence of a sulfonylurea-sensitive organic anion transporter . In the present study, we demonstrate that the sulfonylurea-sensitive transport of acidic dopamine metabolites is unidirectional, ATP dependent, unaffected by ouabain or by tetrodotoxin and blocked by drugs that interact with the multidrug-resistance protein-1 (MRP1) . However, over-expression of MRP1 did not affect transport of the acid metabolites . The pharmacological profile and ion dependence of the transporter also differs from that of known ATP-binding cassette (ABC) family members . Using microdialysis, we also demonstrated a sulfonylurea-sensitive transport process in the striatum of freely moving rats . These results show that acidic dopamine metabolites are actively secreted from dopaminergic cells into surrounding extracellular fluid by a previously undescribed transporter.

J Hum Virol, 2000 May-Jun, 3(3), 150 - 6
Single- and multidrug resistance mutations to reverse transcriptase and protease inhibitors: human immunodeficiency virus type 1-infected patients from two geographical areas in Spain . Spanish Groups for Antiretroviral Resistance Studies; Perez-Alvarez L et al.; OBJECTIVES: To describe the prevalence of genotypic resistance mutations, including single and multidrug resistance (MDR) to reverse transcriptase (RT) and protease (PR) inhibitors in treated and untreated patients from two geographical areas in Spain (Madrid and Galicia) . STUDY DESIGN/METHODS: Resistance mutations to RT inhibitors were studied by line probe assay (LiPA) or by automated sequencing in 468 patients (Madrid, 268; Galicia, 200), and resistance mutations to PR inhibitors were studied by automated sequencing in 295 patients (Madrid, 85; Galicia, 210) . RESULTS: The proportion of resistance mutations in treated and untreated patients results were higher by the LiPA method than by sequencing . By sequencing, we detected resistance mutations to nucleoside analogue RT (NRT) inhibitors and NRT inhibitors plus nonnucleoside RT (NNRT) inhibitors in 35.4% and 17.2% of treated patients, respectively . We also detected MDR to zidovudine plus lamivudine in 13.9% of treated patients from Galicia, in 1.7% from Madrid (p < 0.001), and in 1.5% of untreated patients from Galicia . Also, we detected MDR to NRT inhibitors in 3.8% and to NNRT inhibitors in 9.1% . We found resistance mutations to PR inhibitors in 38.1% of treated patients and in 0.9% of untreated patients . CONCLUSIONS: These findings reinforce the usefulness of testing for resistance mutations in some cases to evaluate their prevalence in a given population and in the follow-up of treated patients.

J Med Liban, 2000 Jan-Feb, 48(1), 18 - 22
Comparative study of antituberculous drug resistance among Mycobacterium tuberculosis isolates recovered at the American University of Beirut Medical Center: 1996-1998 vs 1994-1995; Araj GF et al.; PURPOSE: To study the overall current prevalence of antituberculous drug resistance among M . tuberculosis isolates recovered at the American University of Beirut Medical Center (AUBMC) between 1996-1998 in comparison to those reported on isolates recovered in 1994-1995 . MATERIALS AND METHODS: Seventy-four consecutive M . tuberculosis isolates recovered from the same number of newly diagnosed cases of tuberculosis (TB), between January 1996 and December 1998 (referred to as 1998), were tested against isoniazid (INH), rifampicin (RIF), streptomycin (STM) and ethambutol (ETH), using the BACTEC-TB susceptibility procedure and system . The results were compared to those reported on the isolates recovered in 1994-1995 (referred to as 1995) . RESULTS: A comparison between the results obtained in 1998 vs 1995 showed the following, respectively: The male to female ratio was 3.1:1 vs 2:1 and the mean ages were almost similar in males, 33.4 vs 34.1 years but were slightly higher in females 38.2 vs 32.7 years . Children (< or = 15 yrs) represented 10.8% vs 8.3% of the study population . The prevalence of resistance, to one or more drugs, was almost the same, 24% vs 26% but the overall percentages of single drug resistance were generally higher in 1998 vs 1995 against all the tested drugs except INH: INH (20.2% vs 23.9%), RIF (16.2% vs 12.5%), STM (13.5% vs 7.3%) and ETH (8.1% vs 3%) . Among the resistant isolates, the profiles of resistance indicated decrease in resistance to one and two drugs, 6.7% vs 11.5% and 5.4% vs 10.4%, respectively, but showed increase in resistance to three and four drugs, 8.1% vs 2.1% and 4% vs 2.1%, respectively . Increase in resistance to two or more drugs was also observed, 17.6% vs 14.6%, and the prevalence of multidrug resistance, defined as resistance to at least both INH and RIF, was also increased, 14.7% vs 11.4% . CONCLUSIONS: This study shows a high prevalence and persistence of TB drug resistance tested in our Medical Center in Lebanon . In addition, the shift in the increase of resistance from one and two drugs to three and four drugs are very ominous and should be considered when treating patients in this country . Moreover, such information calls for scrutinizing the existing local TB control programs as part of the global efforts to minimize the incidence of this highly morbid infectious disease.

Cancer Gene Ther, 2000 Jun, 7(6), 893 - 900
Mdr1 promoter-driven tumor necrosis factor-alpha expression for a chemotherapy-controllable combined in vivo gene therapy and chemotherapy of tumors; Walther W et al.; Cancer gene therapy approaches are often designed as single-agent treatments; however, greater therapeutic effect might be obtained if combined with an established conventional treatment regimen such as chemotherapy . In this context, conditional promoters are useful tools, because they may be induced by therapeutic modalities . The human multidrug resistance gene (mdr1) promoter is inducible by cytostatic drugs and can be employed for the chemotherapy-regulated expression of therapeutic genes . In this in vivo study, the human mdr1 promoter fragment (-207 to +158) was used for drug-inducible expression of human tumor necrosis factor-alpha (TNF-alpha) in the vector construct pM3mdr-p-hTNF . The single doxorubicin and vincristine treatment of nude mice xenografted with pM3mdr-p-hTNF-transduced MCF-7 mammary tumors resulted in drug-induced and time-dependent elevation of intratumoral TNF-alpha expression at the mRNA and protein level . The highest drug induction was achieved at 2 days after drug application, as reflected by a maximum 25-fold increase in TNF-alpha secretion in the tumor . This drug-induced TNF-alpha expression is more effective in inhibiting tumor growth compared with the growth of tumors transduced with constitutively TNF-alpha-expressing vectors in combination with chemotherapy.

J Physiol Biochem, 2000 Mar, 56(1), 33 - 8
Multidrug resistance increment in a human colon carcinoma cell line by colchicine; Ruiz Gomez MJ et al.; The most important mechanism in drug resistance is the multidrug resistance (MDR) phenomenon . It is possible to select MDR cells by in vitro exposure to cytotoxic agents . The resistance is due to the hyperexpression of the P-glycoprotein (P-Gp) that take drugs out from the cells . In this study, a colchicine resistant subline (HCA-2/1cch) was selected from a human colon adenocarcinoma after a short period of drug exposure, as an in vitro model of drug resistance selection . These cells showed cross-resistance to other drugs, which were not present in the medium during selection . The relative resistance was 3.32 for colchicine, 3.15 for vinblastine, 2.62 for vincristine and 5.22 for mitomycin C . P-glycoprotein levels were assayed by flow cytometry . It was found that a significant increase of 2.35 and 1.59 had occurred in the peak and mean channel of fluorescence, respectively, indicating an increment of P-glycoprotein expression in relation to the parental line . Moreover, verapamil (10 microg/ml) produced a partial reversion of multidrug resistance . The sensitisation rates were 7.41 for colchicine, 1.25 for vinblastine, 2.36 for vincristine and 1.17 for mitomycin C . The data obtained suggest that colchicine exposure period (10 weeks) and dose (0.5 microg/ml) assayed were sufficient to produce an increment in multidrug resistance . This resistance could be due to higher level of P-Gp expression.

Nature, 2000 Jun 22, 405(6789), 962 - 6
A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis; Stover CK et al.; Mycobacterium tuberculosis, which causes tuberculosis, is the greatest single infectious cause of mortality worldwide, killing roughly two million people annually . Estimates indicate that one-third of the world population is infected with latent M . tuberculosis . The synergy between tuberculosis and the AIDS epidemic, and the surge of multidrug-resistant clinical isolates of M . tuberculosis have reaffirmed tuberculosis as a primary public health threat . However, new antitubercular drugs with new mechanisms of action have not been developed in over thirty years . Here we report a series of compounds containing a nitroimidazopyran nucleus that possess antitubercular activity . After activation by a mechanism dependent on M . tuberculosis F420 cofactor, nitroimidazopyrans inhibited the synthesis of protein and cell wall lipid . In contrast to current antitubercular drugs, nitroimidazopyrans exhibited bactericidal activity against both replicating and static M . tuberculosis . Lead compound PA-824 showed potent bactericidal activity against multidrugresistant M . tuberculosis and promising oral activity in animal infection models . We conclude that nitroimidazopyrans offer the practical qualities of a small molecule with the potential for the treatment of tuberculosis.

J Clin Microbiol, 2000 Jul, 38(7), 2563 - 7
Stability of IS6110 restriction fragment length polymorphism patterns of Mycobacterium tuberculosis strains in actual chains of transmission; Niemann S et al.; The stability of IS6110 restriction fragment length polymorphism (RFLP) patterns of Mycobacterium tuberculosis strains in actual transmission chains has been assessed by analyzing the variability of IS6110 RFLP patterns of strains in fingerprint clusters that have been confirmed by classical epidemiological data . Forty susceptible and 35 drug-resistant (including 17 multidrug-resistant) M . tuberculosis strains obtained from 75 patients living in Germany have been analyzed . The epidemiological relationship among strains within the fingerprint clusters has been verified by family contacts (14 clusters) or by contact tracing of the public health offices (7 clusters) . The time spans between the first and the last isolate of one cluster ranged from less than 1 to 29 months . Of the 75 strains only 1 showed a one-band variation when compared to the other nine isolates grouped in the same cluster, corresponding with a rate of change of approximately 1.9% per possible transmission (one index patient per cluster was subtracted from the total number of isolates) . These results confirm a high degree of stability of IS6110 RFLP patterns of transmitted M . tuberculosis strains . Furthermore, the data presented indicate that isolates with identical IS6110 DNA fingerprint patterns are a good indicator for the rate of recent transmission in a study population.

Jpn J Cancer Res, 2000 Jun, 91(6), 638 - 42
Multidrug resistance reversal activity of taxoids from Taxus cuspidata in KB-C2 and 2780AD cells; Kobayashi J et al.; Some non-taxol-type taxoids having neither an oxetane ring at C-4 and C-5 nor an N-acylphenyl-isoserine group at C-13, such as taxuspine C, 2'-desacetoxyaustrospicatine, and 2-desacetoxytaxinine J, which were isolated from the Japanese yew Taxus cuspidata, increased cellular accu-mulation of vincristine (VCR) in multidrug-resistant 2780AD cells as potently as verapamil, and efficiently inhibited {(3)H}azidopine photolabeling of P-glycoprotein (P-gp) . Taxuspine C, 2'-desacetoxyaustrospicatine, and 2-desacetoxytaxinine J at 10 microM completely reversed the resistance to colchicine, VCR, and taxol in KB-C2 cells, which overexpress P-gp, while taxinine and taxinine M showed no effect . Taxuspine C, 2'-desacetoxyaustrospicatine, and 2-desacetoxytaxinine J may be candidate pharmaceuticals for reversing multidrug resistance (MDR) and also may be good modifiers of MDR in cancer chemotherapy.

Biochem Biophys Res Commun, 2000 Jun 24, 273(1), 333 - 41
Induction of cytochrome P450 (CYP)1A1, CYP1A2, and CYP3A4 but not of CYP2C9, CYP2C19, multidrug resistance (MDR-1) and multidrug resistance associated protein (MRP-1) by prototypical inducers in human hepatocytes; Runge D et al.; Human hepatocytes cultured serum-free for up to 6 weeks were used to study expression and induction of enzymes and membrane transport proteins involved in drug metabolism . Phase I drug metabolizing enzymes cytochrome P450 (CYP)1A1, CYP1A2, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 were detected by Western blot analyses and, when appropriate, by enzymatic assays for ethoxyresorufin-O-deethylase(EROD)-activity and testosterone-6beta-hydroxylase(T6H)-activity . Expression of the membrane transporter multi-drug resistance protein (P-glycoprotein, MDR-1), multidrug resistance-associated protein (MRP-1), and lung-resistance protein (LRP) was maintained during the culture as detected by RT-PCR and Western blot analyses . Model inducers like rifampicin, phenobarbital, or 3-methylcholanthrene and beta-naphtoflavone were able to induce CYP1A or CYP3A4 as well as EROD or T6H activities for up to 30 days . CYP2C9, CYP2C19 and CYP2E1 expression was maintained but not inducible for 48 days . Also, rifampicin and phenobarbital were unable to increase MDR-1 and MRP-1 protein levels significantly .

Clin Cancer Res, 2000 Jun, 6(6), 2456 - 63
Calpain inhibitor II induces caspase-dependent apoptosis in human acute lymphoblastic leukemia and non-Hodgkin's lymphoma cells as well as some solid tumor cells; Zhu DM et al.; Calpain is a calcium-dependent cysteine protease that is implicated in calcium-dependent cell death, and calpain inhibitors are generally considered as inhibitors of apoptosis . To the contrary, in the present study, we found that calpain inhibitor II (CPI-2) triggers rapid apoptosis in acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL) cells . All target cell lines were killed by CPI-2, including: ALL-1, a multidrug-resistant BCR-ABL fusion transcript-positive t(9;22) pro-B ALL cell line; RS4;11, a highly radiation-resistant MLL-AF4 fusion transcript-positive t(4;11) pre-pre B ALL cell line; RAMOS, a highly radiation-resistant and p53-deficient Burkitt's lymphoma cell line; DAUDI, a Burkitt's leukemia/lymphoma cell line; NALM-6, a pre-B ALL cell line; and JURKAT and MOLT-3, two T-lineage ALL/NHL cell lines . CPI-2-induced apoptosis in LYN-deficient and BTK-deficient subclones of the DT-40 lymphoma B cell line as effectively as it did in wild-type DT-40 cells . Thus, CPI-2-induced apoptosis is not dependent on the protein tyrosine kinases LYN or BTK . Notably, caspase inhibitor I effectively inhibited CPI-2-induced apoptosis, suggesting that the inhibition of a CPI-2-susceptible protease results in caspase activation, leading to apoptosis in ALL/NHL cells . Unlike the high calpain-expressing ALL/NHL cell lines, myeloid leukemia cell lines HL-60/AML, K562/CML, and U937/AMML, or solid tumor cell lines BT-20/breast cancer, PC-3/prostate cancer, U373/glioblastoma, and HeLa/epitheloid cancer, were not susceptible to the cytotoxicity of CPI-2 . Taken together, our results identify calpain as a new molecular target for the treatment of ALL and NHL . CPI-2 and its analogues represent a promising new class of antileukemia/lymphoma agents that deserves further development.

Clin Cancer Res, 2000 Jun, 6(6), 2401 - 7
Increased expression of an ATP-binding cassette superfamily transporter, multidrug resistance protein 2, in human colorectal carcinomas; Hinoshita E et al.; The expression of ATP-binding cassette superfamily transporter genes, such as P-glycoprotein/multidrug resistance (MDR) 1 and MDR protein (MRP) 1, is often up-regulated in various tumor types and is involved in responses to some anticancer chemotherapeutic agents . Five human MRP subfamily members (MRP2-6) with structural similarities to MRP1 have been identified . The relationships between MRP2-6 mRNA levels and drug resistance are not well understood . Data on 45 patients with colorectal cancer were analyzed . Of the ATP-binding cassette superfamily genes, we asked whether mRNA levels of MDR1, MRP1, MRP2, and MRP3 correlated with drug resistance to anticancer agents . For this analysis, we used quantitative reverse transcription-PCR, and the sensitivity to anticancer agents in surgically resected colon carcinomas was determined using the in vitro succinate dehydrogenase inhibition test . MDR1, MRP1, and MRP3 were highly expressed in normal colorectal mucosa, and the relative mRNA levels of MDR1, MRP1, and MRP3 in cancerous tissues compared with noncancerous tissues were decreased or unchanged . By contrast, MRP2 mRNA expression was low in normal colorectal mucosa and specifically increased in cancer regions compared with noncancerous regions . Of the anticancer agents prescribed for patients with colorectal cancers, including doxorubicin, mitomycin C, cisplatin, 5-fluorouracil, etoposide, and a camptothecin derivative, mRNA expression of MRP2 was significantly associated with resistance to cisplatin . MRP2 may be important for resistance to cisplatin treatment in colorectal cancer.

Mol Biotechnol, 2000 Feb, 14(2), 165 - 72
High-copy cDNA amplification of minimal total RNA quantities for gene expression analyses; Schwabe H et al.; This protocol describes a PCR-based cDNA amplification technique of small total RNA quantities, optimized for determination and verification of gene expression variations in cells or tissue specimen . A proportional amplification of rare and abundant transcripts is thereby achieved by initial random hexamer-primed reverse transcription of total RNA . Compared to established oligo(dT)-primed techniques, this approach generates shorter than full length copies of long RNAs which leads to a normalized cDNA pool for a more adequate PCR-amplification . Subsequent double oligo(dA) tailing of the synthesized total cDNA strands and the utilization of heteropolymeric primers allow a highly specific, up to 500-fold PCR-amplification of the total cellular RNA amount . Thus, obstacles in availability of RNA from limited sources, such as human biopsies or microdissected histological sections, can be overcome . The amplified total cDNA (atcDNA) is shown to be applicable for confirmation of differential gene expression, as demonstrated in this protocol by expression analysis of the multidrug resistance-associated genes mdr1, mrp1 and lrp, using human cell lines as well as microdissected human tissue sections.

