AIDS, 1997 Aug, 11(10), 1237 - 42 Transmission between HIV-infected patients of multidrug-resistant tuberculosis caused by Mycobacterium bovis; Samper S et al.; OBJECTIVE: To investigate outbreaks of multidrug-resistant tuberculosis (TB) by using DNA fingerprint databases . DESIGN: Investigation of two outbreaks of multidrug-resistant TB in separate hospitals in Spain by restriction fragment length polymorphism (RFLP) and spoligotyping . Outbreak strains were compared with more than 1500 RFLPs of Mycobacterium tuberculosis complex strains isolated in Spain and 6000 RFLPs from 30 different countries . METHODS: Standardized IS6110 DNA fingerprinting and 'spoligotyping' was used to type multidrug-resistant isolates belonging to the M . tuberculosis complex amongst the outbreak cases . The DNA types were matched against DNA fingerprint databases in Spain and The Netherlands . RESULTS: The DNA typing analysis indicated that a single multidrug-resistant Mycobacterium bovis strain was responsible for a nosocomial outbreak in a hospital in Spain involving at least 16 HIV-infected patients with non-treatable to multidrug-resistant TB . Introduction of the fingerprint type of this strain to the international database revealed a single matching strain . This strain was also isolated from an HIV-infected patient in The Netherlands who had died from multidrug-resistant TB . This patient had previously been hospitalized in Spain, where a multidrug-resistant TB nosocomial outbreak involving 20 HIV-infected patients was ongoing . The strains causing this outbreak were also identified as M . bovis with an identical DNA pattern to those strains isolated in the Spanish hospital and the patient in The Netherlands . CONCLUSIONS: The use of centralized DNA databases can help to identify rapidly the origin and transmission routes of multidrug-resistant TB across international boundaries and the potential use of such an early warning surveillance system for investigation of nosocomial multidrug-resistant TB outbreaks between HIV-infected patients . To our knowledge this is the first report of transmission of multidrug-resistant M . bovis between hospitals. Am J Clin Oncol, 1997 Aug, 20(4), 398 - 403 Multidrug-resistant gene expression in small-cell lung cancer; Savaraj N et al.; The development of drug resistance can contribute to treatment failure in small-cell lung cancer (SCLC) . In this report, we investigate p-glycoprotein-mediated multidrug resistance (MDR) in these patients . Tumor tissue was obtained prior to treatment and at relapse if possible, short-term culture was carried out, and these tumor cells were analyzed for MDR gene expression by slot blot and reverse transcriptase polymerase chain reaction (RT-PCR) and northern blot analysis . Three cell lines were also established from short-term cultures . Twenty-four patients with MDR(-) and seven with MDR +(++) were available for survival analysis . Median survival for MDR (-) patients was 10 months, whereas for MDR +(++) patients it was 2 months . This was statistically significance (p < 0.0007) . The presence of MDR1 gene expression also correlated with the lack of response to chemotherapy (p < 0.001) . Increased MDR1 gene expression is usually present in patients with more tumor burden at initial diagnosis . Furthermore, loss of MDR1 gene expression can occur in intrinsically MDR(+) SCLC cells after multiple passages in drug-free media . We concluded that increased MDR1 gene expression is present in a small number of SCLC both before and after chemotherapy and usually signifies poor survival and no response to chemotherapy. Cancer Res, 1997 Aug 1, 57(15), 3208 - 13 Mechanism of action of E7010, an orally active sulfonamide antitumor agent: inhibition of mitosis by binding to the colchicine site of tubulin; Yoshimatsu K et al.; E7010 (N-{2-{(4-hydroxyphenyl)amino}-3-pyridinyl}-4-methoxybenzenesulfonami de), an orally active sulfonamide antitumor agent that is currently in a Phase I clinical trial, showed rather consistent growth-inhibitory activities against a panel of 26 human tumor cell lines (IC50 = 0.06-0.8 microg/ml), in contrast to vincristine (VCR; IC50 = 0.0002-0.04 microg/ml), 5-fluorouracil (IC50 = 0.2-30 microg/ml), Adriamycin (IC50 = 0.002-0.7 microg/ml), mitomycin C (IC50 = 0.007-3 microg/ml), 1-beta-D-arabinofuranoxylcytosine (IC50 = 0.005 to >30 microg/ml), camptothecin (IC50 = 0.002-0.4 microg/ml), and cisplatin (IC50 = 0.5-20 microg/ml) . It caused a dose-dependent increase in the percentage of mitotic cells in parallel with a decrease in cell proliferation, like VCR . It also showed a dose-dependent inhibition of tubulin polymerization, which correlated well with the cell growth-inhibitory activity . 14C-labeled E7010 bound to purified tubulin, and this binding was inhibited by colchicine but not by VCR . However, its binding properties were different from those of colchicine, as well as those of VCR . E7010 was active against two kinds of VCR-resistant P388 cell lines, one of which showed multidrug resistance due to the overexpression of P-glycoprotein (resistant to Taxol), and the other did not show multidrug resistance (sensitive to Taxol) . Furthermore, four E7010-resistant P388 cell lines showed no cross-resistance to VCR, a different pattern of resistance to podophyllotoxin, and collateral sensitivity to Taxol . Therefore, E7010 is a novel tubulin-binding agent that has a wider antitumor spectrum than VCR and has different properties from those of VCR or Taxol. Biochem Biophys Res Commun, 1997 Jul 30, 236(3), 586 - 90 Reversal of heavy metal resistance in multidrug-resistant human KB carcinoma cells; Chen ZS et al.; Human KB carcinoma C-A120 cells that express multidrug resistance-associated protein (MRP) were cross-resistant to trivalent and pentavalent antimonials and arsenicals . Intracellular glutathione (GSH) content was higher in C-A120 than its parental KB-3-1 cell line . Glutathione-S-transferase (GST) was similar in both cell lines . Depletion of cellular GSH by treatment of the cells with the inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS), buthione sulfoximine (BSO), significantly increased the sensitivity of both KB-3-1 and C-A120 cells to heavy metals . A pyridine analog, PAK-104P, almost completely reversed the resistance to antimonials and arsenicals in C-A120 cells . BSO at 100 microM or PAK-104P at 10 microM enhanced the accumulation of antimony potassium tartrate in C-A120 cells to the level of that in KB-3-1 cells without the agents . PAK-104P inhibited the ATP-dependent efflux of antimony potassium tartrate . These findings suggest that MRP transports antimony conjugated with GSH ATP-dependently outside the cells and PAK-104P inhibits the transporting activity of MRP. Biochemistry, 1997 Jul 22, 36(29), 8663 - 70 Binding of two novel bisdaunorubicins to DNA studied by NMR spectroscopy; Robinson H et al.; In the search for new generations of anthracycline drugs, lower cytotoxic side effects and higher activity against resistant cancer cells are two major goals . A new class of bis-intercalating anthracycline drugs has been designed, synthesized, and shown to have promising activity against multidrug-resistant cells . Two daunorubicins symmetrically linked together via a p-xylenyl group, either at their N3' (compound WP631) or N4' sites (compound WP652), exhibit extraordinary DNA binding affinities . We have used high-resolution NRM studies to understand the DNA binding mode of these two new bis-daunorubicin anticancer compounds . The structures of the WP631-d(ACGTACGT)2 and the WP652-d(TGTACA)2 complexes have been determined by NOE-restrained refinement . WP631 binds strongly to the 5'-CG(A/T)(A/T)CG hexanucleotide sequence, with the aglycons intercalated between the two CpG sites at both ends of the hexanucleotide sequence . The overall conformation of the WP631-d(CGTACG)2 part is remarkably similar to the crystal structure of the 2:1 complex of daunorubicin and d(CGTACG)2, as predicted previously {Gao, Y.-G., & Wang, A.H.H . (1996), J . Biomol . Struct . Dyn . 13, 103-117} . In contrast, the related bis-intercalator WP652 prefers the 5'-PyGTPu tetranucleotide sequence, with the aglycons intercalated between the PypG and TpPu sites . The binding of WP652 to DNA results in a severely distroted B-DNA duplex with the p-xylenyl tether moiety significantly protruded away from the bottom of the minor groove . While WP652 in some ways behaves similarly to other anticancer bis-intercalating antibiotics (e.g., triostine A and echinomycin), the detailed interactions between those two classes of bis-intercalators are quite different. FEBS Lett, 1997 Jul 21, 412(1), 201 - 6 MDR-1 gene expression is a minor factor in determining the multidrug resistance phenotype of MCF7/ADR and KB-V1 cells; Kim HM et al.; The relevance of MDR-1 gene expression to the multidrug resistance phenotype was investigated . Drug-resistant cells, KB-V1 and MCF7/ADR, constantly expressed mRNA of the MDR-1 gene and were more resistant to vinblastine and adriamycin than drug-sensitive cells, KB-3-1 and MCF7 . The drug efflux rate of KB-V1 was the same as KB-3-1 although the MDR-1 gene was expressed in only the resistant cell . The higher intracellular drug concentration of KB-3-1 than KB-V1 was due to the large drug influx . In the case of MCF7 and MCF7/ADR, the influx and efflux of the drug had nearly the same pattern and drug efflux was not affected by verapamil . The amount of ATP, cofactor of drug pumping activity of P-glycoprotein, was not changed by the resistance . These observations suggested that drug efflux mediated by MDR-1 gene expression was not a major determining factor of drug resistance in the present cell systems, and that the drug resistance could be derived from the change in drug uptake and other mechanisms. Lancet . 1997 Jul 19;350(9072):192. Beating the malaria parasite at its own game; O'Brien C; PIP: Currently 40% of the world's population is still at risk of becoming infected with malaria, which is an even more worrisome disease because of drug resistance and multidrug resistance . Research on drug resistance is suggesting new ways to attack the parasite by designing selectively toxic compounds . At Washington University, a study has been centering on how malarial parasites developed resistance to antimalarial drugs . A new class of compounds have been developed that block haem polymerization to haemazoin and kill the parasite . These hexadentate metal complexes that bypass the current resistance mechanisms are made from aspirin and are being tested in animals . A new resistance gene also been identified that transports chloroquine out of the parasite or blocks the drug's influx, leaving the parasite unharmed . In addition, a gene associated with mefloquine resistance was isolated at Harvard University . A large number drug transporter inhibitors have also been identified, but none is sufficiently selective for the parasite's transporter . In Oxford, UK, recently, the low-resolution structure of the human multidrug resistant P-glycoprotein was also solved, which might permit the design of modified antimalarial drugs . Exploiting the differences between parasite and human enzymes may also provide new drug targets . The crystal structure of a complex between plasmepsin, a parasite aspartate protease that initiates the digestion of hemoglobin, and an inhibitor has also been revealed . The crystal structure of lactate dehydrogenase (LDH) from Plasmodium falciparum has also been determined finding a big difference around the active site of the malarial enzyme and mammalian LDH . Parasite enzymes are being researched by other scientists elsewhere . At the University of Montpellier, France, the work on the parasite's choline carrier is more advanced . One of three compounds that kill parasites will be chosen for clinical studies . Biochem Biophys Res Commun, 1997 Jul 18, 236(2), 483 - 8 Overexpression of a 40-kDa protein in human multidrug resistant cells; Wang Y et al.; The use of anticancer drugs in the chemotherapeutic treatment of cancer patients frequently results in the emergence of drug resistant tumors . Selection of tumor cell lines in vitro has led to the identification of several proteins that mediate drug resistance to anticancer drugs . In this study, an immuno-dot blot method was used to isolate a monoclonal antibody (IPM96) which recognized a 40 kDa protein (or P-40) co-expressed with P-glycoprotein and MRP in several multidrug resistant cell lines (MCF-7/Adr, SKOV/VLB1.0, H69/Adr, and HL60/AR) . Furthermore, P-40 levels dropped significantly in one revertant cell line (H69/PR) derived from H69/AR cells . Interestingly, the expression of P-40 was also higher in two tumor cell lines (SKTax6a and A2780CP) that were selected with paclitaxel or cisplatin but do not express P-gp or MRP . Immuno-fluorescence staining of cells with IPM96 showed both membrane and cytoplasmic staining . These results were confirmed by Western blot analysis of different subcellular fractions from MCF-7/Adr cells . The membrane bound P-40 was resistant to extraction with high salt, chelating agents, and denaturing agents, but was solubilized with 10 mM CHAPS . Taken together, the overexpression of P-40 in multidrug resistant cells has not been previously determined and therefore could be important in the expression of the drug resistance phenotype. Int J Cancer, 1997 Jul 17, 72(2), 295 - 300 Overcoming CPT-11 resistance by using a biscoclaurine alkaloid, cepharanthine, to modulate plasma trans-membrane potential; Aogi K et al.; Irinotecan, 7-ethyl-10-{4-(1-piperidino)-1-piperidino} carbonyloxycamptothecin, (CPT-11) resistance was overcome by using a biscoclaurine alkaloid, cepharanthine, in CPT-11- and multidrug-resistant 50MT-1 cells . 50MT-1 cells were established from a mouse breast-cancer cell line, FM3A, by subjecting the cells to a low dose of CPT-11 continuously . 