Multidrug resistant

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- dihydro-1,4-dithiinyl)}-2,6-dimethyl-1,4-dihydropyridine-3,5-dicar boxylate (NIK250), was found to overcome MDR in P/VP20 cells . CONCLUSIONS: NIK250 might be useful in reversing MDR, which often develops during chemotherapy of advanced or hormone-resistant prostatic cancer.

Jpn J Cancer Res, 1995 Sep, 86(9), 873 - 8
Inostamycin, an inhibitor of P-glycoprotein function, interacts specifically with phosphatidylethanolamine; Kawada M et al.; The mechanism of inostamycin action was further studied . When multidrug-resistant KB-C4 cells were preincubated with inostamycin for 30 min, the accumulation of {3H}vinblastine was increased for as long as 48 h thereafter . Inostamycin inhibited azidopine binding to P-glycoprotein, even after KB plasma membranes had been preincubated with inostamycin and washed . Carbon 14-labeled inostamycin bound to KB plasma membranes irreversibly, but the binding capacity did not parallel the amount of P-glycoprotein in three KB cell lines . Inostamycin was found to interact specifically with purified phosphatidylethanolamine . These results suggest that inostamycin can inhibit P-glycoprotein irreversibly by binding to plasma membranes irreversibly through phosphatidylethanolamine.

Thorax, 1995 Sep, 50 Suppl 1, S37 - 42
Escalating threat from tuberculosis: the third epidemic; Malin AS et al.; PIP: A 1994 study reported on cases of drug-resistant tuberculosis (TB) at the Chest Service at Bellevue Hospital, in New York City . 20 years of TB laboratory susceptibility tests were reviewed in 4681 cases . Combined resistance to isoniazid and rifampicin rose from 2.5% in 1971 to 16% in 1991 . Over 75% of these cases in 1991 were resistant to rifampicin, isoniazid, streptomycin, and ethambutol . Most of the patients belonged to one or more of the following groups: young, Black or Hispanic, unemployed, homeless, male, HIV-infected, and drug abuser . Clinical characteristics were: anergy, fever, cough, night sweats, weight loss, radiograph bilateral infiltrates, adenopathy, cavities, miliary shadowing, and normal chest radiograph . Overall, in 1993 in the US, 3% of all new cases and 6.9% of recurrent cases were resistant to both rifampicin and isoniazid . This resurgence of TB in industrialized countries has been ascribed to: 1) immigration of foreign populations at high risk of developing TB, 2) coinfection with HIV, and 3) an increase in high risk groups . The WHO stresses the importance of identifying meaningful denominators when discussing both primary and acquired drug resistance rates . Restriction fragment length polymorphism (RFLP) is the molecular technique that differentiates individual strains of Mycobacterium tuberculosis . Three recent studies from New York used RFLP to demonstrate the clustering of multidrug-resistant TB . Solutions to the problem consist of adequate infrastructure, i.e., a national TB program; prescription of combined preparations; inducement or enforcement of compliance using directly observed therapy (DOT) (a DOT protocol employed in Denver, Colorado, used 2 weeks of therapy followed by 24 weeks of twice weekly intermittent therapy); prevention of nosocomial spread by isolation of smear-positive cases during the first 2 weeks of treatment; rapid diagnosis of TB and drug susceptibility (within 10-21 days using radiometric culture, nucleic acid probes, and high performance liquid chromatography of mycolic acids); and treatment by five or six drugs .

J Pharmacol Exp Ther, 1995 Sep, 274(3), 1271 - 7
Complete reversal by thaliblastine of 490-fold adriamycin resistance in multidrug-resistant (MDR) human breast cancer cells . Evidence that multiple biochemical changes in MDR cells need not correspond to multiple functional determinants for drug resistance; Chen G et al.; The emergence of drug resistance is a major obstacle to effective cancer chemotherapy . The identification of novel agents that serve as selective, potent and nontoxic modulators of drug resistance is thus an important goal for improving the success of cancer treatment . Thaliblastine (TBL), a plant alkaloid and P-glycoprotein (P-gp) inhibitor, is presently shown to fully reverse 490-fold resistance to Adriamycin (AdR) in a multidrug-resistant (MDR) human breast cancer cell line (MCF/AdR) that overexpresses P-gp, whereas the same treatment had no effect on AdR cytotoxicity in the drug-sensitive parental MCF-7 cells . Mechanistic studies showed that this striking resistance reversal was achieved without alteration of cellular levels of glutathione and without inhibition of glutathione S-transferase, glutathione peroxidase or P450 reductase by TBL, each of which is significantly altered in MCF/AdR cells, and each of which has been proposed to contribute to AdR resistance in this MDR line . Rather, resistance reversal by TBL can be entirely explained by this drug's capacity to restore the intracellular accumulation of AdR in the resistant cells . These results establish that MDR associated with P-gp overexpression can be fully reversed by the potent P-gp inhibitor TBL . They further indicate that although changes in multiple drug-metabolizing enzymes may accompany the development of MDR, these multiple biochemical alterations need not correspond to multiple functional determinants for drug resistance.

