Biochemistry, 1995 Oct 31, 34(43), 14156 - 62 Bacterial expression of the linker region of human MDR1 P-glycoprotein and mutational analysis of phosphorylation sites; Chambers TC et al.; Phosphorylation may play a role in modulating multidrug resistance by P-glycoprotein (P-gp) . The linker region between the two homologous halves of human P-gp harbors several serine residues which are phosphorylated by protein kinase C (PKC) in vitro . We used the glutathione S-transferase gene fusion system to express and purify a series of fusion proteins containing the relevant portion (residues 644-689) of the linker region of the human MDR1 gene product . The fusion proteins were subjected to in vitro phosphorylation and phosphopeptide mapping analysis to identify specific phosphorylation sites . On the basis of a mutational strategy in which individual serine residues were systematically replaced with nonphosphorylatable alanine residues, Ser-661 and Ser-667 were identified as major PKC sites and Ser-683 was identified as a minor PKC site . Ser-661 and Ser-667 were also found to be the primary sites of phosphorylation for a novel membrane-associated P-gp specific kinase isolated from the multidrug-resistant KB-V1 cell line . Individual phosphorylation sites were recognized independently of each other . These data show that the linker region of P-gp represents a target for multisite phosphorylation not only for PKC but also for the P-gp specific V1 kinase . Specific serine phosphorylation sites are identified, and evidence is presented that the V1 kinase has a specificity which overlaps, but is more restricted than, that of PKC . In addition, these studies also suggest that the use of GST fusion peptides may be applicable for the analysis of multisite and ordered protein phosphorylation in other systems. J Biol Chem, 1995 Oct 27, 270(43), 25468 - 74 Identification and characterization of a hepatoma cell-specific enhancer in the mouse multidrug resistance mdr1b promoter; Song R et al.; The expression of multidrug resistance/P-glycoprotein genes mdr1b(mdr1) and mdr1a(mdr3) is elevated during hepatocarcinogenesis . To investigate the regulation of mdr1b gene expression, we used transient transfection expression assays of reporter constructs containing various 5'-mdr1b flanking sequences in hepatoma and non-hepatoma cells . We found that nucleotides -233 to -116 preferentially enhanced the expression of reporter gene in mouse hepatoma cell lines in an orientation- and promoter context-independent manner . DNase I footprinting using nuclear extracts prepared from hepatoma and non-hepatoma cells identified four protein binding sites at nucleotides -205 to -186 (site A), -181 to -164 (site B), -153 to -135 (site C), and -128 to -120 (site D) . Further analyses revealed that, while site B alone played a major part for the enhancer function, sites A and B combined conferred full enhancer activity . Site-directed mutagenesis results also supported these results . Gel retardation experiments using oligonucleotide competitors revealed that the site B contains a dominant binding protein . This is the first report demonstrating a cell type-specific enhancer in the mdr locus . The role of this enhancer in the activation of mdr1b gene during hepatocarcinogenesis is discussed. Int J Cancer, 1995 Oct 20, 64(5), 322 - 5 Preferential expression of the multidrug-resistance-associated protein (MRP) in adenocarcinoma of the lung; Sugawara I et al.; The expression of multidrug-resistance-associated protein (MRP) was assessed in various types of untreated lung cancer using an immunohistochemical technique . MRP was abundantly expressed in 28 of 59 adenocarcinoma specimens (47%) and its expression was associated with the degree of glandular differentiation of the tumor . MRP expression in well-differentiated adenocarcinomas (56%) was higher than in poorly differentiated adenocarcinomas (22%) (p < 0.01) . lower--20% in squamous-cell carcinomas, 20% in large-cell carcinomas and 0% in small-cell carcinomas and carcinoids . RT-PCR showed that the MRP-positive adenocarcinomas and squamous-cell carcinomas expressed mrp mRNA significantly . Immunoelectron microscopically, MRP was localized in the plasma membrane and rough endoplasmic reticulum . It is thus important to take MRP into account when considering chemotherapy for lung cancers because levels of mdr I gene product, another multidrug-resistance gene family, are low in untreated lung cancers. Cancer Lett, 1995 Oct 20, 97(1), 93 - 8 Flow injection analysis quantifies multidrug resistance phenotype; Venkatesan PV et al.; Flow injection analysis (FIA inverted question mark of DNA damage, repair and drug accumulation was employed to examine the relation between DNA damage, drug resistance and cell survival . Resistant sublines to adriamycin (P388/ADR), mitoxantrone (P388/MTN) and drug sensitive (P388/S) leukemic cells were exposed to different concentrations of adriamycin . The subtle difference in tumor response between sensitive and resistant cells was well differentiated and the results were comparable with other methods of measuring DNA strand-break and repair . Drug concentrations as low as 5 x 10(-11)M could be measured by FIA . In addition the method also enables assessment of cross-resistance/sensitivity . The speed, sensitivity and reproducibility make FIA a good technique for routine monitoring of tumor cell response to DNA damaging agents. Gene, 1995 Oct 16, 164(1), 179 - 84 Increase in mRNA of multiple Eh pgp genes encoding P-glycoprotein homologues in emetine-resistant Entamoeba histolytica parasites; Descoteaux S et al.; With the goal of understanding possible mechanisms of drug resistance by the protozoan parasite Entamoeba histolytica (Eh), two novel Eh P-glycoprotein (Pgp) genes (Eh pgp5 and Eh pgp6) were sequenced, and the expression of four Eh pgp genes determined in wild-type (wt) clone A and emetine-resistant (EmR) clone C2 amebae . The Eh pgp5 gene encodes a 1301-amino acid (aa) protein that is similar to those of Eh pgp1 (64% aa identity), Eh pgp2 (61%), Eh pgp6 (39%) and Homo sapiens MDR (multidrug-resistance-encoding)(Hs MDR1; 38%) genes . The 1282-aa Eh pgp6 open reading frame (ORF), which is 19-28 aa shorter than those encoded by other Eh pgp, is also similar to those of Eh pgp1 (46% aa identity), Eh pgp2 (38%), and Hs MDR1 (39%) . Both Eh pgp5 and Eh pgp6 ORF predict two ATP-binding cassettes and twelve hydrophobic alpha-helices, which form the putative transmembrane channel . EmR clone C2 amebae, growing at all concentrations of drug, show increased amounts of Eh pgp1 and Eh pgp6 mRNA when compared to wt clone A amebae . In contrast, only clone C2 amebae selected for growth at the highest concentrations of emetine (100-200 micrograms/ml) show increased Eh pgp5 mRNA, while mRNA of both clone C2 and clone A Eh amebae fail to bind an Eh pgp2-specific probe . It appears then that multiple Pgp may contribute to amebic Em resistance in vitro. FEBS Lett, 1995 Oct 16, 373(3), 285 - 90 Lack of elevated drug efflux in adriamycin-resistant immunoblastic B lymphoma cells with mdr1 overexpression; Chao CC; A multidrug-resistant (MDR) subline of the immunoblastic B lymphoma cell line was established by sequentially selecting in increasing concentrations of adriamycin . The adriamycin-resistant cell line (HOB1/ADR) demonstrated resistance to a wide spectrum of chemotherapeutic agents including MDR drugs (Vinca alkaloids and anthracycline), antimicrotubule drug (colchicine), and DNA-damaging agents (cisplatin and mitomycin C) . The expression of human mdr1 gene, as analyzed by RT-PCR and Western blotting, revealed a 13-15-fold increase in resistant cells . Unexpectedly, HOB1/ADR cells demonstrated a lack of reduced accumulation and of enhanced efflux of adriamycin . More than 60% adriamycin was effluxed at the same rate in both cell lines within 10 min . In contrast, the initial rate of vincristine accumulation was reduced by 3 fold in this resistant cell line . The maximal level of vincristine accumulation was 50% lower in the resistant cells than the parental cells . The maximal efflux rate was enhanced by 5 fold in the resistant cells . Inhibition of vincristine resistance by verapamil associated with restoration of drug accumulation, suggesting that acquired resistance in these cells is due to P-glycoprotein . These studies demonstrated that immunoblastic B lymphoma cells selected for adriamycin resistance preferentially developed P-glycoprotein-mediated vincristine efflux which plays an important role in vincristine resistance . In contrast, the resistant cells did not elevate adriamycin efflux, suggesting an additional mechanism responsible for adriamycin resistance. Cancer, 1995 Oct 15, 76(8), 1356 - 62 Phase I/II trial of dexverapamil, epirubicin, and granulocyte-macrophage-colony stimulating factor in patients with advanced pancreatic adenocarcinoma; Kornek G et al.; BACKGROUND . The purpose of this study was to determine the maximum tolerated dose (MTD) of a cytotoxic regimen consisting of the second-generation chemosensitizer dexverapamil (DVPM), high dose epirubicin, and recombinant human granulocyte-macrophage-colony stimulating factor (GM-CSF) in pancreatic carcinoma . PATIENTS AND METHODS . Twenty-eight previously untreated patients with locally advanced or metastatic adenocarcinoma of the pancreas were studied . Treatment consisted of oral DVPM at a dose of 1000-1200 mg/day for 3 days, epirubicin administered as an intravenous bolus injection on Day 2 with an initial dose of 90 mg/m2, and a dose of GM-CSF of 400 micrograms administered subcutaneously from Day 5s through 14 . Epirubicin dose escalation levels were 90, 105, 120 and 135 mg/m2 . Consecutive cohorts of four to eight patients were planned at each dose level . Treatment cycles were repeated every 3 weeks . RESULTS . Hematologic toxicity, specifically granulocytopenia, constituted the dose-limiting toxicity with an MTD of 120 mg/m2 for epirubicin . Despite routine supportive therapy with GM-CSF, four, two, and five patients experienced Grade 4 granulocytopenia during their first two treatment courses at levels 105, 120, and 135 mg/m2, respectively . Grade 4 granulocytopenia was observed in two, three, and one additional patients during subsequent courses with these levels . Nonhematologic toxicity was uncommon, generally modest, and did not correlate clearly with the anthracycline dose . Dexverapamil-related cardiovascular symptoms occurred frequently, but they never resulted in serious toxicity requiring active medical intervention or permanent discontinuation of therapy . Nine of 28 patients achieved partial responses to this therapy . Stable disease was observed in nine patients, and tumor progress occurred in 10 . CONCLUSION . The MTD of epirubicin for this regimen with DVPM and GM-CSF was 120 mg/m2 every 3 weeks . Though it remains uncertain whether the encouraging response activity observed in this disease-oriented Phase I study was, in fact, due to successful modulation of multidrug resistance, these results suggest that this regimen is likely to be an effective and tolerable treatment strategy for patients with pancreatic cancer, which should be evaluated further. Eur J Pharmacol, 1995 Oct 15, 291(2), 183 - 9 Opiates inhibit ion conductances elicited by cell swelling and cAMP in cultured cells; Callaghan R et al.; The effect of several opiate compounds on I- efflux was investigated in cultured cell lines . I- efflux was evoked by two distinct stimuli, namely cell swelling and elevation of cellular cAMP levels by prostaglandin E2 . Cells expressing the multidrug resistance P-glycoprotein were found to have increased I- efflux in response to hypo-osmotic challenge . This increased I- efflux in P-glycoprotein containing cells was reduced to levels found in parental cells by the opiates morphine, pentazocine and naloxone . Addition of prostaglandin E2 to T84 cells resulted in elevated cellular cAMP levels and a significant I- efflux . This cAMP stimulated efflux was also inhibited by several opiates . None of the opiates was able to alter cAMP levels or protein kinase A mediated phosphorylation of immunoprecipitated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel in T84 cells . The ability of opiates to alter ion conductances is discussed in relation to the anti-diarrheal effects of these compounds. Biochem Pharmacol, 1995 Oct 12, 50(8), 1245 - 55 Structure-activity relationship of verapamil analogs and reversal of multidrug resistance; Toffoli G et al.; We studied the relationship between the chemical structure and multidrug resistance (MDR) reversal activity of racemic verapamil (VER) and 14 VER analogs (VAs) . The LoVo-R human colon carcinoma cell line was used as an experimental model . This cell line exhibited a typical MDR phenotype and overexpressed the MDR1 gene products . Key structural features were identified as being related to MDR reversal and cytotoxic activity . In particular, we demonstrated that the methoxy groups in the VER molecule structure {1.7-Bis-(3.4-dimethoxyphenyl)-3-methylaza-7-cyan-8-methyl-n onane} prevented cytotoxicity when the VAs were used alone, whereas the 7-cyan-8-methyl groups were important for MDR reversal activity and interaction with P-glycoprotein (P-gp) . Among the VAs tested, the most active compounds were gallopamil, R-isomer of VER (R-VER), and nor-VER, which potentiated doxorubicin (DOX) cytotoxicity by 52.3 +/- 7.2 (n = 3 +/- SD), 38.9 +/- 6.4 (n = 4 +/- SD), and 35.4 +/- 4.3 (n = 3 +/- SD) times, respectively . The reversal activity of these compounds was similar to that of VER, which enhanced DOX cytotoxicity by 41.3 +/- 5.0 (n = 3 +/- SD) times . The potentiation of DOX cytotoxicity was associated with an increase in DOX uptake in LoVo-R cells and with an increased {3H}azidopine P-gp photolabeling inhibition . Some compounds that had a high reversal potency (i.e . R-VER and nor-VER) showed a lower calcium antagonist activity than VER, and seem useful candidates for the treatment of MDR in cancer patients. Biochem Pharmacol, 1995 Oct 12, 50(8), 1135 - 9 Multidrug resistance bypass in cells exposed to doxorubicin-loaded nanospheres . Absence of