Mol Biotechnol, 2000 Feb, 14(2), 131 - 45
Detection of recombinant P-glycoprotein in multidrug resistant cultured cells; Germann UA; The MDR1 multidrug resistance gene encodes a high molecular weight membrane-spanning cell surface protein, P-glycoprotein, that confers multidrug resistance by pumping various cytotoxic drugs, including vinblastine, doxorubicin or paclitaxel, out of cells . Overexpression of P-glycoprotein in human tumors has been recognized as a major obstacle for successful chemotherapy of cancer . Thus, P-glycoprotein represents an important drug target for pharmacological chemosensitizers . Initially, cell culture models to study the multidrug resistance phenotype were established by selecting drug-sensitive cells in step-wise increasing, sublethal concentrations of chemotherapy agents . P-glycoprotein was found to be overexpressed in many of these models . Multidrug resistant cells can also be generated by transfection of cultured cells with the MDR1 gene, followed by selection with cytotoxic drug at a concentration that kills all untransfected host cells . Transfectants expressing wild-type or mutant recombinant P-glycoprotein have significantly contributed to our understanding of the structure of P-glycoprotein and its molecular and cellular functions . Additionally, the MDR1 gene has also been used as a selectable marker for the transfer and coexpression of non-selectable genes . This article details means for detection of P-glycoprotein in DNA-transfected or retrovirally transduced, cultured cells . Different experimental approaches are described that make use of specific antibodies for detection of P-glycoprotein . Strategies to visualize P-glycoprotein include metabolic labeling using 35S-methionine, labeling with a radioactive photoaffinity analog, and non-radioactive immunostaining after Western blotting.

J Pharmacol Exp Ther, 2000 Jul, 294(1), 387 - 95
Detoxication of vinca alkaloids by human P450 CYP3A4-mediated metabolism: implications for the development of drug resistance; Yao D et al.; Vinca alkaloids are important chemotherapeutic agents, and their pharmacokinetic properties display significant interindividual variations, possibly due to CYP3A4-mediated metabolism . We have evaluated the relevance of this metabolism for the chemotherapeutic and the toxicological properties of these drugs . Analysis was performed using Chinese hamster ovary cell lines that expressed either CYP2D6 or CYP3A4 . The latter cells metabolized vinblastine with a turnover number of 0.4 min(-1), resulting in a decreased cytotoxicity of this compound . Whereas vincristine and vinblastine at a concentration of 100 nM killed more than 90% of the parental cells, more than 50 and 35%, respectively, of cells that coexpressed CYP3A4 and cytochrome P450 (P450) reductase survived these treatments . No additional increase in cytotoxicity was noted above 100 nM . Similarly, preincubation of vinblastine with bacterial membranes that contained recombinant CYP3A4 and P450 reductase decreased the cytotoxicity of vinblastine for parental Chinese hamster ovary cells . We also demonstrate that the presence of vinblastine in a coculture of cells that expressed beta-galactosidase together with cells that expressed CYP3A4 strongly selected for the latter cells, resulting in an increased level of CYP3A4 in the surviving cell population . Similarly, treatment of the human colon adenocarcinoma cell line LS174T with vinblastine selected for a cell population with higher levels of endogenous CYP3A4 as revealed by immunohistochemistry without simultaneous increase of multidrug resistance protein 1 (MDR1) . This is the first evidence that tumor P450s have the potential to contribute to the development of drug resistance during chemotherapy.

J Cancer Res Clin Oncol, 2000 Jun, 126(6), 311 - 9
Cytotoxicity, cell-cycle perturbations and apoptosis in human tumor cells by lipophilic N4-alkyl-1-beta-D-arabinofuranosylcytosine derivatives and the new heteronucleoside phosphate dimer arabinocytidylyl-(5'-->5')-N4-octadecyl-1-beta-D-arabinofuranosylcytosi ne; Horber DH et al.; The arabinofuranosylcytosine (AraC) derivative N4-octadecyl-1-beta-D-arabinofuranosylcytosine (NOAC) and its (5'-->5')-heterodinucleoside phosphate analog NOAC-AraC were compared with AraC for cytotoxicity, cell-cycle dependence, phosphorylation by deoxycytidine (dC) kinase and apoptosis induction in native, AraC- or NOAC-resistant HL-60 cells . NOAC was cytotoxic in all cells with three to seven-fold lower IC50 concentrations than those of NOAC-AraC or AraC . In contrast to NOAC-AraC, the lipophilic monomer NOAC overcame AraC resistance, inducing apoptosis in more than 80% of native and AraC-resistant HL-60 cells . This suggests that NOAC-AraC may be cleaved intracellularly only at very slow rates to AraC and NOAC or to the 5'-monophosphates, whereas NOAC exerts different mechanisms of action from AraC . In vitro the dimer was cleaved by phosphodiesterase or human serum to NOAC, AraC and AraC monophosphate . In contrast to AraC, N4-alkylated AraC derivatives with alkyl chains ranging from 6-18 C atoms were not substrates for dC kinase . Furthermore, treatment of the multidrug-resistant cell lines KB-ChR-8-5 and KB-V1 with the N4-hexadecyl-AraC derivative NHAC did not induce P-170 glycoprotein expression, suggesting that the N4-alkyl-AraC derivatives are able to circumvent MDR1 multidrug resistance . The in vivo activity of liposomal NOAC in a human acute lymphatic leukemia xenograft model confirmed the antitumor activity of this representative of the N4-alkyl-arabinofuranosylcytosines.

J Formos Med Assoc, 2000 May, 99(5), 408 - 11
In vitro activity of rifabutin and rifampin against clinical isolates of Mycobacterium tuberculosis in Taiwan; Chien HP et al.; BACKGROUND AND PURPOSE: To determine the in vitro activity of rifabutin against Mycobacterium tuberculosis (MTB) and the cross-resistance rate between rifampin and rifabutin . METHODS: A total of 56 clinical isolates of MTB, including 23 multidrug-resistant (MDR) isolates and 33 susceptible isolates, were tested for susceptibility to rifampin and rifabutin using the absolute concentration method . The concentrations of drugs tested were 2.5 and 5 mg/mL for rifampin and 0.1, 0.5, 1, 2.5, 5, and 10 mg/mL for rifabutin . RESULTS: All 33 MTB isolates that were susceptible to rifampin were also susceptible to rifabutin . None of the 23 MDR-MTB isolates were inhibited by rifabutin at a concentration of 0.1 mg/mL . Among these 23 MDR isolates, three were susceptible to rifabutin at concentrations > or = 0.5 mg/mL, six were susceptible to rifabutin at concentrations > or = 5 mg/mL, 18 were susceptible to rifabutin at concentrations > or = 10 mg/mL and five were not inhibited at any of the concentrations tested . The cross-resistance rate between rifampin and rifabutin was 87% . CONCLUSIONS: Our results indicate that the in vitro activity of rifabutin against drug-susceptible MTB isolates is greater than that of rifampin . For MDR-MTB isolates, the cross-resistance is high between rifampin and rifabutin.

J Formos Med Assoc, 2000 Apr, 99(4), 311 - 6
Verapamil modulation of multidrug resistance in renal cell carcinoma; Yu DS et al.; BACKGROUND AND PURPOSE: Renal cell carcinoma (RCC) is well known for its chemoresistance . The membranous p-glycoprotein (gp-170) is believed to be highly correlated with multidrug resistance (MDR) of cancer cells with energy-dependent pumping efflux of anticancer drugs . Verapamil, a calcium antagonist, inhibits the efflux function of gp-170 and cytoskeletal transportation . The aim of this study was to determine the effect of verapamil on gp-170 expression and intracellular drug accumulation in RCC tumor cells and the modulation of cytotoxicity of various chemotherapeutic drugs on native RCC cell lines and acquired MDR sublines by verapamil . METHODS: Using cultured cell lines of RCC and their MDR sublines as target cells, the effect of verapamil on gp-170 expression was analyzed by immunofluorescence flow cytometry . The influence of verapamil on intracellular drug accumulation in RCC tumor cells was measured by autofluorescence flow cytometry . The modulation of verapamil on cytotoxicity of various chemotherapeutic drugs on native RCC cell lines and acquired MDR sublines was analyzed by the methyl tetrazolium method . RESULTS: From flow cytometric measurement, the expression of gp-170 was significantly decreased in A704 and Caki-1 tumor cells after verapamil treatment . The uptake of adriamycin and maintenance of intracellular drugs were also significantly increased following verapamil treatment in RCC8701 tumor cells . These effects were sustained for as long as 8 hours after verapamil withdrawal . The cytotoxicity of adriamycin and epirubicin on RCC8701 and its MDR subline tumor cells was markedly intensified by verapamil . The verapamil modulation of cytotoxicity was in an immediate-reaction pattern and was dose-dependent, with synergistic effects . Long-term treatment was more effective than short-term treatment in RCC MDR sublines . The cytotoxicity of vinca alkaloid (vinblastine) and alkylators (carboplatin) was also enhanced by verapamil . CONCLUSIONS: These results suggest that verapamil plays an important role in the circumvention of native and acquired chemoresistance of RRC because it suppresses membranous gp-170 expression and cytoplasmic drug transportation.

J Pharmacol Exp Ther, 2000 Jun, 293(3), 717 - 23
Expression and localization of multidrug resistant protein mrp2 in rat small intestine; Mottino AD et al.; The expression of multidrug resistance-associated protein isoform 2 (mrp2), the ATP-dependent export pump that mediates the transport of glucuronic acid-, glutathione-, and sulfate-conjugated derivatives, was studied in rat small intestine . The small intestine was divided into nine equal segments, and mrp2 content was analyzed in homogenate and brush border membrane preparations by Western analysis . mrp2 protein was present mainly in brush border membrane of the proximal segments and gradually decreased from jejunum to the distal ileum . We also analyzed the content of mrp2 in three different populations of proximal enterocytes obtained from the upper and lower villus and the crypt regions . The export pump was mainly expressed in the villus cells and to a lesser degree in the crypt cells of the epithelium . Immunohistochemical analysis performed in duodenum, jejunum, and ileum confirmed in situ the Western blot findings . Analysis of mRNA encoding mrp2 in proximal and distal segments revealed a similar content in both regions, whereas distribution along the villus-crypt axis was similar to the protein gradient . Because conjugating enzymes are distributed similarly to mrp2, we conclude that they may act coordinately to contribute to first-pass metabolism of drugs and other xenobiotics in the proximal small intestine.

Hepatology, 2000 Jul, 32(1), 66 - 72
Mrp2 is essential for estradiol-17beta(beta-D-glucuronide)-induced cholestasis in rats; Huang L et al.; The present study evaluates the roles of the multidrug resistance-1 P-glycoprotein, Mdr1a/1b, the bile salt export pump (Bsep), and the multidrug resistance-associated protein-2 (Mrp2) in mediating cholestasis induced by estradiol-17beta(beta-D-glucuronide) (E(2)17G) . Administration of inverted question mark(3)HE(2)17G (18 nmol/g body weight) gave a similar degree of cholestasis and biliary excretion of E(2)17G-equivalents in wild-type and Mdr1a(-/-)/1b(-/-) mice . When expressed in Sf9 cells, Bsep-mediated adenosine triphosphate (ATP)-dependent transport of taurocholate (TC, 1 micromol/L) in membrane vesicles was 110% +/- 12.5% and 108% +/- 17.3% of control in the presence of 10 and 50 micromol/L E(2)17G, respectively, whereas in rat canalicular membrane, both E(2)17G and the choleretic estradiol-3-beta-D-glucuronide (E(2)3G) inhibited ATP-dependent transport of TC to the same extent . Infusion of inverted question mark(3)HE(2)17G (24 micromol) did not induce cholestasis in Mrp2-deficient TR(-) rats whereas 2 micromol of inverted question mark(3)HE(2)17G inhibited bile flow by 51% in control Wistar rats . The maximal biliary concentration of E(2)17G was 3.5 and 2.5 mmol/L in control and TR(-) rats, respectively . However, 2.2 mmol/L of E(2)17G in bile is associated with inhibition of bile flow in control rats . These data show that (1) Mdr1a/1b are not essential for E(2)17G-mediated cholestasis, (2) direct inhibition of Bsep-mediated bile acid transport is not the mechanism for E(2)17G cholestasis, and (3) accumulation of E(2)17G in bile alone is not sufficient to induce cholestasis . These data indicate that the process of Mrp2-mediated transport of high concentrations of E(2)17G is essential for its induction of cholestasis.

Biochemistry, 2000 Jul 4, 39(26), 7651 - 61
Effects of cholesterol and enantiomeric cholesterol on P-glycoprotein localization and function in low-density membrane domains; Luker GD et al.; Multidrug resistance P-glycoprotein (Pgp) has been reported to localize in low-density, cholesterol-enriched membranes . However, effects of low-density membrane domains on function of Pgp remain unexplored in whole cell systems . In cells that express modest levels of the protein endogenously or through drug selection, Pgp predominantly localized to low-density membranes following separation on a sucrose gradient . When highly overexpressed in NIH 3T3 cells, a prominent amount of Pgp also was detected in high-density membranes . Removing cholesterol from cells with beta-methylcyclodextrin (CD), a sterol acceptor molecule, shifted fractions that contained Pgp from low toward high density, and this effect was reversed to a similar extent by restoring sterols with either cholesterol or enantiomeric cholesterol . However, function of human MDR1 Pgp as probed with Tc-Sestamibi, a transport substrate for Pgp, was not dependent on localization of Pgp in cholesterol-enriched membranes . Specific inhibition of MDR1 Pgp with GF120918 or LY335979 also was independent of cholesterol . Cell-type-specific effects of cholesterol content on function of human Pgp were detected by use of daunomycin, another substrate for Pgp, although efficacy of inhibitors remained independent of cholesterol . Conversely, both function and inhibition of hamster Pgp as measured with Tc-Sestamibi and daunomycin were in part dependent on normal cell content of cholesterol . These data show that Pgp preferentially localizes to low-density, cholesterol-enriched membrane domains, but acute depletion of cholesterol impacts Pgp-mediated drug transport in a substrate- and cell-type-specific manner.

J Cell Physiol, 2000 Aug, 184(2), 263 - 74
Lysosomal accumulation of drugs in drug-sensitive MES-SA but not multidrug-resistant MES-SA/Dx5 uterine sarcoma cells; Wang E et al.; Sequestration of drugs in intracellular vesicles has been associated with multidrug-resistance (MDR), but it is not clear why vesicular drug accumulation, which depends upon intracellular pH gradients, should be associated with MDR . Using a human uterine sarcoma cell line (MES-SA) and a doxorubicin (DOX)-resistant variant cell line (Dx-5), which expresses p-glycoprotein (PGP), we have addressed the relationship between multidrug resistance, vesicular acidification, and vesicular drug accumulation . Consistent with a pH-dependent mechanism of vesicular drug accumulation, studies of living cells vitally labeled with multiple probes indicate that DOX and daunorubicin (DNR) predominately accumulate in lysosomes, whose lumenal pH was measured at < 4.5, but are not detected in endosomes, whose pH was measured at 5.9 . However, vesicular DOX accumulation is more pronounced in the drug-sensitive MES-SA cells and minimal in Dx5 cells even when cellular levels of DOX are increased by verapamil treatment . While lysosomal accumulation of DOX correlated well with pharmacologically induced differences in lysosome pH in MES-SA cells, lysosomal accumulation was minimal in Dx5 cells regardless of lysosomal pH . We found no differences in the pH of either endosomes or lysosomes between MES-SA and Dx5 cells, suggesting that, in contrast to other MDR cell systems, the drug-resistant Dx5 cells are refractory to pH-dependent vesicular drug accumulation . These studies demonstrate that altered endomembrane pH regulation is not a necessary consequence of cell transformation, and that vesicular sequestration of drugs is not a necessary characteristic of MDR .

Int J Tuberc Lung Dis, 2000 Jun, 4(6), 544 - 9
Treatment monitoring and prevalence of drug resistance in tuberculosis patients in Tehran; Bahrmand AR et al.; SETTING: Health care clinics and private practitioners in Tehran . OBJECTIVE: To analyse rates of drug resistance and response to treatment in tuberculosis patients . DESIGN: A prospective study of 257 patients undergoing treatment for whom data were collected on drug susceptibility testing and outcome as well as age, sex and history of treatment . RESULTS: Of 774 initially diagnosed patients, 380 were female and 394 were male; 520 (67%) of the cases had pulmonary disease . The overall rate of primary drug resistance among Mycobacterium tuberculosis isolates resistant to at least one drug was 87/563 (15.5%) . Twenty-three patients were multidrug-resistant . Among 215 patients with drug-susceptible cultures recruited for follow-up, rapid response to short-course chemotherapy was observed in 190 (88%) who were successively both smear and culture negative after 2 and 4 months of treatment . After 6 months of treatment, 12 of the 25 patients with slow response to treatment had positive cultures; one was smear-positive . Of the 42 patients with drug-resistant isolates, satisfactory bacteriological response was observed after 6 months of treatment in 30 (71%) . CONCLUSIONS: These observations support regional recommendations for short-course treatment regimens . Culture rather than smear result could be a key parameter for individually guiding the duration of treatment in patients with poor response to treatment.

Int J Tuberc Lung Dis, 2000 Jun, 4(6), 537 - 43
Human immunodeficiency virus-related tuberculosis and primary drug resistance in Bangkok, Thailand; Punnotok J et al.; SETTING: Central Chest Hospital, a 500-bed referral hospital near Bangkok with a large out-patient department . OBJECTIVES: To determine human immunodeficiency virus (HIV) seroprevalence among patients with pulmonary tuberculosis (TB), and compare HIV-positive and HIV-negative TB patients . DESIGN: From July 1995 through June 1996, a cross-sectional study was conducted of newly registered adults (> or =16 years old) with suspected pulmonary TB . RESULTS: Of 2587 newly registered patients with suspected pulmonary TB, 2019 (78%) received HIV pretest counseling and 1816 (90%) consented to testing . Of these, 364 (20%) were HIV-seropositive . Among 1091 patients with bacteriologically confirmed TB, HIV seroprevalence was 22% . HIV-positive patients were more likely to be young, unemployed, single men and to have a history of injection drug use . HIV-positive patients with first-episode TB were more likely to have Mycobacterium tuberculosis strains resistant to isoniazid (10.9% vs 3.5%; P < 0.001), rifampicin (9.4% vs 2.9%; P < 0.001), and at least isoniazid and rifampicin (multidrug-resistant TB {MDR-TB}; 5.2% vs 0.4%; P < 0.001) . CONCLUSIONS: HIV prevalence is high among TB patients at this Bangkok hospital and is associated with drug resistance, including a 12 times higher risk of MDR-TB . These findings underscore the urgent need to assure adherence to complete, effective TB treatment regimens for all patients, including persons who are potentially difficult to manage such as injection drug users.