50MT-1 cells exhibited resistance to CPT-11 (40-fold in colony-formation assay) and to other drugs such as doxorubicin (11.7-fold) and etoposide (VP-16) (16.8-fold) . The plasma trans-membrane potential was lower in 50MT-1 cells than in FM3A cells, although there were no differences in expressions of P-glycoprotein and of DNA topoisomerase-I and -II proteins . The lower membrane potential in 50MT-1 cells was augmented by co-treatment with a non-toxic dose of cepharanthine . CPT-11 resistance in 50MT-1 cells was overcome (5.0- to 1.4-fold, 6-hr exposure) by the co-treatment with cepharanthine through increasing intracellular accumulation of CPT-11 . Resistance to doxorubicin and VP-16 was also overcome by cepharanthine treatment (2.5- to 0.69-fold and 4.2- to 1.4-fold respectively) . We conclude that the modification of plasma trans-membrane potential by cepharanthine should be effective in overcoming CPT-11 and multidrug resistance in 50MT-1 cells. Int J Cancer, 1997 Jul 3, 72(1), 155 - 9 Mechanism of resistance to cisplatin in a human ovarian-carcinoma cell line selected for resistance to doxorubicin: possible role of p53; Vikhanskaya F et al.; A possible novel mechanism of cross-resistance to cisplatin (CDDP) in the doxorubicin-resistant ovarian-cancer cell line A2780-DX3, which displays atypical multidrug resistance, is presented . A2780-DX3 is found to be more resistant than the parental line A2780 in terms of CDDP-induced cytotoxicity and apoptosis . Resistance is not related to the amount of cross-links . Topoisomerase-II (topII) protein levels were similar in both cell lines, with lower cleavage activity in A2780-DX3 cells . The parental and the doxorubicin-resistant cells expressed the same level of c-erb2, which could be implicated in CDDP resistance . bcl2 was almost undetectable in both cell lines . At the same time, we found strong induction of p53, waf-1 and bax protein levels after CDDP treatment in the A2780, but not in the A2780-DX3, cell line . Treatment of both cell lines with mitomycin C (MMC), which acts with a mechanism different from CDDP, caused equal accumulation of p53 and induction of bax . We found that A2780-DX3 cells exhibit altered cellular localization of p53 protein in comparison with A2780 . A significant proportion of p53 in A2780-DX3 cells was found in the cytoplasmic compartment, and CDDP treatment induced a functional p53 protein in the nucleus of A2780 much more strongly than in A2780-DX3, which coincides with an increase of transcriptional activity of p53 in treated A2780 cells . We propose that the cross-resistance to CDDP in the A2780-DX3 cell line may be due to inactivation of a CDDP-dependent p53-accumulation pathway. J Natl Cancer Inst, 1997 Jul 2, 89(13), 917 - 31 Multidrug resistance in breast cancer: a meta-analysis of MDR1/gp170 expression and its possible functional significance; Trock BJ et al.; BACKGROUND: P-glycoprotein (gp170; encoded by the MDR1 gene {also known as PGY1}) is a membrane protein capable of exporting a variety of anticancer drugs from cells . MDR1/gp170 expression has been studied in breast cancer, but the prevalence of this expression and its role in breast tumor drug resistance are unclear . PURPOSE: We conducted a critical review and meta-analysis of studies examining MDR1/gp170 expression in breast cancer to estimate the likely prevalence and clinical relevance of this expression . We also explored reasons for differences in the findings from individual studies . METHODS: Published papers on MDR1/gp170 expression in breast cancer were identified by searching several literature databases and reviewing the bibliographies of identified papers . Variability across the studies in the proportion of tumors expressing MDR1/gp170 was assessed by use of chi-squared tests of homogeneity, weighted means, and weighted linear regression . Pooled relative risks (RRs) for the association between the induction of MDR1/gp170 expression and prior chemotherapy and associations between MDR1/gp170 expression and several clinical outcomes were estimated by use of Mantel-Haenszel methods . Heterogeneity among the pooled RRs was explored by use of chi-squared tests . Reported P values are two-sided . RESULTS: Thirty-one studies were identified and evaluated . The proportion of breast tumors expressing MDR1/gp170 in all of the studies was 41.2%, but there was substantial heterogeneity in the values across individual studies (P<.0001) . Regression analyses demonstrated that a considerable portion of the observed heterogeneity was a consequence of the change, over time, from RNA hybridization-based assays to immunohistochemistry-based assays of MDR1/gp170 expression . Measuring MDR1/gp170 expression before versus after chemotherapy and use of cytotoxic drugs that are not substrates for gp170 also contributed to the heterogeneity . Treatment with chemotherapeutic drugs or hormonal agents was associated with an increase in the proportion of tumors expressing MDR1/gp170 (RR = 1.77; 95% confidence interval {CI} = 1.46-2.15) . Patients with tumors expressing MDR1/gp170 were three times more likely to fail to respond to chemotherapy than patients whose tumors were MDR1/gp170 negative (RR = 3.21; 95% CI = 2.28-4.51); this RR increased to 4.19 (95% CI = 2.71-6.47) when considering only patients whose tumor expression of MDR1/gp170 was measured after chemotherapy . MDR1/gp170 expression was not associated with lymph node metastases, estrogen receptor status, tumor size, tumor grade, or tumor histology . CONCLUSIONS AND IMPLICATIONS: MDR1/gp170 expression in breast tumors is associated with treatment and with a poor response to chemotherapy . The data are consistent with a contributory role for MDR1/gp170 in the multidrug resistance in some breast tumors. Emerg Infect Dis, 1997 Jul-Sep, 3(3), 373 - 4 Multidrug-resistant enteroaggregative Escherichia coli associated with persistent diarrhea in Kenyan children; Sang WK et al.; To study the association of multidrug-resistant enteroaggregative Escherichia coli with persistent diarrhea in Kenyan children, stool specimens were obtained from 862 outpatients under 5 years of age from July 1991 to June 1993 . E . coli O44 was identified as the sole bacterial pathogen in four patients experiencing at least 14 days of fever, vomiting, and diarrhea . Disk diffusion testing showed E . coli O44 resistance to tetracycline, ampicillin, erythromycin, trimethoprim-sulphamethoxazole, and amoxicillin/clavulanate and sensitivity to chloramphenicol, nalidixic acid, azithromycin, and cefuroxime . Further studies are needed to clarify the epidemiology, clinical spectrum, and pathogenesis of enteroaggregative E . coli infection. Emerg Infect Dis, 1997 Jul-Sep, 3(3), 253 - 9 Recombination in HIV: an important viral evolutionary strategy; Burke DS; Human immunodeficiency virus (HIV) is a diploid virus: each virion carries two complete RNA genomic strands . Homologous recombination can occur when a cell is coinfected with two different but related strains . Naturally occurring recombinant HIV strains have been found in infected patients in regions of the world where multiple genotypic variants cocirculate . One recombinant HIV strain has spread rapidly to millions of persons in Southeast Asia . Recombination is a mechanism whereby high level and multidrug-resistant strains may be generated in individual treated patients . Recombination also poses theoretical problems for the development of a safe HIV vaccine . Certain features of HIV replication, such as syncytium formation and transactivation, may be best understood as components of a sexual reproductive cycle . Recombination may be an important HIV evolutionary strategy. DNA Cell Biol, 1997 Jul, 16(7), 807 - 18 Transcriptional regulation of the murine multidrug resistance gene mdr1b by progesterone occurs via an indirect mechanism; Mallick S et al.; The murine multidrug resistance gene mdr1b is highly induced in the endometrium during pregnancy . Evidence suggests that induction occurs mainly as a result of progesterone action . To study the molecular mechanisms involved in this induction, 5'-flanking sequences between -540 and +97 of the mdr1b gene were fused to the reporter gene, bacterial chloramphenicol acetyltransferase (p540CAT) . Unlike most progesterone-responsive genes, mdr1b is preferentially activated by the A form of the progesterone receptor . We now report that activation is not observed with a DNA-binding domain mutant of progesterone receptor A (PRA) suggesting that induction occurs at the transcriptional level . Time course experiments demonstrated that induction was first observed 12 hr after hormone addition, suggestive of a secondary (or late) response gene . Sequence comparison highlighted the region M1 (-234 to -206), which contains a partially conserved progesterone response element . Its functional significance was evaluated by expression assays and gel shift analysis . Reporter plasmids with modifications of this element were transfected into HeLa cells . Constructs containing the native M1 element, or a mutated element (M1mt) that eliminated any similarity to a progesterone response element, were induced four-fold by progesterone whereas an element containing a consensus progesterone response element (M1PRE) was induced eight-fold . In addition, by gel shift analysis, the M1 element did not bind the progesterone receptor or any other factors . This suggested that the M1 region does not participate in the response to progesterone . 5' Nested deletion analysis, used to identify other regions of the upstream regulatory region that contributed to induction by progesterone, demonstrated that enhancer sequences between -122 and -65, which contain binding sites for C/EBPbeta and NF-Y, were important . Mutations in the binding sites for these factors decreased induction by progesterone . On the basis of our studies using 540 bp of upstream sequence, mdr1b is activated transcriptionally by progesterone, in an indirect manner dependent on basal factors. Eur J Haematol, 1997 Jul, 59(1), 14 - 9 Cytotoxic T-lymphocytes recognizing P-glycoprotein in murine multidrug-resistant leukemias; Azuma E et al.; A multidrug-resistant murine lymphoid leukemia P388/ADR overexpresses P-glycoprotein (P-gp), an active transporter that pumps cytotoxic drugs out of cells and a product of mdr1 gene . Cytotoxic T lymphocytes (CTL) that showed cytotoxicity against P388/ADR were generated from mixed lymphocyte tumor cell culture . CTL do not kill drugsensitive parental P388 (P388/parent) that does not express P-gp . Monoclonal antibody against P-gp inhibited cytotoxic activity . Similar results were obtained in another multidrug-resistant cell line P388/VP-16 . Cytotoxic activity was mediated by Thy1+ CD4- CD8+ T-cells . When P388/ADR was treated with murine IL-4, expression of P-gp was downregulated . Monoclonal antibody against interleukin-4 (IL-4) abrogated the IL-4-induced suppression of P-gp . Cytolytic activity of CTL against IL-4-treated P388/ADR was dose dependently inhibited . These results suggest that P-gp is immunogenic and can be a target of CTL in this murine leukemia model. Curr Opin Hematol, 1997 Jul, 4(4), 256 - 60 Recent advances in the treatment of acute leukemia; Berman E; This review briefly summarizes literature considered noteworthy in the field of adult acute leukemia published during 1996 . Does intensity remains a controversial issue in both acute myelogenous and lymphoblastic leukemia . The most convincing data showing efficacy of high dose fractionated chemotherapy was presented in patients with Burkitt's lymphoma/leukemia; the remainder of clinical studies failed to show a definitive advantage to high-dose therapy . Numerous studies addressed the role of the multidrug resistant phenotype and, at least in adult disease, demonstrated that the presence of this particular phenotype was a poor prognostic indicator . In the pediatric population, the significance of multidrug resistance expression appeared less clear . Discrepancies between protein expression and function were also evaluated in clinical samples and outcomes reported in large clinical series . Among the most interesting scientific investigations were those focused on the molecular mechanisms involved in the specific translocations t(15;17) and t(8;21) in acute myelogenous leukemia and t(12;21) in acute lymphoblastic leukemia . The genes PML and AML1, and ETO were examined in normal hematopoietic progenitors and their fusions proteins, PML/RAR alpha and AML1/ETO, measured in patients in clinical remission, and important data were presented concerning these proteins and measurement of minimal residual disease . Provocative data were also presented suggesting that retinoic acid may induce synthesis of a protein that selectively degrades PML/RAR alpha, and that interferons may regulate PML/RAR alpha expression. Anticancer Res, 1997 Jul-Aug, 17(4A), 2583 - 6 P-glycoprotein mediated multidrug resistance assessment by flow-cytometry in malignant hemopathies; Tatu CA et al.; A relatively common and frequent form of multidrug resistance(MDR) in cancer cells is due to membrane overexpression of P-glycoprotein . Mdr phenotype was investigated by flow-cytometry in several types of malignant hemopathies -chronic lymphocytic leukemia, non-Hodgkin lymphomas, acute lymphoblastic and myeloblastic leukemias . We used daunomycin and fluo-3 as fluorochromes, and verapamil as reversor agent . The method is lacking unitary clinical parametrization and in order to improve it, we tried to establish an optimal concentration of verapamil, which was shown to be 14.92 micrograms/ml . The reliability of results obtained with fluo-3 in culture media containing Ca2+ is questionable, as low variations in the intracellular level of this ion dramatically influences light emission by the fluorochrome and possibly the function of P-gp . To avoid such fluorescence intensity variations, Ca(2+)-free cell culture medium for fluo-3-based flow-cytometric assay is suggested to be used. Anticancer Res, 1997 Jul-Aug, 17(4A), 2435 - 41 Anticancer activities of 2,5,8,9-substituted 6-oxo-1,2,3,4,5,6-hexahydrophenanthridines on multi-drug-resistant phenotype cells; Hua DH et al.; Lycorine is a member of the alkaloid group from the bulb of the amaryllidaceae . This drug has been reported to have anticancer activity . Several synthetic intermediates obtained during the synthetic study of anticancer drugs based on the lycorine structure, were tested for anticancer activity using three cell lines: L1210 and HL60 cell lines which were resistant (R) or sensitive (S) to adriamycin . The two synthetic intermediates, 2-acetoxy- and 2-hydroxy-5-allyl-8,9-methylenedioxy-6-oxo-1,2,3,4,5,6-hexahydr ophenanthridine (1 and 2), both had anticancer activity in all three cell lines . However, the LD50 for the precursors was about 20 fold greater than for the native lycorine . Both 1 and 2 were cytotoxic to the adriamycin-resistant cell line, indicating that these drugs are not affected by the multidrug resistance factors . When low doses of the compounds were used, the HL60R cell line could be induced to differentiate to a cell which expressed a macrophage specific protein . These results suggest that phenanthridines 1 and 2 can be used on cells which are resistant to adriamycin, and that one mechanism of action is the induction of differentiation. Am J Physiol, 1997 Jul, 273(1 Pt 1), L201 - 10 Protein kinase C and Ca2+ activation of mucin secretion in airway goblet cells; Abdullah LH et al.; Airway goblet cells secrete mucin in response to ATP and uridine 5'-triphosphate (UTP), but the underlying signal transduction pathways are poorly understood . Cultures of SPOC1 cells (L . H . Abdullah, S . W . Davis, L . Burch, M . Yamauchi, S . H . Randell, P . Nettesheim, and C . W . Davis . Biochem . J . 