endocytosis; Henry-Toulme N et al.; Multidrug resistance bypass has been achieved in vitro with polyalkylcyanoacrylate nanospheres loaded with doxorubicin as previously shown by Cuvier et al . Fluorescence-imaging experiments were performed to determine if the endocytosis of the particles by the cells could be responsible for this activity . The results obtained with three lines of sensitive and resistant cells (K562, MCF7, and C6) demonstrate that the particles were not internalized by the cells, nor were they adsorbed onto the cell surface . In contrast, these nanospheres were very efficiently endocytosed by phagocytic cells such as macrophages . No correlation was observed between the fate of the particles, endocytosed or not, and their cytotoxic activity . It was concluded that no endocytosis step was involved in the mechanism of the multidrug resistance bypass obtained by associating a drug to polymeric particles. N Engl J Med, 1995 Oct 5, 333(14), 907 - 11 Multidrug-resistant tuberculosis in patients without HIV infection; Telzak EE et al.; BACKGROUND . Investigations of outbreaks of multidrug-resistant tuberculosis have found low rates of treatment response and very high mortality, and they have mainly involved patients with advanced human immunodeficiency virus (HIV) infection . For patients without HIV infection, one study reported an overall rate of response to treatment of 56 percent, and the mortality from tuberculosis was 22 percent . We investigated treatment response and mortality rates in 26 HIV-negative patients in New York with multidrug-resistant tuberculosis . METHODS . We obtained detailed data from seven teaching hospitals in New York City on patients with multidrug-resistant tuberculosis--defined as tuberculosis resistant at least to isoniazid and rifampin--who were HIV-negative on serologic testing . Lengths of times from diagnosis to the initiation of appropriate therapy and from the initiation of appropriate therapy to conversion to negative cultures were assessed . Therapeutic responses were evaluated by both microbiologic and clinical criteria . RESULTS . Between March 1991 and September 1994, 26 HIV-negative patients were identified and treated . Of the 25 patients for whom adequate data were available for analysis, 24 (96 percent) had clinical responses; all 17 patients for whom data on microbiologic response were available had such a response . The median times from diagnosis to the initiation of appropriate therapy and from the initiation of therapy to culture conversion were 44 days (range, 0 to 181) and 69 days (range, 2 to 705), respectively . Side effects requiring the discontinuation of medication occurred in 4 of 23 patients (17 percent) who were treated with second-line antituberculosis medications . The median follow-up for the 23 patients who responded and who received appropriate therapy was 91 weeks (range, 41 to 225) . CONCLUSIONS . In this report from New York City, HIV-negative patients with multidrug-resistant tuberculosis, contrary to previous reports, responded well to appropriate chemotherapy, both clinically and microbiologically. Biochem Biophys Res Commun, 1995 Oct 4, 215(1), 179 - 85 Modulation of P-glycoprotein and mdr1b mRNA expression by growth factors in primary rat hepatocyte culture; Hirsch-Ernst KI et al.; P-glycoproteins encoded by members of the mdr gene family function as membrane-situated transport proteins, isoforms of which are involved in conferring a form of multidrug resistance by participating in secretion of various xenobiotics . In primary rat hepatocytes maintained in serum-free culture, accumulation of immunodetectable P-glycoprotein and mdr1b mRNA occurred in a time-dependent manner and was accompanied by a substantial decrease in retention of the mdr1 substrate rhodamine 123 . However, incubation of cells with epidermal growth factor (EGF) or with insulin-like growth factor I (IGF-I) markedly enhanced time-dependent accumulation of P-glycoprotein and mdr1b mRNA . Furthermore, EGF-treated cells exhibited decreased intracellular rhodamine 123 retention, an effect partially inhibited by the chemosensitizer verapamil . These data suggest that an increase in (a) functional transporter(s) eliciting transport of mdr1 substrates occurs under EGF. Anticancer Drugs, 1995 Oct, 6(5), 681 - 5 Activity of N-benzyl-adriamycin-14-valerate (AD198), a new anthracycline derivate, in multidrug resistant human ovarian and breast carcinoma cell lines; Harstrick A et al.; The new lipophilic anthracycline N-benzyl-adriamycin-14-valerate (AD198) was evaluated for its activity in comparison to doxorubicin in P-glycoprotein (Pgp)-positive and -negative cell lines . AD198 and doxorubicin showed comparable antitumor activity in the Pgp-negative breast cancer cell line MCF-7 and the Pgp-negative ovarian carcinoma cell line A2780 . By contrast, AD198 was significantly more active than doxorubicin in the Pgp-positive breast cancer cell line MCF7AD (IC50 values 2.5 and 0.15 microM for 96 h continuous exposure) and the Pgp-positive ovarian carcinoma cell line A2780 DX5 (IC50 values 0.6 and 0.07 microM, respectively) . Unlike doxorubicin, the activity of AD198 was not increased by concommittant application of cyclosporin A in cell line MCF7AD . Flow cytometry studies showed that, in contrast to doxorubicin, AD198 was not transported by Pgp and that verapamil did not change the intracellular pharmacokinetics of this new anthracycline . These data provide evidence that AD198 possesses high activity in human solid tumor cell lines expressing the classical multidrug resistant phenotype . Its further clinical development appears to be warranted. Anticancer Drugs, 1995 Oct, 6(5), 669 - 80 Decreased uptake of cyclosporin A by P-glycoprotein (Pgp) expressing CEM leukemic cells and restoration of normal retention by Pgp blockers; Didier A et al.; The P-glycoprotein (Pgp) molecules which are expressed on multidrug resistant (MDR) tumor cells can efflux a variety of cytostatics . In both normal and tumoral epitheliums, Pgp molecules are selectively expressed on the apical surface of the epithelial cells . Such a distribution seems to be responsible for the transcellular transport of Pgp substrates, including cyclosporin A (CsA), from the basal to the apical side . Some normal lymphoid cells also express small amounts of Pgp molecules, for as yet unknown functions . Nevertheless, the sensitivity of their mitogen-induced proliferation to cytostatics, including doxorubicin and CsA, could be increased by the Pgp blockers . Using isotopically-labeled CsA and tumoral lymphoid cell lines, we now show a higher CsA retention in Pgp-lacking parental ('Par') cells than in Pgp-expressing MDR cells . The Pgp blockers can restore the CsA retention in the MDR cells to its level in the Par cells. J Pharm Sci, 1995 Oct, 84(10), 1205 - 9 Reversal of drug sensitivity in MDR subline of P388 leukemia by gene-targeted antisense oligonucleotide; Nakashima E et al.; We attempted to reverse multidrug resistance (MDR) by treatment with 25-mer antisense phosphorothioate oligonucleotide . The phosphorothioate analogs, the sequences of which are sense or antisense to the initiation codon of mouse mdr1 mRNA, were tested against murine leukemic P388/S and adriamycin-resistant P388/ADR cell lines . A weak inhibitory effect on the growth of P388/S and P388/ADR cells was observed at a sense and antisense oligonucleotide concentration of 30 microM . Using the monoclonal antibody to P-glycoprotein and a flow cytometry technique, we showed that the level of expression of P-glycoprotein in P388/ADR cells treated with antisense oligonucleotide was lower than when treated with sense oligonucleotide . The antisense oligonucleotide potentiated the growth-inhibitory effect of vinblastine on P388/ADR cells, whereas sense oligonucleotide did not . This was accompanied by an increase in vinblastine retention in the cells . The reversal of the resistance by antisense oligonucleotide was increased by the combination with 1 microM verapamil . These results suggest that the antisense oligonucleotide and low dose verapamil may be useful in circumventing the resistance to anticancer drugs of MDR tumors. Zhonghua Nei Ke Za Zhi, 1995 Oct, 34(10), 655 - 8 {A clinical study on multidrug resistance gene (MDR1) expression in acute leukemia}; Pu J et al.; The authors established a highly sensitive, specific and quantitative method-RT-PCR for measuring the levels of MDR1 mRNA in 91 acute leukemic samples (including 30 untreated cases, 32 remission cases, 29 refractory and relapse cases) . The results showed that MDR1 mRNA positive rate for refractory and relapse cases, untreated cases and remission cases were 82.8%, 40.0%, 28.1% respectively . In untreated patients, it was found that the first complete remission rate differed significantly between MDR1 positive (41.7%) and MDR1 negative groups (88.9%) (P < 0.05) . In remission group, MDR1 positive cases had a higher relapse rate than MDR1 negative case (66.7%; 13.0% P < 0.01) and all these cases relapsed of MDR1 gene overexpression (6 cases) were resistant to further treatment . It is concluded that the expression of MDR1 mRNA might be an unfavorable prognostic factor for patients with acute leukaemia and could predict the efficacy of intensive chemotherapy and serve as a high risk factor for early and resistant relapse. Eur J Clin Microbiol Infect Dis, 1995 Oct, 14(10), 911 - 4 Tuberculosis cutis miliaris disseminata due to multidrug-resistant Mycobacterium tuberculosis in AIDS patients; Antinori S et al.; Two patients with AIDS and disseminated tuberculosis characterized by cutaneous involvement are reported . They developed a maculopapular skin eruption, from which a multidrug-resistant Mycobacterium tuberculosis strain was isolated . In both cases the clinical course was rapidly fatal . Tuberculosis cutis miliaris disseminata should be differentiated from the skin lesions frequently seen in HIV-infected patients, especially from folliculitis . In patients with tuberculosis, the appearance of cutaneous lesions may be due to the haematogenous dissemination of mycobacteria . Therefore, early identification of the causative organism by use of optimal microbiological methods is fundamental. J Chemother, 1995 Oct, 7(5), 449 - 51 Expression of the multidrug-resistance (MDR) gene in breast cancer; Correnti M et al.; Of the approximately 18,000 new cases of cancer in Venezuela each year, only half can be treated with surgery and radiation . The remainder must be treated systematically using chemotherapy or biological response modifiers . It has become evident that any drug resistant human tumors express the MDR1 gene, since MDR1 RNA levels are elevated in many cancers that do not respond to chemotherapy . Human mammary carcinomas have multiple oncogene alterations, the most frequently reported being overexpression of the oncogenes c-myc, int-2, neu and c-myb . Thirteen specimens of mammary cancer were obtained by biopsy of untreated patients in stage IIIB . All these patients received three cycles of FAC or CMF-L+GM-CSF after biopsy . In the slot blot analysis of RNA from invasive carcinomas, MDR1 and c-myc transcripts were detectable at a high level in 30% of tumors . Two patients with increased levels of MDR1 before chemotherapy did not respond to the treatment and distant metastasis and death occurred in these patients . Another patient, MDR1-negative before therapy, did not respond to CMF-1 + GM-CSF and showed high levels of MDR1 transcripts in a second biopsy which was obtained during surgery. Genome Res, 1995 Oct, 5(3), 245 - 58 Functional expression of yeast artificial chromosome-human multidrug resistance genes in mouse cells; Kusaba H et al.; Multidrug resistance (MDR) genes, which are ATP-binding cassette family genes, encode the cell surface glycoprotein, P-glycoprotein, which functions as an energy-dependent drug efflux pump . Two relevant human genes, PGY1 and PGY3, are located on human chromosome 7, and three relevant mouse genes, mdr1a, mdr1b, and mdr2, are located on mouse chromosome 5 . An LMD1 cell line was established after the transfer of a 580-kb yeast artificial chromosome (YAC) clone carrying the human MDR locus into mouse L cells; the cell line was shown to have stably integrated YAC DNA in an apparent intact form . Using LMD1 cells as the parental cell line, five vincristine-resistant sublines, designated LMD1-V50, LMD1-V100, LMD1-V200, LMD1-V500, and LMD1-V1000, were isolated by exposure to increasing concentrations of the drug . LMD1-V50, LMD1-V100, LMD1-V200, LMD1-V500, and LMD1-V1000 showed 3-, 7-, 13-, 45-, and 110-fold higher resistance to the cytotoxic effects of vincristine, respectively, than their parental counterpart, LMD1 . Immunofluorescence, Western blot, and Northern blot analyses revealed that the human PGY1 gene or its product was overexpressed, accompanied by gene amplification . The human PGY3 gene was also overexpressed in the LMD1-V20, LMD1-V100, and LMD1-V1000 cell lines . Southern blot and fluorescence in situ hybridization (FISH) analyses demonstrated that although essentially the entire YAC DNA was integrated in mouse genome and amplified, the endogenous mouse mdr genes were not amplified in these drug-resistant cell lines . Similar results were obtained by the analyses of vincristine-resistant cell lines isolated from four independent subclones of LMD1 cells . Thus, in contrast to their mouse counterparts, the integrated human MDR genes retained susceptibility to both gene activation and amplification, during the selection of drug-resistant mouse cell lines . The possibility that transferred YACs may retain regulatory properties observed in the cells of origin, and may have a chromatin structure that favors augmented expression, is discussed. Genome Res, 1995 Oct, 5(3), 233 - 44 A YAC-based contig of 1.5 Mb spanning the human multidrug resistance gene region and delineating the amplification unit in three human multidrug-resistant cell lines; Torigoe K et al.; A contig of 21 nonchimeric yeast artificial chromosomes (YACs) has been assembled across 1.5 Mb of the multidrug resistance (MDR) gene region located at 7q21, and formatted with four