Int J Tuberc Lung Dis, 2000 Jun, 4(6), 504 - 12
Twenty-five years of tuberculosis in a French university hospital: a laboratory perspective; Robert J et al.; OBJECTIVE: To determine the impact of recent changes in the epidemiology of tuberculosis in France and other industrialised countries on the primary trends of tuberculosis case rates in a French university hospital . DESIGN: Descriptive study of all 4549 culture-positive tuberculosis cases hospitalised at Pitie-Salpetriere Hospital between 1972 and 1996 . RESULTS: From 1972, there was a decline of 5% per year in the tuberculosis case rate, which levelled off in 1983 . The proportion of tuberculosis patients who were human immunodeficiency virus (HIV) positive increased from 2% in 1983 to 28% in 1990, and thereafter remained stable . The proportion of foreign-born tuberculosis patients also increased, from 40% in 1972 to 55% in 1985 . These two changes affected drug resistance patterns . Drug resistance was more common among foreign-born than among French-born patients, whether previously treated or not . Resistance to rifampicin and multidrug resistance among previously untreated patients was highly related to HIV co-infection . Extrapulmonary sites of tuberculosis were more often smear-positive in HIV-positive than in non-HIV-positive patients (22.8% vs 12.6%), and bacteraemia was diagnosed almost exclusively in HIV-positive patients . CONCLUSION: The changes in clinical and bacteriological tuberculosis patterns at the hospital level over the last 25 years have paralleled those observed at national and international level in industrialised countries, including a slowing in the decrease in the case rate, due in part to the HIV epidemic, a higher proportion of foreign-born patients and an increase in drug resistance.

RNA, 2000 Jun, 6(6), 890 - 900
RNA location and modeling of a WD40 repeat domain within the vault; Kong LB et al.; The vault complex is a ubiquitous 13-MDa ribonucleoprotein assembly, composed of three proteins (TEP1, 240 kDa; VPARP, 193 kDa; and MVP, 100 kDa) that are highly conserved in eukaryotes and an untranslated RNA (vRNA) . The vault has been shown to affect multidrug resistance in cancer cells, and one particular component, MVP, is thought to play a role in the transport of drug from the nucleus . To locate the position of the vRNA, vaults were treated with RNases, and cryo-electron microscopy (cryo-EM) was performed on the resulting complexes . Using single-particle reconstruction techniques, 3,476 particle images were combined to generate a 22-A-resolution structure . Difference mapping between the RNase-treated vault and the previously calculated intact vault reconstructions reveals the vRNA to be at the ends of the vault caps . In this position, the vRNA may interact with both the interior and exterior environments of the vault . The finding of a 16-fold density ring at the top of the cap has allowed modeling of the WD40 repeat domain of the vault TEP1 protein within the cryo-EM vault density . Both stoichiometric considerations and the finding of higher resolution for the computationally selected and refined "barrel only" images indicate a possible symmetry mismatch between the barrel and the caps . The molecular architecture of the complex is emerging, with 96 copies of MVP composing the eightfold symmetric barrel, and the vRNA together with one copy of TEP1 and four predicted copies of VPARP comprising each cap.

Int J Pediatr Otorhinolaryngol, 2000 Jun 9, 53(1), 67 - 71
Suspected foreign body aspiration in a child with endobronchial tuberculosis; Park AH et al.; Endobronchial tuberculosis is a form of pulmonary tuberculosis, thought to result from rupture of an infected node through the bronchial wall or from lymphatic spread to the mucosal surface of the bronchial tree . With the presence of multidrug resistant isolates of TB, and its incidence in an increasing number of foreign-born persons immigrating to the US, otolaryngologists must be aware of its often subtle presentation . The following case is an unusual presentation of endobronchial tuberculosis initially diagnosed as an airway foreign body.

Int J Cancer, 2000 Jul 15, 87(2), 172 - 8
Differential expression of sphingolipids in MRP1 overexpressing HT29 cells; Kok JW et al.; We have obtained a novel multidrug resistant cell line, derived from HT29 G(+) human colon carcinoma cells, by selection with gradually increasing concentrations of the anti-mitotic, microtubule-disrupting agent colchicine . This HT29(col) cell line displayed a 25-fold increase in colchicine resistance and exhibited cross-resistance to doxorubicin, VP16, vincristine and taxol . Immunoblotting, combined with RT-PCR showed that the multidrug resistance phenotype was conferred by specific overexpression of the multidrug resistance protein 1 . Confocal scanning laser microscopy revealed that multidrug resistance protein 1 specifically localized in the plasma membrane of HT29(col) cells . In a functional assay, using the fluorescent multidrug resistance protein 1 substrate 5-carboxyfluorescein, an increased efflux activity of HT29(col) cells was measured, as compared to the wild-type HT29 G(+) cells . MK571, a specific inhibitor of multidrug resistance protein 1, blocked the 5-carboxyfluorescein efflux, but only partially reversed resistance to colchicine, indicating that additional multidrug resistance mechanisms operate in HT29(col) cells . In conclusion, these results show for the first time overexpression of a functional multidrug resistance protein 1 under colchicine pressure, indicating that colchicine is not a P-glycoprotein-specific substrate . Colchicine-induced overexpression of multidrug resistance protein 1 is accompanied by a changed sphingolipid composition, i.e., enhanced levels of glucosylceramide and galactosylceramide . In addition, ceramide, a lipid messenger molecule involved in apoptosis-related signal transduction processes, was much more abundant in HT29(col) cells, which is indicative of a stress response .

Mol Pharmacol, 2000 Jul, 58(1), 167 - 74
Cellular response to a glutathione S-transferase P1-1 activated prodrug; Rosario LA et al.; TER286 {gamma-glutamyl-alpha-amino-beta(2-ethyl-N,N,N', N'-tetrakis(2-chloroethyl)phosphorodiamidate)-sulfonyl-propionyl-( R)- (-) phenylglycine} is a novel nitrogen mustard prodrug that is preferentially activated by glutathione S-transferase P1-1 (GSTP1-1) . A human promyelocytic leukemia /TER286-resistant cell line was selected by chronic, long-term exposure to the prodrug . Although resistance was not readily achieved, eventually a 5-fold resistant clone was isolated . Cross-resistance to melphalan occurred, but not to doxorubicin (Adriamycin), taxol, and gamma-glutamyl-S-(benzyl)cysteinyl-R(-)-phenyl glycine diethyl ester, a GSTP1-1 inhibitor . The protein and transcript levels and enzymatic activity of GSTP1-1 were reduced significantly in the selected resistant line . GSTalpha levels were unchanged, and GSTmu was undetectable . Although glutathione levels were elevated in human promyelocytic leukemia/TER286 cells, no changes in the expression of thiol-related genes including gamma-glutamylcysteine synthetase, gamma-glutamyl transpeptidase, or multidrug resistance protein were found . A 7-fold increase in catalase expression in the resistant cell line indicated an adaptive response to oxidative and electrophilic stress, and this was also reflected in the lower prevalence of drug-induced DNA single-strand breaks in the resistant cells . Mouse embryo fibroblast GSTP1-1(-/-) cells exhibited 2-fold resistance to TER286 compared with GSTP1-1(+/+) cells . NIH3T3 cells transfected with combinations of gamma-GCS and multidrug resistance protein exhibited enhanced resistance to TER286, although the degree of resistance was impaired by cotransfection of GSTP1-1 . These results are consistent with responses in the TER286-resistant cells indicative of GSTP1-1-mediated mechanism of activation . In consequence, these data support the rationale that tumors expressing high levels of GSTP1-1 will be more sensitive to the cytotoxic effects of the drug.

Mol Pharmacol, 2000 Jul, 58(1), 37 - 47
Functional analysis of a tryptophan-less P-glycoprotein: a tool for tryptophan insertion and fluorescence spectroscopy; Kwan T et al.; P-glycoprotein (Pgp) functions as an ATP-dependent drug efflux pump to confer multidrug resistance to tumor cells . In the absence of a high-resolution structure for this protein, several important and intriguing aspects of Pgp structure and function remain poorly understood . Fluorescence spectroscopy of endogenous or genetically engineered tryptophan residues represents a potentially powerful method to probe static and dynamic aspects of Pgp at high resolution . We have used site-directed mutagenesis to modify the wild-type (WT) mouse mdr3 Pgp for tryptophan fluorescence spectroscopy by replacement of all 11 tryptophan residues individually with phenylalanine . None of the 11 tryptophans were found to be absolutely essential for Pgp activity, because Chinese hamster ovary cells transfected and overexpressing this mutant Trp-less mdr3 cDNA (mdr3F(1-11)) become multidrug-resistant and can carry out active transport of vinblastine, colchicine, and Calcein-AM . The mdr3F(1-11) mutant has reduced activity compared with WT Mdr3, and shows a unique pattern of drug resistance clearly distinct from WT and, as opposed to the latter, can neither confer FK-506 resistance nor functionally complement ste6 in yeast . Studies with Pgp mutants containing either single or double tryptophan residues or with chimeric molecules constructed between wild-type Pgp and mdr3F(1-11) indicated that no single tryptophan residue was responsible for the reduced activity of the mdr3F(1-11) mutant . Likewise, all but one chimeric Pgp preserved the unique drug resistance profile of the mdr3F(1-11) mutant . Altogether, we show that a Trp-less Pgp is functionally active and can be used as a molecular backbone for insertion of tryptophans in strategic locations to probe various aspects of Pgp function.

Mol Pharmacol, 2000 Jul, 58(1), 1 - 10
Regulation of the MDR1 gene by transcriptional repressors selected using peptide combinatorial libraries; Bartsevich VV et al.; The ability to selectively regulate the expression of genes implicated in cancer or other diseases could have important ramifications for both basic research and for therapy . Using peptide combinatorial libraries expressed in yeast, we have screened for novel zinc finger proteins that selectively bind to an overlapping EGR1/SP1/WT1 regulatory site in the promoter of the MDR1 multidrug resistance gene . The novel proteins were only moderately effective in blocking transcription by simple masking of the target site . However, when coupled to mammalian transactivator or repressor domains, they could selectively modulate the expression of reporter genes having promoters containing the MDR1 target site . Moreover, they could also regulate transcription of the chromosomal MDR1 gene . Thus, in K562 cells, 12-O-tetradecanoylphorbol-13-acetate-inducible expression of P-glycoprotein, the product of MDR1 gene, was strongly and selectively inhibited by the presence of a repressor protein targeted to the MDR1 promoter . These studies potentially provide a novel alternative approach to the control of multidrug resistance . They also provide important insights into strategies for developing selective regulators of gene expression.

Proc Natl Acad Sci U S A, 2000 Jul 5, 97(14), 8180 - 5
Criteria for the control of drug-resistant tuberculosis; Dye C et al.; Antibiotic resistance is a growing impediment to the control of infectious diseases worldwide, tuberculosis (TB) being among them . TB kills two million people each year and foci of multidrug-resistant TB (MDR-TB) have been identified in Eastern Europe, Africa, Asia, and Latin America . A critical question for health policy is whether standardized short-course chemotherapy for TB, based on cheap first-line drugs, can prevent and reverse the spread of drug resistance . Here we use mathematical modeling, in conjunction with treatment results from six countries, to show that best-practice short-course chemotherapy is highly likely to bring strains resistant to either of the two key drugs isoniazid and rifampicin under control and to prevent the emergence of MDR-TB . However, it is not certain to contain MDR-TB once it has emerged, partly because cure rates are too low . We estimate that approximately 70% of prevalent, infectious MDR-TB cases should be detected and treated each year, and at least 80% of these cases should be cured, in order to prevent outbreaks of MDR-TB . Poor control programs should aim to increase case detection and cure rates together for three reasons: (i) these variables act synergistically; (ii) when either is low, the other cannot succeed alone; and (iii) the second-line drugs needed to raise MDR-TB cure rates are few and extremely costly . We discuss the implications of these results for World Health Organization policy on the management of antibiotic resistance.

Biochem Pharmacol, 2000 Aug 1, 60(3), 413 - 26
Effects of MDR1 and MDR3 P-glycoproteins, MRP1, and BCRP/MXR/ABCP on the transport of (99m)Tc-tetrofosmin; Chen WS et al.; Multidrug resistance (MDR1) P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP1), and breast cancer resistance protein (BCRP/MXR/ABCP) are members of the ATP-binding-cassette (ABC) superfamily of membrane transporters and are thought to function as energy-dependent efflux pumps of a variety of structurally diverse chemotherapeutic agents . We herein report the characterization of (99m)Tc-Tetrofosmin, a candidate radiopharmaceutical substrate of ABC transporters . (99m)Tc-Tetrofosmin showed high membrane potential-dependent accumulation in drug-sensitive KB 3-1 cells and low antagonist-reversible accumulation in MDR KB 8-5 and KB 8-5-11 cells in proportion to levels of MDR1 Pgp expression . In KB 8-5 cells, EC(50) values of the potent MDR antagonists 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 (GF120918), (2R)-anti-5- inverted question mark3-{4-(10, 11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl}-2 -hydroxypropoxy inverted question markquinoline trihydrochloride (LY335979), and (3'-keto-Bmt')-{Val(2)}-cyclosporin A (PSC 833) were 40, 66, and 986 nM, respectively . Furthermore, only baculoviruses carrying human MDR1, but not MDR3, conferred both a decrease in accumulation of (99m)Tc-Tetrofosmin in host Spodoptera frugiperda (Sf9) cells and a GF120918-induced enhancement . Transport studies with a variety of stably transfected and drug-selected tumor cell lines were performed with (99m)Tc-Tetrofosmin and compared with (99m)Tc-Sestamibi, a previously validated MDR imaging agent . MDR1 Pgp readily transported each agent . To a lesser extent, MRP1 also transported each agent, likely as co-transport substrates with GSH; neither agent was a substrate for the BCRP/MXR/ABCP half-transporter . In mdr1a(-/-) and mdr1a/1b(-/-) mice, (99m)Tc-Tetrofosmin showed approximately 3 . 5-fold greater brain uptake and retention compared with wild-type, with no net change in blood pharmacokinetics, consistent with transport in vivo by Pgp expressed at the capillary blood-brain barrier . Molecular imaging of the functional transport activity of ABC transporters in vivo with (99m)Tc-Tetrofosmin and related radiopharmaceuticals may enable non-invasive monitoring of chemotherapeutic and MDR gene therapy protocols.

Biochem Pharmacol, 2000 Aug 1, 60(3), 363 - 70
Characterisation of non-P-glycoprotein multidrug-resistant Ehrlich ascites tumour cells selected for resistance to mitoxantrone; Nielsen D et al.; An Ehrlich ascites tumour cell line (EHR2) was selected in vivo for resistance to mitoxantrone (MITOX) . The resistant cell line (EHR2/MITOX) was 6123-, 33-, and 30-fold-resistant to mitoxantrone, daunorubicin, and etoposide, respectively, but retained sensitivity to vincristine . The resistant cells showed moderate sensitisation to mitoxantrone on treatment with verapamil or cyclosporin A . Compared with EHR2, the multidrug resistance-associated protein mRNA was increased 13-fold in EHR2/MITOX . Western blot analysis showed an unchanged, weak expression of P-glycoprotein . Topoisomerase IIalpha was reduced to one-third in EHR2/MITOX relative to EHR2 cells, whereas topoisomerase IIbeta was present in EHR2 but could not be detected in EHR2/MITOX . In the resistant subline, net accumulation of MITOX (120 min) and daunorubicin (60 min) was reduced by 43% and 27%, respectively, as compared with EHR2 . The efflux of daunorubicin from preloaded EHR2/MITOX cells was significantly increased . EHR2/MITOX microsomes had a significant basal unstimulated ATPase activity . The apparent K(i) value for vanadate inhibition of the ATPase activity in EHR2/MITOX microsomes was not significantly different from the K(i) value for P-glycoprotein-positive cells . However, whereas verapamil (50 microM) inhibited the ATPase activity of EHR2/MITOX microsomes, it stimulated the ATPase activity of microsomes derived from P-glycoprotein-positive cells . In conclusion, the resistance in EHR2/MITOX was multifactorial and appeared to be associated with: 1) a quantitative reduction in topoisomerase IIalpha and beta protein; 2) reduced drug accumulation, probably as a result of increased expression of a novel transport protein with ATPase activity; and 3) increased expression of MRP mRNA.

Biochem Pharmacol, 2000 Aug 1, 60(3), 353 - 61
Characterisation of multidrug-resistant Ehrlich ascites tumour cells selected in vivo for resistance to etoposide; Nielsen D et al.; An Ehrlich ascites tumour cell line (EHR2) was selected for resistance to etoposide (VP16) by in vivo exposure to this agent . The resulting cell line (EHR2/VP16) was 114.3-, 5.7-, and 4.0-fold resistant to VP16, daunorubicin, and vincristine, respectively . The amount of salt-extractable immunoreactive topoisomerase IIalpha and beta in EHR2/VP16 was reduced by 30-40% relative to that in EHR2 . The multidrug resistance-associated protein (MRP) mRNA was increased 20-fold in EHR2/VP16 as compared with EHR2, whereas the expression of P-glycoprotein was unchanged . In EHR2/VP16, the steady-state accumulation of {(3)H}VP16 and daunorubicin was reduced by 64% and 17%, respectively, as compared with EHR2 . Deprivation of energy by addition of sodium azide increased the accumulation of both drugs to the level of sensitive cells . When glycolysis was restored by the addition of glucose to EHR2/VP16 cells loaded with drug in the presence of sodium azide, extrusion of {(3)H}VP16 and daunorubicin was induced . Addition of verapamil (25 microM) decreased the efflux of daunorubicin to the level of sensitive cells, but had only a moderate effect on the efflux of {(3)H}VP16 . The resistant cells showed moderate sensitisation to VP16 on treatment with verapamil, whereas cyclosporin A had no effect . Compared with that of sensitive cells, the ATPase activity of plasma membrane vesicles prepared from EHR2/VP16 cells was very low . Vanadate inhibited the ATPase activity of EHR2/VP16 microsomes with a K(i) value of 30 microM . ATPase activity was slightly stimulated by daunorubicin, whereas vinblastine, verapamil, and cyclosporin A had no effect . In conclusion, development of resistance to VP16 in EHR2 is accompanied by a significant reduction in topoisomerase II (alpha and beta) and by increased expression of MRP mRNA (20-fold) . MRP displays several points of resemblance to P-glycoprotein in its mode of action: 1) like P-glycoprotein, MRP causes resistance to a range of hydrophobic drugs; 2) MRP decreases drug accumulation in the cells and this decrease is abolished by omission of energy; and 3) MRP increases efflux of drug from cells . However, compared with that of P-glycoprotein-positive cells, the ATPase activity of MRP-positive cells is found to be low and not able to be stimulated by verapamil.