316: 943-951, 1996) secreted mucin on exposure to phorbol 12-myristate 13-acetate (PMA) {apparent affinity (K0.5) approximately 100 nM} and ionomycin (K0.5 approximately 5 microM) almost fivefold over baseline . Thapsigargin also elicited secretion (K0.5 approximately 20 nM) . Ionomycin and PMA together elicited approximately twice the secretion of either agent alone . Overnight exposure to half-maximal PMA abolished the response to maximal doses of UTP and PMA, whereas ionomycin was fully effective . Protein kinase C (PKC) activity in the membrane fraction was increased by maximal doses of PMA and UTP, whereas ionomycin had no effect . PKC inhibitors were relatively ineffective against PMA- and UTP-induced mucin secretion . Human and canine goblet cells in epithelial explants, by video microscopy, underwent exocytosis with ionomycin (1 microM) and PMA (0.1 or 1 microM) . SPOC1 cell mucin secretion was not stimulated by forskolin, 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate, or 8-bromoguanosine 3',5'-cyclic monophosphate . Cystic fibrosis transmembrane conductance regulator was not detected in SPOC1 cells by Western blotting, and its mRNA was detected by reverse transcriptase polymerase chain reaction (PCR) only as a very weak band and after 55 PCR cycles . Multidrug resistance (MDR1), however, was readily detected by Western blotting, and its mRNA was detected as a major band after 35 PCR cycles . Thus airway goblet cell mucin secretion, distal to receptor activation, may be regulated independently by Ca(2+)- and PKC-dependent pathways . Cystic fibrosis transmembrane conductance regulator and cyclic nucleotides, however, may not play a major role in this secretion. J Interferon Cytokine Res, 1997 Jul, 17(7), 419 - 23 Poly ICLC enhances the antimalarial activity of chloroquine against multidrug-resistant Plasmodium yoelii nigeriensis in mice; Awasthi A et al.; Swiss mice infected with multidrug-resistant Plasmodium yoelii nigeriensis were treated with polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethyl cellulose (Poly ICLC), a potent interferon (IFN) inducer and immune enhancer, in combination with chloroquine (CQ), which completely eliminated the malaria parasite from these animals . The enhancement of the antimalarial activity of poly ICLC was found to be completely reversed by the cytochrome P-450 inducer, phenobarbitone . No effect of Nw nitro-L-arginine (NLA), an inhibitor of nitric oxide, was seen on the enhancement of the antimalarial activity of CQ by Poly ICLC . These results suggest the possible involvement of cytochrome P-450 enzyme-mediated mechanism in the enhancement of the antimalarial activity of CQ by Poly ICLC. Br J Urol, 1997 Jul, 80(1), 11 - 7 Is the expression of multidrug resistance gene product a prognostic indicator for the clinical outcome of patients with renal cancer? Hofmockel G, Bassukas ID, Wittmann A, Dammrich J. OBJECTIVE: To determine the significance of the expression of the multidrug resistance gene product (MDR-1) for the aggressiveness of renal cell carcinoma (RCC) . PATIENTS AND METHODS: The study comprised 31 patients with clinically locally confined RCC treated with radical nephrectomy (mean age 64.1 years, range 41-78; mean follow-up 5.4 years, range 4.3-7.3) . Their survival time and disease-free period were evaluated retrospectively . The expression of the MDR-1 gene product was determined immunohistochemically using the JSB-1 antibody in formalin-fixed paraffin-embedded tumour samples from these patients . The significance of this variable and tumour stage and malignancy grade was assessed for predicting the survival and disease-free period using Kaplan-Meier plots (log-rank test or Tarone's test) and the Cox multiple hazard regression analysis . RESULTS: In a univariate analysis, tumour stage (P < 0.002), malignancy grade (P < 0.007) and MDR-1 (P < 0.03) were significant prognostic variables for both survival and disease-free period . Lower MDR-1 expression was correlated with poorer prognosis . On multivariate analysis, MDR-1 and tumour stage were significant factors for predicting the disease-free period, whereas tumour stage and malignancy grade were the most relevant factors for survival time . Calculating prognostic indices based on the results of the Cox analysis, MDR-1 could replace malignancy grade, resulting in a better prediction of survival and disease-free period (P < 0.001 vs 0.0031, P < 0.001 vs 0.021, respectively) . CONCLUSION: MDR-1, an established predictor for chemo-resistance, may also be a potent prognostic factor for outcome in patients with locally confined RCC . Moreover, MDR-1 expression seems to correlate with the differentiation of the RCC and thus its value as an objective measure of the degree of differentiation should be further explored. Chest, 1997 Jul, 112(1), 63 - 70 A cost-effectiveness analysis of directly observed therapy vs self-administered therapy for treatment of tuberculosis; Burman WJ et al.; STUDY OBJECTIVES: To compare the costs and effectiveness of directly observed therapy (DOT) vs self-administered therapy (SAT) for the treatment of active tuberculosis . DESIGN: Decision analysis . SETTING: We used published rates for failure of therapy, relapse, and acquired multidrug resistance during the initial treatment of drug-susceptible tuberculosis cases using DOT or SAT . We estimated costs of tuberculosis treatment at an urban tuberculosis control program, a municipal hospital, and a hospital specializing in treating drug-resistant tuberculosis . OUTCOME MEASURES: The average cost per patient to cure drug-susceptible tuberculosis, including the cost of treating failures of initial treatment . RESULTS: The direct costs of initial therapy with DOT and SAT were similar ($1,206 vs $1,221 per patient, respectively), although DOT was more expensive when patient time costs were included . When the costs of relapse and failure were included in the model, DOT was less expensive than SAT, whether considering outpatient costs only ($1,405 vs $2,314 per patient treated), outpatient plus inpatient costs ($2,785 vs $10,529 per patient treated), or outpatient, inpatient, and patients' time costs ($3,999 vs $12,167 per patient treated) . Threshold analysis demonstrated that DOT was less expensive than SAT through a wide range of cost estimates and clinical event rates . CONCLUSION: Despite its greater initial cost, DOT is a more cost-effective strategy than SAT because it achieves a higher cure rate after initial therapy, and thereby decreases treatment costs associated with failure of therapy and acquired drug resistance . This cost-effectiveness analysis supports the widespread implementation of DOT. J Nucl Med, 1997 Jul, 38(7), 1003 - 8 Clinical validation of the influence of P-glycoprotein on technetium-99m-sestamibi uptake in malignant tumors; Kostakoglu L et al.; We prospectively studied 48 patients with either breast cancer (30 patients) or lung cancer (18 patients) to ascertain the relationship between the degree of accumulation of 99mTc-sestamibi and the expression of p-glycoprotein in tumor tissues . METHODS: During initial presentation (37 patients) or post-therapy evaluation (11 patients), the patients underwent contemporaneous 99mTc-sestamibi imaging and biopsy (30 patients) or surgery (18 patients) . The interval between surgery/biopsy and imaging varied between 3 and 15 days . All patients had radiologically detectable tumors . Immunohistochemical studies were performed on paraffin sections using a monoclonal antibody, JSB-1, developed against the internal epitope of p-glycoprotein . Tumor-to-background ratios were correlated with the level of p-glycoprotein expression determined by immunohistochemical studies . RESULTS: Our results showed an inverse correlation between the tumor-to-background ratios of 99mTc-sestamibi and the density of p-glycoprotein expression in tumor tissues . The values for the tumor-to-background ratios were significantly lower for those tumors expressing p-glycoprotein at high levels than those with scattered and no expression (p < 0.01 and p < 0.001, respectively) . CONCLUSION: Although our results warrant further studies at the molecular level using PCR techniques after the extraction of mRNA, our data strongly suggest that 99mTc-sestamibi imaging is useful to noninvasively determine the presence of multidrug resistance in patients with malignant tumors. Am J Respir Cell Mol Biol, 1997 Jul, 17(1), 51 - 9 Interaction with type II estrogen binding sites and antiproliferative activity of tamoxifen and quercetin in human non-small-cell lung cancer; Caltagirone S et al.; The antiestrogen tamoxifen is thought to antagonize the effects of estrogens by competing with them for estrogen receptor (ER) binding . However, tarnoxifen can also reverse multidrug resistance, synergize with cisplatin cytotoxicity, and inhibit growth in ER-negative lung cancer cells . In addition to ERs, rat and human target tissues contain a second binding macromolecule termed the type II estrogen binding site (type II EBS) . It has been shown that tamoxifen and flavonoids, a widely distributed class of natural substances with a variety of biologic actions, bind to type II EBS and inhibit the growth of several tumor cell types . At present, conflicting data about ERs and an absence of data about type II EBSs exist for lung tumors . We have tested non-small-cell lung carcinoma cell lines and primary tumor cells for the presence of ERs and type II EBSs and have evaluated the effects of tamoxifen and quercetin (pentahydroxyflavone) on the growth of these cells . Using a whole-cell assay and nuclear and cytosolic radiobinding experiments with {3H}estradiol as tracer, we have found that SK-LU1, SW900, ChaGo-K-1, H441, H661, and A549 cells, as well as primary tumors, bind estrogen specifically . This binding results mainly from the presence of a large number of type II EBSs, whereas ERs are absent or present at low concentrations . Type II EBSs bound tamoxifen and quercetin with similar affinity . Cell counts and a thymidine incorporation assay showed that both compounds inhibit cell growth in a concentration-dependent manner at concentrations ranging from 10 nM to 1 microM . Neither ipriflavone, an isoflavone, nor rutin, the 3-rhamnosylglucoside of quercetin, bound type II EBSs or inhibited cell growth . These findings suggest that tamoxifen and quercetin could regulate lung cancer cell growth through a binding interaction with type II EBSs . This mechanism could also be active in vivo, in that we have observed that nuclear and cytosolic type II EBSs were present in all primary lung cancers tested (n = 12), and that tamoxifen and quercetin were effective in inhibiting in vitro bromodeoxyuridine (BrdU) incorporation and proliferation-cell nuclear antigen expression by neoplastic cells in these cancers. Antimicrob Agents Chemother, 1997 Jul, 41(7), 1449 - 54 Potentiation of an antimalarial oxidant drug; Winter RW et al.; In a previous report we described the synergistic antimalarial interaction between two structurally similar compounds, rufigallol and exifone . To explain this phenomenon, we proposed that exifone is transformed inside the parasitized erythrocyte into a xanthone with potent antimalarial properties . We speculated that the transformation process was induced by the prooxidant activity of rufigallol . On the basis of this model we hypothesized that exifone would act synergistically with other oxidant drugs . In the present study we have found a similar synergistic interaction between exifone and ascorbic acid (vitamin C) against both chloroquine-susceptible and multidrug-resistant strains of Plasmodium falciparum . The prooxidant activity of ascorbic acid against Plasmodium-infected erythrocytes is believed to result from an intraerythrocytic Fenton reaction occurring in the acidic food vacuole of the parasite . The hydroxyl radicals produced during this process are believed to attack exifone, which undergoes cyclodehydration to become 2,3,4,5,6-pentahydroxyxanthone (X5) . Evidence presented to support this "xanthone hypothesis" includes the demonstration that the exifone ==> X5 transformation occurs readily in vitro under mildly acidic conditions in the presence of iron, ascorbic acid, and oxygen. J Infect Dis, 1997 Jul, 176(1), 265 - 8 Antagonism between human immunodeficiency virus type 1 protease inhibitors indinavir and saquinavir in vitro; Merrill DP et al.; Human immunodeficiency virus type 1 (HIV-1) protease inhibitors are a promising class of antiretroviral agents that compromise enzymatic function through substrate mimicry . The in vitro susceptibility of a panel of HIV-1 clinical isolates demonstrating various drug resistance phenotypes to combinations of the HIV-1 protease inhibitors saquinavir