previously reported probes, six newly isolated probes, and three sequence-tagged sites (STSs) from internal and end fragments of YACs . A physical map of rare cutter restriction enzyme sites across the region was also constructed by pulsed-field gel electrophoretic (PFGE) analysis of four overlapping YAC clones . The amplification unit of this region in different cell lines was then determined by Southern blot analysis on the basis of the physical map and probes . Amplified DNA was located in extrachromosomal elements in human MDR cell lines studied here, and the size of the amplification unit was determined to be discrete in one MDR amplification but variable in others. Eur Respir J, 1995 Oct, 8(10), 1688 - 93 Rapid drug susceptibility testing of Mycobacterium tuberculosis using conventional solid media; Schaberg T et al.; Radiometric methods for M . tuberculosis drug susceptibility testing yield much faster results than standard techniques; however, these methods require sophisticated equipment and are expensive . We investigated a rapid drug susceptibility testing method for isoniazid, rifampin, ethambutol, streptomycin and pyrazinamide in specimens from 197 patients with pulmonary tuberculosis using a simplified agar-dilution method . Middlebrook 7H11 agar solid medium and microcolony detection were used to test sputum from 64 smear-positive, and from 70 culture-positive but smear-negative patients . Culture-positive material from bronchoscopy, surgical biopsy, pleural fluid or gastric fluid of 63 patients was tested . In 64 smear-positive patients, the median time for final susceptibility results was 11 days (95% confidence interval (95% CI) 10-12 days) compared to 62 days (95% CI 56-66 days) with the standard method . In 133 smear-negative patients, results were available after a median of 35 days (95% CI 32-40 days) in contrast to 72 days (95% CI 62-83 days) with the standard method, regardless of whether or not sputum or other materials were used for primary culture . The rapid method detected all cases of single-drug resistance (n = 20) and multidrug resistance (n = 14) within 13 days (95% CI 9-17 days) in smear-positive patients (n = 8), or within 38 days (95% CI 35-48 days) in smear-negative patients (n = 26) . Only one discrepancy was encountered in 985 resistance tests . Moreover, contamination was not observed . Our rapid susceptibility testing method for M . tuberculosis on Middlebrook 7H11 agar is fast, practical and inexpensive . It provides an alternative when more sophisticated techniques are not available or affordable. Genetika, 1995 Oct, 31(10), 1449 - 51 {Relationship between amplicon composition and cytologic type of structures containing amplified DNA in murine P388 cells with multiple drug resistance}; Il'inskaia GV et al.; Previously, we showed that, development of multidrug resistance (MDR) in mouse P388 leukemia cells, is often associated with the appearance of newly-formed chromosome-like structures that contain amplified copies of the mdrl gene . In the present study, we compared amplicon content in P388 sublines showing different types of these structures . A strong correlation between the formation of specific acentric markers consisting of two identical arms and the absence of the sorcin gene co-amplification was found . In all the sublines containing other types of chromosome-like structures, the sorcin gene is co-amplified. Eur J Cancer, 1995 Oct, 31A(11), 1862 - 8 Adding a reverser (verapamil) to combined chemotherapy overrides resistance in small cell lung cancer xenografts; Arvelo F et al.; Small cell lung carcinomas (SCLC) are characterised by chemosensitivity to diverse antitumoral compounds . However, responses are transitory and relapses are commonly observed . We examined the ability of verapamil, a reverser of P-glycoprotein (Pgp)-related resistance, to improve the efficacy of CyCAV combined chemotherapy (Cy, cyclophosphamide (CPA); C, cisplatin (CDDP); A, doxorubicin (ADM);V, etoposide (VP16)), as currently administered to SCLC patients at Institut Gustave-Roussy, France, and adapted to the treatment of nude mice implanted with these tumours . Although Pgp encoded by the MDR1 (multidrug resistance) gene is not the only mechanism for multidrug resistance (MDR), and not all drugs included in this regimen are recognised by Pgp, we anticipated a therapeutic benefit . Four different SCLC lines, expressing the MDR1 gene and recently grafted into nude mice, were used . SCLC-75, SCLC-6 and SCLC-41 originated from untreated patients, and SCLC-74T was derived from a patient treated with a combination of ADM, CPA and VP16 . SCLC-41% and SCLC-6T tumours were used after having undergone, respectively, five and nine cycles of in vivo passage and CyCAV treatment of the tumour-bearing nude mice, to reinforce their chemoresistance . The efficacy of the CyCAV regimen, associated with or without verapamil (given 24 h before CyCAV on days 1-5), was tested on the growth of these SCLC . Verapamil (25 mg/kg) improved the antitumour effect of CyCAV in mice bearing SCLC-6T, SCLC-41T and SCLC-75 tumours, although toxicity was observed . Verapamil modestly delayed the plasma clearance of ADM . Two daily injections of 10 mg/kg of verapamil, administered at a 3 h interval, proved to be effective, whereas the same total dose administered as a bolus was not . These results indicate that the association of some reversers of MDR, including drugs possibly interacting with Pgp, might potentiate SCLC combined chemotherapy. Drugs, 1995 Oct, 50(4), 714 - 41 Artesunate . A review of its pharmacology and therapeutic efficacy in the treatment of malaria; Barradell LB et al.; Artesunate is an antimalarial agent, available in oral, rectal and parenteral formulations, that provides a rapid clinical effect in patients with Plasmodium falciparum malaria . The rapidity of effect, availability of an intravenous and intramuscular formulation and convenient dosage regimen make artesunate an ideal candidate for the treatment of severe malaria, including cerebral disease . While some results have been promising, there is no clear evidence to date that artesunate reduces mortality in patients with cerebral malaria to any greater extent than standard quinine therapy . When given as monotherapy, treatment should be continued for at least 5 to 7 days to prevent recrudescence . Combination therapy with mefloquine allows artesunate to be administered over 3 days or less, with a satisfactory clinical outcome maintained . Although optimal dosages remain to be determined, this combination continues to provide the rapid onset of clinical effect observed with artesunate monotherapy, but decreases the rate of recrudescence to 2% (i.e . radical cure rate of 98%) when used as treatment in patients with uncomplicated malaria from areas with a high risk of multidrug-resistance falciparum malaria . Although assessment of tolerability is complicated by the difficulty of distinguishing between disease- and treatment-related events, artesunate and artesunate-mefloquine combinations appear to be well tolerated in adults and children . Indeed, it is possible that prior administration of artesunate may reduce the incidence of mefloquine-induced vomiting . Clinical findings to date have not revealed any pattern of resistance to artesunate after use of the drug . However, given the history of the development of resistance to other antimalarial drugs, the use of artesunate should be restricted to areas of multidrug resistance, the drug should be used in combination with a longer acting agent such as mefloquine, and it should be used in regimens that provide radical cure rates of 90 to 100% . If used according to these treatment principles, artesunate will provide a well tolerated and valuable addition to the current extremely limited treatment options for multidrug-resistant falciparum malaria, a widespread parasitic disease associated with considerable mortality. Acta Paediatr Jpn, 1995 Oct, 37(5), 610 - 3 Effect of cyclosporin A on human bone marrow granulocyte-macrophage progenitors with anti-cancer agents; Ishida Y et al.; Cyclosporin A (CyA) overcomes P-glycoprotein (P-gp) associated multidrug resistance (MDR) . P-gp expression is frequently observed among, not only various cancer cells, but also several normal tissues including bone marrow progenitor cells . These findings lead us to examine whether CyA enhances the myelotoxicity of anti-cancer agents . Bone marrow mononuclear cells were incubated with anti-cancer agents (vincristine, VCR; doxorubicin, ADM; etoposide, VP-16; cytarabine, Ara-C; methotrexate, MTX) and a concentration of CyA (0.5, 5.0 micrograms/mL) . The methylcellulose assay for granulocyte-macrophage progenitors (CFU-GM) was conducted using the post-treated cells . There was no significant toxicity for marrow CFU-GM formation after 72 h incubation with CyA (84-108% of control) . The inhibitory concentration that reduced colonies by 50% (IC50) was 12 nmol/L for VCR, 6 nmol/L for ADM, 220 nmol/L for VP-16, 15 nmol/L for Ara-C and 35 nmol/L for MTX, respectively . For VCR, ADM and VP-16, the number of CFU-GM was unchanged with the addition of CyA at 0.5 microgram/mL concentration . In contrast at 5 micrograms/mL CyA, the number of CFU-GM (% of control) was reduced significantly (P < 0.05 or P < 0.01) . With MTX and Ara-C, the number of CFU-GM was unchanged after addition of CyA, even at 5 micrograms/mL concentration . We conclude CyA may therefore enhance cytotoxic drug sensitivity in MDR tumor cells at a clinically achievable concentration (0.5 microgram/mL) without marrow toxicity. Nippon Rinsho, 1995 Oct, 53(10), 2604 - 11 {Structure and function of P-glycoprotein in antitumor agent resistance; implication for clinical setting}; Tsuruo T; Resistance of tumors to a variety of chemotherapeutic agents presents a major problem in cancer treatment . The gene responsible for multidrug resistance, termed mdr1, encodes a membrane glycoprotein (P-glycoprotein) that acts as a pump to transport various cytotoxic agents . The P-glycoprotein has been shown to bind anticancer drugs and several resistance-reversing agents including calcium channel blockers, and to be an ATPase . The P-glycoprotein was found to function in blood-brain barrier . The implication of the P-glycoprotein in relation to therapy is discussed. Cancer Res, 1995 Oct 1, 55(19), 4352 - 60 Increased rate of adenosine triphosphate-dependent etoposide (VP-16) efflux in a murine leukemia cell line overexpressing the multidrug resistance-associated protein (MRP) gene; Lorico A et al.; WEHI-3B/NOVO is a cloned murine leukemia cell line selected for resistance to novobiocin that is cross-resistant to the cytotoxic action of etoposide (VP-16) and to a lesser extent to a variety of other topoisomerase II (topo II)-reactive drugs . We have reported previously (Cancer Res . 52: 2782-2790, 1992) that WEHI-3B/NOVO cells exhibit a pronounced decrease in VP-16 induced DNA-topo II cross-link formation compared to the parental WEHI-3B/S cell line in intact cells, in the absence of a significant difference in the P4 unknotting activity of topo II assayed in nuclear extracts . Because the pattern of cross-resistance was suggestive of a topo II-mediated mechanism, we have ascertained whether a change in topo II can account for the multidrug-resistant phenotype of WEHI-3B/NOVO cells . No differences existed between WEHI-3B/S and WEHI-3B/NOVO cells in topo II mRNA and protein levels, as well as in the amount of topo II associated with the nuclear matrix . Neither sensitive nor resistant cells expressed detectable levels of the MDR1 gene; however, VP-16 accumulation in WEHI-3B/NOVO cells was 3-4-fold less than that present in WEHI-3B/S cells, whereas doxorubicin accumulation was the same in both cell lines . Over the first 60 s, no difference existed in the rate of uptake of VP-16 between parental and resistant cells; however, beyond the first 60 s of incubation, {3H}VP-16 accumulated to a greater extent in parental sensitive cells . Thus, an increased rate of efflux of VP-16 was responsible for the lower steady-state concentration of the drug in resistant cells . The efflux Km for VP-16 in WEHI-3B/NOVO cells was 254.7 microM and the Vmax was 10.4 pmol/s/10(7) cells . In the presence of the inhibitors of energy metabolism, sodium azide and deoxyglucose, the efflux of VP-16 was markedly inhibited; readdition of glucose restored the original efflux rate . Northern blot analyses using the human 10.1 probe for the 3'-terminal region of the multidrug-resistance protein (MRP) cDNA revealed a mRNA species of approximately 6 kb in WEHI-3B/NOVO cells but not in WEHI-3B/S cells . Overexpression was associated with amplification of the cognate gene . To ascertain whether the overexpressed gene in WEHI-3B/NOVO cells was the murine MRP or a different member of the same superfamily of ATP-binding ABC cassette transporters, a 341-bp MRP cDNA probe was generated from a murine genomic library.