Biochim Biophys Acta, 2000 Jun 26, 1486(1), 128 - 44
ABC transporters in lipid transport; Borst P et al.; Since it was found that the P-glycoproteins encoded by the MDR3 (MDR2) gene in humans and the Mdr2 gene in mice are primarily phosphatidylcholine translocators, there has been increasing interest in the possibility that other ATP binding cassette (ABC) transporters are involved in lipid transport . The evidence reviewed here shows that the MDR1 P-glycoprotein and the multidrug resistance (-associated) transporter 1 (MRP1) are able to transport lipid analogues, but probably not major natural membrane lipids . Both transporters can transport a wide range of hydrophobic drugs and may see lipid analogues as just another drug . The MDR3 gene probably arose in evolution from a drug-transporting P-glycoprotein gene . Recent work has shown that the phosphatidylcholine translocator has retained significant drug transport activity and that this transport is inhibited by inhibitors of drug-transporting P-glycoproteins . Whether the phosphatidylcholine translocator also functions as a transporter of some drugs in vivo remains to be seen . Three other ABC transporters were recently shown to be involved in lipid transport: ABCR, also called Rim protein, was shown to be defective in Stargardt's macular dystrophy; this protein probably transports a complex of retinaldehyde and phosphatidylethanolamine in the retina of the eye . ABC1 was shown to be essential for the exit of cholesterol from cells and is probably a cholesterol transporter . A third example, the ABC transporter involved in the import of long-chain fatty acids into peroxisomes, is discussed in the chapter by Hettema and Tabak in this volume.

Eur J Cancer, 2000 Jun, 36(9), 1149 - 60
Altered cell cycle response of drug-resistant lung carcinoma cells to doxorubicin; O'Loughlin C et al.; The effect of doxorubicin treatment on cell cycle parameters in asynchronous populations of multidrug-resistant human lung carcinoma cell lines was investigated . A sensitive (DLKP-SQ) and three resistant (DLKP-SQ A250 10p#7, DLKP-A2B and DLKP-A5F) variants of a human lung carcinoma cell line DLKP were exposed to equitoxic concentrations of doxorubicin . The latter three were 8-fold, 30-fold and 300-fold resistant to doxorubicin, respectively . Irreversible G2/M arrest in sensitive (DLKP-SQ) cells was observed 24 h after initiation of doxorubicin treatment . In resistant variants, G2/M arrest occurred at 12-16 h with a subsequent bypass of the G2/M arrest to re-emerge and accumulate in G1 . This transient G2/M arrest and subsequent progression into G1 indicated an inefficient checkpoint for monitoring DNA damage induced by doxorubicin treatment . Caffeine treatment could bypass the G2/M block in DLKP-SQ cells . Doxorubicin treatment did not alter cyclin B or cdc2 protein levels, the ability of cdc2 to form complexes with cyclin B or the levels of cyclin B bound to cdc2 . The G2/M arrest seen in sensitive cells was associated with an increase in inhibitory phosphorylation of Tyr15 on cdc2 . In contrast, tyrosine 15 phosphorylation did not change in resistant variants after drug treatment and a general increase in cdc2 kinase activity was seen . Cdc25C levels were not altered following drug treatment.

Oncol Rep, 2000 Jul-Aug, 7(4), 859 - 66
Establishment of new multidrug-resistant human osteosarcoma cell lines; Oda Y et al.; Multidrug-resistant clones of human osteosarcoma MNNG/HOS and MG63 cells were isolated by stepwise selection on exposure to increasing doses of doxorubicin (DXR) . The final clones MNNG/HOS/DXR1000 and MG63/DXR1000, established after ethylmethane sulfonate mutagenesis, showed 96-fold and 121-fold higer resistance to DXR than their parental cell lines . They were also cross-resistant to vincristine, but not to cisplatinum or methotrexate . The levels of multidrug-resistance-1 (MDR1) mRNA expression increased gradually according to the concentration of DXR in both cell lines . Although the parental MNNG/HOS cells expressed a low level of MDR1 mRNA, the parental MG63 cells showed no MDR1 expression . The IC50 values of MNNG/HOS and its resistant variant to DXR were higher than those of MG63 and its resistant clone . Multidrug-resistant associated protein (MRP) mRNA expression was detected in MNNG/HOS or MG63 parental cell lines, and in their resistant variants . MG63 and its resistant variants revealed stable expression of MRP, whereas the resistant phenotype of MNNG/HOS showed decreased MRP expression, compared to its parental cell line . No alteration in the levels of hepatocyte growth factor (HGF) or its receptor c-MET was recognized between parental lines and their resistant variants . The results indicate that our DXR-resistant variants of MNNG/HOS and MG63 reveal a classical MDR phenotype and can offer a model with which to investigate the mechanisms of multidrug resistance in osteosarcoma.

Eur J Intern Med, 2000 Jun, 11(3), 145 - 150
Extrapulmonary tuberculosis in the northeastern suburbs of Paris: 141 cases; Fain O et al.; Background: Big cities were particularly affected by tuberculosis in the 1990s . Methods: We studied 141 cases of extrapulmonary tuberculosis in patients not infected by HIV in the northeastern suburbs of Paris . Results: A total of 84 men and 57 women were included in the study . Their average age at diagnosis was 42.2 years . Some 73.6% of the patients were foreign-born . A total of 182 sites were identified in 141 patients . There was an association with pulmonary tuberculosis in 38 cases . The sites were: lymph node (48.9%), pleural (25.5%), skeletal (22.7%), genitourinary (5.7%), and meninges (5%) . Unfavorable social conditions were frequently observed . The average duration of treatment was 10 months . Twenty-four adverse drug effects were noted . Sixty-eight strains of Mycobacterium tuberculosis were isolated . Five cases of primary resistance to at least one antituberculous drug and only one case of multidrug resistance were observed . Some 95.7% of the 93 patients who were not lost to follow up were cured . Conclusion: Independently of HIV infection, extrapulmonary tuberculosis is still present, particularly in the suburbs of big cities, where social conditions are poor . The significant number of patients lost to follow-up demands that measures be adapted for the therapeutic management of these patients.

J Bacteriol, 2000 Jun, 182(12), 3331 - 5
Genetic antagonism and hypermutability in Mycobacterium smegmatis; Karunakaran P et al.; Multidrug-resistant strains of Mycobacterium tuberculosis are a serious and continuing human health problem . Such strains may contain as many as four or five different mutations, and M . tuberculosis strains that are resistant to both streptomycin and rifampin contain mutations in the rpsL and rpoB genes, respectively . Coexisting mutations of this kind in Escherichia coli have been shown to interact negatively (S . L . Chakrabarti and L . Gorini, Proc . Natl . Acad . Sci . USA 72:2084-2087, 1975; S . L . Chakrabarti and L . Gorini, Proc . Natl . Acad . Sci . USA 74:1157-1161, 1977) . We investigated this possibility in Mycobacterium smegmatis by analyzing the frequency and nature of spontaneous mutants that are resistant to either streptomycin or rifampin or to both antibiotics . Mutants resistant to streptomycin were isolated from characterized rifampin-resistant mutants of M . smegmatis under selection either for one or for both antibiotics . Similarly, mutants resistant to rifampin were isolated from streptomycin-resistant strains . The second antibiotic resistance mutation occurred at a lower frequency in both cases . Surprisingly, in both cases a very high rate of reversion of the initial antibiotic resistance allele was detected when single antibiotic selection was used; the majority of strains resistant to only one antibiotic were isolated by this process . Determinations of rates of mutation to antibiotic resistance in M . smegmatis showed that the frequencies were enhanced up to 10(4)-fold during stationary phase . If such behavior is also typical of slow-growing pathogenic mycobacteria, these studies suggest that the generation of multiply drug-resistant strains by successive mutations may be a more complex genetic phenomenon than suspected.

Mol Gen Genet, 2000 May, 263(4), 702 - 11
Tagging of genes involved in multidrug resistance in Aspergillus nidulans; de Souza CC et al.; We have used a plasmid containing the Neurospora crassa pyr4 gene to transform an Aspergillus nidulans pyrG89 mutant strain in the presence of Bam-HI, and isolated multidrug-sensitive mutants among the transformants . Using this approach, we hoped to identify genes whose products are important for drug resistance by analyzing gene disruptions that alter the drug sensitivity of the cell . About 1300 transformants isolated following transformation were screened for sensitivity to drugs or various stress agents with different and/or the same mechanism of action . Seventy-seven of these transformants showed sensitivity to at least one drug, while fourteen transformants showed a complex phenotype of sensitivity to different drugs . The pyr4 marker was shown to be tightly linked to the mutant phenotype in only 36% of the pleiotropic mutants analyzed in sexual crosses . Genetic crosses between our multidrug-sensitive transformants and cycloheximide-sensitive and imazalil-resistant mutants of A nidulans were performed to determine whether mutations were present at the same loci . We have shown that the gene imaD that confers resistance to imazalil may also be involved in cycloheximide and hygromycin sensitivity, since this mutation is allelic to scyB (mutant scy290) . In addition, the cross between the transformant R223 and the imazalil-resistant mutant ima535 showed that both mutations are in the same complementation group, suggesting that the gene imaG could also be involved in cycloheximide and itraconazole sensitivity.

Cancer Biother Radiopharm, 1998 Oct, 13(5), 369 - 73
Loss of VLA-3 (CD49c/CD29) expression in two multidrug resistant Burkitt's lymphoma cell lines; Duensing S et al.; Expression of P-glycoprotein (P-gp) mediated multidrug resistance (MDR) has been suggested to be associated with an impaired clinical outcome in several malignancies . In contrast to P-gp itself, further phenotypical and functional alterations related to MDR are poorly characterized . In this in vitro study, we analyzed two Burkitt's lymphoma cell lines (Raji and Daudi) for the beta 1 integrin phenotype prior to and after induction of MDR via co-cultivation with vincristine . A significant loss of the VLA-3 (CD49c/CD29) adhesion receptor was observed whereas all other intergins analyzed lacked considerable changes . We conclude that induction of P-gp mediated MDR does not only affect resistance to cytotoxic drugs but also induces cellular changes with potential relevance for migratory and/or adhesive properties of malignant cells.

Prog Neurobiol, 1999 Dec, 59(6), 663 - 90
Trophic effects of purines in neurons and glial cells; Rathbone MP et al.; In addition to their well known roles within cells, purine nucleotides such as adenosine 5' triphosphate (ATP) and guanosine 5' triphosphate (GTP), nucleosides such as adenosine and guanosine and bases, such as adenine and guanine and their metabolic products xanthine and hypoxanthine are released into the extracellular space where they act as intercellular signaling molecules . In the nervous system they mediate both immediate effects, such as neurotransmission, and trophic effects which induce changes in cell metabolism, structure and function and therefore have a longer time course . Some trophic effects of purines are mediated via purinergic cell surface receptors, whereas others require uptake of purines by the target cells . Purine nucleosides and nucleotides, especially guanosine, ATP and GTP stimulate incorporation of {3H}thymidine into DNA of astrocytes and microglia and concomitant mitosis in vitro . High concentrations of adenosine also induce apoptosis, through both activation of cell-surface A3 receptors and through a mechanism requiring uptake into the cells . Extracellular purines also stimulate the synthesis and release of protein trophic factors by astrocytes, including bFGF (basic fibroblast growth factor), nerve growth factor (NGF), neurotrophin-3, ciliary neurotrophic factor and S-100beta protein . In vivo infusion into brain of adenosine analogs stimulates reactive gliosis . Purine nucleosides and nucleotides also stimulate the differentiation and process outgrowth from various neurons including primary cultures of hippocampal neurons and pheochromocytoma cells . A tonic release of ATP from neurons, its hydrolysis by ecto-nucleotidases and subsequent re-uptake by axons appears crucial for normal axonal growth . Guanosine and GTP, through apparently different mechanisms, are also potent stimulators of axonal growth in vitro . In vivo the extracellular concentration of purines depends on a balance between the release of purines from cells and their re-uptake and extracellular metabolism . Purine nucleosides and nucleotides are released from neurons by exocytosis and from both neurons and glia by non-exocytotic mechanisms . Nucleosides are principally released through the equilibratory nucleoside transmembrane transporters whereas nucleotides may be transported through the ATP binding cassette family of proteins, including the multidrug resistance protein . The extracellular purine nucleotides are rapidly metabolized by ectonucleotidases . Adenosine is deaminated by adenosine deaminase (ADA) and guanosine is converted to guanine and deaminated by guanase . Nucleosides are also removed from the extracellular space into neurons and glia by transporter systems . Large quantities of purines, particularly guanosine and, to a lesser extent adenosine, are released extracellularly following ischemia or trauma . Thus purines are likely to exert trophic effects in vivo following trauma . The extracellular purine nucleotide GTP enhances the tonic release of adenine nucleotides, whereas the nucleoside guanosine stimulates tonic release of adenosine and its metabolic products . The trophic effects of guanosine and GTP may depend on this process . Guanosine is likely to be an important trophic effector in vivo because high concentrations remain extracellularly for up to a week after focal brain injury . Purine derivatives are now in clinical trials in humans as memory-enhancing agents in Alzheimer's disease . Two of these, propentofylline and AIT-082, are trophic effectors in animals, increasing production of neurotrophic factors in brain and spinal cord . Likely more clinical uses for purine derivatives will be found; purines interact at the level of signal-transduction pathways with other transmitters, for example, glutamate . They can beneficially modify the actions of these other transmitters.

Oncol Res, 1999, 11(10), 471 - 8
Cytotoxic effects toward human hematopoietic progenitor cells and tumor cell lines of paclitaxel, docetaxel, and newly developed analogues IDN5109, IDN5111, and IDN5127; Ferlini C et al.; The growth inhibitory effect of paclitaxel, docetaxel, and newly developed taxanes IDN5109, IDN5111, and IDN5127 was assessed on peripheral blood (PB) CD34+ maintained in liquid culture and on three human cancer cell lines (MDA-MB231, MCF-7 ADRr, CEM VBLr) . Concomitantly, DNA analysis was also performed . For unfractionated peripheral blood progenitor cells (PBPC) toxicity was also assessed by clonogenic assay . The cytotoxic effects induced by taxanes toward PBPC as measured by clonogenic assay were correlated with those found for multidrug resistance (MDR)-positive cell lines (IDN5109 > IDN5111 > IDN5127 > docetaxel > paclitaxel) . We established a therapeutic index (TI) between the antitumor activity in MDR-positive cells and the toxicity toward PBPC . Paclitaxel and IDN5109, as determined by TI, showed the best value in MDR-negative and MDR-positive cells, respectively . The ranking of the cytotoxic effects observed in PB CD34+ was not correlated with that obtained in clonogenic assay and in cancer cells (IDN5127 > IDN5109 > docetaxel > IDN5111) . Remarkably, in DNA analysis docetaxel induced the maximal cell cycle blocking activity . Newly developed taxanes IDN5109 and IDN5111 are endowed of a profile of anticancer activity in MDR-bearing cells and toxicity toward hematopoietic progenitors better than that of docetaxel . However, mechanism(s) underlying toxicity toward hematopoietic progenitors could be, at least in part, different from that of docetaxel and likely dependent on the interaction with P-glycoprotein function in PB CD34+ cells.

Urol Res, 2000 Apr, 28(2), 86 - 92
Establishment and characterization of renal cell carcinoma cell lines with multidrug resistance; Yu DS et al.; Many of the discoveries of multidrug resistance (MDR) have resulted from studies using drug-resistant cultured tumor cell lines as experimental models . To date, there has been no report on the detailed characterization of such a cell line from renal cell carcinoma (RCC) . By long-term exposure of an established RCC (RCC8701) to increasing concentrations of adriamycin, we established a series of subcultures that were considerably more resistant to the cytotoxic effect of this drug . Biological morphology and cell cycles were analyzed by morphometry and flow cytometry . The chemoresistance index of cells were measured by methyl tetrazolium assay . For evaluation of the expression of MDR-related protein (MRP), mdr-1, glutathione transferase (GST-pi), and topoisomerase II mRNAs, the reverse transcription-polymerase chain reaction was used . Membranous expression of mdr-1-related p-glycoprotein was analyzed by immunofluorescence cytometry . The intracellular content of both glutathione (GSH) and glucose-6-phosphate dehydrogenase (G-6-PDH) were measured using a capillary electrophoresis method . Compared with parent cells, the resistant sublines had a slower growth rate and lower confluent density . They were smaller and mixed with giant cells in different sizes and with different numbers of nucleoli . Flow cytometric analyses showed that resistant cells had a greater percentage of cells in the G2/M phase . The resistant cells, RCC8701/ADR800, were 122 times more resistant to adriamycin and 238 times more resistant to epirubicin than the parent cells . The resistant cells also demonstrated cross-resistance to cisplatin and 5-fluorouracil . In addition to MRP, the contents of mRNA coding for mdr-1, GST-pi, and topoisomerase II in the MDR sublines were higher than in the native cell line . A higher content of cytoplasmic GSH and G-6-PDH were found in the resistant cells; however, the expression of the MDR-related membranous glycoprotein, p-glycoprotein, was not raised . The adriamycin-induced MDR sublines may be used as an experimental system for the search of a means to overcome drug resistance and elucidate possible mechanisms of acquired MDR involved in human renal cancer.

Cancer Res, 2000 Jun 1, 60(11), 2964 - 72
Discovery and characterization of OC144-093, a novel inhibitor of P-glycoprotein-mediated multidrug resistance; Newman MJ et al.; OC144-093 is a novel substituted diarylimidazole (Mr 495) generated using the OntoBLOCK system, a solid-phase combinatorial chemistry technology, in combination with high-throughput cell-based screening . OC144-093 reversed multidrug resistance (MDR) to doxorubicin, paclitaxel, and vinblastine in human lymphoma, breast, ovarian, uterine, and colorectal carcinoma cell lines expressing P-glycoprotein (P-gp) with an average EC50 of 0.032 microM . Inhibition of MDR by OC144-093 was reversible, but the effect persisted for at least 12 h after removal of compound from the culture medium . OC144-093 had no effect on the response to cytotoxic agents by cells in vitro lacking P-gp expression or expressing a multidrug resistance-associated protein (MRP-1) . OC144-093 was not cytotoxic by itself against 15 normal, nontransformed, or tumor cell lines, regardless of P-gp status, with an average cytostatic IC50 of >60 microM . OC144-093 blocked the binding of {3H}azidopine to P-gp and inhibited P-gp ATPase activity . The compound was >50% p.o . bioavailable in rodents and dogs and did not alter the plasma pharmacokinetics of i.v.-administered paclitaxel . OC144-093 increased the life span of doxorubicin-treated mice engrafted with MDR P388 leukemia cells by >100% and significantly enhanced the in vivo antitumor activity of paclitaxel in MDR human breast and colon carcinoma xenograft models, without a significant increase in doxorubicin or paclitaxel toxicity . The results demonstrate that OC144-093 is an orally active, potent, and nontoxic inhibitor of P-gp-mediated multidrug resistance that exhibits all of the desired properties for treatment of P-gp-mediated MDR, as well as for prevention of MDR prior to selection and/or induction of refractory disease.