Carcinogenesis, 1995 Sep, 16(9), 2051 - 5
Effects of single doses of irradiation on the expression of resistance-related proteins in murine NIH 3T3 and human lung carcinoma cells; Stammler G et al.; In this report the effects of single doses of ionizing radiation on the mRNA expression of several proteins involved in multiple drug resistance were analyzed . Murine NIH 3T3 cells treated with single doses of 5, 10 and 20 Gy during the time interval from 1.5 to 72 h after irradiation were compared with their corresponding controls at the same points of time . The glutathione S-transferase-pi (GST pi) level was elevated in cells treated with 10 or 20 Gy from 24 to 72 h after irradiation compared with the control . Topoisomerase II alpha and thymidylate synthase were decreased in irradiated cells 24-72 h after exposure . These down-regulations were associated with cellular proliferation, determined by mRNA expression of the proliferation marker histone 3 . Irradiated cells exhibited no alteration in the P-glycoprotein or glutathione peroxidase mRNA content . The finding that GST pi mRNA was overexpressed after irradiation was validated by investigations on a human lung carcinoma cell line (LXF 289) on the mRNA and protein level . Thus, our results indicate that irradiation alters the expression of proteins involved in multidrug resistance and may, therefore, play a role in clinical drug response.

J Surg Oncol, 1995 Sep, 60(1), 50 - 4
Overexpression of P-glycoprotein in untreated AFP-producing gastric carcinoma; Dhar DK et al.; P-glycoprotein (P-gly), which is responsible for the phenotypic expression of multidrug resistance in cancerous tissue was stained immunohistochemically in previously untreated alpha-fetoprotein (AFP)-producing (n = 20) and nonproducing gastric cancers (n = 20) . P-gly, AFP, and carcinoembryonic antigen(CEA) were stained in formalin-fixed paraffin-embedded tissue sections immunohistochemically using the monoclonal antibody JSB-1, anti-AFP, and anti-CEA, respectively . DNA ploidy pattern was determined by Fluorescence Activated Cell Sorter (FACS) analyzer . P-gly was significantly overexpressed in AFP producing gastric cancers (60%) than in AFP nonproducing ones (20%) (P < 0.01) . When the result of P-gly staining was analyzed among the AFP-positive cases, P-gly positivity did not emerge either as a significant prognostic factor or as a predictor of the metastatic potentiality of the tumor . The intrinsic overexpression of P-gly in AFP producing gastric cancers proves its biological and morphological similarities to hepatocellular carcinoma . The significantly (P < 0.05) higher incidence of P-gly in diploid tumors indicate that expression of this phenotype might be related to the differentiation of the tumor . P-gly was overexpressed in AFP producing gastric carcinoma and the existing drug resistance, frequent recurrence, and poor prognosis might be explained by presence of P-gly in this carcinoma.

Eur J Cancer, 1995 Sep, 31A(10), 1682 - 8
Flow cytometric functional analysis of multidrug resistance by Fluo-3: a comparison with rhodamine-123; Koizumi S et al.; Using four cell lines including drug-sensitive K562/Parent cells, P-glycoprotein (Pgp)-mediated multidrug resistant (MDR) K562/VCR, K562/ADR and revertant K562/ADR-R cells, two fluorescent agents, Fluo-3 and rhodamine-123 (Rh-123), were compared as indicators in a functional assay of MDR . Cells were incubated with 4 microM Fluo-3 or 1 microM Rh-123 for 45 min and then the intracellular accumulation of the agent was measured using a flow cytometer . Verapamil (20 microM) or cepharanthine (biscoclaurine alkaloid, 10 microM) was added just before the fluorescent agents . Efflux patterns were also studied 60 min after incubation with or without verapamil and cepharanthine . Increased intracellular accumulation and a delayed efflux pattern of Fluo-3 by verapamil and cepharanthine were demonstrated in multidrug resistant K562/VCR and K562/ADR cells, indicating that Fluo-3 is another good indicator of MDR . However, a similar, but lower, increase in uptake and a delayed efflux pattern of Fluo-3 by verapamil and cepharanthine were also demonstrated even in Pgp-non-overexpressed K562/Parent cells . In contrast, accumulation of Rh-123 was not affected by verapamil and cepharanthine . To further study the Pgp dependency of Fluo-3, another cell line, K562/NC16 expressing minimum MDR1 mRNA, was cloned . Increased uptake and a delayed efflux pattern of Fluo-3, but not Rh-123, with verapamil or cepharanthine were again demonstrated in K562/NC16 cells, indicating that intracellular accumulation of Fluo-3 may be non-specifically influenced by verapamil and cepharanthine at very low levels of Pgp-related MDR, while the influx and efflux patterns of Rh-123 may be specifically affected by Pgp overexpression.