(ABSTRACT TRUNCATED AT 400 WORDS) Cancer Res, 1995 Oct 1, 55(19), 4214 - 9 The LRP gene encoding a major vault protein associated with drug resistance maps proximal to MRP on chromosome 16: evidence that chromosome breakage plays a key role in MRP or LRP gene amplification; Slovak ML et al.; A cDNA encoding the novel drug resistance gene, LRP (originally termed lung resistance-related protein), was isolated from HT1080/DR4, a 220-fold doxorubicin-resistant human fibrosarcoma cell line which displays a multidrug resistance phenotype and overexpresses the multidrug resistance protein (MRP) but does not overexpress P-glycoprotein encoded by the MDR1 gene . Using the full-length 2.8-kb cDNA probe, the gene for LRP was regionally localized to the 16p13.1-16p11.2 chromosomal segment in human metaphases . Dual color fluorescence in situ hybridization studies refined the localization of LRP to 16p11.2, a location approximately 27 cM proximal to MRP (16p13.1) . Two color hybridization studies indicated that HT1080/DR4 fibrosarcoma cells contain amplification of both the MRP and LRP genes in a striking striped pattern in the homogeneously staining region, hsr(7)(p12p15) . In contrast, only amplified MRP gene sequences were contained within the homogeneously staining region, hsr(18q) . Amplification of LRP was not identified in any of seven other drug-resistant tumor cell lines characterized by 20-300-fold levels of doxorubicin resistance, including two cell lines known to overexpress LRP (SW1573/2R120 and GLC4/ADR) . Amplified MRP gene sequences were identified in H69AR, GLC4/ADR, and HL-60/AR whereas only MDR1 gene amplification was observed in the S1B120 colon carcinoma cell line . These data indicate that although both the MRP and LRP genes map to the short arm of chromosome 16, they are rarely coamplified and are not normally located within the same amplicon . A key role for chromosome breakage in gene amplification is supported by the presence of non-random karyotypic anomalies near the MRP and LRP normal cellular loci. J Clin Oncol, 1995 Oct, 13(10), 2508 - 16 Anaphylactoid reactions in children receiving high-dose intravenous cyclosporine for reversal of tumor resistance: the causative role of improper dissolution of Cremophor EL; Theis JG et al.; PURPOSE: An unusually high incidence of anaphylactoid reactions was observed during a phase I/II trial of high-dose intravenous cyclosporine (CsA) therapy to attenuate tumor multidrug resistance (MDR) . Five of 21 children experienced severe anaphylactoid reactions shortly after initiation of the first or second CsA infusion . We hypothesized that improper dissolution of the vehicle Cremophor EL may have been a cause for these anaphylactoid reactions . METHODS: All nurses who had administered intravenous CsA were interviewed regarding their technique of preparing the infusion and the occurrence of an anaphylactoid reaction . The responses were statistically analyzed . The effect of various mixing techniques on the distribution of Cremophor EL in the infusion was experimentally evaluated . Different mixing techniques were used to assess their effect on the distribution of Cremophor EL in the solution . RESULTS: Analysis of the preparation techniques of the CsA infusion showed significant correlation between suboptimal mixing of CsA by nurses and the occurrence of anaphylactoid reactions (P = .02) . Experimental simulation showed that suboptimal mixing results in an uneven distribution of Cremophor EL, which subsequently sinks to the bottom of the vial . CONCLUSION: Improper mixing of high-dose CsA infusions causes nonsolubilized Cremophor EL to sink to the outflow area of the bottle . An initial bolus infusion of highly concentrated Cremophor EL may produce an anaphylactoid-like response . This mechanism of toxicity is important to recognize, because it is easily preventable by proper preparation of the infusion, thus reducing the incidence of potentially life-threatening anaphylactoid reactions. Differentiation, 1995 Oct, 59(3), 179 - 92 Parallel patterns of cell-specific gene expression during enterocyte differentiation and maturation in the small intestine of the rabbit; Freeman TC; Enterocytes are the major epithelial cell type of the small intestine . Their capacity to secret, absorb and digest specific ions and nutrients is dependent on their position along the length of the small intestine as well as their stage of development as they migrate and differentiate along the crypt-villus axis . In order to further understand the molecular processes that regulate enterocyte differentiation and function, this study has compared the levels of six mRNA species produced by genes expressed in rabbit enterocytes; specifically, the multidrug resistance (MDR1) gene encoding the 170-kDa P-glycoprotein, CaBP 9k, which encodes a putative intracellular calcium buffer, calbindin, LPH, APN, and AP which encode the brush-border hydrolases lactase-phlorizin hydrolase, aminopeptidase N and alkaline phosphatase, respectively, and SGLT1, encoding the brush border Na(+)-glucose cotransporter . The level of each mRNA species has been mapped along the small intestine using quantitative in situ hybridisation . This has revealed characteristic regional variations in the abundance of each of the mRNAs, supporting the opinion that there is a strong genetic component to the maintenance of gradients in epithelial function along the length of the small intestine . Analysis of the cellular accumulation of mRNA during enterocyte migration along the crypt-villus axis, over gut-associated lymphoid tissue, and at epithelial boundaries, has, by contrast, established a clear correlation in the expression of these genes . These data illustrate the dynamics of enterocyte gene expression, thereby providing an insight into the molecular mechanisms which co-ordinate the events of cell transformation that underlie functional differences between the epithelial populations of the small intestine. Leukemia, 1995 Oct, 9(10), 1661 - 6 Expression of multidrug resistance-associated protein (MRP) and multidrug resistance (MDR1) genes in acute myeloid leukemia; Zhou DC et al.; The frequency, prognostic value and interrelation of MRP and MDR1 gene expressions were investigated by quantitative reverse transcription polymerase chain reaction (RT-PCR) in 91 cases of de novo acute myeloid leukemia (AML), of which 51 were newly diagnosed, 21 were relapsed, and 19 were refractory patients . As compared with normal bone marrow cells and peripheral granulocytes, an overexpression of MRP gene was found in 24% (22 of 91) cases of de novo AML . The incidence of MRP gene overexpression tended to be higher in relapsed patients than in newly diagnosed patients (38 vs 18%, P = 0.063) . In 52 evaluable newly diagnosed and relapsed patients treated with MDR-related drugs, both MRP and MDR1 gene overexpressions correlated to a higher rate of emergence of clinical drug resistance (83 vs 22%, P = 0.005; and 67 vs 24%, P = 0.045, respectively) . A positive correlation was found between MRP and MDR1 gene overexpressions (R = 0.53, P < 0.001) . Analysis of 46 evaluable MDR1-negative cases revealed a trend for higher resistant disease rate in MRP-positive patients as compared with MRP-negative patients (100 vs 20%, P = 0.053) . These data suggest that MRP, like MDR1, may have an important negative impact on the outcome of chemotherapy, and that there may be a common mechanism of induction for the overexpression of these two genes. Leukemia, 1995 Oct, 9(10), 1653 - 60 Topoisomerase II alpha gene expression in childhood acute lymphoblastic leukemia; Klumper E et al.; Previously, we showed that in vitro resistance to daunorubicin (DNR) at initial diagnosis was related to a poor long-term clinical outcome in childhood acute lymphoblastic leukemia (ALL), and that cells of relapsed ALL were in vitro more resistant to DNR than cells of untreated ALL . Topoisomerase II (Topo II) is an intracellular target for anthracyclines and epipodophyllotoxins . Decreased levels and/or activity of Topo II have been associated with multidrug resistance in cell lines . We investigated Topo II alpha gene expression in fresh leukemic samples from 19 children with untreated and 14 children with relapsed ALL using a sensitive RNase protection assay . The in vitro cytotoxicity of the Topo II inhibitors DNA and teniposide (VM26) was measured using the MTT assay, and the cell cycle distribution of leukemic samples was analyzed by DNA flow cytometry . Results showed that (1) relapsed ALL samples were more resistant to DNR, but not to VM26 compared to untreated samples; (2) large interpatient variations existed in both Topo II alpha gene expression and in vitro cytotoxicity results; (3) Topo II alpha gene expression was detectable in 29/33 childhood ALL samples with a median expression of 5% the level of a relatively chemosensitive human small cell lung cancer cell line; (4) Topo II alpha gene expression did not differ between untreated and relapsed ALL; (5) Topo II alpha gene expression was positively correlated with the percentage of ALL cells in S- and G2M-phase, but not with the in vitro cytotoxicity of the drugs tested . In conclusion, resistance to DNR in childhood ALL can not be explained by decreased levels of Topo II alpha gene expression, but additional Topo II activity studies in fresh leukemia samples may need further exploration. Leukemia, 1995 Oct, 9(10), 1631 - 7 Phase I trial of high-dose tamoxifen as a modulator of drug resistance in combination with daunorubicin in patients with relapsed or refractory acute leukemia; Berman E et al.; Tamoxifen and its main metabolite N-desmethyltamoxifen (NDMTmx) have been shown to increase intracellular daunorubicin (DNR) levels in human leukemia cell lines that display the multidrug resistant (MDR) phenotype . We designed a phase I dose escalation study of Tmx (200-700 mg/day p.o . for 7 days) in combination with a fixed dose of DNR (50 mg/m2 intravenously on days 5, 6 and 7) in patients with advanced leukemia to determine whether this combination could be given safely and whether plasma levels of 10 microM, the effective in vitro MDR modulator concentration, could be achieved . Pharmacologic studies of Tmx, NDMTmx and DNR, and its main metabolite daunorubicin-ol (DNR-ol) were performed as was determination of P-glycoprotein (Pgp) using a monoclonal antibody that recognizes an external epitope of the molecule . A total of 14 patients (median age 50, range 22-67) were treated at the following dose levels: 200 mg/day: three patients; 400 mg/day: four patients; 550 mg/day: three patients; and 700 mg/day: four patients . Two patients with relapsed AML achieved remission . Toxicity of the combination was similar to that seen with DNR alone and no severe hepatic, cardiac or retinal toxicity was noted . Plasma Tmx levels approached 7 microM at the two highest dose levels studied; plasma levels of NDMTmx were slightly less . The area under the curve for DNR and its main metabolite daunorubicin-ol (DNR-ol) did not show significant changes with escalation of Tmx dose . This phase I study suggests that concentrations of Tmx high enough to reverse the MDR phenotype can be approached and that the combination of high-dose Tmx with a standard dose of DNR has an acceptable toxicity profile . More evaluation in phase II studies is necessary to define further its role as an MDR modulator. J Pharmacol Exp Ther, 1995 Oct, 275(1), 73 - 8 Cepharanthin, a multidrug resistant modifier, is a substrate for P-glycoprotein; Hirai M et al.; P-glycoprotein modulators are respected to be multidrug resistance reversing agents in cancer chemotherapy . Some calcium channel blockers, calmodulin inhibitors or immunosuppressive agents have been used in clinical studies, although the dose of these drugs required to test in vitro experimental data might cause potent pharmacological effects which are not desirable in patients . By using LLC-GA5-COL150 cells that express P-glycoprotein specifically on the apical membranes, we examined the transport of anticancer drugs mediated by P-glycoprotein . Cepharanthin, a biscoclaurine alkaloid, potently inhibits the transport of vinblastine and daunorubicin, both commonly used anticancer agents . The 50% inhibitory concentration of cepharanthin on daunorubicin transport was 2.06 microM . Combined inhibitory effects on daunorubicin transport were observed when cepharanthin was used together with cyclosporin A, a potent immunosuppressive agent and P-glycoprotein modulator . Cepharanthin itself was transported by P-glycoprotein . Transcellular transport of cepharanthin across LLC-GA5-COL150 cell monolayers was saturable when its concentration was under 5 microM, and the transport was inhibited by P-glycoprotein modulators . These results indicate that cepharanthin can reverse multidrug resistance, and proper combination with other P-glycoprotein modulators could potentiate its inhibitory effect on expelling the anticancer drugs out of the cell via P-glycoprotein. Tuber Lung Dis, 1995 Oct, 76(5), 425 - 30 Rapid detection of rifampicin resistance in sputum and biopsy specimens from tuberculosis patients by PCR and line probe assay; De Beenhouwer H et al.; SETTING: Multidrug resistant Mycobacterium tuberculosis strains are threatening TB control in the world . Rapid diagnosis of resistance is essential for adequate treatment and optimal control of the disease . OBJECTIVE: Evaluation of a new technique (Line Probe Assay, LiPA) for easy and rapid detection of Rifampicin resistance (RMPR) of M . tuberculosis . DESIGN: After amplification of the region of the RNA polymerase, involved in RMPR, the amplified product is hybridized with a set of 10 oligonucleotides immobilized onto a membrane strip . From the pattern obtained the presence or absence of RMPR M . tuberculosis can be assessed . 67 clinical samples positive in culture for M . tuberculosis were analyzed with LiPA and results were compared with classical susceptibility testing . RESULTS: In vitro drug sensitivity testing identified 46 rifampicin sensitive and 21 resistant strains . In 65 of the 67 specimens LiPA results matched classical testing . In two RMPR cases LiPA showed a sensitive pattern . CONCLUSION: In contrast to culture and sensitivity testing, where results take on average 6 weeks, LiPA testing is an easy and rapid (< 48 h) method of detecting RMPR M . tuberculosis in clinical samples . Results correlated in 97% of the samples . In the two RMPR samples with a sensitive LiPA pattern another mechanism of resistance is suspected. Jpn J Cancer Res, 1995 Oct, 86(10), 969 - 77 Multidrug resistance-associated protein-mediated multidrug resistance modulated by cyclosporin A in a human bladder cancer cell line; Kim WJ et al.; A doxorubicin-resistant subline (5637/DR5.5) from human bladder cancer cells (5637) was induced by stepwise increase in the doxorubicin concentration . 5637/DR5.5 cells were cross-resistant to vinblastine and etoposide but not to mitomycin C and cisplatin . We analyzed the mdr1, MRP (multidrug resistance-associated protein), and DNA topoisomerase II gene expression using the reverse transcription polymerase chain reaction assay (RT-PCR) and investigated possible differences in the accumulation and efflux of radiolabeled daunorubicin . 