Cancer Biother Radiopharm, 1998 Apr, 13(2), 81 - 7
Chemosensitivity of advanced larynx carcinoma cells in vitro and significance of multidrug resistance markers in these tumors; Juvekar AS et al.; Thirty cases of previously untreated advanced larynx carcinoma were checked for in vitro chemosensitivity and presence of the resistance markers viz . P-glycoprotein (P-gp) glutathione-S-transferase-pi (GST-pi) and protein kinase C (PKC) overexpression . The cytotoxicity testing was done using MTT assay and the resistance markers were checked by immunohistochemical methods using monoclonal antibodies . The drug combinations employed in MIT assay were 5FU* + MTX*, 5FU + cisPt*, 5FU + Mito*, cisPt + Mito and MTX + Mito (*5FU = 5Fluorouracil, MTX-methotrexate, cisPt-cisplatin and Mito = mitomycin C) . No statistically significant correlation was observed between resistance to the above drug combinations and presence of the resistance markers under consideration . A statistically significant correlation was observed between node positivity and expression of resistance markers which indicates that presence of one or more of these markers in these tumors may be considered as a negative prognosis marker . CisPt-Mito was found to be the most effective drug combination in vitro, in the cases studied.

Cancer Biother Radiopharm, 1998 Apr, 13(2), 71 - 80
A phase I and pharmacokinetic study of CBT-1 as a multidrug resistance modulator in the treatment of patients with advanced cancer; Oldham RK et al.; CBT-1, a natural product, was studied in an escalating dose Phase I clinical trial with doxorubicin at 60 mg/m2 . CBT-1 was administered by mouth at doses from 200 mg/m2 to 600 mg/m2 . The drug was given for 7 days and doxorubicin administered intravenously on day 6 . The MTD was determined to be 500 mg/m2 although some patients did tolerate 600 mg/m2 with moderate nausea and occasional vomiting . Side effects were otherwise mild in the 23 patients treated . Pharmacokinetic determinations in an additional 11 patients demonstrated that CBT-1 did not significantly alter the pharmacokinetics of doxorubicin . In this Phase I study, 25 of 34 patients were evaluable for response and 5 patients demonstrated tumor shrinkage.

Cancer Biother Radiopharm, 1999 Jun, 14(3), 221 - 30
Angiogenesis and growth of murine colon carcinoma are dependent on infiltrating leukocytes; Yoneda J et al.; We determined whether the angiogenesis and growth of murine colon carcinomas growing in the wall of the cecum is dependent on infiltrating leukocytes . Syngeneic BALB/c or SCID mice were treated with a myelosuppressive, maximally tolerated dose of doxorubicin . Parental or multidrug resistant CT-26 colon carcinoma cells were implanted into the cecal wall 3 days after the second intravenous injection of doxorubicin . Control mice developed large, well-vascularized tumors, whereas doxorubicin-pretreated mice did not . Intravenous injection of spleen cells from normal BALB/c or SCID mice one day prior to tumor cell implantation reversed the decreased vascularity and tumorigenicity . The production of proangiogenic molecules and microvessel density in tumors directly correlated with the number of infiltrating leukocytes, suggesting that tumor-infiltrating leukocytes are essential to angiogenesis of murine colon carcinomas.

J Viral Hepat, 1999 Jan, 6(1), 17 - 34
Gene therapy of viral hepatitis and hepatocellular carcinoma; Ruiz J et al.; Gene therapy represents an attractive approach to treat a great variety of diseases, both inherited and acquired, and it is moving slowly from a proof-of-principle phase to a wide application in most medical fields . Liver cancer and viral hepatitis are natural targets for this new therapeutic alternative due to the lack of success of conventional antitumoral and antiviral treatments and the ominous prognosis related with liver tumours . Gene therapy for viral hepatitis is aimed to boost the patient immune response against viral antigens or to make cells resistant to infection by blocking the viral life cycle . Gene transfer techniques applied to the treatment of hepatocellular carcinoma include drug sensitization by suicide genes, genetic immunotherapy, normal tissue protection by transfer of the multidrug resistance gene, replacement of tumour suppressor genes, inhibition of oncogenes and modifications of the biology of the tumour (antiangiogenesis) . However, major advances in our understanding of the regulation of gene expression, design of the expression cassettes and development of more efficient gene transfer vectors are mandatory before gene therapy can become a widely used therapeutic modality.

Bull Cancer, 1995 Apr, 82(4), 265 - 85
{Pharmacology of camptothecin and its derivatives}; Rivory LP et al.; 20 (S) Camptothecin was discovered in the early 60's as a result of the intensive screening of natural products by the NCI . Camptothecin lactone was poorly water soluble and was administered as the sodium salt in phase I trials . Despite some encouraging responses in early studies, continued evaluation of this compound revealed severe and unpredictable toxicities such as haemorrhagic cystitis and diarrhoea . The reversible opening of the lactone ring of a camptothecin is pH-dependent and yields a ring-opened carboxylate form which has greatly reduced activity in vivo and in vitro . Under physiological conditions, the carboxylate form predominates, but the exact position of this equilibrium in vivo also depends on other factors such as protein-binding and differential metabolism and elimination . The site of action of camptothecin is a complex formed by the nuclear enzyme topoisomerase I and DNA, which represents a novel target for cancer chemotherapy . The principal role of topoisomerase I is the relaxation of DNA required for transcription and replication . The transient covalent complexes formed by the linking of the enzyme and the 3' extremity of a nicked DNA strand are stabilised in the presence of camptothecin and involved in collisions with replication forks . The ensuing arrest of the fork is accompanied by the generation of permanent double-strand breaks which are thought to be responsible for the antiproliferative properties of camptothecin . The acquisition of resistance to camptothecin in cell culture appears, in general, to be due to a reduction in content and activity of topo-isomerase I . Single-point mutations of the gene of the enzyme have been detected in a number of these resistant variants . Camptothecin appears to be a poor substrate of P-glycoprotein and its intracellular accumulation is not appreciably reduced in cells expressing the multidrug-resistant phenotype . Several water soluble and active derivatives of camptothecin have been synthetized of which CPT-11 and topotecan are the most advanced in clinical trials . These compounds represent two different approaches to the problem of the poor water solubility of camptothecin lactone . CPT-11 is a soluble prodrug which is converted in vivo to the highly active SN-38, whereas topotecan itself is water-soluble due to the presence of a tertiary amine substitution which is charged at physiological pH . These two compounds present different pharmacological properties in the clinical setting.

Bull Cancer, 1995 Jan, 82(2), 114 - 6
Flow cytometric evaluation of multidrug-resistance circumvention; Leonce S et al.; Flow cytometry, through the measurement of P-gp expression and function, is an important tool in the characterization of multidrug resistant cell lines used in pharmacology . Furthermore, as a complement to classical in vitro tests, this technique allows rapid evaluation of the capacity of new modulator agents to reverse the resistance to cytotoxic drugs and contributes a deeper insight into the mechanism of action of these new molecules.

Breast Cancer Res Treat, 2000 Mar, 60(2), 153 - 66
Beta-adrenoceptor signaling and its control of cell replication in MDA-MB-231 human breast cancer cells; Slotkin TA et al.; MDA-MB-231 human breast cancer cells express high beta-adrenoceptor levels, predominantly the beta2 subtype . Receptor stimulation by isoproterenol evoked immediate reductions in DNA synthesis which were blocked completely by propranolol and were of the same magnitude as effects elicited by high concentrations of 8-Br-cAMP . Isoproterenol-induced inhibition of DNA synthesis was maintained throughout several days of exposure, resulting in a decrement in total cell number, and the effects were augmented by cotreatment with dexamethasone; an even greater effect was seen when cAMP breakdown was inhibited by theophylline, with or without addition of isoproterenol . Despite the persistent effect of isoproterenol, receptor downregulation was evident with as little as 1 h of treatment, and over 90% of the receptors were lost within 24 h . Receptor downregulation was paralleled by homologous desensitization of the adenylyl cyclase response to beta-adrenoceptor stimulation . Dexamethasone augmented the effects of isoproterenol on DNA synthesis but did not prevent receptor downregulation or desensitization . These results indicate that beta-adrenoceptors are effectively linked, through cAMP, to the termination of cell replication in MDA-MB-231 human breast cancer cells, and that activation of only a small number of receptors is sufficient for a maximal effect . Novel pharmacologic strategies that focus on cell surface receptors operating through adenylyl cyclase may offer opportunities to combat cancers that are unresponsive to hormonal agents, or that have developed multidrug resistance.

Mol Microbiol, 2000 May, 36(4), 955 - 61
Increased sensitivity to the antimalarials mefloquine and artemisinin is conferred by mutations in the pfmdr1 gene of Plasmodium falciparum; Duraisingh MT et al.; The declining efficacy of chloroquine and pyrimethamine/sulphadoxine in the treatment of human malaria has led to the use of newer antimalarials such as mefloquine and artemisinin . Sequence polymorphisms in the pfmdr1 gene, the gene encoding the plasmodial homologue of mammalian multidrug resistance transporters, have previously been linked to resistance to chloroquine in some, but not all, studies . In this study, we have used a genetic cross between the strains HB3 and 3D7 to study inheritance of sensitivity to the structurally unrelated drugs mefloquine and artemisinin, and to several other antimalarials . We find a complete allelic association between the HB3-like pfmdr1 allele and increased sensitivity to these drugs in the progeny . Different pfmdr1 sequence polymorphisms in other unrelated lines were also associated with increased sensitivity to these drugs . Our results indicate that the pfmdr1 gene is an important determinant of susceptibility to antimalarials, which has major implications for the future development of resistance.

J Natl Cancer Inst, 2000 Jun 7, 92(11), 898 - 902
Blockage of drug resistance in vitro by disulfiram, a drug used to treat alcoholism; Loo TW et al.; BACKGROUND: P-glycoprotein (P-gp) pumps a wide range of cytotoxic drugs out of cells . Inhibiting maturation of P-gp would be a novel method for circumventing P-gp-mediated multidrug resistance, which complicates cancer chemotherapy and treatment of patients infected with human immunodeficiency virus . We examined the effect of disulfiram (Antabuse(TM)) on the maturation and activity of P-gp . METHODS: Embryonic kidney cells were transfected with a complementary DNA for the P-pg gene, and the effects of disulfiram on the sensitivity of the transfected cells to cytotoxic agents were determined . Enzyme assays were used to determine the effects of disulfiram on the verapamil-stimulated adenosine triphosphatase (ATPase) activity of P-gp . Disulfiram modifies cysteine residues, and mutant forms of P-gp that lack individual cysteines were used to determine whether particular cysteine residues mediate disulfiram's effects on P-gp activity . Maturation of recombinant P-gp was followed on immunoblots . RESULTS: Disulfiram increased the sensitivity of P-gp-transfected cells to vinblastine and colchicine and inhibited P-gp's verapamil-stimulated ATPase activity . Half-maximal inhibition of ATPase activity occurred at 13.5 microM disulfiram . Disulfiram (at 100 microM) inhibited a P-gp mutant by 43% (95% confidence interval {CI} = 37%-48%) when cysteine was present at position 431 only and by 72% (95% CI = 66%-77%) when cysteine was present at position 1074 only . Treatment of P-gp-transfected cells with 50 nM disulfiram blocked maturation of recombinant P-gp . CONCLUSIONS: Disulfiram can potentially reduce P-gp-mediated drug resistance by inhibiting P-gp activity (possibly via cysteine modification) and/or by blocking its maturation . These results suggest that disulfiram has the potential to increase the efficacy of drug therapies for cancer and acquired immunodeficiency syndrome.

J Med Chem, 2000 Jun 1, 43(11), 2285 - 9
Topoisomerase I-mediated antiproliferative activity of enantiomerically pure fluorinated homocamptothecins; Lavergne O et al.; Homocamptothecin (hCPT) is an E-ring modified camptothecin (CPT) analogue bearing a methylene spacer between the alcohol and carboxyl functions of the CPT lactone . Combining pronounced inhibitory activity of topoisomerase I (Topo I) with enhanced plasma stability, hCPT constitutes an attractive template for the elaboration of new anticancer agents . Fluorinated hCPT analogues, prepared in enantiomerically pure form, were assayed by their stimulation of Topo I-mediated DNA cleavage . Translation into cytotoxicity against tumor cells was evaluated on HT29 human colon adenocarcinoma and on the multidrug resistant lung and bladder tumor cell lines, A549 and T24r . Good correlation is observed between the ability of the drugs to stimulate Topo I-mediated DNA cleavage and the respective 50% inhibitory concentrations (IC(50) values) of the HT29, A549, and T24r cell growth . Fluorine substitution in the A-ring of hCPT was found to have a pronounced influence on biological activity, providing several compounds which are up to 100-fold more potent than CPT in terms of IC(50) . Among these, 10,11-difluoro-hCPT has been selected for further development.

Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6775 - 9
Ecteinascidin 743, a transcription-targeted chemotherapeutic that inhibits MDR1 activation; Jin S et al.; Ecteinascidin 743 (ET-743), a highly promising marine-based antitumor agent presently in phase II clinical trials, has been shown to interfere with the binding of minor-groove-interacting transcription factors, particularly NF-Y, with their cognate promoter elements in vitro . We have shown that NF-Y is a central mediator of activation of transcription of the human P glycoprotein gene (MDR1) by a variety of inducers and that NF-Y functions by recruiting the histone acetyltransferase PCAF to the MDR1 promoter . In the present study, we tested whether ET-743 could block activation of the MDR1 promoter by agents that mediate their effect through the NF-Y/PCAF complex . We report that physiologically relevant concentrations of ET-743 abrogate transcriptional activation of both the endogenous MDR1 gene and MDR1 reporter constructs by the histone deacetylase inhibitors as well as by UV light, with minimal effect on constitutive MDR1 transcription . Notably, this inhibition does not alter the promoter-associated histone hyperacetylation induced by histone deacetylase inhibitors, suggesting an in vivo molecular target downstream of NF-Y/PCAF binding . ET-743 is therefore the prototype for a distinct class of transcription-targeted chemotherapeutic agents and may be an efficacious adjuvant to the treatment of multidrug-resistant tumors.

Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7476 - 81
Multidrug-resistance protein 5 is a multispecific organic anion transporter able to transport nucleotide analogs; Wijnholds J et al.; Two prominent members of the ATP-binding cassette superfamily of transmembrane proteins, multidrug resistance 1 (MDR1) P-glycoprotein and multidrug resistance protein 1 (MRP1), can mediate the cellular extrusion of xenobiotics and (anticancer) drugs from normal and tumor cells . The MRP subfamily consists of at least six members, and here we report the functional characterization of human MRP5 . We found resistance against the thiopurine anticancer drugs, 6-mercaptopurine (6-MP) and thioguanine, and the anti-HIV drug 9-(2-phosphonylmethoxyethyl)adenine (PMEA) in MRP5-transfected cells . This resistance is due to an increased extrusion of PMEA and 6-thioinosine monophosphate from the cells that overproduce MRP5 . In polarized Madin-Darby canine kidney II (MDCKII) cells transfected with an MRP5 cDNA construct, MRP5 is routed to the basolateral membrane and these cells transport S-(2,4-dinitrophenyl)glutathione and glutathione preferentially toward the basal compartment . Inhibitors of organic anion transport inhibit transport mediated by MRP5 . We speculate that MRP5 might play a role in some cases of unexplained resistance to thiopurines in acute lymphoblastic leukemia and/or to antiretroviral nucleoside analogs in HIV-infected patients.

Biochem J, 2000 Jun 15, 348 Pt 3, 597 - 606
Determinant of the extracellular location of the N-terminus of human multidrug-resistance-associated protein; Zhang JT; Multidrug-resistance-associated protein (MRP) is a member of the ATP-binding cassette (ABC) membrane-transport superfamily and is responsible for multidrug resistance in cancer cells . Distinct from other members of the ABC superfamily, MRP has three membrane-spanning domains (MSDs) and the N-terminus is located extracellularly . It has been shown that the first MSD (MSD1) with an extracellular N-terminus is important for MRP function . To address what ensures the generation of this structural organization of MRP and to understand in general the molecular mechanism of membrane folding of polytopic proteins with extracellular N-termini, the biogenesis of MSD1 in human MRP1 was examined using an in vitro expression system . Surprisingly, the second transmembrane segment (TM2) in MSD1 was found to play a critical role in the correct membrane translocation and folding of MSD1 in human MRP1 . TM2 not only plays an essential role to ensure the N-terminus-outside/C-terminus-inside orientation of TM1 with an extracellular N-terminus, it can also translocate into membranes post-translationally in a signal-recognition particle and ribosome-dependent manner to provide an additional insurance for correct folding of MSD1 in MRP . These findings suggest that TM2 in a polytopic membrane protein with an extracellular N-terminus may play a critical role in controlling correct membrane translocation and folding of the protein in general.

Br J Cancer, 2000 Jun, 82(11), 1851 - 9
Deficient activation of CD95 (APO-1/Fas)-mediated apoptosis: a potential factor of multidrug resistance in human renal cell carcinoma; Ramp U et al.; The pronounced resistance of human renal cell carcinoma (RCC) to anticancer-induced apoptosis has primarily been related to the expression of P-glycoprotein and effective drug detoxification mechanisms . Because the CD95 system has recently been identified as a key mediator of anticancer drug-induced apoptosis, we analysed the contribution of the CD95 system to chemotherapy-induced apoptosis in four newly established RCC cell lines . Here, we demonstrate that all RCC cell lines expressed CD95-receptor and -ligand . Exposure to agonistic anti-CD95 antibodies resulted in induction of apoptosis and significant (P < 0.05) reduction of cell number in three out of four cell lines, indicating that the essential components for CD95-mediated apoptosis were present and functionally intact in the majority of these RCC cell lines . Moreover, treatment of cultures with bleomycin or topotecan, a novel topoisomerase I inhibitor with little substrate affinity for P-glycoprotein, led to induction of apoptosis and significant (P < 0.05) dose-dependent reduction of cell number in all RCC cell lines . Both anticancer drugs also induced upregulation of CD95 ligand expression in all cell lines . Additionally, augmentation of CD95 receptor expression was found in three RCC cell lines, including one p53-mutated cell line, whereas another p53-mutated cell line showed no or only a weak CD95 receptor upregulation after exposure to topotecan or bleomycin, respectively . Despite this upregulation of CD95 receptor and ligand, antagonistic antibodies directed against CD95 receptors or ligands could not inhibit induction of apoptosis by topotecan and bleomycin in any cell line . Thus, although a functionally intact CD95 signalling cascade is present in most RCC cell lines, the anticancer drugs topotecan and bleomycin that induce upregulation of CD95 receptor and ligand fail to effectively activate CD95-mediated apoptosis . This deficient activation of CD95-mediated apoptosis might be an important additional factor for the multidrug resistance phenotype of human RCCs.