Eur J Cancer, 1995 Sep, 31A(10), 1611 - 4
Intrapatient comparison of single-agent epirubicin with or without lonidamine in metastatic breast cancer; Lopez M et al.; The aim of this study was to determine if lonidamine (LND) supplementation to single-agent epirubicin (EPI) could reverse anthracycline resistance in patients with metastatic breast cancer . 45 patients with metastatic breast cancer were treated with EPI 120 mg/m2 by intravenous (i.v.) bolus every 3 weeks . Patients who progressed were given the same chemotherapy regimen on day 4 in combination with oral LND, 150 mg on day 1, 300 mg on day 2 and 450 mg on days 3-5 . Among the 40 evaluable patients, 6 complete responses (CR) and 14 partial responses (PR) were achieved with EPI treatment alone for an overall response rate of 50% . The median duration of response was 6.5 months . Among the 25 patients treated with EPI+LND, 5 PR (21% of 24 evaluable patients) were observed with a median duration of response of 7 months . The median survival in patients receiving both treatments was 20 months . The survival for all patients was 18 months . The survival of patients receiving LND was not significantly longer than for the other patients . Myelotoxicity was the most common side-effect followed by alopecia, nausea and vomiting, and stomatitis . LND-related toxic effects were mild-to-moderate epigastralgia and myalgia . Anthracycline-related toxicity was the same in the two treatment groups . This study indicates that LND may circumvent clinical resistance to EPI without altering the pattern or severity of the toxicity of this anthracycline . Continued investigation of the clinical modulation of EPI resistance by LND in breast cancer is warranted, hopefully in patients with known multidrug resistance status.

J Med Chem, 1995 Aug 18, 38(17), 3282 - 6
Synthesis of (dialkylamino)alkyl-disubstituted pyrimido{5,6,1- de}acridines, a novel group of anticancer agents active on a multidrug resistant cell line; Antonini I et al.; A series of pyrimidoacridine derivatives with two basic side chains, 7a-e, was synthesized, as potential antitumor drugs, starting from 2-{2-(dimethylamino)ethyl}-6-chloropyrimido{5,6,1-de}acridine-1,3, 7- trione (6) and a suitable (alkylamino)alkylamine . The products 6 and 7a-e showed significant cytotoxic activity in vitro against L1210 leukemia . Compounds 7a,d were 2 orders of magnitude more cytotoxic than ametantrone . All compounds were also examined for their activity on LoVo and resistant LoVo/Dx cell lines . Unlike ametantrone, the compounds have shown to be able to overcome the multidrug resistance . Compounds 7a,d, the two most active in vitro, were tested in vivo against murine P388 leukemia showing good activity.

J Biol Chem, 1995 Aug 18, 270(33), 19383 - 90
P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site; Urbatsch IL et al.; P-glycoprotein (Pgp or multidrug-resistance protein) shows drug-stimulated ATPase activity . The catalytic sites are known to be of low affinity and specificity for nucleotides . From the sequence, two nucleotide sites are predicted per Pgp molecule . Using plasma membranes from a multidrug-resistant Chinese hamster ovary cell line, which are highly enriched in Pgp, we show that vanadate-induced trapping of nucleotide at a single catalytic site produces stably inhibited Pgp, with t 1/2 for reactivation of ATPase activity of 84 min at 37 degrees C and >30 h at 4 degrees C . Reactivation of ATPase correlated with release of trapped nucleotide . Concentrations of MgATP and MgADP required to produce 50% inhibition were 9 and 15 microM, respectively, thus the apparent affinity for nucleotide is greatly increased by vanadate-trapping . The trapped nucleotide species was ADP . Divalent Cation was required, with magnesium, manganese, and cobalt all effective: cobalt yielded a very stable inhibited species, t1/2 at 37 degrees C = 18 h . No photocleavage of Pgp was observed after vanadate trapping with MgATP, nor was UV-induced photolabeling of Pgp by trapped adenine nucleotide observed . Vanadate-trapping with 8-azido-ATP followed by UV irradiation caused permanent inactivation and specific labeling of Pgp . Vanadate-induced inhibition was also shown with pure, reconstituted Pgp, with similar characteristics to those in plasma membranes . Vanadate trapping overcomes technical difficulties posed by lack of high affinity nucleotide-binding site(s) or a covalent enzyme-phosphate catalytic intermediate in Pgp . The finding that vanadate trapping of nucleotide at just one site/Pgp is sufficient to give full inhibition at ATPase activity shows that the two predicted nucleotide sites can not function independently as catalytic sites.

Biochim Biophys Acta, 1995 Aug 17, 1245(1), 57 - 61
P-glycoprotein is hyperphosphorylated in multidrug resistant HOB1 lymphoma cells treated with overdose of vincristine; Lee WP; Two proteins with M(r) values of 170 kDa and 200 kDa, respectively, were identified in HOB1 lymphoma cells resistant to 1.0 microM vincristine (designated HOB1/VCR1.0) by Western blot . Using anti-P-glycoprotein monoclonal antibody to immunoprecipitate the protein from the 32P-labeled extract of HOB1/VCR1.0 cells, the major form of the protein was in the area of 200 kDa . When the cells were cultured in 0.5 microM vincristine for 72 h, the major form shifted to the area of 170 kDa . Northern blot analysis of the mdr transcription showed the gene had been overexpressed to a maximum in the cells resistant to 0.5 microM vincristine . {3H}Vincristine uptake study showed the cells with hyperphosphorylated P-glycoprotein accumulated only half the amount of the agent after 60 min of incubation as compared with those with hypophosphorylated protein . The current study suggests hyperphosphorylated P-glycoprotein is a form by which the cells can effectively exploit the protein when it can not be induced any more.