5637/DR5.5 cells do not express the mdr1 gene, but the expression levels of MRP are markedly higher than in drug-sensitive 5637 cells . The intracellular accumulation of radiolabeled daunorubicin was markedly decreased in the 5637/DR5.5 cells in comparison with the parent cells . This reduced drug accumulation was associated with an enhanced drug efflux, but was reversed when cells were incubated with cyclosporin A . Cyclosporin A at the concentration of 5 microM caused 3.4-fold enhancement of daunorubicin-sensitivity in the 5637/DR5.5 cells . On the other hand, there was no difference in DNA-topoisomerase II activity between the parent and resistant cells . The resistance of the 5637/DR5.5 cells is therefore associated with an enhanced drug efflux mediated by the MRP gene overexpression, as distinct from P-glycoprotein, and is modulated by cyclosporin A. Am J Trop Med Hyg, 1995 Oct, 53(4), 388 - 91 In vitro activity of atovaquone against the African isolates and clones of Plasmodium falciparum; Basco LK et al.; The in vitro activity of atovaquone (566C80) was evaluated and compared with that of chloroquine, quinine, mefloquine, halofantrine, artemether, pyrimethamine, and cycloguanil against African isolates and clones of Plasmodium falciparum using an isotopic, semimicro, drug susceptibility test . Atovaquone was highly active against the chloroquine-susceptible L-3 (geometric mean 50% inhibitory concentration {IC50} = 0.978 nM) and L-16 clones (mean IC50 = 0.680 nM) and against the multidrug-resistant FCM 29 clone (mean IC50 = 1.76 nM) . Similar low IC50 values for atovaquone were observed against the chloroquine-susceptible isolates (n = 35; geometric mean IC50 = 0.889 nM) and the chloroquine-resistant parasites (n = 26; geometric mean IC50 = 0.906 nM) . The in vitro responses between atovaquone and the other antimalarial drugs were not correlated, indicating the absence of in vitro cross-resistance . The high in vitro activity of atovaquone without any in vitro evidence for cross-resistance with other antimalarial drugs against the naturally occurring malaria parasites is a factor that favors further development of the drug for clinical use. Planta Med, 1995 Oct, 61(5), 409 - 13 Reversal of daunomycin and vinblastine resistance in multidrug-resistant P388 leukemia in vitro through enhanced cytotoxicity by triterpenoids; Hasegawa H et al.; Examined in vitro were the effects of some triterpenoids from Panax (Araliaceae) and Glycyrrhiza (Leguminosae) spp . on the sensitivity to daunomycin (DAU) and vinblastine (VBL) of adriamycin (ADM)-resistant P388 leukemia cells (P388/ADM), which were resistant to multiple anticancer drugs . Quasipanaxatriol, 20(S)-protopanaxatriol, ginsenoside Rh2, and compound K greatly enhanced the cytotoxicity of the anti-cancer drugs in P388/ADM cells . The extent of enhancement was different among the triterpene compounds; the 4- to 46-fold increase in DAU cytotoxicity was observed in P388/ADM cells in the presence of non-toxic or marginally toxic concentrations of individual compounds, while those for VBL were in the ratios of 2- to 37-fold . The maximum increase in cytotoxicity was observed with 50 microM quasipanaxatriol; the resistance indices defined to be the ratios of the IC50 values for P388/ADM and P388 parental cells decreased from 79 to 1.7 and from 180 to 4.9 in the cases of DAU and VBL, respectively . The reversal of DAU resistance in P388/ADM by quasipanaxatriol could be explained by the effective accumulation of the drugs mediated by the DAU-efflux blockage. Mol Pharmacol, 1995 Oct, 48(4), 682 - 9 A structure-function relationship among reserpine and yohimbine analogues in their ability to increase expression of mdr1 and P-glycoprotein in a human colon carcinoma cell line; Bhat UG et al.; We previously showed that there is a structure-function relationship among reserpine and yohimbine analogues in their ability to inhibit the function of P-glycoprotein (P-gp) and reverse multidrug resistance (MDR) . Because some P-gp inhibitors (e.g., verapamil and nifedipine) can increase mdr1 and P-gp expression in human colon carcinoma cell lines, we used our reserpine/yohimbine analogues to determine whether there was a structural requirement for this induction . We found that 10 microM reserpine increased both mdr1 and P-gp expression by 4-10-fold in 48 hr in a human colon carcinoma cell line that expresses moderate levels of mdr1 (LS180-Ad50) but not in several other cell lines that expressed no mdr1 . The reserpine/yohimbine analogues rescinnamine, trimethoxybenzoylyohimbine, and LY191401 (compound G), all of which contain the three structural elements used to describe the MDR pharmacophore, also increased both mdr1 and P-gp expression significantly . Despite some exceptions, we found that there was a good association between the ability of these analogues to induce mdr1 and P-gp expression and their ability to reverse vinblastine and doxorubicin resistance, revealing a structure-function relationship for this phenomenon . The increased P-gp expressed by these cells appeared to be functional, as determined by flow cytometric detection of rhodamine 123 retention . The increased expression was suppressed by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an RNA synthesis inhibitor, whereas the protein synthesis inhibitor cycloheximide enhanced the expression several-fold, suggesting that induction of mdr1 by these analogues is regulated at both the transcriptional and post-transcriptional levels.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1995 Sep 29, 270(39), 22859 - 65 Characterization of prenylcysteines that interact with P-glycoprotein and inhibit drug transport in tumor cells; Zhang L et al.; Prenylcysteine methyl esters that represent the C-terminal structures of prenylated proteins demonstrate specific substrate-like interactions with P-glycoprotein (Zhang, L., Sachs, C . W., Fine, R . L., and Casey, P . J . (1994) J . Biol . Chem . 269, 15973-15976) . The simplicity of these compounds provides a unique system for probing the structural specificity of P-glycoprotein substrates . We have further assessed the structural elements of prenylcysteines involved in the interaction with P-glycoprotein . Carboxyl group methylation, a modification in many prenylated proteins, plays an essential role of blocking the negative charge at the free carboxylate . Substitution of the methyl ester with a methyl amide or simple amide does not change the ability of the molecule to stimulate P-glycoprotein ATPase activity, but substitution with a glycine is not tolerated unless the carboxyl group of glycine is methylated . The presence of a nitrogen atom, which is found in many P-glycoprotein substrates and modifiers, is also essential for prenylcysteines to interact with P-glycoprotein . The structure at the nitrogen atom can, however, influence the type of interaction . Acetylation of the free amino group of prenylcysteine/results in a significant loss in the ability of prenylcysteines to stimulate P-glycoprotein ATPase activity . Instead, certain acetylated prenylcysteines behave as inhibitors of this activity . In studies using MDR1-transfected human breast cancer cells, the acetylated prenylcysteine analogs inhibit P-glycoprotein-mediated drug transport and enhance the steady-state accumulation of {3H}vinblastine, {3H}colchicine, and {3H}taxol . These inhibitors do not, however, affect drug accumulation in parental cells . These studies provide a novel approach for designing P-glycoprotein inhibitors that could prove effective in reversing the phenotype of multidrug resistance in tumor cells. Biochem Pharmacol, 1995 Sep 28, 50(7), 967 - 74 The P-glycoprotein-mediated relative decrease in cytosolic free drug concentration is similar for several anthracyclines with varying lipophilicity; Mulder HS et al.; We have used a new methodology to measure the activity of P-glycoprotein (P-gp) in multidrug-resistant (MDR) tumor cells . This activity leads to a lower cytosolic concentration and a lower cytotoxicity of the classical anthracyclines, daunorubicin (DNR), and doxorubicin (DOX) . It has been reported that the anthracycline idarubicin (IDA), which is more lipophilic, has a higher clinical efficacy in acute myeloid leukemias (AML) than DNR and DOX . In our study, the aim was to determine for a series of anthracyclines how variations in the passive drug influx rate as well as the P-gp-mediated drug pumping rate affect their cytosolic free drug concentrations and how these parameters are related to drug cytotoxicity . We selected six anthracyclines: DOX, DNR, epidoxorubicin (EPI), IDA, cyano-morpholino-doxorubicin (CMD), and carminomycin (CAR), ordered according to their increasing octanol/PBS buffer concentration ratios, respectively . To measure the passive permeation coefficient, the P-gp-mediated drug pumping rate, and the cytosolic free drug concentration, we used a flow-through system in which cells were exposed to a flowing medium containing drugs . We used the MDR P-gp-containing cell line KB8-5 . It was shown that the passive drug permeation coefficient as well as the drug pumping rate of P-gp increased with increasing lipophilicity in this series of anthracyclines . The cytosolic free drug concentration was lowered by P-gp to a similar extent in KB8-5 cells for all drugs tested (40-50% of the extracellular drug concentration) . CMD, IDA, and CAR had lower IC50 values and lower resistance factors in comparison to DOX, DNR, and EPI . Verapamil reversed the resistance for all anthracyclines tested . In conclusion, for several anthracyclines the activity of P-gp leads to a similar relative decrease in the cytosolic free drug concentration; consequently, the reported lower resistance factor of IDA compared to that of DNR is not due to the inability of P-gp to export IDA from cells. JAMA, 1995 Sep 27, 274(12), 945 - 51 Eleven years of community-based directly observed therapy for tuberculosis; Chaulk CP et al.; OBJECTIVE--To evaluate community-based directly observed therapy (DOT) for tuberculosis (TB) control . DESIGN--Ecological study . METHODS--Three comparisons were made in this descriptive study . (1) An 11-year retrospective comparison of TB case rates, sputum conversion rates (SCRs), rates of therapy completion, and confounding factors (acquired immunodeficiency syndrome {AIDS}, immigration, unemployment, and poverty) in Baltimore, Md, with those of the five major US cities having the highest TB incidence in 1981 but which did not have comprehensive DOT programs . (2) An 11-year trend of TB in Baltimore and the 19 major US cities with the highest TB incidence in 1981 . (3) A 7-year trend in TB in both city groups between 1985 and 1992 . SETTING--Twenty US metropolitan cities with more than 250,000 residents . RESULTS--Since 1981, Baltimore experienced the greatest decline in TB incidence (35.6 cases per 100,000 population, 1981; 17.2 cases per 100,000 population, 1992 {-51.7%}), and city rank for TB (sixth in 1981, 28th in 1992) . Conversely, the average incidence of TB increased 2.1% in the five-city cohort and increased 1.8% in the 19-city cohort . Since 1985, TB incidence increased 35.3% in the five-city cohort and 28.5% in the 19-city cohort, but declined 29.5% in Baltimore . From 1986 through 1992, Baltimore's DOT-managed cases had the highest annual SCRs at 3 months (mean, 90.7%), and the highest completion rates for standard anti-TB therapy (mean, 90.1%) when compared with the five cities . These trends could not be attributed to differentials in AIDS, immigration, poverty, or unemployment . Increasingly, more Baltimore cases were treated under DOT (86.5%, 1993) over time . Disease relapse rates remained low, even among HIV-infected patients . Within Baltimore, the documented SCR was significantly higher among DOT-managed cases compared with non-DOT-managed cases (P < .05); multidrug resistance remains rare (0.57%) . Within Maryland, Baltimore accounted for 44.4% of all TB cases in 1981, compared with 28.7% in 1992 (P < .001) . CONCLUSIONS--In contrast to the national TB upswing during the 1980s, Baltimore experienced a substantial decline in TB following implementation of community-based DOT, despite highly prevalent medicosocial risk factors . Directly observed therapy facilitated high treatment completion rates and bacteriologic evidence of cure . Directly observed therapy could help reduce TB incidence in the United States, particularly in cities with high case rates. Biochemistry, 1995 Sep 26, 34(38), 12210 - 20 Characterization of multidrug resistance P-glycoprotein transport function with an organotechnetium cation; Piwnica-Worms D et al.; Multidrug resistance (MDR) in mammalian cells and tumors is associated with overexpression of an approximately 170 kDa integral membrane efflux transporter, the MDR1 P-glycoprotein . Hexakis (2-methoxyisobutyl isonitrile)technetium(I) (Tc-SESTAMIBI), a gamma-emitting lipophilic cationic metallopharmaceutical, has recently been shown to be a P-glycoprotein transport substrate . Exploiting the negligible lipid membrane adsorption properties of this organometallic substrate, we studied the transport kinetics, pharmacology, drug binding, and modulation of P-glycoprotein in cell preparations derived from a variety of species and selection strategies, including SW-1573, V79, Alex, and CHO drug-sensitive cells and in 77A, LZ-8, and Alex/A.5 MDR cells . Rapid cell accumulation (t1/2 approximately 6 min) of the agent to a steady state was observed which was inversely proportional to immunodetectable levels of P-glycoprotein . Many MDR cytotoxic agents inhibited P-glycoprotein-mediated Tc-SESTAMIBI efflux, thereby enhancing organometallic cation accumulation . Median effective concentrations (EC50; microM) were as follows: vinblastine, 13; daunomycin, 55; idarubicin, 65; actinomycin D, 235; colchicine, minimal inhibition; adriamycin, no effect . P-glycoprotein modulators generally demonstrated significantly greater potency (EC50; microM): SDZ PSC 833, 0.08; cyclosporin A, 1.3; verapamil, 4.1; quinidine, 6.4; prazosin, > 300 . Modulator-induced enhancement up to 100-fold was observed with Hill coefficients approximately 1, consistent with simple Michaelis-Menten kinetics . Vanadate was an efficacious transport inhibitor, while agents usually not