and indinavir was determined . Antiviral effect was assessed by an HIV-1 p24 antigen reduction assay in phytohemagglutinin-stimulated peripheral blood mononuclear cells after harvesting of cell-free supernatant fluids at peak antigen production (days 4-7) . Drug interactions were determined by median-dose-effect analysis, with the combination index (CI) calculated at several inhibitory concentrations (IC50, IC75, IC90, IC95, IC99) . The interactive effects ranged from synergy at low efficacy doses to antagonism at higher doses against a pan-susceptible clinical isolate of HIV-1 . Against a zidovudine-resistant isolate as well as a multidrug-resistant isolate, the combination of saquinavir and indinavir demonstrated antagonism at all doses. Gastroenterology, 1997 Jul, 113(1), 255 - 64 The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic and obstructive cholestasis; Trauner M et al.; BACKGROUND & AIMS: The excretion of various organic anions into bile is mediated by an adenosine triphosphate-dependent conjugate export pump, which has been identified as the canalicular isoform of the multidrug resistance protein (Mrp2) . Mrp2 function is impaired in various experimental models of intrahepatic and obstructive cholestasis, but the underlying molecular mechanisms are unclear . The aim of this study was to investigate these molecular mechanisms . METHODS: The effects of endotoxin, ethinylestradiol, and common bile duct ligation (CBDL) on Mrp2 protein, messenger RNA (mRNA) expression, and Mrp2 tissue localization were determined in rat livers by Northern blotting, Western analysis, and tissue immunofluorescence . To assess whether changes were specific for Mrp2, we also examined the expression of canalicular ecto-adenosine triphosphatase (ecto-ATPase) and mdr P-glycoproteins (P-gp) . RESULTS: All three cholestatic models resulted in a marked decrease in Mrp2 protein (P < 0.01) and its tissue localization at the canalicular membrane . Mrp2 mRNA levels diminished profoundly after endotoxin (P < 0.0005) and CBDL (P < 0.05), but did not change after ethinylestradiol . In contrast to Mrp2, protein expression of ecto-ATPase and P-gp remained unchanged in endotoxin- and ethinylestradiol-treated animals, whereas P-gp levels increased after CBDL (P < 0.05) . CONCLUSIONS: Down-regulation of Mrp2 expression may explain impaired biliary excretion of amphiphilic anionic conjugates in these models of cholestasis. Leukemia, 1997 Jul, 11(7), 1180 - 6 Apoptosis and resistance to daunorubicin in human leukemic cells; Efferth T et al.; We compared test methods based on specific mechanisms of daunorubicin (DNR) resistance to more global procedures . Assessment of P-glycoprotein (P-gp) expression and function by means of immunocytochemistry, DNR accumulation, and modulation of resistance and accumulation by the P-gp inhibitor cyclosporin A (CsA) were selected as parameters for multidrug resistance (MDR) . On the other hand, we used the MTT assay and measured apoptosis and proliferative activity (S- and G2M-phases of the cell cycle) by flow cytometry . Validation of test methods was achieved for four leukemic cell lines (HL-60, KG-1a, K562/WT, K562/ADM) . This battery of tests was then applied to mononuclear cells (MNC) from 18 leukemic patients . Low proficiency of MNC to undergo apoptosis and low proliferative activity rather than P-gp-mediated MDR correlated with DNR resistance as measured by the MTT assay . Bell-shaped dose-response curves for apoptosis, however, which reflect a switch from the apoptotic to the necrotic death mode with increasing cellular damage tend to limit practicability in clinical testing, because appropriate dose range and time points need to be explored . Thus, measurement of apoptosis by flow cytometry may be less convenient than the MTT assay for determination of chemosensitivity, if clinical samples with unknown patterns of responsiveness are to be tested . Spontaneous apoptosis in untreated MNC following 24 h incubation in vitro correlated significantly with DNR sensitivity in the MTT assay . A lack of essential viability factors (eg growth factors or cytokines) in vitro which are known to prevent apoptosis may contribute to DNR sensitivity. Leukemia, 1997 Jul, 11(7), 1170 - 9 Anthracycline subcellular distribution in human leukemic cells by microspectrofluorometry: factors contributing to drug-induced cell death and reversal of multidrug resistance; Morjani H et al.; There is a large discrepancy between the changes in drug accumulation and the changes in drug cytotoxicity that accompany development of anthracycline in multidrug-resistant cells . Moreover, although different molecular targets for anthracyclines such as DNA, cell membranes, or enzymes like topoisomerases could be involved, mechanisms by which these compounds exert their cytotoxic and differentiating effects remain unclear . Studies of correlation between the biological effects of anthracyclines and drug uptake have given conflicting conclusions . For example, a decrease in drug cytotoxicity for different incubation temperatures has been observed in spite of the same intracellular anthracycline amount, suggesting that temperature-dependent cytotoxic effects may be mediated by drug interaction with the cell membrane . What we propose in this review are results of our laboratory which are in agreement with an action mechanism targeted to the nucleus . In fact, we have shown by using microspectrofluorometry, that identical nuclear anthracycline concentration induces the same degree of cytotoxicity, independent of cellular MDR phenotype and the anthracycline structure . Thus, we could acquire information on the mechanisms of drug resistance related to drug transport . We could also give evidence that this accumulation is increased when MDR modulators, such as verapamil and S9788 and cyclosporin A or anthracyclines are used . For clinical applications, our studies have already dealt with nuclear concentration measurements of doxorubicin in leukocytes of treated patients, and in vitro measurements of drug efflux from nuclei of acute leukemic cells and its correlation with P-glycoprotein expression . However, in these studies, there was no correlation between anthracycline nuclear accumulation in vitro and P-glycoprotein expression . In addition, from preliminary results, we have shown that some modulators such as quinine do not significantly increase nuclear accumulation of anthracyclines in MDR cells but are able to restore anthracycline sensitivity . Other authors have recently shown that quinine has a relatively weak effect on cellular doxorubicin accumulation in MDR cells but is able to completely restore doxorubicin sensitivity . They concluded that quinine has essentially intracellular targets involved in drug distribution (cytoplasm --> nucleus) from sequestration compartments . Our data contradict this and we believe that such modulator modifies the molecular environment of anthracyclines and/or their binding to a possible cytoplasmic target leading to different cell death . Thus, we conclude that mechanisms by which anthracyclines induce cell death, and ways by which chemotherapy fails in resistant cells remain complex and are related to more than one target. Leukemia, 1997 Jul, 11(7), 1166 - 9 Detection of multidrug resistance gene expression in multiple myeloma; Dalton WS; Multiple myeloma is a disease which is generally considered responsive to chemotherapy; however, essentially all patients who respond to drug treatment will relapse and die of drug-resistant disease . This disease is therefore considered a paradigm for studying the development of acquired drug resistance in the clinic . Natural product agents are frequently used in the treatment of myeloma, especially vincristine and doxorubicin . Studies using human myeloma cell lines have shown that the MDR1 gene product, P-glycoprotein (Pgp), is responsible for conferring drug resistance to natural products and glucocorticoids . We have developed assays to measure the expression of MDR1/Pgp in human myeloma specimens . These assays include immunocytochemistry, flow cytometry, and RT/PCR . Human myeloma cell lines, 8226/Dox, that are resistant to natural product agents and overexpress MDR1/Pgp are important for standardizing results and offer a means of comparing inter- and intra-patient results . Assays which measure both the presence and function of Pgp are necessary to determine the role of Pgp in clinical drug resistance in patients with myeloma. Leukemia, 1997 Jul, 11(7), 1160 - 5 Assays for the analysis of P-glycoprotein in acute myeloid leukemia and CD34 subsets of AML blasts; Sonneveld P et al.; Expression of the multidrug resistance (MDR) phenotype is an independent prognostic variable in acute myeloid leukemia . Approximately 43-57% of the patients have P-glycoprotein (P-gp) expression . A major drawback with the interpretation of P-gp data in AML is the lack of coherence with different analytical assays . We have focused our efforts of P-gp detection on flow cytometry using a dual technique of P-gp staining with antibodies for the extracellular epitope (MRK16) and a functional analysis of P-gp using the rhodamine efflux assay and the effect of P-gp inhibitors such as SDZ PSC 833 . This technique was combined with the staining of lineage-specific antigens such as CD34, CD56 and c-kit . In this way, various subsets of AML cells can be identified such as MRK 16+/-, CD34+/- blasts . These cells can be sorted for further analysis, such as the molecular expression of P-gp and other pleiotropic drug resistance genes. Leukemia, 1997 Jul, 11(7), 1156 - 9 Accumulation of simple organic cations correlates with differential cytotoxicity in multidrug-resistant and -sensitive human and rodent cells; Lampidis TJ et al.; Structure/functional studies previously reported showed that in a series of simple organic cations in which the charge is delocalized, an aromatic ring and a minimal degree of lipophilicity (log P > -1) were required for recognition by murine cells which express P-glycoprotein (p-gp)-mediated multidrug resistance (MDR) . In the present report we find that 3H-octylpyridinium, the simple aromatic cation which has been shown to be preferentially toxic to MDR- as compared to MDR+ cells, accumulates 4.7-fold greater in the MDR- cell line . In contrast, we find that 3H-guanidinium which displays no selective toxicity between MDR+ and MDR- cells, shows no significant uptake differences between these two cell types . We also present data which demonstrate that other organic cations which contain aromatic rings, a minimal degree of lipophilicity (log P> -1) and carry a delocalized (Rho 123) or shielded (triphenylmethyl phosphonium) positive charge, also accumulate to a greater degree in MDR- vs MDR+ cells . Additionally, we find that human cells which express p-gp MDR, have similar requirements for recognition of these simple compounds . In fact, the sensitivity profiles of these compounds closely correlate between murine and human cell lines . It was also found that none of the series of simple organic compounds tested showed modulatory activity in MDR+ cells, as assayed by monitoring retention of Rho 123 . Thus, the requirements for MDR recognition vs those for MDR modulation are clearly distinguished with these simple structured compounds . In comparison, the calcium channel antagonist, verapamil, and a calcium channel agonist, Bay K 8644, both showed modulatory activity by increasing Rho 123 retention in MDR+ cells, further supporting the interpretation that verapamil's modulation of MDR is unrelated to its action on calcium flux . Overall, the data presented here add further information for defining the structural requirements of compounds for their recognition by, or modulation of, human cells expressing p-gp-mediated MDR. Leukemia, 1997 Jul, 11(7), 1147 - 55 Functional assay of multidrug resistant cells using JC-1, a carbocyanine fluorescent probe; Kuhnel JM et al.; Multidrug resistance (MDR) is characterized by a decrease in the efficiency of chemotherapeutic agents correlated with the expression and activity of a membrane protein: the permeability-glycoprotein (Pgp 170) . Clinically, detection of MDR can be performed by functional tests based on the accumulation of fluorescent compounds such as rhodamine 123 . With the aim of improving the sensitivity of such analysis, we have evaluated JC-1, a fluorescent lipophilic carbocyanine dye . Above a critical concentration, JC-1 aggregates in a 'liquid crystal' form . Aggregates display a specific red emission band centered at 597 nm whereas the monomers display a green emission band centered at 540 nm . JC-1 was avidly accumulated in sensitive K562 cells where it displayed both a green cytoplasmic and red mitochondrial fluorescence . In contrast, JC-1 was poorly accumulated in resistant K562 cells, which displayed only a slight green fluorescence . The level of JC-1 accumulation was correlated with the level of Pgp expression detected by MRK16 and UIC2 antibodies on a set of K562 subclones with increasing resistance levels . The specific fluorescence properties of JC-1 allow accurate discrimination between low-level resistant cells and sensitive cells . Chemosensitizers such as verapamil, cyclosporine A or S9788 restored JC-1 accumulation in resistant cells . The fluorescence properties of JC-1 could therefore be used for monitoring the effects of reversing agents. Leukemia, 1997 Jul, 11(7), 1131 - 7 Characterization of functional assays of multidrug resistance P-glycoprotein transport activity; Bosch I et al.; P-glycoprotein-mediated multidrug resistance has emerged as one of the most attractive targets to improve anticancer therapy . The P-glycoprotein functions as an energy-dependent, membrane transport pump capable of decreasing the intracellular concentration of a broad range of chemotherapeutic agents . Pharmaceuticals which inhibit P-glycoprotein transport activity are currently being evaluated in clinical trials . Characterization of P-glycoprotein functional activity is critical in determining if these multidrug resistance reversal agents improve therapeutic responses of tumors expressing