Cancer Lett, 1995 Aug 16, 95(1-2), 195 - 200
Heat shock (hsp70) and resistance proteins in non-small cell lung carcinomas; Volm M et al.; The aim of the study was to prove whether or not an association exists between the heat shock protein 70 (hsp70) and drug resistance . Tumor samples of 90 patients with previously untreated non-small lung carcinomas were investigated immunohistochemically for expression of resistance related proteins . Additionally, resistance to doxorubicin was determined using a short term test . No association between resistance related proteins . Additionally, resistance to doxorubicin was determined using a short term test . No association between resistance to doxorubicin and hsp70 was found . Of 63 resistant tumors, 33 showed low and 30 high hsp70 expression . Of the 26 sensitive tumors, 11 had low and 16 had high hsp70 expression . No relationship could be found between P-glycoprotein which is related to multidrug resistance and hsp70 expression or between hsp70 expression and expression of topoisomerase II, thymidylate synthase and metallothionein . On the other hand, a trend was noted for tumors with high glutathione S-transferase-pi expression to show high hsp70 expression . In addition, there was a significant relationship between hsp70 and catalase positivity . These data indicate that heat shock and stress promote intracellular oxidative damage and catalase is necessary for protection.

Cancer Lett, 1995 Aug 16, 95(1-2), 135 - 8
Expression of multidrug resistance-associated protein (MRP) in thyroid cancers; Sugawara I et al.; It was found that the mechanism of anti-cancer drug resistance in anaplastic carcinoma of the thyroid was not explicable only in terms of expression of mdr1 and its gene product, P-glycoprotein . The multidrug resistance-associated protein (MRP), another member of the mdr gene family, may be involved in anti-cancer drug resistance of this carcinoma . The MRP expression was examined immunohistochemically in 8 cell lines and 73 thyroid cancer tissues; its frequency in anaplastic carcinoma (52%) was significantly higher than that in other thyroid cancer types.

J Natl Cancer Inst, 1995 Aug 16, 87(16), 1230 - 7
Drug resistance-associated marker Lrp for prediction of response to chemotherapy and prognoses in advanced ovarian carcinoma; Izquierdo MA et al.; BACKGROUND: Drug resistance is a major impediment to the successful treatment of ovarian carcinoma . None of the earlier-identified resistance mechanisms, such as overexpression of the MDR1 gene product, P-glycoprotein (Pgp), has been shown to be a major determinant of clinical response to chemotherapy and survival for ovarian cancer patients . The multidrug resistance-associated protein (Mrp) and the lung resistance protein (Lrp, also called the p110 major vault protein), are newly described proteins associated with multidrug resistance in vitro . PURPOSE: The aim of this retrospective study was to investigate the expression of Mrp and Lrp, in addition to Pgp, in advanced ovarian carcinoma and to determine whether such expression was predictive of response to chemotherapy and survival . METHODS: Fifty-seven banked frozen specimens, previously collected and frozen at the time of diagnosis from an equal number of patients with International Federation of Gynecology and Obstetrics (FIGO) stage III or IV ovarian carcinoma, were immunostained for Pgp (with monoclonal antibodies {MAbs} MRK-16 and JSB-1), Mrp (with MAb MRPrl), and Lrp (with MAb LRP-56) . All patients had received platinum- or alkylating-based chemotherapy after debulking surgery . Clinicopathologic parameters determined at diagnosis were retrospectively assessed for their relationship with Pgp, Mrp, and Lrp expression . Response to treatment and survival were compared between Pgp, Mrp, and Lrp expression groups . Qualitative variables were analyzed using Fisher's exact test or the chi-squared test . All reported P values are two-tailed . RESULTS: Nine (16%), 39 (68%), and 44 (77%) of the 57 tumor specimens examined showed positive immunostaining for Pgp, Mrp, and Lrp, respectively . Positive immunostaining for these proteins was not associated with any other prognostic factor examined . No association was found between Pgp and Mrp expression and response to chemotherapy and survival . In contrast, patients with Lrp-positive tumors had poorer response to chemotherapy (P = .004) and shorter progression-free (P = .003) and overall (P = .007) survival than Lrp-negative patients . Multivariate analysis of Lrp expression, FIGO stage, residual tumor after initial surgery, tumor grade, and presence or absence of ascites showed that only Lrp status was independently related to both progression-free survival and overall survival . CONCLUSIONS: Positive Lrp immunostaining in advanced ovarian carcinoma appears to be an indicator of poor response to standard chemotherapy (platinum or alkylating agents) and of adverse prognoses . IMPLICATIONS: The functional characterization of Lrp and related proteins may reveal new approaches to modulate Lrp-associated drug resistance . A large prospective study is warranted to confirm the prognostic value of Lrp.