Reprod Toxicol, 2000 May-Jun, 14(3), 217 - 24
Functional expression of P-glycoprotein in primary cultures of human cytotrophoblasts and BeWo cells; Utoguchi N et al.; The objective of this study was to investigate the functional expression of the efflux transporter, P-glycoprotein (P-gp), in primary cultures of human cytotrophoblasts and BeWo cell monolayers . Uptake studies with primary cultures of human cytotrophoblasts or BeWo cells were conducted with calcein-AM and vinblastine (P-gp markers) or fluorescein (MRP marker) in the presence of specific P-gp or MRP inhibitors . Results showed that the accumulation of P-gp substrates calcein-AM and vinblastine by BeWo cells or primary cultures of human cytotrophoblasts was significantly enhanced in the presence of a typical P-gp inhibitor, cyclosporin-A, or other inhibitors such as quinidine, verapamil, and dipyridamole . MRP inhibitors had no effect on the accumulation of calcein or fluorescein by BeWo cells . Western blots confirmed the presence of multidrug resistant gene product 1 (MDR1) in both primary cultures of human cytotrophoblasts and BeWo cells . This study demonstrates functional P-gp in term human trophoblasts and further supports the use of primary cultures of human cytotrophoblasts and BeWo cells as in vitro models of the trophoblast to investigate mechanisms regulating drug distribution across the placenta.

Am J Physiol Cell Physiol, 2000 Jun, 278(6), C1256 - 65
Role of multidrug resistance P-glycoprotein in the secretion of aldosterone by human adrenal NCI-H295 cells; Bello-Reuss E et al.; We determined the role of the multidrug resistance (MDR1) gene product, P-glycoprotein (PGP), in the secretion of aldosterone by the adrenal cell line NCI-H295 . Aldosterone secretion is significantly decreased by the PGP inhibitors verapamil, cyclosporin A (CSA), PSC-833, and vinblastine . Aldosterone inhibits the efflux of the PGP substrate rhodamine 123 from NCI-H295 cells and from human mesangial cells (expressing PGP) . CSA, verapamil, and the monoclonal antibody UIC2 significantly decreased the efflux of fluorescein-labeled (FL)-aldosterone microinjected into NCI-H295 cells . In MCF-7/VP cells, expressing multidrug resistance-associated protein (MRP) but not PGP, and in the parental cell line MCF7 (expressing no MRP and no PGP), the efflux of microinjected FL-aldosterone was slow . In BC19/3 cells (MCF7 cells transfected with MDR1), the efflux of FL-aldosterone was rapid and it was inhibited by verapamil, indicating that transfection with MDR1 cDNA confers the ability to transport FL-aldosterone . These results strongly indicate that PGP plays a role in the secretion of aldosterone by NCI-H295 cells and in other cells expressing MDR1, including normal adrenal cells.

J Photochem Photobiol B, 2000 Feb, 54(2-3), 136 - 44
Preferential cytotoxicity for multidrug-resistant K562 cells using the combination of a photosensitizer and a cyanine dye; Kanofsky JR et al.; The cyanine dye 1,1',3,3,3',3'-hexamethylindodicarbocyanine iodide (HIDC) protects K562 leukemia cells from photodynamic membrane damage caused by cis-di(4-sulfonatophenyl)diphenylporphine (TPPS2) and 420 nm light . This wavelength of light is chosen because it is absorbed by TPPS2, but not by HIDC . The photodynamic system studied may be useful as a model for antineoplastic therapy . A subline of K562 leukemia (K562/DOX), expressing the multidrug-resistance (MDR) phenotype, is found to accumulate smaller amounts of HIDC than the parent cell line and thus has less photoprotection . In the absence of added HIDC, the K562/DOX cell line is more resistant to photodynamic cytotoxicity than the K562 cell line . The resistance of the K562/DOX cell line is not due to a smaller accumulation of TPPS2 than the K562 cell line . However, when both cell lines are incubated with HIDC and TPPS2, and then exposed to light, the K562/DOX cell line becomes more sensitive to photodynamic cell damage than the K562 cell line . The combination of a photosensitizer with a cationic or lysomorphotropic photoprotector represents a novel strategy for the eradication of malignant cells expressing the MDR phenotype.

Nat Med, 2000 Jun, 6(6), 652 - 8
Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells; Abonour R et al.; Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin . In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation . There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296 . The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery . There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector . Gene marking has persisted more than a year at levels higher than previously reported in humans.

Nat Genet, 2000 Jun, 25(2), 223 - 7
Mutations in a gene encoding an ABC transporter cause pseudoxanthoma elasticum; Le Saux O et al.; Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by calcification of elastic fibres in skin, arteries and retina that results in dermal lesions with associated laxity and loss of elasticity, arterial insufficiency and retinal haemorrhages leading to macular degeneration . PXE is usually found as a sporadic disorder, but examples of both autosomal recessive and autosomal dominant forms of PXE have been observed . Partial manifestations of the PXE phenotype have also been described in presumed carriers in PXE families . Linkage of both dominant and recessive forms of PXE to a 5-cM domain on chromosome 16p13.1 has been reported (refs 8,9) . We have refined this locus to an 820-kb region containing 6 candidate genes . Here we report the exclusion of five of these genes and the identification of the first mutations responsible for the development of PXE in a gene encoding a protein associated with multidrug resistance (ABCC6).

Jpn J Cancer Res, 2000 May, 91(5), 543 - 50
Establishment and characterization of 6-{{2-(Dimethylamino)ethyl}amino}-3-hydroxy-7H-indeno{2,1-c}quinolin-7-one dihydrochloride (TAS-103)-resistant cell lines; Aoyagi Y et al.; 6-2-(Dimethylamino)ethylamino-3-hydroxy-7H-indeno2, 1-cquinolin-7-one dihydrochloride (TAS-103) is a novel anticancer agent that was developed to target both topoisomerase (Topo) I and Topo II . To elucidate its mechanism of action, we have established and characterized TAS-103-resistant cells, derived from mouse leukemia (P388), human colon cancer (DLD-1), and human lung adenocarcinoma (A549) cell lines, by exposure to stepwisely increasing concentrations of TAS-103 in the culture medium . P388 / TAS cells showed only cross-resistance to VP-16 and adriamycin (ADR) . The Topo II activity in these cells was decreased to below one-fourth of that in the parental cells, while the Topo I activity remained unchanged . DLD / TAS cells appeared to be cross-resistant to VP-16, ADR, camptothecin (CPT), SN-38 and vincristine (VCR) . The enzymatic activities of both Topo I and Topo II in these cells were decreased to one-fourth of that observed in the parental cells . Furthermore, the decreased activities were accompanied by lower expression at the mRNA and protein levels . A549 / TAS cells acquired cross-resistance to VP-16, ADR and VCR, though the Topo activities were virtually unchanged . In this cell line, the intracellular accumulation of TAS-103 was significantly decreased and the expression of multidrug resistance associated protein (MRP) was elevated when compared with the parental cells . The results indicate that the affected activities of Topo I and / or Topo II, and in some instances decreased accumulation of TAS-103, are associated with the development of resistance to TAS-103, although the main mechanism of resistance to TAS-103 varied among cell lines.

EMBO J, 2000 Jun 1, 19(11), 2503 - 14
The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism; van Veen HW et al.; The bacterial LmrA protein and the mammalian multidrug resistance P-glycoprotein are closely related ATP-binding cassette (ABC) transporters that confer multidrug resistance on cells by mediating the extrusion of drugs at the expense of ATP hydrolysis . The mechanisms by which transport is mediated, and by which ATP hydrolysis is coupled to drug transport, are not known . Based on equilibrium binding experiments, photoaffinity labeling and drug transport assays, we conclude that homodimeric LmrA mediates drug transport by an alternating two-site transport (two-cylinder engine) mechanism . The transporter possesses two drug-binding sites: a transport-competent site on the inner membrane surface and a drug-release site on the outer membrane surface . The interconversion of these two sites, driven by the hydrolysis of ATP, occurs via a catalytic transition state intermediate in which the drug transport site is occluded . The mechanism proposed for LmrA may also be relevant for P-glycoprotein and other ABC transporters.

Breast Cancer Res Treat, 2000 Feb, 59(3), 231 - 44
Role of specific apoptotic pathways in the restoration of paclitaxel-induced apoptosis by valspodar in doxorubicin-resistant MCF-7 breast cancer cells; Chadderton A et al.; Paclitaxel (Taxol) kills tumor cells by inducing both cellular necrosis and apoptosis . A major impediment to paclitaxel cytotoxicity is the establishment of multidrug resistance whereby exposure to one chemotherapeutic agent results in cross-resistance to a wide variety of other drugs . For example, selection of MCF-7 breast cancer cells for resistance to doxorubicin (MCF-7ADR cells) results in cross-resistance to paclitaxel . This appears to involve the overexpression of the drug transporter P-glycoprotein which can efflux both drugs from tumor cells . However, MCF-7ADR cells possess a deletion mutation in p53 and have considerably reduced levels of the Fas receptor, Fas ligand, caspase-2, caspase-6, and caspase-8, suggesting that paclitaxel resistance may also stem from a bona fide block in paclitaxel-induced apoptosis in these cells . To address this issue, we examined the ability of the P-glycoprotein inhibitor valspodar to restore paclitaxel accumulation, paclitaxel cytotoxicity, and paclitaxel-induced apoptosis . Compared to drug sensitive MCF-7 cells, MCF-7ADR cells accumulated >6-fold less paclitaxel, were approximately 100-fold more resistant to killing by the drug, and were highly resistant to paclitaxel-induced apoptosis . In contrast, MCF-7ADR cells pretreated with valspodar were indistinguishable from drug-sensitive cells in their ability to accumulate paclitaxel, in their chemosensitivity to the drug, and in their ability to undergo paclitaxel-induced apoptosis . Valspodar, by itself, did not affect these parameters . This suggests that the enhancement of paclitaxel toxicity in MCF-7ADR cells involves a restoration of apoptosis and not solely through enhanced drug-induced necrosis . Morever, it appears that changes in the levels/activity of p53, the Fas receptor, Fas ligand, caspase-2, caspase-6, or caspase-8 activity have little effect on paclitaxel-induced cytotoxicity and apoptosis in human breast cancer cells.

Gan To Kagaku Ryoho, 2000 May, 27(5), 717 - 22
{Histoculture drug response assay on non-small cell lung cancer}; Yoshimasu T et al.; We examined the chemosensitivity of non-small cell lung cancer (NSCLC) tissues to CDDP, 5-FU, ADM, MMC, ETP and SN38 using histoculture drug response assay (HDRA) . One-hundred and thirty surgical specimens from NSCLC patients who were not given preoperative chemotherapy were used . The inhibition indexes of CDDP, 5-FU, MMC, ADM, ETP and SN38 were 39.1 +/- 18.2%, 48.0 +/- 19.7%, 63.3 +/- 17.7%, 47.6 +/- 22.0%, 36.9 +/- 21.1%, and 37.9 +/- 25.2%, respectively . Inhibition indexes were above the cutoff level, i.e., 'judged sensitive,' in 40 cases (31.3%) for CDDP, 34 cases (27.4%) for 5-FU, 54 cases (44.3%) for MMC, 36 cases (33.0%) for ADM, 29 cases (29.8%) for ETP, and 34 cases (37.4%) for SN38, respectively . In almost one-third of patients, the inhibition indexes of all drugs were under cutoff levels . Correlations between in vitro chemosensitivity data and patient responses to chemotherapy were obtained from 16 evaluable patients, and a 44.4% true positive rate and a 100% true negative rate were observed . Our results with HDRA for NSCLC showed a high incidence of intrinsic multidrug resistance . HDRA may help doctors to avoid non-effective chemotherapy for NSCLC patients.

Nucl Med Biol, 2000 Apr, 27(3), 299 - 307
Involvement of the glutathione S-conjugate compounds and the MRP protein in Tc-99m-tetrofosmin and Tc-99m-sestamibi uptake in glioma cell lines; Perek N et al.; The objective of this study was to compare the accumulation of Tc-99m-tetrofosmin and Tc-99m-sestamibi in four grade IV glioma cell lines and to correlate their accumulation with the multidrug resistance of the cells . Tc-99m-tetrofosmin in all glioma cell lines showed slightly higher uptake and more efficient release beyond 150 min than Tc-99m-sestamibi and the retention of both tracers in the cells was to a certain extend inversely proportional to their degree of multidrug resistance . The results obtained showed that the efflux of both tracers was carried out only in part through the MRP/GS-X pump system . Tc-99m-tetrofosmin showed good potential as a marker of recurrent malignant glioma and in vivo studies are currently underway to confirm these observations.

Exp Parasitol, 2000 Mar, 94(3), 190 - 3
Plasmodium yoelii nigeriensis (MDR)-efficacy of oral pyronaridine against multidrug-resistant malaria in Swiss mice; Tripathi R et al.; A multidrug-resistant strain of Plasmodium yoelii nigeriensis (MDR) showing a wide spectrum of resistance to chloroquine, amodiaquine, mepacrine, mefloquine, halofantrine, quinine, and quinidine was used in this study for in vivo evaluation of the blood schizontocidal activity of pyronaridine, a topoisomerase II inhibitor, in Swiss mice . The parasite produces 100% lethal infection in mice . The drug was administered orally once a day from day 0 onward . The initial studies showed that low doses of pyronaridine (0.625 to 5.0 mg base/kg x9 days) did not completely control blood-induced P . yoelii nigeriensis infection . Finally a series of doses of pyronaridine ranging from 1.25 to 30.0 mg/kg administered orally for 7 consecutive days were evaluated and in spite of high level of resistance to standard antimalarials, the parasite P . yoelii nigeriensis has shown complete susceptibility to pyronaridine (15 mg/kg dose x7 days) . The present paper also compares the merits of a single MDR strain vs a battery of different resistant lines for quick antimalarials screening .

Anticancer Drugs, 2000 Mar, 11(3), 217 - 24
Efficacy of MGI 114 (HMAF) against the MRP+ metastatic MV522 lung carcinoma xenograft; Kelner MJ et al.; This study is part of an effort to evaluate efficacy of the novel agent MGI 114 (HMAF) against tumors resistant to conventional chemotherapeutic agents . MGI 114 is a novel semisynthetic anticancer agent currently in chemotherapeutic phase II trials to evaluate activity against various solid tumors . Previous studies indicate MGI 114 was active against human MDR1/gp170+ solid tumor xenografts . Recent evidence suggests overexpression of the MRP protein may also be clinically relevant to development of drug resistance in solid tumors . We evaluated the efficacy of MGI 114 against a human MRP+ lung carcinoma xenograft . Parent MV522 lung carcinoma cells were transfected with a MRP cDNA expression vector and resistant cells selected by exposure to vinblastine (30-fold resistance) . Analysis of resistant clones indicated 20- to 40-fold increases in expression of both MRP mRNA and MRP protein . Administration of MGI 114 at the maximum tolerated dose (7 mg/kg, 5 x/week for 3 weeks) to MRP tumor-bearing mice demonstrated this novel agent was active against MRP+ tumors and significantly extended their lifespan (p<0.001) . In contrast, other cytotoxic agents had minimal activity against this MRP+ xenograft . These results indicate MGI 114 should retain activity in vivo against MRP+ tumor types . The development of this MRP+ xenograft model, in conjunction with the parent MV522 and MDR1/gp170+ xenograft models, will be useful for screening new classes of agents for activity against multidrug-resistant tumors.

Anticancer Drugs, 2000 Mar, 11(3), 193 - 200
Low-dose twice-daily fractionated X-irradiation of ovarian tumor cells in vitro generates drug-resistant cells overexpressing two multidrug resistance-associated proteins, P-glycoprotein and MRP1; Hill BT et al.; Failure of chemotherapy is frequently observed in patients previously treated with radiotherapy . To establish a cellular model for examining this resistance phenotype a series of mammalian tumor cell lines were exposed in vitro to fractionated X-irradiation and were then shown to express resistance to multiple antitumor drugs, including vincristine, etoposide and cisplatin . In these experiments the radiation was delivered as 10 fractions of 5 Gy (dose resulting in 1 log cell kill) given intermittently over several months . We now report that a comparable multidrug-resistance profile is expressed by human SK-OV-3 human ovarian tumor cells exposed in vitro to low dose (2 Gy) twice-daily fractions of X-rays given for 5 days on two consecutive weeks, essentially mimicking clinical practice, involving an overexpression of two MDR-associated proteins, P-glycoprotein and the multidrug resistance protein 1 (MRP1), with the latter being readily detectable by immunocytochemistry.

FEBS Lett, 2000 May 26, 474(1), 107 - 10
Regulation of {Ca(2+)}(i) homeostasis in MRP1 overexpressing cells; Filipeanu CM et al.; Regulation of capacitative Ca(2+) entry was studied in two different multidrug resistance (MDR) protein (MRP1) overexpressing cell lines, HT29(col) and GLC4/ADR . MRP1 overexpression was accompanied by a decreased response to thapsigargin . Moreover, inhibition of capacitative Ca(2+) entry by D, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) was abolished in MRP1 overexpressing cells . Both PDMP and the MRP1 inhibitor MK571 greatly reduced InsP(3)-mediated (45)Ca(2+) release from intracellular stores in HT29 cells . Again, these effects were virtually abolished in HT29(col) cells . Our results point to a modulatory role of MRP1 on intracellular calcium concentration ({Ca(2+)}(i)) homeostasis which may contribute to the MDR phenotype.