P-glycoprotein . In this report, we directly compare and characterize assays using rhodamine 123, dimethyloxadicarbocyanine iodide (DiOC2), {3H}daunorubicin and hexakis(2-methoxyisobutyl isonitrile)technetium(I) ({(99m)Tc}Sestamibi) as P-glycoprotein transport probes to quantitate functional activity . The accumulation of certain substrates is concentration dependent and the parameters which determine probe accumulation are impacted by the level of P-glycoprotein expression . In addition, higher concentrations of reversal agents are required to inhibit multidrug resistance in cell lines expressing higher levels of P-glycoprotein . Furthermore, the concentration of reversal agents required to inhibit completely P-glycoprotein transport activity is higher than generally recognized . Thus, the level of P-glycoprotein expression may confound intersample comparisons unless sensitive probes are used in combination with saturating concentrations of potent reversal agents . These results highlight the importance of carefully characterizing assay systems under uniform conditions to quantitate P-glycoprotein function. Leukemia, 1997 Jul, 11(7), 1119 - 23 Monoclonal antibodies specific for P-glycoprotein; Okochi E et al.; Multidrug resistance (MDR) is one of the major obstacles to successful cancer chemotherapy . Since P-glycoprotein (P-gp) encoded by the MDR-1 gene plays a key role in MDR, many P-gp-specific monoclonal antibodies (MAbs) have been generated for characterization and analysis of P-gp . Among those antibodies, MRK16 has been widely used not only for elucidation of the mechanisms of P-gp-mediated MDR but also for diagnostic and therapeutic studies . Two types of magnetic cell sorting assays, termed MRK16-MACS and MRK16-MACS-FACS, have been established by us and may offer a useful tool to quantitate low levels of P-gp expression . This article describes the characteristics of the antibodies against P-gp and discuss the diagnostic implications of the antibodies. Leukemia, 1997 Jul, 11(7), 1110 - 8 Theoretical and practical considerations for the measurement of P-glycoprotein function in acute myeloid leukemia; Broxterman HJ et al.; This paper summarizes experimental data and theoretical considerations, that are important for the measurement of P-glycoprotein (Pgp) function in acute myeloid leukemia (AML) . The data are presented in subdivisions based on the techniques used, which will facilitate finding specific information . Based on our extensive experience with Pgp analysis, which includes radioactive assays, flow cytometry and fluorescence microscopy, we recommend a flow cytometry-based assay, that measures the effect of 2 microM PSC 833 on rhodamine 123 (R123) accumulation as the most practical and sensitive functional Pgp test . In combination with the flow cytometric measurement of Pgp using an antibody against an extracellular epitope (eg MRK16), this offers a sensitive and reproducible method for Pgp detection in AML, which is also rapid and practical . Furthermore, an R123 accumulation assay is specific for Pgp, because R123 is transported much less efficiently by the multidrug resistance protein (MRP) than by Pgp . Another probe of similar sensitivity and specificity is 3,3'-diethyloxacarbocyanine iodide . Alternatively, especially for the analysis of small numbers of cells (for example sorted subpopulations of leukemic cells), convenient and sensitive procedures are being developed by using DNA-binding Pgp substrates which remain fixed in the nuclei of the cells upon formaldehyde exposure for quantitative fluorescence laser scanning microscopy with image analysis . Less experimental data have been published to establish the optimal conditions for dual parameter flow cytometry (Pgp function, in eg Pgp+ or CD34+ cells) . However, laboratories with flow cytometry experience will be able to implement this useful option to analyze subpopulations of cells. Leukemia, 1997 Jul, 11(7), 1107 - 9 Methods to detect P-glycoprotein and implications for other drug resistance-associated proteins; Beck WT et al.; The problem of tumor cell drug resistance remains a barrier to the successful treatment of many neoplastic diseases . Problems of tumor cell heterogeneity and expression of multiple mechanisms of drug resistance complicate treatment strategies . Indeed, even that form of resistance to natural product anticancer agents, multidrug resistance (MDR), can have multiple mechanisms . Compounding these problems is the use of different methodologies and different reagents to assess expression of the most widely studied form of MDR, that due to increased expression of the MDR1 gene and its product, P-glycoprotein (Pgp) . In this paper, we discuss problems associated with assay variability and accurate measurement of markers of drug resistance, and summarize consensus findings of the St Jude Workshop on methods to detect Pgp in tumors. Leukemia, 1997 Jul, 11(7), 1095 - 106 French multicentric evaluation of mdr1 gene expression by RT-PCR in leukemia and solid tumours . Standardization of RT-PCR and preliminary comparisons between RT-PCR and immunohistochemistry in solid tumours . French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris; Chevillard S et al.; Since there is no consensus on the techniques for multidrug resistance (MDR) phenotype evaluation, many discrepancies concerning the importance and frequency of mdr1 gene expression in leukemias and solid tumors are observed in the literature . In order to establish an inter-laboratory consensus in France, a multicenter study was carried out to propose further guidelines for MDR phenotype evaluation . The techniques used by the 38 laboratories participating in the trial were: immunodetection (immunohisto and/or cytochemistry, flow cytometry), functional tests, reverse transcription-polymerase chain reaction (RT-PCR) or Northern blot . We present the results obtained by 19 laboratories concerning the measurement of mdr1 gene expression assessed by RT-PCR or Northern blot in: (1)19 samples of tumor cells obtained from leukemic patients; (2) six solid tumor samples obtained at surgery; (3) eight cell lines exhibiting variable levels of resistance, and; (4)10 preparations of RNA and of cDNA obtained from solid tumors . Standardization of the RT-PCR technique and preliminary results comparing RT-PCR with immunohistochemistry in solid tumors are also reported. Leukemia, 1997 Jul, 11(7), 1086 - 94 Multicentric evaluation of the MDR phenotype in leukemia . French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris; Marie JP et al.; The wide discrepancies in the frequency of 'positive' samples for multidrug resistance (MDR) phenotype within the same type of tumor observed in the literature justified the need for the definition of consensus recommendations . To define standard techniques of MDR phenotype measurement, we ran a large multicentric evaluation of the different methods available . Thirty-six French centers participated in the study, and 742 samples of 2-10 x 10(6) viable cells were sent by overnight express mail between December 1993 and February 1996 . The same batches of MRK16, 4E3 and UIC2 were used . Nineteen samples of leukemia (12 AML, 1 ALL, 6 lymphoproliferative syndromes) and six leukemic cell lines with different levels of MDR expression were tested . Five meetings reached agreement concerning the guidelines for each technique, except immunocytochemistry . The 19 fresh samples were tested by each center using one to four techniques among cytofluorometry, immunocytochemistry, functional tests and RT-PCR . Five samples were diagnosed as 'negative' according to local criteria, with few discordant results (0 to 16% of 'positive' results) . For all the 14 remaining samples, large discrepancies were observed from center to center, and from one technique to another . No correlations could be found between techniques . Flow cytometric analysis of cells already exposed to MRK16 or control IgG2A, fixed in paraformaldehyde and sent to centers did not reduce the discrepancies between centers in two of the four samples with moderate expression, emphasizing the role of histogram interpretation . The use of alternative monoclonal antibodies (4E3 and UIC2) did not reduce the discrepancies observed . In a second step, the K562 parental cell line, a low resistant subline (K562/HHT100, x7 resistance index to DNR) and a high resistant subline (K562/HHT300, x125 resistance index to DNR) were sent blindly three times, with an increasing level of recommendations for flow cytometry . Dramatic improvements were observed in cytometric results when the result was expressed as the ratio of arithmetic mean of fluorescence of antibody (10 microg of MRK16)/arithmetic mean of fluorescence of control (10 microg IgG2A): the proportion of expected results increased from 61 to 100% for K562, and from 37 to 85% for K562/HHT100 . For uptake and drug efflux measurements, the use of 1 h uptake of 0.1 microM of rhodamine, followed by 1 h efflux +/-10 microM of verapamil, permitted an increased reproducibility of the technique from 71 to 100% for K562 and K562/HHT100 . Whatever the technique used, concordant results were obtained for K562/HHT300 . The immunocytochemistry, using several antibodies (MRK16, JSB1 and C219) gave many non-interpretable results (44%), due to a frequent high background and discordant results between antibodies in the same centers, and discordant conclusions between centers . The group does not recommend this technique for circulating tumoral cells. Leukemia, 1997 Jul, 11(7), 1078 - 85 Optimal immunocytochemical and flow cytometric detection of P-gp, MRP and LRP in childhood acute lymphoblastic leukemia; Den Boer ML et al.; The clinical relevance of multidrug resistance (MDR)-related proteins in childhood acute lymphoblastic leukemia (ALL) is largely unknown . The diversity of techniques, fixation methods, storage of cells (fresh or cryopreserved) etc, may contribute to discrepancies observed between several studies . We therefore optimized the detection of P-glycoprotein (P-gp), MDR-associated protein (MRP) and lung resistance-related protein (LRP) by immunocytochemistry and flow cytometry in childhood ALL cells . Thirteen fixation methods were compared using six antibodies in both immunocytochemistry and flow cytometry . The optimal fixation for P-gp (C219, MRK16), MRP (MRPr1) and LRP (LRP56) was a mixture of 2% (v/v) formaldehyde solution and acetone incubated for only 10 s at room temperature (FAc) . For MRP recognized by MRPm6, the optimal fixation condition was acetone for 5 min at room temperature in immunocytochemistry, and methanol for 15 min at -20 degrees C in flow cytometry . P-gp staining by 4E3 was strongly antibody batch-dependent; on cytospins FAc fixation was optimal, but inconclusive data were obtained by flow cytometry . The optimized fixation conditions on fresh samples revealed a day-to-day variation in staining (both increasing and decreasing) in one third of the immunocytochemical tests . In flow cytometry the day-to-day variation in the fluorescence index was -1 +/- 22% . In both techniques, staining was comparable between fresh and cryopreserved cells . We recommend the use of the above mentioned fixation methods in order to study the clinical relevance of P-gp, MRP and LRP in childhood ALL. Leukemia, 1997 Jul, 11(7), 1073 - 7 Immunocytochemical detection of the multidrug resistance-associated protein and P-glycoprotein in acute myeloid leukemia: impact of antibodies, sample source and disease status; Filipits M et al.; Immunocytochemical detection of the expression of the MRP gene and the MDR1 gene in clinical specimens might be affected by several factors . Thus, we studied the impact of monoclonal antibodies, sample source (peripheral blood vs bone marrow) and disease status on the expression of multidrug resistance-associated protein (MRP) as well as P-glycoprotein (P-gp) in leukemic cells of patients with acute myeloid leukemia (AML) . MRP expression was determined by means of anti-MRP antibodies (QCRL-1, QCRL-3, QCRL-1/QCRL-3 or MRPr1) . In the case of P-gp, monoclonal antibodies C219 and MRK16 were used . High MRP expression ranged from 5 to 35% and high P-gp expression from 5 to 14% of the specimens . A fair correlation between results obtained with QCRL-1/QCRL-3 and those obtained with MRPr1, as well as a moderate correlation between C219 and MRK16, were seen . MRP and P-gp expression of peripheral blood blasts were similar to those of bone marrow blasts in the majority of cases . The degrees of MRP expression at the time of diagnosis were also similar to the degrees of expression at relapse, albeit an analysis of sequential MRP expression in 13 patients indicated an increase of expression at relapse in six patients as compared to the time of diagnosis. Leukemia, 1997 Jul, 11(7), 1067 - 72 Multidrug resistance in acute leukemia: a comparison of different diagnostic methods; Pall G et al.; Accurate measurement of P-glycoprotein (P-170) expression in clinical samples still remains a controversial issue . In this study tumor cell P-170 expression was assessed in 29 patients suffering from acute leukemia (17 acute myeloid leukemia (AML) and 12 acute lymphoblastic leukemia (ALL)) using three different techniques: flow cytometry measuring rhodamine 123 (Rh123) efflux (functional level), immunocytochemistry (protein level) and RT-PCR (mRNA level) . Rh123 efflux was detectable in 10/29 (34%) of all cases, in 9/17 (53%) of AML and in 1/12 (8%) of ALL samples . In AML patients a significant association of CD34 expression and P-170 activity was observed (P < 0.02) . All AML patients with the FAB subtype M5 were Rh123 negative (P < 0.007) . Cytospin preparations were analyzed for staining with monoclonal antibodies JSB1 and MM4.17 . Eight of 16 (50%) AML and 0/9 (0%) ALL cases expressed the multidrug resistance (MDR) protein assessed by JSB1 . With MM4.17 87% of AML and 50% of ALL patients were scored positive . Agreement between both antibodies was found in only 13/23 (57%) samples . Extracted RNA from 12 patients was analyzed by RT-PCR to evaluate the expression of MDR1 and multidrug resistance-associated protein (MRP) mRNA . An increased level of MDR1 mRNA was detectable in 4/7 AML and 0/5 ALL cases . MRP expression was found in 3/7 AML and 0/5 ALL patients . Comparison of Rh123 assay and immunocytochemistry revealed a very good correlation when using MoAb JSB1 (P < 0.004) but not with MM4.17 (not significant (NS)) . JSB1 also showed a much better association with the PCR results (P < 0.05) than MM4.17 (NS) . Finally, we compared the results of the functional Rh123 assay and RT-PCR and observed a high correlation for Rh123/MDR1 (r = 0.819, P < 0.001) but low for Rh123/MRP (r = 0.562, NS) . We conclude that measurement of Rh123 efflux and immunocytochemical staining of cytospin preparations with JSB1 allows the accurate monitoring of P-170 expression in acute leukemia . The simplicity of these two MDR assays suggests their use for routine MDR screening. Leukemia, 1997 Jul, 11(7), 1040 - 4 A new human cell line with pre-B cell phenotype and t(5;12); Ingles-Esteve J et al.; A new cell line (LR10.6) with pre-B cell phenotype has been established from bone marrow cells obtained from a child with B lineage acute lymphoblastic leukemia in complete clinical remission . The line expresses nuclear TdT enzyme, cytoplasmic Ig lambda-chain and membrane mu-chain and other B but no T or myeloid markers . The cells also show activation antigens CD69 and CD71, adhesion molecules CD54, CD50 and CD56 and the tyrosine kinase receptor CD117 . No expression of multidrug resistance phenotype MDR-1 is observed on these cells which nevertheless express the transcriptional factor p53 protein in a mutant form . Cytogenetic study shows a translocation t(5;12)(q31;p13) involving breakpoints which contain the growth factor interleukin 3 gene (5q31) and the recently identified TEL/ETV6 gene (12p13) . Activation of the cells with phorbol-12 myristate 13-acetate (PMA) up-regulates the expression of the CD69 activation antigen and down-regulates the CD117 molecule . In addition, PMA fails to induce the CD20 B cell antigen. Leukemia, 1997 Jul, 11(7), 950 - 7 Sensitization of multidrug-resistant human leukemia cells with MDR1-targeted antisense and inhibition of drug-mediated MDR1 induction; Li X et al.; Increased expression of MDR1 is strongly implicated in the appearance of chemotherapeutic drug resistance in cancer, especially hematological malignancies . We therefore examined the potential of antisense oligonucleotides to inhibit MDR1 and restore sensitivity to drug-resistant human lymphoblastic cells (CCRF-CEM) . Treatment with two different phosphorothioate-modified antisense sequences as well as a DNA-RNA hybrid sequence resulted in a 30 to 45% decrease in MDR1 expression as determined by staining with the monoclonal antibody MRK16 followed by flowcytometry (FCM) analysis . Further, inhibition of MDR1 expression persisted for 3 days after removal of oligonucleotides . Increased accumulation of rhodamine 123 and nearly a three-fold sensitization of cells to vincristine paralleled the reduction in staining with MRK16 . Reversed or scrambled control sequences had no effect in any of the assays . During the course of these studies, we observed a 25 to 75% increase in MRK16 staining of cells treated with the chemotherapeutic agents daunorubicin and vincristine as well as by the resistance reversal agents verapamil and cyclosporin . Treatment of cells with antisense oligonucleotides prior to exposure to daunorubicin or cyclosporin reduced the increase in MRK16 staining . These results indicate that antisense targeted to MDR1 can sensitize drug-resistant leukemia cells and suggest that antisense treatment may prevent the emergence of MDR1-mediated drug resistance. Leukemia, 1997 Jul, 11(7), 945 - 9 Bcl-x(L) is heterogenously expressed by acute myeloblastic leukaemia cells and is associated with autonomous growth in vitro and with P-glycoprotein expression; Pallis M et al.; The cells from approximately 70% patients with acute myeloblastic leukaemia exhibit autonomous growth characteristics in vitro, which have been associated with a poor response to therapy . We have previously shown that leukaemic cells with autonomous growth characteristics express high levels of bcl-2 and are relatively resistant to apoptosis . As bcl-x(L) is a bcl-2-related gene with anti-apoptotic activity which also confers resistance to cytotoxic drugs we have studied its expression in AML in relation to cellular growth characteristics and to the expression of P-glycoprotein . Cells from 15 patients were studied . Immunoblotting demonstrated bands at 31 kDa corresponding to bcl-x(L) from the cells of all patients . Bcl-x(S) was not detected in any sample . Using standardised, quantitative flow cytometry, bcl-x(L) expression ranged from 0.25 x 10(5) to 4.24 x 10(5) bound FITC molecules, (median 1.35 x 10{5}) . AML blasts with autonomous growth in vitro expressed more bcl-x(L) (median 1.76 x 10{5}) than those which did not (median 0.86 x 10(5), P=0.01) . Quantitative bcl-x(L) expression strongly correlated with that of P-glycoprotein, also measured by quantitative flow cytometry using the MRK16 antibody (r=0.95, P < 0.001), but not with MRPr1 . These results provide a further explanation for the poor prognosis associated with autonomous in vitro growth of AML blasts and illustrate that these cells may coexpress different modalities of resistance to cytotoxic drug therapy involving both anti-apoptotic pathways (bcl-x(L), bcl-2) and classic multidrug resistance (MDR1) . The implication of these findings is that the use of agents to reverse MDR1 function in AML may be unsuccessful in the absence of strategies to reduce resistance to apoptosis. Biochemistry, 1997 Jul 1, 36(26), 8180 - 8 Functional interactions between synthetic alkyl phospholipids and the ABC transporters P-glycoprotein, Ste-6, MRP, and Pgh 1; Ruetz S et al.; The ABC superfamily of transporters includes the mammalian P-glycoprotein family (Class I and Class II P-gps), the multidrug resistance-associated protein (MRP), the Pgh-1 product of Plasmodium falciparum gene pfmdr1, all of which are associated with cellular pleiotropic drug resistance phenomena . STE6, the yeast transporter for the farnesylated peptide pheromone a, is also a member of this family . Structural similarities in this family translate into functional homology as expression of mouse Mdr3S (P-gp), P . falciparum Pgh-1, and human MRP partially restore mating in a sterile yeast mutant lacking a functional STE6 gene . The demonstration that Class II P-gps function as phosphatidylcholine (PC) translocators raise the possibility that other ABC transporters may also interact with physiological lipids . We report the identification of the synthetic lipid and PC analog ET-18-OCH3 (edelfosine) as a substrate for not only Class II P-gp but also for Class I P-gps and surprisingly for the other ABC transporters MRP, Pgh-1, and STE6 . Expression of these proteins in the yeast Saccharomyces cerevisiae JPY201 was found to confer cellular resistance to cytotoxic concentrations of this lipid by a factor of 4-20-fold in a growth inhibition assay . The noted activity of ABC transporters toward this synthetic lipid was specific as a mutant variant of Mdr3 (Mdr3F) with reduced activity could not convey cellular resistance to ET-18-OCH3 . ET-18-OCH3 was also found capable of blocking a-peptide pheromone transport and STE6 complementation by these ABC proteins . The inhibitory effect of ET-18-OCH3 on cell growth and a-factor transport could be abrogated by incubation with the lipid acceptor protein BSA or by enzymatic cleavage by microsomal alkylglycerol mono-oxygenase (MAMO) . MAMO and BSA reversal of the ether lipid effect was only seen in the presence of a functional transporter . These results suggest that the group of cytotoxic synthetic PC analogs studied reveal possible structural and functional aspects common to the ABC transporters tested . Furthermore, the studies with BSA and MAMO suggest that the mechanism of transport of ET-18-OCH3 by these ABC transporters may be related to the flippase mechanism of PC transport by Mdr2. Biochem Biophys Res Commun, 1997 Jun 27, 235(3), 849 - 53 Function of P-glycoprotein expressed in placenta and mole; Nakamura Y et al.; We examined the expression of P-glycoprotein in human placentas and hydatidiform moles . Trophoblasts in all the examined placentas and moles expressed P-glycoprotein, and the size of the P-glycoprotein was smaller than that in multidrug-resistant human epidermoid carcinoma KB cells . The P-glycoprotein in the placenta and mole was photolabeled with {3H}azidopine, and {3H}vincristine was transported in an ATP-dependent manner into membrane vesicles prepared from trophoblasts that expressed P-glycoprotein . These findings indicate that P-glycoprotein expressed in trophoblasts has a drug binding site(s) and the ability to transport vincristine, suggesting that P-glycoprotein in the placenta protects the fetus from xenobiotics and confers drug resistance on moles. Cancer Lett, 1997 Jun 24, 116(2), 205 - 11 Expression and intracellular localization of heat shock proteins in multidrug resistance of a cisplatin resistant human ovarian cancer cell line; Kamishima T et al.; TYK-R10 is a cisplatin resistant human ovarian carcinoma cell line and showed a cross resistance to various anti-cancer drugs including adriamycin (ADR), vincristine (VCR) and etoposide, despite a lack of multidrug phenotype . Under normal conditions, various heat shock proteins (HSPs) were expressed in TYK-R10 but not in parental line (TYK-nu) . Non-lethal short-term heat shock treatment induced a high tolerance for cisplatin and VCR in TYK-R10 and ADR, and VCR in TYK-nu . This treatment induced and/or enhanced the expression of various types of HSPs in various intracellular localizations in both TYK-R10 and TYK-nu, with minor differences . These findings indicate that combined expression and intracellular localization of HSPs may play an important role in drug resistance of TYK-R10. J Med Chem, 1997 Jun 20, 40(13), 2102 - 6 Structure-activity relationships of diverse Annonaceous acetogenins against multidrug resistant human mammary adenocarcinoma (MCF-7/Adr) cells; Oberlies NH et al.; Fourteen structurally diverse Annonaceous acetogenins, representing the three main classes of bis-adjacent, bis-nonadjacent, and single-THF ring(s), were tested for their ability to inhibit the growth of adriamycin resistant human mammary adenocarcinoma (MCF-7/Adr) cells . This cell line is resistant to treatment with adriamycin, vincristine, and vinblastine and is, thus, multidrug resistant (MDR) . Among a series of bis-adjacent THF ring acetogenins, those with the stereochemistry of threo-trans-threo-trans-erythro (from C-15 to C-24) were the most potent with as much as 250 times the potency of adriamycin . A spacing of 13 carbons between the flanking hydroxyl of the THF ring system and the gamma-unsaturated lactone seems to be optimum with a spacing of 11 and 9 carbons being significantly less active . Several single-THF ring compounds were also quite potent with gigantetrocin A (11) being the most potent compound tested . The acetogenins may, thus, have chemotherapeutic potential, especially with regard to MDR tumors. J Med Chem, 1997 Jun 20, 40(13), 2047 - 52 Structure-activity relationship of newly synthesized quinoline derivatives for reversal of multidrug resistance in cancer; Suzuki T et al.; The effect of 24 newly synthesized quinoline derivatives on tumor cell multidrug resistance (MDR) was examined in vitro . At low concentrations, these compounds enhanced the accumulation of {3H}vincristine in K562/ADM cells and reversed tumor cell MDR . The results of the structure-activity relationship analysis indicate that in highly active compounds the two aryl rings in the hydrophobic moiety deviate from a common plane, so they are capable of interacting with hydrogen bond donors of P-170 glycoprotein (P-gp) via pi-hydrogen-pi interactions . Other major structural features which influence the MDR-reversing activities of these compounds are a quinoline nitrogen atom and a basic nitrogen atom in piperazine . Furthermore, in highly active compounds, the distance between the hydrophobic moiety and the basic nitrogen atom (an atom connected to 2-hydroxypropoxyquinoline) must be at least 5 A . Several compounds were found to reverse vincristine resistance in K562/ADM cells in vitro, and compound 16 (MS-209) was selected for clinical studies. Biochem Pharmacol, 1997 Jun 15, 53(12), 1855 - 66 Cross-resistance to antifolates in multidrug resistant cell lines with P-glycoprotein or multidrug resistance protein expression; van Triest B et al.; Resistance to some (lipophilic) antifolates has been associated with P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) . A possible relationship with non-P-gp MDR has not been established . We studied resistance to antifolates in SW-1573 human lung carcinoma cells, a P-gp overexpressing variant SW-1573/2R160 and a multidrug resistance protein (MRP) overexpressing variant SW-1573/2R120 . In this study, thymidylate synthase (TS) inhibitors with different properties concerning the efficiency of membrane transport and the efficiency of polyglutamylation were tested for cross-resistance in SW-1573/2R120 and SW-1573/2R160 cells . Growth inhibition patterns in this cell line panel were measured by the Sulforhodamine B (SRB) assay . Resistance factors for TS inhibitors were: 2.4 and 0.4 for 5-fluorouracil (5FU), 18.8 and 8.8 for ZD1694, 17 and 0.7 for AG337, and 40 and 8.3 for BW1843U89 in SW-1573/2R160 and SW-1573/2R120, respectively . This study showed changes in the TS enzyme kinetics during the induction of doxorubicin resistance in both SW-1573 variants, resulting in 2-fold lower Km values for 2'-deoxyuridine-5'-monophosphate (dUMP) in both resistant variants compared to the parental cell line . TS activity, TS protein induction and TS mRNA expression all had 2-fold increased in the SW-1573/2R120 compared to the SW-1573/2R160 . 