Proc Natl Acad Sci U S A, 1995 Aug 15, 92(17), 7690 - 4
Role of glutathione in the export of compounds from cells by the multidrug-resistance-associated protein; Zaman GJ et al.; Multidrug-resistance-associated protein (MRP) is a plasma membrane glycoprotein that can confer multidrug resistance (MDR) by lowering intracellular drug concentration . Here we demonstrate that depletion of intracellular glutathione by DL-buthionine (S,R)-sulfoximine results in a complete reversal of resistance to doxorubicin, daunorubicin, vincristine, and VP-16 in lung carcinoma cells transfected with a MRP cDNA expression vector . Glutathione depletion had less effect on MDR in cells transfected with MDR1 cDNA encoding P-glycoprotein and did not increase the passive uptake of daunorubicin by cells, indicating that the decrease of MRP-mediated MDR was not due to nonspecific membrane damage . Glutathione depletion resulted in a decreased efflux of daunorubicin from MRP-transfected cells, but not from MDR1-transfected cells, suggesting that glutathione is specifically required for the export of drugs from cells by MRP . We also show that MRP increases the export of glutathione from the cell and this increased export is further elevated in the presence of arsenite . Our results support the hypothesis that MRP functions as a glutathione S-conjugate carrier.

Blood, 1995 Aug 15, 86(4), 1515 - 24
Expression of mdr-1 in refractory lymphoma: quantitation by polymerase chain reaction and validation of the assay; Kang YK et al.; Measurement of P-glycoprotein and the gene that encodes it, mdr-1, is an important tool for assessing the impact of multidrug resistance in clinical cancer . We evaluated mdr-1 expression by a quantitative polymerase chain reaction (PCR) assay in 78 biopsy samples from 48 patients with refractory lymphoma enrolled on a trial of infusional chemotherapy (EPOCH) in which R-verapamil was added as an antagonist of P-glycoprotein in a subset of patients whose tumors were unresponsive to treatment . Expression of mdr-1 was detectable in all biopsies at the time of enrollment on study, and a fourfold or greater increase in mdr-1 expression was noted in 42% of patients at the time of treatment failure . Expression of mdr-1 was also detectable in biopsies from patients at the time of diagnosis of lymphoma . An endogenous control gene, beta 2-microglobulin, was quantitated for normalization of the mdr-1 values . The use of beta 2-microglobulin expression for normalization was validated in a subset of samples by comparing Northern blots detecting beta 2-microglobulin, beta actin, and GAPDH gene expression . Immunoblot analysis suggested that no major discrepancy was present between mRNA expression and protein level . Immunophenotyping of lymphomatous lymph nodes showed that infiltration of tumor cells ranged from 8% to 95% and of normal T cells from 1% to 83% . Expression of mdr-1 in normal T cells and monocytes was also shown to be low . The mdr-1 levels in patient samples were independent of T-cell contamination, suggesting that the presence of normal cells has at best a small impact on mdr-1 measurements . Expression of mdr-1 in lymphoma can be quantitated by PCR, and wide variations in expression can be observed . Increased expression in patients with refractory disease supports an important role for Pgp in drug resistance in lymphoma . These studies will aid in the design and interpretation of clinical trials in lymphoma.

Int J Cancer, 1995 Aug 9, 62(4), 436 - 42
Comparison of solutol HS 15, Cremophor EL and novel ethoxylated fatty acid surfactants as multidrug resistance modification agents; Buckingham LE et al.; Some well-known fatty acid ester surfactants, e.g., Cremophor EL and Solutol HS 15, are modulators of multidrug resistance in vitro and in vivo . Because they are polydisperse, and their active component(s) have not been identified, the therapeutic potential of such surfactants is unclear . To better define the active components of Solutol HS 15 and to make more potent surfactant multidrug resistance modulators, highly purified C-18 fatty acids were esterified with ethylene oxide at 5-200 molar ratios . Unexpectedly, ethylene oxide esters of pure 12-hydroxy stearic acid, the major components of Solutol HS 15, displayed negligible resistance modification activity compared with Solutol HS 15 itself or to stearic and oleic acid esters synthesized under identical conditions . Since oleic acid esters appeared to have good activity, a series of these compounds was prepared to determine the optimal ethylene oxide/fatty acid ratio . The optimal ratio was found to be 20 mole ethylene oxide: I mole fatty acid, with a steep decline in activity for products made with ratios above and below the optimum . The most active oleic acid ester, designated CRL 1337, was 8.4-fold as potent as Solutol HS 15 and over 19-fold as potent as Cremophor EL in promoting rhodamine 123 accumulation in multidrug-resistant KB 8-5-11 cells in vitro . Our results show that the structure of the hydrophobic domain (fatty acid) of surfactants as well as its hydrophile-lipophile balance are critical in determining the potency of surfactants as reversing agents.