Blood, 2000 Jun 1, 95(11), 3514 - 9
Deletion of the multidrug resistance protein MRP1 gene in acute myeloid leukemia: the impact on MRP activity; van Der Kolk DM et al.; Deletion of the multidrug resistance gene MRP1 has been demonstrated in acute myeloid leukemia (AML) patients with inversion of chromosome 16 (inv{16}) . These AML patients are known to have a relatively favorable prognosis, which suggests that MRP1 might play an important role in determining clinical outcome . This study analyzed MRP1 deletion by fluorescent in situ hybridization (FISH), with a focus on inv(16) AML patients . Functional activity of multidrug resistance protein (MRP) was studied in a flow cytometric assay with the use of the MRP substrate carboxyfluorescein (CF) and the inhibitor MK-571 . MRP1, MRP2, and MRP6 messenger RNA (mRNA) expression was determined with reverse transcriptase-polymerase chain reaction (RT-PCR) . The results were compared with normal bone marrow cells . MRP1 deletion was detected in 7 AML patients; 2 cases showed no MRP1 FISH signals, and 5 cases had 1 MRP1 signal, whereas in 4 AML patients with inv(16) no MRP1 deletions were observed . A variability in MRP activity, expressed as CF efflux-blocking by MK-571, was observed (efflux-blocking factors varied between 1.2 and 3.6); this correlated with the number of MRP1 genes (r = 0.91, P < . 01) . MRP activity in the AML cases was not different from normal hematopoietic cells . MRP1 mRNA was detected in patients with 1 or 2 MRP1 FISH signals, but not in patients with no MRP1 signals . MRP2 and MRP6 mRNA were expressed predominantly in AML samples with 1 MRP1 signal, whereas in normal bone marrow cells no MRP2 and MRP6 mRNA was observed . In conclusion, this study shows that MRP activity varies among inv(16) AML cases and does not differ from that in normal hematopoietic cells; this might be in part due to the up-regulation of other MRP genes.

Hepatology, 2000 Jun, 31(6), 1285 - 95
Cholestasis with altered structure and function of hepatocyte tight junction and decreased expression of canalicular multispecific organic anion transporter in a rat model of colitis; Kawaguchi T et al.; Cholestasis is frequently associated with inflammatory bowel disease . Because some cholestasis is resulted from altered hepatocyte tight junctions (TJs) or the canalicular multispecific organic anion transporter, we have investigated the following topics in a rat model of inflammatory bowel disease: (1) alterations in hepatocyte TJs and in the canalicular multispecific organic anion transporter, (2) etiologic factors for cholestasis, and (3) effects of antibiotics on cholestasis . Rats with trinitrobenzene sulfonic acid-induced colitis were studied 24 hours after treatment . Hepatocyte TJs and the canalicular multispecific organic anion transporter were evaluated by immunostaining for TJ-associated proteins, 7H6 and ZO-1, and multidrug resistance protein 2 (mrp2) . To investigate etiologic factors causing cholestasis, portal endotoxin and proinflammatory cytokines were examined . The effects of polymyxin B, penicillin G, or metronidazole on immunostaining for 7H6, ZO-1, mrp2, and cholestasis were investigated . (1) Immunostaining for 7H6 and ZO-1 colocalized outlining the bile canaliculi and immunostaining for mrp2 localized on the canalicular membrane in controls . Treatment with trinitrobenzene sulfonic acid induced significant cholestasis and caused translocation of immunostaining for 7H6, but not that for ZO-1, to the cytoplasm and diminished immunostaining for mrp2 on the canaliculus membrane . (2) The levels of portal endotoxin, but not proinflammatory cytokines, was increased . (3) Polymyxin B, but not the other antibiotics, prevented alterations in immunostaining for both 7H6 and mrp2, and cholestasis . We described that both hepatocyte TJs and the canalicular multispecific organic anion transporter were altered and that gut-derived endotoxin levels in the portal blood were increased in this rat colitis model.

Biochem Pharmacol, 2000 Jul 15, 60(2), 233 - 9
Association of tamoxifen biliary excretion rate with prior tamoxifen exposure and increased mdr1b expression; Riley J et al.; ATPase transporter proteins are commonly found in the hepatocyte canalicular membrane . Some of these, in particular the multidrug resistance (mdr1b) gene, have been previously demonstrated to be inducible genes . In this study, we found that tamoxifen induced expression of the mdr1b gene in the liver up to 40-fold after 14 days' exposure to tamoxifen in the diet at a concentration of 420 ppm . As tamoxifen and its metabolites are primarily excreted into the bile, we investigated if the increased expression of mdr1b in the liver following tamoxifen exposure had any effect on its excretion in rats . We found that the excretion of tamoxifen and its metabolites into bile was increased from 8 +/- 1% to 51 +/- 18% (mean +/- SD) of an administered dose of 180 nmol/kg over a collection period of 3 hr in rats that had received tamoxifen (35 mg/kg) orally for 12 days (plus a 3-day rest) prior to the experiment . These data suggest that prolonged treatment with tamoxifen may result in lower serum and tumour concentrations, due to a self-mediated enhancement of excretion via mdr1b gene-encoded P-glycoprotein . This may have implications for other drugs sharing the same route of excretion and co-administered with tamoxifen.

Cancer Res, 2000 May 15, 60(10), 2589 - 93
Breast cancer resistance protein is localized at the plasma membrane in mitoxantrone- and topotecan-resistant cell lines; Scheffer GL et al.; Tumor cells may display a multidrug resistant phenotype by overexpression of ATP-binding cassette transporters such as multidrug resistance (MDRI) P-glycoprotein, multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP) . The presence of BCRP has thus far been reported solely using mRNA data . In this study, we describe a BCRP-specific monoclonal antibody, BXP-34, obtained from mice, immunized with mitoxantrone-resistant, BCRP mRNA-positive MCF-7 MR human breast cancer cells . BCRP was detected in BCRP-transfected cells and in several mitoxantrone- and topotecan-selected tumor cell sublines . Pronounced staining of the cell membranes showed that the transporter is mainly present at the plasma membrane . In a panel of human tumors, including primary tumors as well as drug-treated breast cancer and acute myeloid leukemia samples, BCRP was low or undetectable . Extended studies will be required to analyze the possible contribution of BCRP to clinical multidrug resistance.

Clin Infect Dis, 1999 Apr, 28(4), 808 - 15
The effects of mefloquine treatment in pregnancy; Nosten F et al.; We investigated the relationship between mefloquine antimalarial treatment and the outcome of pregnancy in Karen women living in an area along the western border of Thailand where multidrug-resistant Plasmodium falciparum infections are common . Of 3,587 pregnancies investigated, 208 (5.8%) were exposed to mefloquine, 656 (18.3%) to quinine only, and 909 (25.3%) to other antimalarials, and 2,470 (68.9%) had no documented malaria . There were 61 stillbirths and 313 abortions . Women who received mefloquine treatment during but not before pregnancy had a significantly greater risk of stillbirth than did women treated with quinine alone (odds ratio {OR}, 4.72; 95% confidence interval {CI}, 1.7-12.7), women exposed to other treatments (OR, 5.10; 95% CI, 2-13.1), and women who had no malaria (OR, 3.50; 95% CI, 1.6-7.6) (P < .01) . This association remained after adjustment for all identified confounding factors . Mefloquine was not associated with abortion, low birth weight, neurological retardation, or congenital malformations . Mefloquine treatment during pregnancy was associated with an increased risk of stillbirth.

Biochemistry, 2000 May 23, 39(20), 6094 - 102
The multidrug resistance protein is photoaffinity labeled by a quinoline-based drug at multiple sites; Daoud R et al.; Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP) . The MRP has been shown to mediate the transport of cytotoxic natural products, in addition to glutathione-, glucuronidate-, and sulfate-conjugated cell metabolites . However, the mechanism of MRP drug binding and transport is at present not clear . In this study, we have used a photoreactive quinoline-based drug, N-(hydrocinchonidin-8'-yl)-4-azido-2-hydroxybenzamide (IACI), to show the photoaffinity labeling of the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells . The photoaffinity labeling of the 190 kDa protein by IACI was saturable and specific . The identity of the IACI-photolabeled protein as the MRP was confirmed by immunoprecipitation with the monoclonal antibody QCRL-1 . Furthermore, a molar excess of leukotriene C(4), doxorubicin, colchicine, and other quinoline-based drugs, including MK571, inhibited the photoaffinity labeling of the MRP . Drug transport studies showed lower IACI accumulation in MRP-expressing cells which was reversed by depleting ATP levels in H69/AR cells . Mild digestion of the purified IACI-photolabeled MRP with trypsin showed two large polypeptides ( approximately 111 and approximately 85 kDa) . The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody epitopes corresponds to the C-terminal half of the MRP (amino acids approximately 900-1531) containing the third multiple spanning domain (MSD3) and the second nucleotide binding site . The 111 kDa polypeptide which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the MRP sequence (amino acids 1-900) containing the MSD1 and MSD2 plus the first nucleotide binding domain . Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and approximately 6 plus 4 kDa photolabeled peptides, respectively . In addition, resolution of the exhaustively digested IACI-photolabeled MRP by HPLC showed two major and one minor radiolabeled peaks that eluted late in the gradient (60 to 72% acetonitrile) . Taken together, the results of this study show direct binding of IACI to the MRP at physiologically relevant sites . Moreover, IACI photolabels three small peptides which localize to the N- and C-halves of the MRP . Finally, IACI provides a sensitive and specific probe for studying MRP-drug interactions.

J Neurosci Res, 2000 Jun 1, 60(5), 594 - 601
Functional expression of P-glycoprotein and multidrug resistance-associated protein (Mrp1) in primary cultures of rat astrocytes; Decleves X et al.; Although it has been well established that the drug efflux pump P-glycoprotein (P-gp) protects the brain against the entry of cytotoxic drugs, its real in situ localization, i.e., at brain capillary endothelial cells or on astrocyte foot processes, is still controversial . The aim of this study was to compare the expression of P-gp and of multidrug resistance-associated protein (Mrp1), another drug efflux pump, in cultured neonatal rat brain astrocytes and in cultured brain capillary endothelial cells . Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the mdr1b gene was preferentially expressed in astrocytes, whereas both mdr1a and mdr1b mRNA were detected in endothelial cells . Moreover, the mrp1 gene encoding Mrp1 was expressed in both cell types . Western blotting analysis revealed higher expression of P-gp in endothelial cells as compared with astrocytes, but higher expression of Mrp1 in astrocytes . Moreover, P-gp and Mrp1 expression was not modified in more differentiated astrocytes obtained when cultured with db-cAMP for 48 hr . Our functional analysis of P-gp showed a modest effect of P-gp modulators (CsA, verapamil, PSC 833) on the uptake of colchicine (a substrate of P-gp) by astrocytes, whereas they increased by about 50% the uptake of vincristine (a common substrate of P-gp and MRP) by astrocytes . MRP modulators (genistein, probenecid, and sulfinpyrazone) did not modify the uptake of colchicine but increased that of vincristine with a major effect found for sulfinpyrazone . Moreover, indomethacin, probenecid, and sulfinpyrazone increased the uptake of fluorescein (a substrate of MRP but not of P-gp) . Taken together, our results provide the first biochemical and functional evidence supporting the expression of P-gp and Mrp1 in rat cultured astrocytes .

Antivir Chem Chemother, 2000 Mar, 11(2), 135 - 40
N-{2-(4-methylphenyl)ethyl}-N'-{2-(5-bromopyridyl)}-thiourea as a potent inhibitor of NNRTI-resistant and multidrug-resistant human immunodeficiency virus type 1; Uckun FM et al.; The composite non-nucleoside reverse transcriptase inhibitor (NNRTI) binding pocket model was used to study a number of thiourea analogues with different substitutions at the 4-phenyl position including N-{2-(4-methylphenyl)ethyl}-N'-{2-(5-bromopyridyl)}-thiourea (compound HI-244), which inhibited recombinant RT better than trovirdine or compound HI-275 with an unsubstituted phenyl ring . HI-244 effectively inhibited the replication of HIV-1 strain HTLV(IIIB) in human peripheral blood mononuclear cells with an IC50 value of 0.007 microM, which is equal to the IC50 value of trovirdine . Notably, HI-244 was 20 times more effective than trovirdine against the multidrug-resistant HIV-1 strain RT-MDR with a V106A mutation (as well as additional mutations involving the RT residues 74V, 41L and 215Y) and seven times more potent than trovirdine against the NNRTI-resistant HIV-1 strain A17 with a Y181C mutation.

Zhongguo Yao Li Xue Bao, 1999 May, 20(5), 435 - 9
Circumvention of tumor multidrug resistance by a new annonaceous acetogenin: atemoyacin-B; Fu LW et al.; AIM: To explore the effect of atemoyacin-B (Ate) on overcoming multidrug resistance (MDR) . METHODS: Bullatacin (Bul) was used as a positive control . Cytotoxic effects of Bul and Ate were studied with cell culture of human MDR breast adenocarcinoma cells, MCF-7/Dox and human KBv200 cells, and their parental sensitive cell lines MCF-7 and KB . Cytotoxicity was determined by tetrazolium (MTT) assay . The function of P-glycoprotein (P-gp) was examined by Fura 2-AM assay . Cellular accumulation of doxorubicin (Dox) was determined by fluorescence spectrophotometry . Apoptosis was measured by flow cytometry . RESULTS: IC50 of Ate for MCF-7/Dox, MCF-7, KBv200, and KB cells were 122, 120, 1.34, and 1.27 nmol.L-1, respectively . IC50 of Bul for MCF-7/Dox, MCF-7, KBv200, and KB cells were 0.60, 0.59, 0.04, and 0.04 nmol.L-1, respectively . The cytotoxicities of Bul and Ate to MDR cells were similar to those to parental sensitive cells . Bul and Ate markedly increased cellular Fura-2 and Dox accumulation in MCF-7/Dox cells, but not in MCF-7 cells . The rates of apoptosis in MDR cells were similar to those in sensitive cells induced by Ate . CONCLUSION: There was no cross-resistance of P-gp positive MCF-7/Dox and KBv200 cell lines to Bul and Ate as compared with their sensitive P-gp negative MCF-7 and KB cell lines . The mechanism of the circumvention of MDR was associated with the decrease of P-gp function and the increase of cellular drug accumulation in MDR cells.

Br J Cancer, 2000 May, 82(10), 1732 - 9
Isolation and characterization of an IGROV-1 human ovarian cancer cell line made resistant to Ecteinascidin-743 (ET-743); Erba E et al.; By exposing Igrov-1 human ovarian cancer cells to increasing concentrations of Ecteinascidin-743 (ET-743), either for a short or prolonged time, we obtained sublines resistant to ET-743 which overexpress Pgp . The most resistant clone (Igrov-1/25 ET) was evaluated for biological and pharmacological characterizations . The increased Pgp levels of Igrov-1/25 ET were not due to amplification of the mdr-1 gene but to increased mRNA levels . No increase in other multidrug resistance-related proteins such as MRP or LRP was observed in Igrov-1/25 ET . The IC50 values of ET-743 against Igrov-1/25 ET was approximately 50 times higher than the parental cell line . Resistance was not reversed while maintaining the cell line in drug-free medium for at least 24 months . Igrov-1/25 ET was cross-resistant to Doxorubicin and VP16 while it was equally sensitive to L-PAM, MNNG, CPT and only marginally less sensitive to Cis-DDP and Oxaliplatin compared to the parental cell line . Igrov-1/25 ET exposed to Doxorubicin retained this drug much less, mainly because of a more efficient drug efflux . The cyclosporine analogue SDZ PSC-833 reversed the resistance of Igrov-1/25 ET to ET-743, without any enhancement of the drug activity against the parental Igrov-1 cell line . Igrov-1/25 ET exhibits typical features of cell lines overexpressing the mdr-1 gene and can be a potentially useful tool in selecting ET-743 non-cross-resistant analogues as well as to investigate methods to counteract resistance to this drug.

J Biol Chem, 2000 Aug 11, 275(32), 24970 - 6
Regulation of multidrug resistance 1 (MDR1)/P-glycoprotein gene expression and activity by heat-shock transcription factor 1 (HSF1); Vilaboa NE et al.; Infection of HeLa cells with adenovirus-carrying HSF1(+) cDNA, which encodes a mutated form of HSF1 with constitutive transactivation capacity, increased multidrug resistance 1 (MDR1) mRNA level and P-glycoprotein (P-gp) cell surface content and stimulated rhodamine 123 accumulation and vinblastine efflux activity . On the other hand, infection with adenovirus-carrying HSP70 and HSP27 cDNAs did not increase MDR1/P-gp expression . HSF1 regulates MDR1/P-gp expression at the transcriptional level, since HSF1(+) bound the heat-shock consensus elements (HSEs) in the MDR1 gene promoter and also activated the expression of an MDR1 promoter-driven reporter plasmid (pMDR1(-1202)) . In addition, heat-shock increased pMDR1(-1202) promoter activity but not the activity of a similar reporter plasmid with point mutations at specific HSEs, and the heat-induced increase was totally inhibited by co-transfection with an expression plasmid carrying HSF1(-), a dominant negative mutant of HSF1 . The stress inducers arsenite, butyrate, and etoposide also increased pMDR1(-1202) promoter activity, but the increase was not inhibited (in the case of butyrate) or was only partially inhibited (in the case of arsenite and etoposide) by HSF1(-) . These results demonstrate that HSF1 regulates MDR1 expression, and that the HSEs present in the -315 to -285 region mediate the heat-induced activation of the MDR1 promoter . However, other factors may also participate in MDR1 induction by stressing agents.

Biochem Soc Trans, 2000 Feb, 28(2), 27 - 32
Chemical modulation of chemotherapy resistance in cultured oesophageal carcinoma cells; Sheehan D et al.; Oesophageal carcinoma is a common form of cancer in developing countries, especially in the Caspian littoral and northern China . In contrast, it has a much lower incidence in Japan, the U.S.A . and western Europe . Certainly in the case of squamous cell oesophageal carcinoma, dietary composition, smoking, alcohol and exposure to nitrosamines are major risk factors that may partly explain the disease's geographical distribution . The prognosis for oesophageal carcinoma is generally poor, due to the high incidence of distant metastasis and local recurrence . Combination treatment with both cisplatin and 5-fluorouracil is the most common chemotherapy regime used . We have carried out a detailed study of sensitivity of two oesophageal cell lines: OC1 cells from a squamous carcinoma of a male patient, and OC2, a squamous carcinoma obtained from a female patient . Both cell lines are sensitive to Vinca alkaloids and doxorubicin, while being quite resistant to alkylating agents such as cisplatin and 1,3-bis-(2-chloroethyl)-1-nitrosourea . This pattern of resistance suggests a possible role for glutathione S-transferase (GST) and/or glutathione (GSH) in resistance, and would seem to exclude the multidrug resistance phenotype . Both cell lines possess mainly Pi-class GSTs, and have distinct levels of GSH, with OC2 possessing some 25% of the level of OC1 cells . Effects of a variety of modulating agents on the pattern of resistance, such as the GSH depleter, buthionine sulphoximine, and the GST inhibitor, ethacrynic acid, were determined . An unexpected observation was that ethacrynic acid appears to increase the level of GSH in both cell lines.