included in the MDR phenotype were without effect . Scatchard analysis showed quinidine to be a noncompetitive inhibitor of P-glycoprotein-mediated Tc-SESTAMIBI transport, indicating allosteric effector sites on P-glycoprotein . The lipid bilayer adsorbing agents tetraphenyl borate and phloretin induced large increases in final Tc-SESTAMIBI accumulation, showing maximal accumulations 2-fold greater than classic MDR modulators and Hill coefficients >> 2 . In V79 and 77A cells, modulators of PKC activity altered Tc-SESTAMIBI accumulation, while there was no indication of modulation of P-glycoprotein-mediated Tc-SESTAMIBI transport by hypotonic buffer, extracellular ATP, Cl-, or K+ (membrane potential) . While recognized and avidly transported by the P-glycoprotein at buffer concentrations as low as 7 pM, Tc-SESTAMIBI at up to 100 microM only minimally modulated the cytotoxic action of colchicine, doxorubicin, or vinblastine in MDR cells . In conclusion, transport analysis with Tc-SESTAMIBI is a sensitive assay for detecting functional expression of low levels of P-glycoprotein and for the quantitative characterization of transporter modulation and regulation . The biochemical data favor a high Km, high capacity allosterically modulated translocation mechanism for P-glycoprotein-mediated transport of this organometallic cation. J Biol Chem, 1995 Sep 22, 270(38), 22393 - 8 Verapamil reversal of chloroquine resistance in the malaria parasite Plasmodium falciparum is specific for resistant parasites and independent of the weak base effect; Martiney JA et al.; Verapamil increases the net uptake and cytotoxicity of structurally diverse hydrophobic molecules in many multidrug-resistant mammalian cell lines . This compound has also been reported to reverse chloroquine resistance in the human malaria parasite Plasmodium falciparum (Martin, S.K., Oduola, A.M.J., and Milhous, W.K . (1987) Science 235, 899-901) . Although the mechanism of this reversal is unknown, it apparently involves an increase in the amount of chloroquine present in erythrocytes infected with the resistant parasites . Chloroquine is a diprotic weak base that accumulates in acidic organelles as a function of the pH gradient present between the organelle and the external medium . By changing the external medium pH, this property of chloroquine was used to alter the cytotoxicity phenotype of genetically chloroquine-sensitive and -resistant trophozoites . Verapamil was also found to be toxic for malaria trophozoites, although this toxicity was independent of external pH and consistently about 3-4-fold higher against resistant strains . When verapamil was tested for its effects on chloroquine cytotoxicity under conditions of phenotypic reversal, it was still found to exert only a measurable effect on the genetically resistant trophozoites . In short time incubations, verapamil was found to increase net chloroquine accumulation in erythrocytes infected with both chloroquine-sensitive and -resistant organisms, but only to affect the chloroquine susceptibility of the latter . Analysis of our data demonstrates that verapamil works independently of the overall pH gradient concentrating chloroquine into a trophozoite's lysosome . Instead, we propose that it inhibits the activity of a membrane ion channel indirectly responsible for determining chloroquine transit within the parasite's cytoplasm. Anal Biochem, 1995 Sep 20, 230(2), 239 - 47 Molecular study of P-glycoprotein in multidrug resistance using surface plasmon resonance; Demeule M et al.; P-Glycoprotein is an integral membrane protein which mediates the energy-dependent efflux of various antitumor agents from multidrug-resistant cancer cells . Surface plasmon resonance was used for the detection of P-glycoprotein after solubilization from drug-resistant and drug-sensitive Chinese hamster ovary cells and for the analysis of its interaction with cyclosporin A, a competitive inhibitor of drug efflux . Detection of P-glycoprotein relied on its binding to the monoclonal antibody C219 which was immobilized on a sensor chip . Binding of Zwittergent 3-14-solubilized P-glycoprotein to the antibody was concentration-dependent and reflected the relative abundance of P-glycoprotein in both cell lines . It was abolished when C219 was omitted or replaced by a rabbit anti-mouse IgG antibody and considerably reduced after precipitation of P-glycoprotein with wheat germ agglutinin . Preincubation of solubilized proteins with cyclosporin A increased the amount of protein bound to the antibody by approximately 30% . These results indicate that surface plasmon resonance is well suited to the detection of P-glycoprotein from biological samples and shows promise as a tool for the study of its interaction with different drugs. Cancer Res, 1995 Sep 15, 55(18), 4073 - 8 Transduction of NIH 3T3 cells with a retrovirus carrying both human MDR1 and glutathione S-transferase pi produces broad-range multidrug resistance; Doroshow JH et al.; In the experiments, we examined the ability of a retroviral vector, pHaMASV, to encode two potential chemoprotective genes on separate transcription units . We previously described the pHaMSV vector, which includes the human MDR1 gene as a selectable marker and chemoprotective gene, plus an internal SV40 promoter for expressing a second heterologous gene along with MDR1 {M . E . Metz, D . M . Best, and S . E . Kane . Virology, 208: 634-643, 1995} . To test the ability of this vector to deliver two therapeutic genes simultaneously, the cDNA for human glutathione S-transferase pi (GST pi, the most abundant member of the glutathione S-transferase family in human tumor cells) was inserted into pHaMASV, and this plasmid was transfected into ecotropic packaging cells . The resulting pHaMASV.GST pi ecotropic retrovirus, which was produced at a titer of 2 x 10(6) colony-forming units/ml, was used to transduce NIH 3T3 cells . After initial selection in 60 ng/ml colchicine, a population of transduced cells was exposed to stepwise increasing colchicine concentrations to select for amplified expression of MDR1 . As MDR1 expression increased, the expression of GST pi increased in concert, as demonstrated by Northern analysis, Western analysis, and measurement of glutathione S-transferase activity . Transduced cells growing in 1280 ng/ml colchicine had about 3-fold higher total glutathione S-transferase activity than nontransduced cells and 2.5-fold higher activity than transduced cells growing in 60 ng/ml colchicine . Northern hybridizations demonstrated a 3-5-fold increase in both the full-length retroviral message encoding MDR1 and the subgenomic mRNA encoding GST pi after amplification of resistance from 60 to 1280 ng/ml colchicine . The cytotoxic effects of several xenobiotics were evaluated in NIH 3T3 cells transfected with MDR1 (3T3.MDR) or transduced with the MDR1-GST pi retrovirus (3T3.GST640 or 3T3.GST1280) to evaluate the ability of our vector to produce a spectrum of drug resistances specific for the genes expressed . 3T3.MDR and 3T3.GST1280 cells expressing equivalent levels of MDR1 had identical levels of resistance to doxorubicin or colchicine . These results suggest that GST pi expression did not contribute to doxorubicin resistance in this model system . However, 3T3.GST640 cells were about 4-fold resistant to ethacrynic acid and 1-chloro-2,4-dinitrobenzene compared to cells expressing MDR1 alone, consistent with the ability of GST pi to conjugate both of these cytotoxins . Increases in drug resistance paralleled increases in gene-specific mRNA and recombinant protein levels in all cases.4+ chemotherapy. Cancer Res, 1995 Sep 15, 55(18), 4004 - 9 Cross-resistance to camptothecin analogues in a mitoxantrone-resistant human breast carcinoma cell line is not due to DNA topoisomerase I alterations; Yang CJ et al.; We have previously described a mitoxantrone-resistant human breast carcinoma cell line, MCF7/MX, in which resistance was associated with a defect in the energy-dependent accumulation of mitoxantrone in the absence of P-glycoprotein overexpression (M . Nakagawa et al., Cancer Res . 52: 6175-6181, 1992) . We now report that this cell line is highly cross-resistant to the camptothecin analogues topotecan (180-fold), 9-aminocamptothecin (120-fold), CPT-11 (56-fold), and SN38 (101-fold), but is only mildly cross-resistant to the parent compound camptothecin (3.2-fold) and 10,11-methylenedioxy-camptothecin (2.9-fold) . Topotecan accumulation was decreased in MCF7/MX cells compared to parental MCF7/WT cells, and there was a corresponding reduction in topotecan-mediated stimulation of the enzyme/DNA complex formation in MCF7/MX cells compared to MCF7/WT cells . No overexpression of the multidrug resistance-associated protein was detected compared to parental MCF7/WT cells . Furthermore, both sensitive MCF7/WT and mitoxantrone-resistant MCF7/MX cells contain equal amounts of DNA topoisomerase I protein, and DNA relaxation activities were equal in both cell lines and inhibited to the same extent by topotecan and camptothecin . Thus, these results suggest a novel mechanism of resistance to topoisomerase I inhibitors in cancer cells. Blood, 1995 Sep 15, 86(6), 2329 - 42 Correlation of multidrug resistance (MDR1) protein expression with functional dye/drug efflux in acute myeloid leukemia by multiparameter flow cytometry: identification of discordant MDR-/efflux+ and MDR1+/efflux- cases; Leith CP et al.; Resistance to chemotherapy is a major factor limiting successful treatment of acute myeloid leukemia (AML); one of the best characterized drug resistance mechanisms is extrusion of drugs by the energy-dependent multidrug resistance (MDR1) transport protein . Expression of MDR1 is common in AML and has been linked to lower remission induction rates and decreased remission durations . Because MDR1 efflux function may be modified by drugs such as cyclosporin A, accurate identification of MDR1+/efflux+ AML cases will be critical to identify patients who may benefit from therapies that contain such MDR1 modulators . We have optimized single and multiparameter flow cytometric assays to detect efflux of drugs or fluorescent dyes by previously cryopreserved AML blasts . These assays allowed precise identification of efflux by leukemic blasts, and correlation with CD34 and MDR1 expression . We subsequently studied a series of 60 previously untreated AML cases . Functional efflux was identified in 39 cases and was significantly correlated with MDR1 expression (P = .0002) . However, discrepant cases were identified; 10 cases were efflux+ without significant MDR1 expression, whereas 6 MDR1+ cases were efflux- . There was also a highly significant correlation of efflux with CD34; 31 (79%) of the 39 efflux+ cases were CD34+ in comparison with only 5 (24%) of the 21 efflux- cases (P < .0001) . Multivariate analysis showed that efflux was significantly associated with independent effects of both CD34 (P = .0011) and MDR1 expression (P = .034); the majority of efflux+ cases were CD34+, whereas 5 of the 6 MDR1+ efflux- cases lacked CD34 expression . Cyclosporin A blocked efflux in all but 2 cases regardless of MDR1 expression . Functional efflux in AML is frequently detected without the classic MDR1+ phenotype indicating that alternate non-MDR1-mediated efflux mechanisms may be important . Efflux assays may better identify patients who would benefit from therapies that include efflux modulators. Biochim Biophys Acta, 1995 Sep 13, 1238(2), 147 - 55 The cationic lipid stearylamine reduces the permeability of the cationic drugs verapamil and prochlorperazine to lipid bilayers: implications for drug delivery; Webb MS et al.; The therapeutic activity of a wide variety of drugs is significantly improved when their longevity in the circulation is extended by encapsulation in liposomes . To improve the retention of cationic drugs in liposomes, we have investigated the effect of the cationic lipid stearylamine on the permeability of the calcium channel blocker verapamil and the antipsychotic drug prochlorperazine, both of which are also multidrug resistance modulators . Both drugs were efficiently incorporated into liposomes composed of DSPC/cholesterol that possessed a transmembrane pH gradient (inside acidic) . However, the efflux of the loaded drugs was relatively rapid (i.e., 50% of the encapsulated verapamil was released after 4 h at 37 degrees C), despite the presence of a 3 unit pH gradient (pHi = 4.0, pHo = 7.5) . Drug retention within the liposomes was improved by increasing the magnitude of the transmembrane pH gradient to approx . 5 units (pHi = 2.0, pHo = 7.5) . Further improvements in drug retention were achieved by the addition of 10 mol% of the cationic lipid stearylamine in the DSPC/cholesterol liposomes . The combination of the 5 unit pH gradient and stearylamine resulted in increases of the retention of verapamil and prochlorperazine by approx . 20- and 5-fold, respectively . Calculation of the permeability coefficients for the charged (cationic) and neutral forms of the drugs indicated that the neutral forms of both drugs were approx . 