Biochem Pharmacol, 1995 Aug 8, 50(4), 475 - 80
Expression of multidrug resistance in response to differentiation in the K562 human leukaemia cell line; Marks DC et al.; With the increasing use of inducers of cellular differentiation in the treatment of leukaemia, it is essential to understand the relationship between differentiation and the expression of the multidrug resistance . Using the K562 human leukaemia cell line and its multidrug resistant subline K562/E15B, differentiation was examined along two different pathways, megakaryocyte in response to treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), and erythroid in response to treatment with sodium butyrate, in the same cell line . P-glycoprotein expression was increased in the multidrug resistant K562/E15B subline, but not induced in the parental K562 cell line . However, both treatments conferred a different phenotype on the drug resistant subline . TPA treatment caused an increase in P-glycoprotein, increased drug resistance and decreased rhodamine-123 accumulation which was verapamil sensitive, demonstrating that TPA induced a fully functional P-glycoprotein . However, sodium butyrate treatment caused an increase in P-glycoprotein without increased drug resistance or without decreased rhodamine-123 accumulation suggesting that the P-glycoprotein induced by sodium butyrate was nonfunctional . These results stress the importance of examining not only the expression of P-glycoprotein in cells, but also the function of the P-glycoprotein induced.

Biochem Pharmacol, 1995 Aug 8, 50(4), 459 - 64
Cross-resistance studies on two K562 sublines resistant to diaziridinylbenzoquinones; Ward TH et al.; Two resistant K562 sublines have been developed by treatment with AZQ (2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone) and BZQ (2,5-bis(2-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone) . The ID50 values of for AZQ on K562, the AZQ-resistant sublines (AZQR) and the BZQ-resistant sublines (BZQR) were 0.063, 1.47 and 0.244 microM, respectively . The relative ID50 values for BZQ on the same cell lines were 0.2, 0.67 and 0.83 microM, respectively . Although it is generally believed that these two quinones function by different mechanisms, the two sublines have similar decreased levels of cytochrome P-450 reductase and DT-diaphorase and increased levels of glutathione and superoxide dismutase, compared to the parent cell line . The sublines are also cross-resistant to adriamycin, mitozolamide, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and mitomycin C . This work indicates the potential multifactorial mechanisms by which drug resistance can be induced in cell lines in the absence of conventional 'P'-glycoprotein multidrug resistance.

Biochem Pharmacol, 1995 Aug 8, 50(4), 451 - 7
Reversal of multidrug resistance by verapamil analogues; Pereira E et al.; The basic distinguishing feature of multidrug resistant (MDR) cells is a decrease in steady-state drug levels as compared to drug-sensitive controls . It is well-known that verapamil increases the sensitivity of MDR cells to drugs, thus reverting drug resistance . A limiting factor for its clinical use is the pronounced cardiovascular effects of the calcium channel antagonist which occur at the high plasma concentrations required to block P-glycoprotein transport efficiently . From a clinical point of view, it is important to find verapamil derivatives with low calcium channel blocking activity and high reverting activity . This was the aim of the present study . In this context we have investigated the ability of 20 verapamil analogues with restricted molecular flexibility to increase cellular accumulation of anticancer drugs and overcome resistance, and their inotropic, chronotropic, and slow calcium channel antagonistic activity . In this study an anthracycline derivative 4'-O-tetrahydropyranyl adriamycin, and an erythroleukaemia K562 cell line were used . Three of the 20 derivatives checked were completely devoid of calcium channel blocking activity while exhibiting MDR reverting ability comparable to that of verapamil . These derivatives could be useful for the treatment of MDR in cancer patients and for the design and development of other verapamil derivatives.

Biochem Pharmacol, 1995 Aug 8, 50(4), 443 - 50
Influx of daunorubicin in multidrug resistant Ehrlich ascites tumour cells: correlation to expression of P-glycoprotein and efflux . Influence of verapamil; Nielsen D et al.; Classic multidrug resistance is characterized by a decrease in the intracellular concentration of drugs in resistant cells as compared to sensitive cells . This is correlated with the presence of P-glycoprotein in the membrane . P-glycoprotein is responsible for an active efflux of drug . In this study we investigated the correlation between P-glycoprotein and influx of daunorubicin . Four Ehrlich ascites tumour cell lines selected in vivo for resistance to daunorubicin were investigated . The sublines EHR2/0.1, EHR2/0.2, passage no . 12 of EHR2/0.8, EHR2/0.4, and passage no . 72 of EHR2/0.8 were 6-, 6-, 5-, 33-, and 35-fold resistant to daunorubicin, respectively . All sublines overexpressed P-glycoprotein as determined with Western blot . Influx was measured over 40 sec . In glucose-enriched medium influx was significantly decreased in all but one of the resistant sublines . A correlation between P-glycoprotein, degrees of resistance, and influx was demonstrated in four sublines . Comparing influx experiments with efflux experiments (Nielsen et al., Biochem Pharmacol 1994, 47, 2125-2135) we found a linear relationship between influx and efflux in the resistant sublines (r = 0.97) . Verapamil (5.5 microM, 11.0 microM) increased influx significantly in all resistant sublines, whereas the drug had no effect on sensitive cells . Verapamil (3.3 microM) increased influx in the EHR2/0.8 (passage no . 72) subline to the level of sensitive cells . Comparing this result with efflux experiments, verapamil was found to increase influx preferentially . Depletion of energy (medium without glucose including Na(+)-azide) increased influx in all resistant sublines . In EHR2/0.4 and EHR2/0.8 (passage no . 72) the influx, however, was still significantly decreased after depletion of energy . In these cells further addition of verapamil increased influx to the level of EHR2 . These data were consistent with the hypothesis that P-glycoprotein effluxes drug directly from the plasma membrane.