Clin Cancer Res, 2000 May, 6(5), 2075 - 86
Distinctive potentiating effects of cisplatin and/or ifosfamide combined with etoposide in human small cell lung carcinoma xenografts; Nemati F et al.; Combined modalities are currently used for cancer therapy, although their mechanisms of activity remain incompletely deciphered . The design of new drug combinations suffers from our inability to anticipate accurately their efficacy or toxicity . They can be evaluated in vivo, using human tumors grafted into immunodeficient mice, as we did here with combined protocols used in the clinical setting . Xenografts of small cell lung carcinoma (SCLC) from eight patients were used to test the tumor sensitivity to etoposide (VP16; 12-16 mg/kg/days, days 1, 2, and 3), cisplatin (CDDP; 6-9 mg/kg/day, day 1) and ifosfamide (IFO; 90-210 mg/kg/day, days 1, 2, and 3) as single agents and to evaluate the efficacy of the two-drug or three-drug combinations . Five xenografts came from untreated patients (SCLC-61, SCLC-6, SCLC-10, SCLC-41, and SCLC-96) and three after treatment (SCLC-74, SCLC-101, and SCLC-108) . p53 was inactivated in all of them . Tumor growth inhibition, growth delay, and the survival rate of tumor-bearing mice reflected individual SCLC chemosensitivity . As single agents, IFO inhibited tumor growth in a dose-dependent manner, whereas CDDP and VP16 had little or no effect . Both CDDP and IFO potentiated VP16, inducing complete regressions in the most sensitive SCLCs; VP16-IFO was more effective than VP16-CDDP, with complete regressions in six versus three of the eight tumors tested, respectively . CDDP-IFO was less effective than VP16-IFO, with three of eight SCLCs giving complete regressions . The three-drug combination led to modest improvement over the best two-drug combination but only for sensitive SCLCs . Because drug-responses distinguished two classes of SCLCs, as sensitive or refractory, MDR1, glutathione S-transferase pi, lung-related multidrug resistance protein, multidrug resistance protein, and topoisomerase IIalpha mRNA expression was studied by semiquantitative reverse transcription . There was no correlation with SCLC sensitivity; topoisomerase IIalpha and multidrug resistance protein was expressed in all cases, lung-related multidrug resistance protein and glutathione S-transferase pie in seven of eight, and MDR1 gene in four of eight . In conclusion, these SCLC xenografts displayed a pattern of chemotherapy response close to that observed in patients . This model confirmed that in two-drug combinations, each component potentiated the effects of the other, with VP16-IFO tending to be the best two-drug combination, both of which were more effective than VP16-CDDP and better tolerated than CDDP-IFO . The addition of a third agent gave a modest, if any, therapeutic benefit in the responders but none in refractory SCLCs . There was no correlation between the extent of response and resistance markers.

Clin Cancer Res, 2000 May, 6(5), 1949 - 57
Nuclear delivery of doxorubicin via folate-targeted liposomes with bypass of multidrug-resistance efflux pump; Goren D et al.; Folic acid, attached to polyethyleneglycol-derivatized, distearoyl-phosphatidylethanolamine, was used to target in vitro liposomes to folate receptor (FR)-overexpressing tumor cells . Confocal fluorescence microscopic observations demonstrated binding and subsequent internalization of rhodamine-labeled liposomes by a high FR-expressing, murine lung carcinoma line (M109-HiFR cells), with inhibition by free folic acid . Additional experiments tracking doxorubicin (DOX) fluorescence with DOX-loaded, folate-targeted liposomes (FTLs) indicate that liposomal DOX is rapidly internalized, released in the cytoplasmic compartment, and, shortly thereafter, detected in the nucleus, the entire process lasting 1-2 h . FR-mediated cell uptake of targeted liposomal DOX into a multidrug-resistant subline of M109-HiFR cells (M109R-HiFR) was unaffected by P-glycoprotein-mediated drug efflux, in sharp contrast to uptake of free DOX, based on verapamil-blockade experiments with quantitation of cell-associated DOX and flow cytometry analysis . Delivery of DOX by FTLs to M109R-HiFR cells increased continuously with time of exposure, reaching higher drug concentrations in whole cells and nuclei compared with exposure to free DOX . The in vitro cytotoxic activity obtained with DOX-loaded FTLs was 10-fold greater than that of the nontargeted liposome formulation, but was not improved over that of free DOX despite the higher cellular drug levels obtained with the targeted liposomes in M109R-HiFR cells . However, if M109R-HiFR cells were exposed to drugs in vitro and tested in an in vivo adoptive assay for tumor growth in syngeneic mice along a 5-week time span, FTL DOX was significantly more tumor inhibitory than free DOX . It is suggested that the biological activity of liposomal DOX released inside the cellular compartment is reduced in vitro due to the aggregated state of DOX, resulting from the liposome drug-loading process, and requires a long period of time and/or an in vivo environment for full expression.

Int J Tuberc Lung Dis, 2000 May, 4(5), 481 - 4
Detection of rifampicin resistance in Mycobacterium tuberculosis isolates from diverse countries by a commercial line probe assay as an initial indicator of multidrug resistance; Traore H et al.; The line probe assay (LiPA), a rapid molecular method for detecting rifampicin resistance (RMPr) in Mycobacterium tuberculosis, correctly identified all 145 rifampicin-sensitive (RMPs) and 262 (98.5%) of 266 RMPr strains among 411 isolates collected from diverse countries . If used as a marker of multidrug-resistant tuberculosis (MDR-TB), detection of RMPr by LiPA would have detected 236 of the 240 MDR strains in this study but would have wrongly suggested the presence of MDR in 26 RMP-monoresistant isolates (sensitivity 98.3%, specificity 84.8%) . Hence, the reliability of using LiPA (or any other rapid RMPr-detection method) as a surrogate marker of MDR-TB largely depends on the prevalence of RMP-monoresistance in the study population . This approach must therefore be validated in each local situation.

Int J Tuberc Lung Dis, 2000 May, 4(5), 433 - 40
Drug-resistant tuberculosis in South African gold miners: incidence and associated factors; Churchyard GJ et al.; SETTING: A gold mining company in the Free State Province of South Africa . OBJECTIVE: To document the incidence of and factors associated with drug-resistant tuberculosis (TB) in South African gold miners . DESIGN: Review of Mycobacterium tuberculosis drug susceptibility records for the period from 1 July 1993 to 30 June 1997 . RESULTS: Over the study period, 2241 miners had culture-positive M . tuberculosis pulmonary disease where isolates were tested for drug susceptibility to the four primary anti-tuberculosis drugs . The proportions of primary and acquired drug resistance were respectively 7.3% and 14.3% for isoniazid and 1.0% and 2.8% for resistance to at least isoniazid and rifampicin (multidrug resistance) . Resistance to streptomycin and ethambutol was uncommon, and rifampicin monoresistance was rare . No significant factors for primary drug resistance were identified . Patients with retreatment pulmonary TB who failed primary TB treatment (versus cure) were significantly more likely to have TB with resistance to any TB drug or MDR (odds ratios respectively 9.82, 95%CI 2.97-33.5, and 18.74, 95%CI 1.76-475) . Human immunodeficiency virus (HIV) infection was not significantly associated with primary or acquired drug resistance, and there was no trend of increasing resistance over time . CONCLUSION: Anti-tuberculosis drug resistance has remained stable despite the HIV epidemic and increasing TB rates . Directly observed therapy may have contributed to containing the level of drug resistance . Adherence to and completion of treatment are essential to prevent drug resistance and treatment failure, including in situations with high HIV prevalence.

Int J Tuberc Lung Dis, 2000 May, 4(5), 427 - 32
Acquired anti-tuberculosis drug resistance in Yaounde, Cameroon; Kuaban C et al.; SETTING: Tuberculosis centre of Hopital Jamot, Yaounde, Cameroon . OBJECTIVES: To determine the prevalence of acquired resistance (ADR) to the main anti-tuberculosis drugs, and to identify risk factors associated with its occurrence in Yaounde . DESIGN: A total of 111 previously treated adults admitted consecutively to the tuberculosis centre with sputum smear-positive pulmonary tuberculosis between June 1996 and July 1997 were included in the study . Information on potential risk factors for ADR was obtained from each patient, and human immunodeficiency virus (HIV) serostatus was determined . Drug susceptibility testing to the main anti-tuberculosis drugs was performed on cultures of Mycobacterium tuberculosis complex isolated from sputum samples of each patient by the indirect proportion method . All patients whose isolates tested resistant to at least one anti-tuberculosis drug were defined as having ADR . RESULTS: Growth of M . tuberculosis complex was obtained from sputum specimens of 98 (88.3%) of the 111 patients studied; 57 (58.2%) of these were resistant to at least one anti-tuberculosis drug . Resistance to isoniazid was the most common (54.1%), followed by resistance to rifampicin (27.6%), streptomycin (25.5%) and ethambutol (12.2%) . Multidrug resistance was observed in 27 (27.6%) of the cases . In a multivariate logistic regression analysis, ADR was significantly associated only with monotherapy use in previous tuberculosis treatment(s) (P = 0.03) . CONCLUSION: The rate of ADR of M . tuberculosis is quite high in Yaounde . Acquired resistance to rifampicin alone or in combination with isoniazid is also high . Monotherapy in previous anti-tuberculosis treatment(s) is a significant predictor of ADR in previously treated patients in Yaounde . These results underscore the urgent need for the re-establishment of a tuberculosis control programme, using the DOTS strategy, in Cameroon.

Int J Tuberc Lung Dis, 2000 May, 4(5), 387 - 94
Redefining MDR-TB transmission 'hot spots'; Becerra MC et al.; Halting further spread of multidrug-resistant tuberculosis (MDR-TB) requires both new resources and a renewed discussion of priority setting informed by estimates of the existing burden of this disease . The 1997 report of the first phase of the global survey by the World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Disease (IUATLD) uses the indicator of the proportion of TB cases that are MDR-TB to identify MDR-TB 'hot spots' . We sought to refine the definition of MDR-TB transmission 'hot spots' . For this purpose, we obtained estimates of two additional indicators for regions where data are available: MDR-TB incidence per 100,000 population per year, and expected numbers of new patients with MDR-TB per year . There is generally much agreement in the three indicators considered, and some differences also appear . We conclude that it is useful, when defining indicators of MDR-TB transmission 'hot spots', to include estimates of underlying TB incidence rates and of absolute numbers of MDR-TB cases . Estimating the force of morbidity of MDR-TB in a population is important for comparing this burden across settings with very different underlying TB incidence rates; estimating the absolute number of MDR-TB patients will be critical for planning the delivery of directly observed MDR-TB therapy and the rational procurement of second-line drugs . Through this exercise, we aim to initiate discussion about improved methods of quantifying and comparing current MDR-TB transmission 'hot spots' that require intervention.

JAMA, 2000 May 17, 283(19), 2537 - 45
Standard short-course chemotherapy for drug-resistant tuberculosis: treatment outcomes in 6 countries; Espinal MA et al.; CONTEXT: No large-scale study has investigated the impact of multidrug-resistant tuberculosis (TB) on the outcome of standard short-course chemotherapy under routine countrywide TB control program conditions in the World Health Organization's (WHO) directly observed treatment short-course strategy for TB control . OBJECTIVE: To assess the results of treatment with first-line drugs for patients enrolled in the WHO and the International Union Against Tuberculosis and Lung Disease's global project on drug-resistance surveillance . DESIGN AND SETTING: Retrospective cohort study of patients with TB in the Dominican Republic, Hong Kong Special Administrative Region (People's Republic of China), Italy, Ivanovo Oblast (Russian Federation), the Republic of Korea, and Peru . PATIENTS: New and retreatment TB cases who received short-course chemotherapy with isoniazid, rifampicin, pyrazinamide, and either ethambutol or streptomycin between 1994 and 1996 . MAIN OUTCOME MEASURE: Treatment response according to WHO treatment outcome categories (cured; died; completed, defaulted, or failed treatment; or transferred) . RESULTS: Of the 6402 culture-positive TB cases evaluated, 5526 (86%) were new cases and 876 (14%) were retreatment cases . A total of 1148 (20.8%) new cases and 390 (44.5%) retreatment cases were drug resistant, including 184 and 169 cases of multidrug-resistant TB, respectively . Of the new cases 4585 (83%) were treated successfully, 138 (2%) died, and 151 (3%) experienced short-course chemotherapy failure . Overall, treatment failure (relative risk {RR}, 15.4; 95% confidence interval {CI}, 10.6-22.4; P<.001) and mortality (RR, 3.73; 95% CI, 2.13-6.53; P<.001) were higher among new multidrug-resistant TB cases than among new susceptible cases . Even in settings using 100% direct observation, cases with multidrug resistance had a significantly higher failure rate than those who were susceptible (9/94 {10%} vs 8/1410 {0.7%}; RR, 16.9; 95% CI, 6.6-42.7; P<.001) . Treatment failure was also higher among patients with any rifampicin resistance (n=115) other than multidrug resistance (RR, 5.48; 95% CI, 3.04-9.87; P<.001), any isoniazid resistance (n=457) other than multidrug resistance (RR, 3 . 06; 95% CI, 1.85-5.05; P<.001), and among patients with TB resistant to rifampicin only (n=76) (RR, 5.47; 95% CI, 2.68-11.2; P<.001) . Of the retreatment cases, 497 (57%) were treated successfully, 51 (6%) died, and 124 (14%) failed short-course chemotherapy treatment . Failure rates among retreatment cases were higher in those with multidrug-resistant TB, with any isoniazid resistance other than multidrug resistance, and in cases with TB resistant to isoniazid only . CONCLUSIONS: These data suggest that standard short-course chemotherapy, based on first-line drugs, is an inadequate treatment for some patients with drug-resistant TB . Although the directly observed treatment short-course strategy is the basis of good TB control, the strategy should be modified in some settings to identify drug-resistant cases sooner, and to make use of second-line drugs in appropriate treatment regimens . JAMA . 2000;283:2537-2545

Int J Oncol, 2000 Jun, 16(6), 1137 - 9
Expression of TRAIL (Apo2L), DR4 (TRAIL receptor 1), DR5 (TRAIL receptor 2) and TRID (TRAIL receptor 3) genes in multidrug resistant human acute myeloid leukemia cell lines that overexpress MDR 1 (HL60/Tax) or MRP (HL60/AR); Kim CH et al.; Previously we have reported a differential expression of CD95/CD95L and Bcl-2 family of genes in multidrug resistant tumor cells . TRAIL, a member of the TNF receptor family, induces apoptosis in many tumor cells by binding to DR4 (TRAIL receptor 1) and DR5 (TRAIL receptor 2) . In contrast, TRAIL-induced apoptosis is prevented by a decoy receptor (DcR1, TRID or TRAIL receptor 3) . In the present study, we compared the expression of TRAIL, DR4, DR5, and TRID between a drug sensitive HL60, a myeloid leukemia cell line, and its multidrug resistant (MDR) sublines that either overexpressed MDR 1 gene (HL60/Tax) or MRP gene (HL60/AR), using RT-PCR . TRAIL mRNA was expressed in HL60 cells but was present in low levels in HL60/AR cells and was completely lacking in HL60/Tax cells . Both DR4 and DR5 were undetectable in HL60/Tax but were present at comparable levels in HL60/AR and drug sensitive HL60 cells . TRID were absent in HL60 and HL60/Tax cells, but was present in low but comparable levels in peripheral blood mononuclear cells and HL60/AR cells . These data suggest that the multidrug resistance in MDR HL60 cell lines, regardless of overexpression of MDR 1 or MRP, may be due to different mechanisms . In HL60/AR cells it appears that MDR may be due to decreased expression of TRAIL and constitutive expression of TRID, whereas in HL60/Tax cells, MDR could be due to the absence of TRAIL and/or DR4 and DR5.

Leuk Lymphoma, 2000 Jun, 38(1-2), 59 - 70
Alternative pathways of cell death to circumvent pleiotropic resistance in myeloma cells: role of cytotoxic T-lymphocytes; Shtil AA et al.; Pleiotropic resistance to treatment remains one of the major reasons for therapeutic failures in patients with multiple myeloma . Myeloma cells are frequently resistant to physiological inducers of cell death prior to chemotherapy . Moreover, in the course of treatment cells acquire a multidrug resistant (MDR) phenotype, making eradication of the tumor even more difficult . A necessary prerequisite for circumventing complex pleiotropic resistance is therefore defining the signaling pathways that execute death in myeloma cells . This review discusses evidence that cytokine-expressing autologous tumor cell vaccine may be an efficient tool for elimination of both intrinsically resistant myeloma cells as well as cells with acquired MDR in murine models . The vaccine was similarly potent against wild type cells that were resistant to several death receptor ligands, and their isogenic sublines selected for P-glycoprotein-mediated MDR . The anti-myeloma effect of the vaccine was mediated by granzyme B/perforin-secreting cytotoxic T-lymphocytes . This is an example of therapeutic strategy directed at utilizing death pathways that are preserved in pleiotropically resistant tumor cells.

Leuk Lymphoma, 2000 Jun, 38(1-2), 1 - 11
A role for P-glycoprotein in regulating cell death; Johnstone RW et al.; P-glycoprotein (P-gp) is an energy dependent drug pump responsible for multidrug resistance (MDR) in human cancers . While it is irrefutable that P-gp can efflux xenobiotics out of cells, the biological function of P-gp in multicellular organisms has yet to be firmly established . The question of what, if anything, P-gp does when not effluxing drugs has been raised by recent reports indicating that P-gp may regulate apoptosis, chloride channel activity, cholesterol metabolism and immune cell function . There is now a lively debate regarding the possible role of P-gp in regulating cell differentiation, proliferation and survival.

What Is Yeast?, What Is Cell Biology?, What is Food Microbiology?, What Is Activated Sludge?, What Is Environmental Microbiology?, r, Bacteria, s, Microorganisms, c, Microbe, s, Bacterium, c, Microorganism, i, Ps. putida, n, Bacillus subtilis, n, Vibriosis, o, E coli O157, i, Bacillus, a, Bacillus, o, Antibiotics, n, Microorganisms, o, Thermophiles, r, Growth media, i, Multidrug resistant, n, Escherichia coli, a, Clostridia, r, Haemophilus, e, Pseudomonas aeruginosa, o, Bacillus, o, Sepsis, r, Antibiotics, o, Escherichia coli, o, Cellulomanas, o, Enterobacters

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005