10(4)-fold more permeable than were the cationic forms of the drugs . Further, the presence of stearylamine reduced the permeability coefficient for the cationic species of the drugs by approximately an order of magnitude, but had no effect on the neutral species of the drugs . The efflux curves observed for both verapamil and prochlorperazine could be mathematically modeled by assuming that the primary influence of stearylamine was on the development of a positive surface charge density on the inner monolayer of the liposome . Taken in sum, these results indicate that stearylamine is effective at decreasing the leakage of cationic drugs from liposomes, and may prove to be a valuable component of liposomal drug formulations. Biochim Biophys Acta, 1995 Sep 13, 1238(2), 137 - 46 Verapamil competes with doxorubicin for binding to anionic phospholipids resulting in increased internal concentrations and rates of passive transport of doxorubicin; Speelmans G et al.; It is well documented that the Ca2+ channel antagonist verapamil can reverse multidrug resistance in cancer cells by decreasing P-glycoprotein mediated drug efflux . However, less information is available about effects of verapamil on drug-phospholipid interactions and on passive diffusion of drugs across the membrane, which both may play an important role in resensitizing cells to anti-cancer drugs . Therefore we studied the binding of verapamil to model membranes (large unilamellar vesicles) composed of various phospholipids and biological membranes . An increase of the amount of anionic phospholipids resulted in an enhanced binding of verapamil . Competition between verapamil and the anti-cancer drug and P-glycoprotein substrate doxorubicin for binding to anionic phospholipids was observed in model membranes composed of synthetic lipids, or composed of native Escherichia coli phospholipid mixtures, and in cytoplasmic membrane vesicles of this organism . Furthermore, verapamil specifically increased the rate of passive diffusion of doxorubicin across model membranes containing anionic phospholipids . It can be concluded that besides the decrease of P-glycoprotein mediated efflux at least two other effects may account for an increase of the internal (free and DNA-bound) doxorubicin concentration in the presence of verapamil; (i) a decrease of binding to anionic phospholipids in plasma-and intracellular membranes and (ii) an increase of the rate of passive import of doxorubicin across the plasma membrane. Immunol Lett, 1995 Sep, 47(3), 223 - 6 P-170 glycoprotein (P-170) is involved in the impairment of natural killer cell-mediated cytotoxicity in HIV+ patients; Lucia MB et al.; In the present study we analyze peripheral blood lymphocytes (PBL) from patients with human immunodeficiency virus (HIV) infection for both phenotypic expression and function of P-glycoprotein (P-170) . This transmembrane efflux pump is known to be one of the mechanisms responsible for the multidrug resistance (MDR) in cancer therapy and it is also constitutively expressed in normal PBL . P-170 function, evaluated as Rhodamine 123 (Rh123) efflux in flow cytometry, was found to be significantly reduced in CD16+ natural killer (NK) cells from patients with HIV infection . Interestingly, this reduced efflux significantly correlates with the decreased NK cytotoxicity observed in HIV+ patients, as evaluated against the NK-specific K562 target cell line . These results support a possible role of the P-170-related pump in specific immunological lymphocyte function such as NK cell-mediated cytotoxicity. Zhonghua Zhong Liu Za Zhi, 1995 Sep, 17(5), 340 - 2 {Study on the reversing effect of tripiperaquine on human multidrug resistant leukemic cell line K562/A02}; Yang R et al.; K562/A02 is a Cell line with multi-drug resistance established in our laboratory bey long term induction with adriamycin . In this paper, reversal of MDR in K562/A02 cell line by tripiperaquine is reported . The cytotoxicity and intracellular concentration of daunorubicin (DNR) in K562/A02 were measured by MTT colorimetric assay and spectrofluorimetry . The results showed that the sensitivity of K562/A02 to DNR was greatly enhanced by tripiperaquine at 10 micrograms/ml, with an 11-fold increase in cytotoxic activity . The intracellular concentration of DNR in K562/A02 was significantly increased after coincubation with 20 mumol/L tripiperaquine for 3 hours . Our results suggest that tripiperaquine might be used in clinical trial to reverse MDR. Ann Oncol, 1995 Sep, 6(7), 651 - 7 Resistance to cytotoxic therapy: a speculative overview; Preisler HD; While most recent studies have focused on the MDR1 gene and other similar types of multidrug resistance, two other phenomena (inhibition of the apoptotic pathway and regrowth resistance) have the potential for producing a much broader type of resistance to cytotoxic therapy . This speculative review discusses the potential contribution of these types of resistance and the possible interrelationships between the various types of resistance to cytotoxic therapy . The need for new approaches to assess the effects of biological agents is discussed. Mol Microbiol, 1995 Sep, 17(5), 989 - 99 Molecular characterization and transcriptional analysis of a multidrug resistance gene cloned from the pristinamycin-producing organism, Streptomyces pristinaespiralis; Blanc V et al.; A multidrug resistance gene (mdr) has been cloned from Streptomyces pristinaespiralis, a producer of two antibiotics having synergistic activities together known as pristinamycin . This gene, ptr, provides resistance not only to two structurally dissimilar compounds (pristinamycin I, PI; pristinamycin II, PII) and the natural pristinamycin mixture but also to rifampicin . Mutagenesis and subcloning of ptr localized it to a 2 kb region which was sequenced and analyzed . It contained an open reading frame of 1506 bp which encoded a putative membrane protein with 14 hydrophobic domains, and showed sequence similarity to a superfamily of bacterial proteins that employ transmembrane electrochemical gradients to catalyse active efflux of various antibiotics and toxic compounds . Ptr was most similar to a subfamily which included other mdr genes and antibiotic transport genes associated with antibiotic biosynthetic gene clusters in actinomycetes . In vitro coupled transcription-translation experiments were used to identify the ptr gene product . Analysis of the upstream region did not reveal a divergently transcribed repressor gene, as is the case for several related resistance determinants involved in antibiotic transport, suggesting that ptr is regulated by a different mechanism . Transcriptional analyses of this gene, carried out in both S . pristinaespiralis and Streptomyces lividans, indicated the same transcriptional start point and predicted -10 and -35 hexamers which were somewhat similar to Streptomyces vegetative-type promoters. Mol Microbiol, 1995 Sep, 17(5), 1001 - 12 Stress-activated expression of a Streptomyces pristinaespiralis multidrug resistance gene (ptr) in various Streptomyces spp . and Escherichia coli; Salah-Bey K et al.; A promoter which controls expression of the pristinamycin multidrug resistance gene (ptr) in Streptomyces pristinaspiralis could be induced by physiological stresses in both Streptomyces spp . and Escherichia coli . In S . pristinaspiralis, the ptr promoter (Pptr) was induced by pristinamycin I (PI) or pristinamycin II (PII) . Streptomyces lividans was adopted as a convenient heterologous host for studies of Pptr regulation since it has no known pristinamycin biosynthetic genes . Two key regulatory features were documented in these studies: many (19 of 70) antibiotics and chemicals with no common targets or structural features induced the Pptr; induction with PI was most efficient during a transition phase when antibiotic biosynthetic genes are switched on . In Streptomyces coelicolor, Pptr activity was similarly inducible by PI and not dependent on sigma factors HrdA, HrdC, or HrdD . In E . coli, Pptr cloned in the bifunctional promoter probe vector pIJ2839 was functional and activated upon entry into stationary phase in the absence of exogenous inducer . Finally, gel-retardation studies demonstrated a Pptr-binding protein in S . lividans (where its activity was PI-inducible), S . coelicolor and S . pristinaespiralis . The fact that this activity was not detected in E . coli suggested the existence of another regulatory system perhaps also present in Streptomyces. Mol Microbiol, 1995 Sep, 17(6), 1109 - 19 Unusual regulatory mechanism for a Streptomyces multidrug resistance gene, ptr, involving three homologous protein-binding sites overlapping the promoter region; Salah-Bey K et al.; A promoter controlling expression of the pristinamycin multidrug resistance gene (ptr), originally isolated from Streptomyces pristinaespiralis, is inducible by many toxic compounds in various Streptomyces species . Studies of ptr promoter control were carried out in the heterologous host, Streptomyces lividans . In S . lividans, a regulatory protein or a protein complex (Pip), identified by its ability to bind to the ptr promoter in gel-retardation experiments, was induced by pristinamycin I (PI) . In situ copper-phenanthroline footprinting analysis identified three (A, B, and C) similar Pip-binding sites having the sequence GTACA(C/G)CGTA(C/T) . These sites overlapped with functionally important regions of the promoter: the 'A' site overlapped with the -35 hexamer, 'B' overlapped with the -10 hexamer and 'C' was located between the transcription start site and the Shine-Dalgarno sequence . A GT-AG dinucleotide mutation was introduced at positions 8-9 of the consensus sequence to generate seven variant promoters: three mutated in one of the three sites, three mutated in two sites, and one mutated in all three sites . Whereas these promoters had reduced antibiotic (PI)-induced activity, their levels of expression in the absence of PI was higher . This suggested an unusual regulatory mechanism in which Pip could act either as an activator or repressor . Gel shift experiments revealed Pip or its homologues in many other Streptomyces species, suggesting that it is widely employed in the regulation of antibiotic resistance genes and perhaps secondary metabolism. Eur Respir J Suppl, 1995 Sep, 20, 714s - 718s New drugs for tuberculosis; Grassi C et al.; Since the late 1960s, tuberculosis has been successfully cured with antibiotics . With the introduction of rifampin, "short course" regimens using isoniazid and rifampin together with either streptomycin, ethambutol or pyrazinamide, for 6-9 months, have been successfully adopted . The spread of drug resistant M . tuberculosis strains in large urban areas has made this armamentarium of drugs insufficient, calling for the development of new drugs . Among rifamycin derivatives, rifabutin is more active than rifampin in vitro and in experimental animals, and allows sputum conversion rats of 95-100% . It is effective in treating multidrug-resistant tuberculosis . Rifapentine is more active than rifampin in vitro and has a longer half-life, but it is not active against rifampin-resistant strains . Fluoroquinolones concentrate within macrophages, are effective against M . tuberculosis and act synergistically with rifampin and isoniazid . Ofloxacin, ciprofloxacin, sparfloxacin and lomefloxacin have been evaluated as antimycobacterial agents, and no cross-resistance with major antituberculous drugs has been found . Several other drugs, including new inhibitors of beta-lactamase and new beta-lactamase-resistant antibiotics, the aminoglycoside antibiotic, paromomycin, and the new nitroimidazole, 2-ethyl-5-intro-2.3-dihydro imidazo-oxazole, have been found to be active in vitro against M . tuberculosis. Eur Respir J Suppl, 1995 Sep, 20, 701s - 713s Drug resistance in Mycobacterium tuberculosis; Cole ST et al.; During the last decade, there has been a marked increase in the number of gravity of tuberculosis cases both in developing countries and in industrialized nations . This is, in part, due to the acquired immune deficiency syndrome (AIDS) pandemic, but global economic depression, increased homelessness and declining control programmes have also contributed . One of the more insidious consequences of this resurgence has been the recent emergence and nosocomial transmission of multidrug-resistant strains of Mycobacterium tuberculosis, thus raising the possibility that untreatable forms of the disease may become widespread . Somewhat surprisingly, given the difficulties of working with this slow-growing pathogen, remarkable progress has been made in a relatively short time, in understanding the molecular epidemiology, the genetic basis, and the biochemical mechanisms of drug resistance . Furthermore, a number of promising molecular tools are now available to help counter tuberculosis and to further understanding. Leuk Lymphoma, 1995 Sep, 19(1-2), 159 - 63 VAD-cyclosporine therapy for VAD-resistant multiple myeloma; Weber D et al.; Few effective treatments are available for patients with multiple myeloma that is resistant to vincristine-doxorubicin by continuous infusion with high dose dexamethasone (VAD) . In order to modulate p-glycoprotein, the multidrug resistance gene product, we administered a VAD-cyclosporine combination to patients with confirmed resistance to VAD . Twenty-five patients with multiple myeloma resistant to VAD received cyclosporine 4 mg/kg infused over 2 hours followed by a continuous infusion of 10 mg/kg/24 hrs for a total of 108 hours . VAD was given concurrently as a continuous infusion of vincristine 0.3 mg and doxorubicin 9 mg/m2 daily for 4 days with oral dexamethasone 20 mg/m2/day for 4 days beginning on days 1, 9 and 17 . Clinical response and toxicity were correlated with MDR expression in plasma cells and the effects of cyclosporine on liver function . Six of 25 patients responded (24%; 95% CI 9-45%) with a median remission time of 7 months . Clinical response did not correlate with either the measured or the calculated MDR expression in plasma cells . Responses occurred more frequently in patients who developed high cyclosporine blood levels and paralytic ileus . The occasional benefit from VAD-cyclosporine for resistant multiple myeloma appeared to be due to a higher bioeffective dose of VAD rather than successful modulation of MDR. Leuk Lymphoma, 1995 Sep, 19(1-2), 135 - 40 Analysis of MDR1 and MDR3 multidrug resistance gene expression and amplification in consecutive samples in patients with acute leukaemias; MacFarland A et al.; White blood cells from a total of 19 patients diagnosed as having acute lymphoblastic (ALL) or acute myeloid (AML) leukaemia were analysed (36 samples) for amplification and expression of the mdr1 and mdr3 genes . Nine of the patients had samples analysed at presentation and at subsequent stages of the disease (24 samples, including 4 at second relapse) . Patients received standard MRC UK Trial remission-induction treatment protocols appropriate to disease and age . No amplification of either the mdr1 or mdr3 gene was found in any of the samples, and neither were mdr3 transcripts detected by dot-blot analysis using gene-specific probes . Transcripts of the mdr1 gene were