Arch Intern Med, 1995 Aug 7-21, 155(15), 1595 - 600
Health-care expenditures for tuberculosis in the United States; Brown RE et al.; BACKGROUND: The resurgence of tuberculosis (TB) and the increase in multidrug-resistant TB prompted this study, which estimates direct expenditures for TB treatment and public health activities in the United States . METHODS: This retrospective cost of illness study estimated 1991 direct expenditures for TB-related outpatient and inpatient diagnosis and treatment, screening, preventive therapy, contact investigations, surveillance, and outbreak investigations . Existing databases at the Centers for Disease Control and Prevention (Atlanta, Ga) and the Codman Research Group, Lebanon, NH, were supplemented by surveys of state and local TB programs and interviews of organizations that conduct large-scale screening . No estimates of indirect costs were made . RESULTS: The direct medical expenditures for TB in 1991 were estimated at $703.1 million . This cost includes $423.8 million for inpatient care, $182.3 million for outpatient care, $72.1 million for screening, $3.4 million for contact investigations, $17.9 for preventive therapy, and $3.6 million for surveillance and outbreak investigations . Sensitivity analyses yielded a range of expenditures between $515.7 million and $934.5 million . CONCLUSIONS: Treatment accounted for more than 86% of all TB-related expenditures; inpatient treatment accounted for 60% of the total . Prevention activities made up only 14% of all costs . Direct medical expenditures may be underestimated because of limitations in the database on hospital expenditures and health department cost-accounting systems and because of the lack of a national database on screening activities . Greater emphasis should be placed on outpatient treatment and prevention in high-risk populations, and improved cost-accounting systems should be developed in state and local health department TB control programs to facilitate economic evaluation and improve the allocation of health dollars.

Lancet . 1995 Aug 5;346(8971):328.
Foreign aid and TB control policy in Nepal; Fryatt RJ; PIP: Since 1987, Nepal's national tuberculosis (TB) project has received considerable support from the Nepal-Japan technical cooperation project and has introduced short-course chemotherapy (SCC) into almost half of Nepal's districts . Application of SCC has been limited, however, by lack of supervision, inadequate training, and poor drug supply logistics . Problems continue with drug distribution despite a grant from the Japanese pharmaceutical industry of three to four years' supply of rifampicin . These supply difficulties have led to concerns about the increase of multidrug-resistant TB, which is already seen in Nepal where 5-24% of patients have TB which is resistant to at least one drug . This problem is compounded because the Japanese government will not supply the rifampicin in combination with other drugs . This has led to calls for limiting SCC to areas where its use can be directly observed . Meanwhile, as a compromise, the rifampicin will be combined in blister packs with the other drugs (with packaging technology developed in Nepal) . Measures should be taken to insure that the rifampicin will not be used alone and, thus, be responsible for a major epidemic of multidrug-resistant TB .

J Natl Cancer Inst, 1995 Aug 2, 87(15), 1169 - 75
Paclitaxel in metastatic breast cancer: a trial of two doses by a 3-hour infusion in patients with disease recurrence after prior therapy with anthracyclines; Gianni L et al.; BACKGROUND: To date, anthracyclines are the most active drugs against breast tumors, and the taxane paclitaxel (Taxol) looks very promising . Both classes of drugs are affected by cellular multidrug-resistance mechanisms, and therefore their sequential use raises the possibility of clinical cross-resistance . It is therefore important to assess the activity of paclitaxel in patients with clinical resistance to anthracyclines . PURPOSE: We assessed the safety and efficacy of paclitaxel administered by the logistically convenient 3-hour infusion to breast cancer patients who had disease progression within 12 months since prior therapy with anthracyclines . METHODS: Fifty-one patients with metastatic breast cancer who had all relapsed or whose disease had progressed within 12 months from completion of an anthracycline-containing chemotherapy protocol (six receiving adjuvant therapy, 19 receiving neoadjuvant therapy, and 26 receiving treatment for metastatic disease) were enrolled in this phase II trial from June 1992 to May 1994 . After medication to prevent type I acute hypersensitivity reactions, paclitaxel was given intravenously over 3 hours at 175 mg/m2 to the first 15 patients and at 225 mg/m2 to the next 36 patients . The median age was 50 years (range, 31-62 years), and the median Eastern Cooperative Oncology Group performance status was 0 (range, 0-2) . RESULTS: Patients received a median of five cycles (range, one to 11 cycles) . After initial doses of 175 and 225 mg/m2, paclitaxel could be increased by 25 mg/m2 in 73% and 58% of cycles, respectively . Among 50 assessable patients, seven achieved a complete response and 12 achieved a partial response (response rate, 38% {95% confidence interval = 25%-53%}) . The median duration of response was 7 months (range, 4-16 months), and the median time to disease progression for all patients was 5 months . Grade 4 neutropenia occurred in 3% of the cycles and in 12% of the patients and was never associated with fever and infection . Common toxic effects were myalgia and arthralgia (94% of the patients; 4% grade 3), peripheral neuropathy (92% of the patients; 8% grade 3), and alopecia (all patients) . Pruritus and neuropathy were significantly more frequent and severe, respectively, with the higher dose (P < .01 by chi 2 test) . Frequency and severity of other toxic effects were similar at either starting dose . Ten patients had symptoms of neuro-optic toxicity . Only one patient had a grade 2 hypersensitivity reaction . CONCLUSIONS: Paclitaxel at starting doses of 175 and 225 mg/m2 given as a 3-hour infusion can safely be administered to, and is active in women whose disease has progressed after prior treatment with anthracyclines . There was evidence of increased toxicity at the higher dose but no suggestion of better efficacy . IMPLICATION: Paclitaxel by a 3-hour infusion in combination with doxorubicin should be investigated in patients with metastatic breast cancer . Unless randomized trials demonstrate greater efficacy of the more toxic higher dose, it is suggested that a dose of 175-200 mg/m2 be administered with the 3-hour infusion schedule.