3H-MTX influx was 2-fold lower in SW-1573/2R160 cells compared to SW-1573/2R120 and SW-1573 cells . In the SW-1573/2R160 cell line, an aberrant intracellular trafficking towards the target TS was observed, compared to SW-1573/2R120 and SW-1573 cells as measured by the TS in situ assay . The rate of TS inhibition by the TS inhibitors used in this study was similar in all cell lines . In conclusion, collateral sensitivity to 5FU and the lipophilic AG337 and cross-resistance to other antifolates were observed in non-P-gp MDR SW-1573/2R120 cells, as well as resistance to all antifolates in P-gp SW-1573/2R160 cells . The mechanism of resistance in SW-1573/2R160 cells possibly involves reduced influx and changes in intracellular trafficking routes . For the SW-1573/2R120 cell line, several changes related to the TS enzyme possibly play a role in the observed cross-resistance and collateral sensitivity pattern. Cancer Res, 1997 Jun 15, 57(12), 2325 - 30 Multidrug resistance protein (MRP) expression in retinoblastoma correlates with the rare failure of chemotherapy despite cyclosporine for reversal of P-glycoprotein; Chan HS et al.; Failure of chemotherapy associated with expression of the multidrug resistance protein p170 frequently occurs in retinoblastoma (RB) . Despite using cyclosporine, which inhibits p170 and improves our chemotherapy results, rare failures occur . In nonmetastatic primarily enucleated RBs, we show expression of p170 in 3 of 18 samples and expression of multidrug resistance protein (MRP), the second protein associated with resistance to chemotherapy, in 1 of 18 samples . All three RBs that failed chemotherapy without cyclosporine expressed MRP with p170 . All three RBs that were enucleated immediately when chemotherapy failed despite the addition of cyclosporine expressed only MRP . One RB enucleated 2 years after failing chemotherapy with cyclosporine, despite radiation and salvage chemotherapy, expressed both p170 and MRP . Two metastatic RBs that expressed both p170 and MRP at diagnosis and at recurrence failed chemotherapy without cyclosporine, whereas one metastatic RB that expressed neither protein was cured by chemotherapy without cyclosporine . MRP may result in failure of chemotherapy despite the elimination of p170-expressing clones by cyclosporine. J Cell Biochem, 1997 Jun 15, 65(4), 513 - 26 Reduced drug accumulation and multidrug resistance in human breast cancer cells without associated P-glycoprotein or MRP overexpression; Lee JS et al.; MCF-7 human breast cancer cells selected in Adriamycin in the presence of verapamil developed a multidrug resistant phenotype, which was characterized by as much as 100,000-fold resistance to mitoxantrone, 667-fold resistance to daunorubicin, and 600-fold resistance to doxorubicin . Immunoblot and PCR analyses demonstrated no increase in MDR-1 or MRP expression in resistant cells, relative to parental cells . This phenotype is similar to one previously described in mitoxantrone-selected cells . The cells, designated MCF-7 AdVp, displayed a slower growth rate without alteration in topoisomerase II alpha level or activity . Increased efflux and reduced accumulation of daunomycin and rhodamine were observed when compared to parental cells . Depletion of ATP resulted in complete abrogation of efflux of both daunomycin and rhodamine . No apparent alterations in subcellular daunorubicin distribution were observed by confocal microscopy . No differences were noted in intracellular pH . Molecular cloning studies using DNA differential display identified increased expression of the alpha subunit of the amiloride-sensitive sodium channel in resistant cells . Quantitative PCR studies demonstrated an eightfold overexpression of the alpha subunit of the Na+ channel in the resistant subline . This channel may be linked to the mechanism of drug resistance in the AdVp cells . The results presented here support the hypothesis that a novel energy-dependent protein is responsible for the efflux in the AdVp cells . Further identification awaits molecular cloning studies. J Biol Chem, 1997 Jun 13, 272(24), 15174 - 83 NF-kappaB-mediated induction of mdr1b expression by insulin in rat hepatoma cells; Zhou G et al.; The expression of P-glycoproteins encoded by the mdr gene family is associated with the emergence of multidrug resistance phenotype in animal cells . However, the mechanisms controlling the expression of these genes have not been well elucidated . Here, we report that the expression of rat mdr1b gene in cultured H-4-II-E hepatoma cells can be induced by insulin . Transient transfection assays using reporter gene constructs containing various 5' mdr1b sequences showed that the sequence located between base pairs -243 and -163 is important for insulin's induction of mdr1b promoter activity . Further analyses revealed that a NF-kappaB-binding site (located between base pairs -167 and -158) is required for insulin-induced promoter activity . Gel mobility shift assay demonstrated that insulin stimulates the binding of nuclear p50/p65 subunits to the mdr1b NF-kappaB sequence . Cotransfection of plasmids expressing either the p50/p65 NF-kappaB subunits or Raf-1 kinase or both resulted in increased expression of the gene containing wild-type but not NF-kappaB site-mutated mdr1b promoter . Finally, expression of either the antisense p65 subunit of NF-kappaB or dominant negative Raf-1 kinase blocked insulin's induction of the mdr1b promoter activity . Taken together, our results suggest that the insulin-induced mdr1b expression is mediated by transcription factor NF-kappaB via the Raf-1 kinase signaling pathway. J Natl Cancer Inst, 1997 Jun 4, 89(11), 807 - 13 Tumor necrosis factor-alpha and expression of the multidrug resistance-associated genes LRP and MRP; Stein U et al.; BACKGROUND AND PURPOSE: Cancer cells that express P-glycoprotein, multidrug resistance-associated protein (MRP), or lung resistance protein (LRP) have demonstrated resistance to a wide variety of chemotherapeutic drugs . Recently, we reported that human colon carcinoma cells that express all three proteins exhibit reduced P-glycoprotein gene expression and a loss of multidrug resistance after exposure to tumor necrosis factor-alpha, a hormone-like protein produced by cells of the immune system . In this study, we examined the effects of tumor necrosis factor-alpha on MRP and LRP gene expression in the same colon carcinoma cells . METHODS: HCT15 and HCT116 colon carcinoma cells were incubated with tumor necrosis factor-alpha at 100 U/mL for 2, 12, 24, 48, or 72 hours; alternatively, cells transfected with an expression vector containing a human tumor necrosis factor-alpha complementary DNA were studied . The effects of tumor necrosis factor-alpha on MRP and LRP messenger RNA expression were evaluated by means of reverse transcription and the polymerase chain reaction; effects on MRP and LRP protein expression were examined by use of specific monoclonal antibodies and flow cytometry . The flow cytometry data were analyzed by use of the two-sided, nonparametric Mann-Whitney rank sum test . RESULTS: Treatment with exogenous tumor necrosis factor-alpha reduced the level of LRP messenger RNA in both cell types in an apparently time-dependent fashion; in HCT15 cells, almost no LRP messenger RNA was detected after 48 hours of treatment . In contrast, the level of MRP messenger RNA was increased in HCT116 cells by such treatment, but the level in HCT15 cells was unchanged . Treatment with exogenous tumor necrosis factor-alpha induced changes in LRP and MRP protein expression in the two cell types that paralleled the changes found for messenger RNA . In transfected cells, the endogenous production of tumor necrosis factor-alpha reduced LRP gene expression (both messenger RNA and protein) and increased MRP gene expression (both messenger RNA and protein), regardless of cell type . CONCLUSION: In human colon carcinoma cells, tumor necrosis factor-alpha influences MRP and LRP gene expression in opposite ways . The findings for LRP gene expression parallel our earlier findings for P-glycoprotein expression in these cells . IMPLICATION: In developing strategies for overcoming multidrug resistance in tumor cells, the possibility that an agent can suppress one or more mechanisms of drug resistance and enhance others should be considered. Biochemistry, 1997 Jun 3, 36(22), 6847 - 53 Inhibition of P-glycoprotein ATPase activity by beryllium fluoride; Sankaran B et al.; ATPase activity of P-glycoprotein (multidrug-resistance protein) was found to be potently inhibited by beryllium fluoride (BeFx) in combination with MgATP, MgADP, or corresponding Mg-8-azido-nucleotides . Inhibition was due to trapping of nucleoside diphosphate at catalytic sites . Full inhibition was achieved on trapping of 1 mol of nucleotide per mol of Pgp . Reactivation was slow (t(1/2) = 32 min at 37 degrees C), and release of trapped nucleotide correlated with recovery of ATPase . Trapping of 8-azido-ADP followed by UV irradiation yielded permanent inactivation and specific labeling of Pgp in plasma membranes . Both N- and C-terminal nucleotide binding sites were labeled . These findings give strong confirmation of the concepts that in intact Pgp both nucleotide sites are active in MgATP hydrolysis, and that they interact strongly . The characteristics of inhibition by BeFx were similar in general to those seen with vanadate . However, PPi gave strong protection against BeFx inhibition, and in this respect, inhibition by BeFx was clearly different from vanadate inhibition. Cancer Lett, 1997 Jun 3, 116(1), 33 - 9 Lack of a point mutation of human DNA topoisomerase II in multidrug-resistant anaplastic thyroid carcinoma cell lines; Satake S et al.; DNA topoisomerases are major defined targets for a large variety of clinically important anticancer agents, including etoposide, adriamycin, and mitoxantrone . Mutations at amino acids 439, 450 and 803 of DNA topoisomerase II were examined in multiple anticancer drug-resistant anaplastic thyroid carcinomas (ten cell lines and three cancerous tissues) by reverse transcriptase-polymerase chain reaction (RT-PCR) and subsequent DNA sequencing . No mutation was found in these cell lines and tissues, but mdr1, mrp and/or lrp mRNA were expressed to a varying degree, and there was no significant difference in DNA topoisomerase IIalpha content among the cell lines and tissues as evaluated by Western blotting . Our experimental data indicate that overexpression of multidrug resistance-related mRNA is sufficient to confer drug resistance. FEBS Lett, 1997 Jun 2, 409(1), 67 - 73 Xanthones as antimalarial agents; studies of a possible mode of action; Ignatushchenko MV et al.; We recently demonstrated that 2,3,4,5,6-pentahydroxyxanthone (X5) inhibits the in vitro growth of both chloroquine-sensitive and multidrug-resistant strains of P . falciparum . To study the molecular basis of its antimalarial action, we tested X5 and selected hydroxyxanthone analogs as inhibitors of in vitro heme polymerization in a low ionic strength phosphate solution at mildly acidic pH . We found that addition of 1 Eq . of X5 resulted in complete inhibition of polymerization in this system whereas addition of up to 40 Eqs . of standard antimalarial compounds (chloroquine, primaquine, quinacrine, artemisinin and methylene blue) had no such effect although these compounds did co-precipitate with heme . The antimalarial potency of the hydroxyxanthones correlated well with their ability to inhibit in vitro heme polymerization in our assay, suggesting that these compounds exert their antimalarial action by preventing hemozoin formation . Based on the observed structure-activity relationships, we propose a model displaying possible interactions between hydroxyxanthones and heme. Cytokines Cell Mol Ther, 1997 Jun, 3(2), 91 - 9 Multidrug resistance: molecular and clinical aspects; Dicato M et al.; Clinical drug resistance, a common and compromising side-effect during anticancer chemotherapy, is an acquired cellular resistance simultaneously to several cytotoxic drugs . Expression of the multidrug resistance gene (mdr) is one of the most-studied potential underlying mechanisms . The human mdr gene family encompasses two homologous members, the first of which, called the mdr1 gene, is the best-characterized so far . The human mdr1 gene has been shown to encode a membrane P-170 glycoprotein that, on the basis of its structure, is considered to act as a drug-efflux pump excreting various drugs from cells . The human mdr1 gene is thus a major regulated gene playing an important role in the molecular mechanism of multidrug resistance . Its bipartite structure of two similarly organized halves is explained by a gene fusion event during evolution . However, the clinical significance of this particular feature, if it seemed obvious in the 1980s as a factor producing chemoresistance, is currently revised-being a marker of tumor aggressiveness rather than the cause of drug resistance. Biochem Pharmacol, 1997 Jun 1, 53(11), 1597 - 604 Regulation of the function of P-glycoprotein by epidermal growth factor through phospholipase C; Yang JM et al.; Many multidrug-resistant (MDR) cell lines overexpress the epidermal growth factor receptor (EGFR) as well as P-glycoprotein (P-gp) . However, the role of the increased EGFR in P-gp-mediated drug resistance remains unclear . Since recent studies suggest that activation of phospholipase C (PLC) could increase the phosphorylation of P-gp, and activation of the EGFR would also activate PLC, we investigated whether the effect of epidermal growth factor (EGF) on the phosphorylation of P-gp was mediated through PLC . Treatment of the human MDR breast cancer cell line, MCF-7/AdrR, with EGF increased the phosphorylation of P-gp by 20-50% . The increased phosphorylation of P-gp was accompanied by stimulation of PLC activity, as measured by the production of inositol, 1,4,5-trisphosphate and diacylglycerol, products of phosphatidylinositol-4,5-bisphosphate hydrolysis . Treatment of MDR cells with EGF also had detectable effects on P-gp function . For example, following incubation of MCF-7/AdrR cells with ECF, we observed a consistent decrease in total vinblastine (VBL) accumulation . Kinetic analysis revealed this change to be due to an increase in membrane efflux . The latter was