found in only 2 ALL samples (of 10) . However, mdr1 transcripts were detected in all AML patients and there was a significant increase in the transcript levels in these patients who went on to first and second relapse, compared with levels measured at presentation (P < 0.001) . The results support the hypothesis that P-glycoprotein-mediated drug resistance may be a significant factor in tumour cell resistance to chemotherapy at relapse following initial induction-remission therapy for acute myeloid leukemia. Trans R Soc Trop Med Hyg, 1995 Sep-Oct, 89(5), 523 - 7 Artesunate versus artemether in combination with mefloquine for the treatment of multidrug-resistant falciparum malaria; Price RN et al.; To compare the therapeutic efficacy of oral artesunate and artemether in combination with mefloquine for the treatment of multidrug resistant malaria, a trial was conducted in 540 adults and children on the Thai-Myanmar border . Three regimens were compared: artesunate (4 mg/kg/d for 3 d), artemether (4 mg/kg/d for 3 d), both in combination with mefloquine (25 mg/kg), and a single dose of mefloquine (25 mg/kg) . The artesunate and artemether regimens gave very similar clinical and parasitological responses, and were both very well tolerated . There was no significant adverse effect attributable to the artemisinin derivatives . Fever and parasite clearance times with mefloquine alone were significantly longer (P < 0.001) . After adjusting for reinfections the failure rates were 13.9% for the artesunate combination, 12.3% for the artemether combination and 49.2% for mefloquine alone (P < 0.0001; relative risk 3.8 {95% confidence interval 2.6-5.4}) . Mefloquine should no longer be used alone for the treatment of multidrug resistant falciparum malaria in this area . Three-day combination regimens with artesunate or artemether are well tolerated and more effective. Bull Cancer, 1995 Sep, 82(9), 687 - 97 {Use of rhodamine 123 for the detection of multidrug resistance}; Canitrot Y et al.; Multidrug resistance (MDR) is characterized by the overexpression of P-glycoprotein (Pgp), which is responsible for decreasing drug uptake and/or increasing drug efflux in resistant cells . Although Pgp has a broad-spectrum specificity, this protein seems to react preferentially with amphiphilic and cationic molecules . Rhodamine 123 (R123) is widely used as a marker for mitochondria in living cells and its uptake is dependent on plasma and mitochondrial membrane potential . More recently, cross-resistance to R123 in cells resistant to adriamycin has been demonstrated and a correlation between expression of Pgp and reduced intracellular accumulation of R123 has been shown . The measurement of R123 uptake or efflux allows the characterization of cells displaying a MDR phenotype with overexpression of Pgp, even with low levels of resistance . Other proteins have now been identified which play a role in resistance and in drug transport, including MRP . For this reason we need to determine if R123 is transported only by Pgp or if R123 is a substrate for transport by other drug resistance proteins as well . We also discuss the possibilities of using several techniques based on fluorescence with R123 in order to fully characterize cells by measuring both Pgp activity and its presence/localization. Biophys J, 1995 Sep, 69(3), 883 - 95 Overexpression of the cystic fibrosis transmembrane conductance regulator in NIH 3T3 cells lowers membrane potential and intracellular pH and confers a multidrug resistance phenotype; Wei LY et al.; Because of the similarities between the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance (MDR) proteins, recent observations of decreased plasma membrane electrical potential (delta psi) in cells overexpressing either MDR protein or the CFTR, and the effects of delta psi on passive diffusion of chemotherapeutic drugs, we have analyzed chemotherapeutic drug resistance for NIH 3T3 cells overexpressing different levels of functional CFTR . Three separate clones not previously exposed to chemotherapeutic drugs exhibit resistance to doxorubicin, vincristine, and colchicine that is similar to MDR transfectants not previously exposed to chemotherapeutic drugs . Two other clones expressing lower levels of CFTR are less resistant . As shown previously these clones exhibit decreased plasma membrane delta psi similar to MDR transfectants, but four of five exhibit mildly acidified intracellular pH in contrast to MDR transfectants, which are in general alkaline . Thus the MDR protein and CFTR-mediated MDR phenotypes are distinctly different . Selection of two separate CFTR clones on either doxorubicin or vincristine substantially increases the observed MDR and leads to increased CFTR (but not measurable MDR or MRP) mRNA expression . CFTR overexpressors also exhibit a decreased rate of 3H -vinblastine uptake . These data reveal a new and previously unrecognized consequence of CFTR expression, and are consistent with the hypothesis that membrane depolarization is an important determinant of tumor cell MDR. Br J Cancer, 1995 Sep, 72(3), 550 - 4 Expression of the multidrug resistance-associated protein (MRP) gene in non-small-cell lung cancer; Ota E et al.; We examined the levels of expression of the multidrug resistance-associated protein (MRP) gene quantified by Northern blot analysis in comparison with those of the MDR1 gene determined by reverse transcription-polymerase chain reaction (RT-PCR) in 104 non-small-cell lung cancer (NSCLC) specimens {59 adenocarcinoma (Ad), 40 squamous cell carcinoma (Sq), four large cell carcinoma (La) and one adeno-squamous carcinoma (AdSq)} . Thirty-three (31.7%) of the 104 NSCLC expressed the MRP gene at various levels . The NSCLC showing high (++) levels of MRP gene expression (19 out of 33, 57.6%) were predominantly squamous cell carcinomas (Ad, 5; Sq, 13; La, 1) (P < 0.05) . Six of the eight NSCLCs expressing high levels of MRP mRNA and no MDR1 (MRP ++, MDR1-) were squamous cell carcinomas . Sixty-one of the 104 NSCLC patients received chemotherapy with MRP-related anti-cancer drugs {vindesine (VDS) and etoposide (VP-16)} . Twenty-three patients (37.7%) with tumour expressing high or moderate levels of MRP showed significantly worse prognoses than those with non- or low-MRP-expressing tumours (P < 0.05) . These results suggest that the level of MRP gene expression is related to the histopathology and prognosis of NSCLC. Br J Cancer, 1995 Sep, 72(3), 543 - 9 Functional detection of MDR1/P170 and MRP/P190-mediated multidrug resistance in tumour cells by flow cytometry; Feller N et al.; Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma membrane drug transporter P-glycoprotein (P-gp) or the recently discovered multidrug resistance-associated protein (MRP) . In this study we investigated the specificity and sensitivity of the fluorescent probes rhodamine 123 (R123), daunorubicin (DNR) and calcein acetoxymethyl ester (calcein-AM) in order to detect the function of the drug transporters P-gp and MRP, using flow cytometry . The effects of modulators on the accumulation and retention of these probes were compared in several pairs of sensitive and P-gp- as well as MRP-overexpressing cell lines . R123, in combination with the modulator PSC833, provided the most sensitive test for detecting P-gp-mediated resistance . Moreover, in a 60 min drug accumulation assay R123 can be regarded as a P-gp-specific probe, since R123 is not very efficiently effluxed by MRP . In contrast to R123, a 60 min DNR or calcein-AM accumulation test could be used to detect MRP-mediated resistance . The MRP-specific modulator genistein could be used in combination with DNR, but not with calcein-AM . Vincristine (VCR) can be used to increase the cellular uptake of calcein-AM in MDR cells, but is not specific for MRP . Thus, although the combination of DNR with genistein appeared to be as sensitive as the combination of calcein-AM with VCR, the former may be used to probe specific MRP activity whereas the latter provides a combined (P-gp + MRP) functional MDR parameter . With these functional assays the role and relative importance of P-gp and MRP can be studied in, for example, haematological malignancies. Br J Cancer, 1995 Sep, 72(3), 535 - 42 An etoposide-resistant lung cancer subline overexpresses the multidrug resistance-associated protein; Doyle LA et al.; We have characterised an etoposide-resistant subline of the small-cell lung cancer cell line, UMCC-1, derived at our centre . Subline UMCC-1/VP was developed by culturing the parent line in increasing concentrations of etoposide over 16 months . UMCC-1/VP is 20-fold resistant to etoposide by MTT assays, relative to the parent line, and is cross-resistant to doxorubicin, vincristine and actinomycin D, but not to taxol, cisplatin, melphalan, thiotepa or idarubicin . Topoisomerase II immunoblotting demonstrates a 50% reduction of the protein in the resistant subline . The UMCC-1/VP subline demonstrates a marked decrease in the accumulation of {3H}etoposide relative to the parent line, as well as a modest reduction in the accumulation of daunorubicin . Reverse transcription-polymerase chain reaction assays demonstrate no detectable mdr1 expression but marked expression of the multidrug resistance-associated protein (MRP) gene in the resistant subline . Northern blotting with an MRP cDNA probe confirms marked overexpression of the MRP gene only in the UMCC-1/VP subline . Western blotting with antisera against MRP peptide confirms a 195 kDa protein band in the UMCC-1/VP subline . Southern blotting experiments demonstrate a 10-fold amplification of the MRP gene in the resistant subline . Depletion of glutathione with buthionine sulphoximine sensitised UMCC-1/VP cells to daunorubicin and etoposide . Our studies indicate that MRP gene expression may be induced by etoposide and may lead to reduced accumulation of the drug. Dimens Crit Care Nurs, 1995 Sep-Oct, 14(5), 236 - 44 Multidrug-resistant tuberculosis: a new era in prevention and control; Serkey JM; Failure to recognize Multidrug-Resistant Tuberculosis (MDR-TB) has been the cause of explosive outbreaks . To avoid this devastating consequence of the disease, especially among HIV-infected persons, critical care staff must use preventive strategies . This article provides information on the pathogenesis of MDR-TB, its epidemiology, and some case management problems associated with the disease . Also provided are checklists to identify the risk of infection and instructions on how to combat infection if it is diagnosed. Chest, 1995 Sep, 108(3), 712 - 7 Chemoprophylaxis of multidrug-resistant tuberculous infection in HIV-uninfected individuals using ciprofloxacin and pyrazinamide . A decision analysis; Stevens JP et al.; STUDY OBJECTIVE: To evaluate the use of ciprofloxacin and pyrazinamide for the prophylaxis of individuals infected with multiply drug-resistant strains of Mycobacterium tuberculosis . DESIGN: Decision analysis, using software (SMLTREE) . Probabilities based on published studies, with sensitivity analysis for each probability . SETTING: Health-care workers infected with multidrug-resistant strains of M tuberculosis . INTERVENTIONS: Prophylaxis with ciprofloxacin and pyrazinamide . MEASUREMENTS AND RESULTS: We calculated the utilities of taking or not taking prophylaxis with ciprofloxacin and pyrazinamide . The decision analysis favored the use of ciprofloxacin-pyrazinamide prophylaxis by a small margin . CONCLUSION: Ciprofloxacin-pyrazinamide prophylaxis should be considered for health-care workers infected with multiply drug-resistant M tuberculosis. Blood, 1995 Sep 1, 86(5), 1903 - 10 Expression of bcl-xL can confer a multidrug resistance phenotype; Minn AJ et al.; It has been suggested that genes that regulate apoptotic cell death may play an important role in determining the sensitivity of tumor cells to chemotherapy . We have recently cloned a member of the bcl-2 family, bcl-x . To test whether bcl-XL expression affects the sensitivity of tumor cells to chemotherapy, we have created stable cell lines overexpressing bcl-XL and have tested these cells for resistance to cell death induced by metabolic inhibitors and chemotherapeutic agents . Bcl-XL expression dramatically reduces the cytotoxicity of bleomycin, cisplatin, etoposide, vincristine, hygromycin B, and mycophenolic acid for up to 4 days in culture . Bcl-XL does not prevent cells from undergoing cell cycle arrest in response to these drugs, but rather prevents treated cells from undergoing apoptosis . Cell-cycle analysis on cells treated with the chemotherapeutic agents bleomycin, cisplatin, etoposide, and vincristine, show that the drugs cause growth arrest in different positions within the cell cycle . Bcl-XL expressing cells treated with chemotherapeutic drugs retain their proliferative ability after the drugs are removed . Interestingly, vincristine-treated cells expressing bcl-XL become polyploid after drug removal . These data show that bcl-XL protects cells from a wide variety of apoptotic stimuli, acts in multiple positions within the cell cycle, and confers a multidrug resistance phenotype . The ability of bcl-XL to prevent apoptotic cell death in response to chemotherapy-induced DNA damage and cell-cycle arrest may contribute to the accumulation of chromosomal aberrations within tumors . The expression of bcl-XL in tumor cells is likely to be an important indicator of chemotherapeutic efficacy. J Urol, 1995 Sep, 154(3), 1210 - 6 Reversal by a dihydropyridine derivative of non-P-glycoprotein-mediated multidrug resistance in etoposide-resistant human prostatic cancer cell line; Tasaki Y et al.; PURPOSE: We have isolated etoposide-resistant prostatic cancer cell lines, P/VP10 and P/VP20, to investigate the multidrug resistance (MDR) mechanism and to find MDR reversal agents . MATERIALS AND METHODS: We examined expression of MDR-related genes and screened reversal agents of MDR in P/VP20 cells . RESULTS: These cells demonstrated a non-P-glycoprotein (P-gp)-mediated MDR phenotype with overexpression of MDR-associated protein (MRP) mRNA due to MRP DNA amplification . A 1,4-dihydropyridine derivative, bis(4-pyridylmethyl)4-{2-(3-methyl-5,6- dih