measured by the initial uptake velocity, which was inhibited by EGF . VBL uptake measured at 0-320 sec was inhibited by 20-40%, which was associated with a similar increase in VBL efflux . EGF had no effect on drug accumulation, uptake, or efflux in sensitive MCF-7 cells . These data indicate that EGF can modulate the phosphorylation and function of P-gp, and suggest that this effect may be initiated by the activation of PLC. Environ Health Perspect, 1997 Jun, 105 Suppl 4, 855 - 60 A new type of hazardous chemical: the chemosensitizers of multixenobiotic resistance; Kurelec B; The purpose of this overview is to introduce the property of a new class of hazardous chemicals-the inhibitors of multixenobiotic resistance (MXR) in aquatic organisms, referred to as chemosensitizers . Aquatic organisms possess MXR, a mechanism similar to the well-known P-glycoprotein extrusion pump in multidrug resistant (MDR) tumor cells . MXR in aquatic organism moves from cells and organisms both endogenous chemicals and xenobiotics, including also some man-made chemicals . MXR in aquatic organisms represents a general biological first-line defense mechanism for protection against environmental toxins . Many chemical agents, the chemosensitizers, may after the function of this fragile mechanism . It is this new, MXR-inhibiting property, unrecognized as yet, that classifies these chemicals among top-rank hazardous water pollutants . The knowledge that the presence of one xenobiotic may block the pumping out of other xenobiotic(s), and hence accelerate their accumulation, may have important implications on environmental parameters like exposure, uptake, bioaccumulation, and toxicity . In this overview we present the evidence for the expression of MXR-phenotype in aquatic organisms, the demonstration of toxic consequences caused by MXR inhibitors, and the description of methods for measurement of concentration of MXR inhibitors in environmental samples. J Mol Med, 1997 Jun, 75(6), 420 - 8 The canalicular multispecific organic anion transporter and conjugated hyperbilirubinemia in rat and man; Paulusma CC et al.; The human Dubin-Johnson syndrome is an autosomal recessive liver disease characterized by a chronic conjugated hyperbilirubinemia . Patients have impaired hepatobiliary transport of many endogenous and xenobiotic compounds . A similar disease phenotype has been described for a naturally occurring mutant Wistar rat strain, the TR- rat, which is defective in the, functionally defined, canalicular multispecific organic anion transporter (cMOAT) . The complementary DNA encoding this protein has been cloned from rat and recently from human liver . cMOAT is a new member of the ATP-binding cassette transporter family, and homologous to the multidrug resistance-associated protein 1 . A mutation in the cMOAT gene is responsible for the phenotype observed in TR- rats . This information should soon lead tc a complete genetic characterization of the human Dubin-Johnson syndrome. Gene Ther, 1997 Jun, 4(6), 544 - 52 Employment of the mdr1 promoter for the chemotherapy-inducible expression of therapeutic genes in cancer gene therapy; Walther W et al.; Numerous approaches in gene therapy of human cancers are focused on the establishment of cell type specific or inducible expression vectors allowing the targeted and regulated expression of therapeutic genes . Various conditionally active vectors have been created carrying promoters responding to certain factors or therapeutic modalities (eg hormones, irradiation) . The promoter of the multidrug resistance gene (mdr1) harbors such responsive elements and two of these elements have been related to drug responsiveness . In earlier studies we and others have characterized the mdr1 drug responsive-element in CAT reporter assays demonstrating its inducibility by MDR-associated drugs . To exploit this property, we linked the mdr1 promoter sequence to the human tumor necrosis factor alpha (TNF) cDNA in a retroviral vector and transduced the vector into human mammary and colon carcinoma cell lines . These cells were treated with various mdr1-associated drugs to induce TNF expression in vitro . We have shown that the mdr1 promoter-driven TNF expression is drug-inducible and that this induction is drug concentration and time dependent . The studies demonstrate the feasibility of the novel vector system for a chemotherapy-inducible expression of a chemosensitizing cytokine that is successful at enhancing cytotoxicity of drugs in cancer therapy. Am J Trop Med Hyg, 1997 Jun, 56(6), 613 - 7 Open randomized trial of oral artemether alone and a sequential combination with mefloquine for acute uncomplicated falciparum malaria; Looareesuwan S et al.; One hundred fifty-one patients with acute uncomplicated falciparum malaria were enrolled in a randomized, open-label study of oral artemether given alone for five or seven days or a sequential treatment of oral artemether followed by mefloquine . Forty patients received oral artemether, 100 mg initially, then 50 mg every 12 hr for a total dose of 500 mg over a five-day period: Group I . Fifty-eight patients received oral artemether, 100 mg initially, then 50 mg every 12 hr for a total dose of 750 mg over a seven-day period: Group II . Fifty-three patients received oral artemether, 200 mg every 8 hr for a total dose of 600 mg, followed 8 hr later with mefloquine (1,250 mg divided into two doses given 6 hr apart: Group III . All patients were admitted to the hospital for 28 days to exclude reinfection and 131 patients remained through the 28-day follow-up . Only two, nine, and nine patients in Groups I, II, and III, respectively, left the hospital prior to study completion for reasons unrelated to their treatment . Cure rates for the three groups were 74% (28 of 38) for Group I, 98% (48 of 49) for Group II, and 98% (43 of 44) for Group III . Mean fever and parasite clearance times were not significantly different (32.8, 27.5, and 31.4 hr for fever clearance times and 40.2, 40.6, and 36.7 hr for parasite clearance times of Groups I, II, and III, respectively) nor were any adverse effects seen . In vitro drug susceptibility testing of admission and recrudescent parasite isolates was conducted for 10 patients . These data showed no decreased response to artemether or dihydroartemisinin in recrudescent isolates when compared with admission isolates . The results of this study suggest that sequential treatment for two days with oral artemether (600 mg) followed by mefloquine (1,250 mg) is effective and well-tolerated in patients with acute uncomplicated falciparum malaria and may be an alternative treatment for multidrug-resistant falciparum malaria, particularly useful for treating patients in rural areas where the period of admission to the hospital should be as short as possible . A seven-day regimen of artemether alone (750 mg) is also very effective, yet requires prolonged administration of drug after malaria symptoms disappear. Gen Diagn Pathol, 1997 Jun, 142(5-6), 317 - 25 P-glycoprotein expression in high grade central osteosarcoma and normal bone cells . An immunohistochemical study; Posl M et al.; One important mechanism by which multidrug resistance is mediated is the mdr1 gene product, P-glycoprotein (Pgp) . Even though chemotherapy, in the treatment of high grade central osteosarcoma (hgc-OS), has led to dramatic improvements in survival rate, a certain percentage of patients still show only a poor response to chemotherapy . To further characterize a potential connection between Pgp and chemotherapy as well as the role of Pgp in tumorigenesis of osteosarcoma, we analyzed Pgp-expression in hcg-OS . Immunohistochemistry was performed on 68 hgc-OS samples from 58 patients using the monoclonal antibody JSB-1; in addition, Pgp-expression in normal bone cells was studied in 5 human epiphyseal growth plates . 70.5% of all cases stained positive for P-glycoprotein, while 29.5% of the cases were negative . Cases investigated after chemotherapy showed a higher incidence (82.9%) of positive P-glycoprotein immunostaining than cases prior to chemotherapy (64.4%) . The Pgp-expression of 34 biopsies was compared with chemotherapy, as determined at the surgical specimen . In these cases, however, no correlation could be established between P-glycoprotein expression of the biopsy and the later response to chemotherapy . 48.4% of the cases with biopsies, initially positive for Pgp, showed a good response in the surgical specimen, while only 27.2% of Pgp-positive biopsies were later classified as non-responders . In the normally growing skeleton, positive immunostaining was detected in the area of mineralization of epiphyseal growth plates . Osteoclasts, hypertrophic chondrocytes, and cuboidal osteoblasts showed Pgp-expression, while there was a lack of Pgp in the majority of osteocytes and chondrocytes in the resting and proliferating zone . These data therefore suggest that P-glycoprotein expression in hgc-OS resembles, at least in part, the phenotype of active bone cells . These results may explain why P-glycoprotein, by using immunohistochemistry, in biopsies of osteosarcomas is insufficient to predict the response to chemotherapy. Acta Med Okayama, 1997 Jun, 51(3), 121 - 7 Growth inhibitory effects of antifolates against an adriamycin-resistant human small cell lung cancer cell line; Matsuo K et al.; We have established an Adriamycin (ADM)-resistant small cell lung cancer (SCLC) cell line, SBC-3/ADM 100, which shows multifactorial mechanisms of resistance to ADM, such as over-expression of P-glycoprotein, an enhanced detoxifying system and a decrease in topoisomerase II activity . In the present study, we confirmed that SBC-3/ADM 100 showed collateral sensitivity to methotrexate and TNP-351, a new antifolate, though this cell line showed a typical multidrug resistance (MDR) pattern . We also demonstrated a faster uptake and higher accumulation (1.3-fold) of TNP-351 in the SBC-3/ADM 100 cells than those in the parent SBC-3 cells . These results explain one of the mechanisms for collateral sensitivity in the resistant cells . Furthermore, this cell line was found to have no cross-resistance to edatrexate and minimal cross-resistance to trimetrexate, 254-S (cisplatin analog), 5-fluorouacil and 4-hydroperoxyifosfamide . These drugs will have clinical importance in patients with SCLC who were previously treated with an ADM-containing regimen . Thus, antifolates, especially TNP-351 and edatrexate, can be expected to eradicate residual multidrug resistant SCLC cells selected by ADM. Am J Physiol, 1997 Jun, 272(6 Pt 1), G1285 - 303 Molecular aspects of hepatobiliary transport; Muller M et al.; Generation of bile flow is a regulated, ATP-dependent process and depends on the coordinated action of a number of transporter proteins in the sinusoidal and canalicular domains of the hepatocyte . Dysfunction of any of these proteins leads to retention of substrates, with conjugated hyperbilirubinemia or cholestasis as a result . In recent years many of the transport proteins involved in bile formation have been identified, cloned, and functionally characterized . The hepatocyte sinusoidal membrane contains transport proteins for the hepatic uptake of organic anions and cations and for the uptake of bile acids . The multispecific organic anion transporting polypeptide (OATP) mediates the hepatic uptake of organic anions and a variety of organic amphiphilic compounds, including organic cations . The organic cation transporter OCT1 more specifically transports small organic cations . NTCP is the Na(+)-bile acid cotransporting protein that mediates the hepatic uptake of bile acids . The canalicular transport proteins are able to transport endogenous and exogenous metabolites into the bile against steep concentration gradients . Most of these transporters are members of the large ATP-binding cassette (ABC) superfamily, and their transport function directly depends on the hydrolysis of Mg2+/ATP . At least five ABC transporter proteins have been characterized so far: 1) the human multidrug resistance protein MDR1 mediates the excretion of hydrophobic, mostly cationic, metabolites; 2) MDR3 is involved in phosphatidylcholine secretion; 3) the canalicular bile acid transporter cBAT mediates secretion of monovalent bile salts and provides the molecular basis of bile acid-dependent bile flow; 4) SPGP, product of the P-glycoprotein sister gene, is exclusively expressed in the liver but its function is currently unknown; and 5) the human multidrug resistance protein MRP2 mediates the excretion of multivalent anionic conjugates. Am J Physiol, 1997 Jun, 272(6 Pt 1), E1050 - 8 Intestinal function in mice with small bowel growth induced by glucagon-like peptide-2; Brubaker PL et al.; Glucagon-like peptide-2 (GLP-2) stimulates small intestinal growth through induction of intestinal epithelial proliferation . To examine the physiology of GLP-2-induced bowel, mice were treated with GLP-2 (2.5 micrograms) or vehicle for 10 days . Small intestinal weight increased to 136 +/- 2% of controls in GLP-2-treated mice, in parallel with 1.4 +/- 0.1- and 1.9 +/- 0.5-fold increments in duodenal RNA and protein content, respectively (P < 0.05-0.001) . Similarly, the activities of duodenal maltase, sucrase, lactase, glutamyl transpeptidase, and dipeptidyl-peptidase IV (215 +/- 28% of controls; P < 0.001) were increased by GLP-2 . Oral or duodenal administration of glucose or maltose did not reveal any differences in the ability of GLP-2-treated mice to absorb these nutrients, possibly because of decreases in expression of the glucose transporters sodium-dependent glucose transporter-1 (SGLT-1) and GLUT-2 . In contrast, absorption of leucine plus triolein was increased after duodenal administration in GLP-2-treated mice (P < 0.01-0.001) . Finally, GLP-2 did not alter other markers of intestinal or pancreatic gene expression, including levels of mRNA transcripts for ornithine decarboxylase, multidrug resistance gene, amylase, proglucagon, proinsulin, and prosomatostatin . Thus induction of intestinal growth by GLP-2 in wild-type mice results in a normal-to-increased capacity for nutrient digestion and absor