Leuk Lymphoma, 1995 Aug, 18(5-6), 515 - 20
Expression of multidrug resistant gene (mdr-1/P-glycoprotein) in a megakaryoblastic cell line, CMK, and its enhancement during megakaryocytic differentiation; Sato T et al.; Multidrug resistance is a severe clinical problem in the chemotherapy of malignant disease . Acute megakaryoblastic leukemia (AMKL) is a rare form of childhood leukemia, and is often resistant to many anti-cancer chemotherapeutic drugs . Here we report the expression of the mdr-1/P-glycoprotein in a cell line, CMK, established from a patient with AMKL . Expression of mdr-1 mRNA in CMK11-5 cells, a well differentiated subline, was higher than in CMK6 cells, a poorly differentiated subline . The level of P-glycoprotein was also higher in CMK11-5 cells . The cytokines interferon-gamma (IFN-gamma), GM-CSF and IL-3, which were shown to induce megakaryocytic differentiation of CMK cells, enhanced the expression of the mdr-1 mRNA and levels of P-glycoprotein . These results imply that differentiated megakaryocytic cells may have higher levels of the P-glycoprotein expression, suggesting a possible normal physiological function of P-glycoprotein in mature megakaryocytes.

Leuk Lymphoma, 1995 Aug, 18(5-6), 435 - 42
Multidrug resistance (Mdr1) gene expression in peripheral blasts from patients with acute leukemia only rarely increases during disease progression after combination chemotherapy; Gruber A et al.; Multidrug resistance gene (mdr1) RNA levels were determined in 55, and P-glycoprotein expression in 37 samples of peripheral leukemic cells from 17 patients with acute myeloblastic leukemia (AML) and 7 patients with acute lymphocytic leukemia (ALL) . Between sample collections, patients were treated with various chemotherapy regimens . Mdr1 RNA levels were quantified by a RNA-RNA solution hybridization assay . P-glycoprotein was determined by Western blot analysis . Samples from 14 patients (9 AML, 5 ALL) had undetectable mdr1 RNA levels at initial analysis . Only two of these had detectable levels after chemotherapy . Ten patients (8 AML, 2 ALL) had detectable mdr1 RNA levels at initial analysis (median 1.0 transcript per cell, range 0.2-1.4) . Increase of mdr1 RNA levels after chemotherapy were observed in cells from 3 patients, one patient had a lower level after chemotherapy and the 6 remaining patients had essentially unchanged mdr1 RNA levels in their leukemic cells . Samples from 13 patients were sequentially analysed for P-glycoprotein expression . In one patient, no P-glycoprotein was detectable at initial analysis but was weakly positive after chemotherapy . In the remaining 12 patients, P-glycoprotein levels stayed stable during disease progression . In conclusion, combination chemotherapy seems only rarely to be associated with an increase of mdr1 gene expression in residual leukemic cells . The addition of resistance modifiers to chemotherapy in order to overcome P-glycoprotein mediated resistance might therefore be more effective in chemotherapy naive patients since it is possible that during later disease progression additional mechanisms of resistance may be more operative.

Stem Cells, 1995 Aug, 13 Suppl 2, 64 - 71
Chemoresistance and multiple myeloma: from biological to clinical aspects; Rossi JF; Resistance to chemotherapy represents a major cause for cancer treatment failure . Several biological mechanisms implicated in chemoresistance have been described, including multidrug resistance (MDR1/P-glycoprotein {P-gp} or p170), resistance-related proteins (p95 and p110), multidrug resistance-associated protein (p190), proteins implicated in cell detoxification such as glutathione S-transferase and genes affecting DNA structure (topoisomerases) . MDR1 has been the most studied in hematological malignancies, particularly in lymphoma and multiple myeloma (MM), diseases generally considered as overexpressing such mechanisms in relapse . Overexpression of chemoresistance is generally an induced phenomenon caused or amplified by the drugs, as demonstrated by the development of drug-resistant cell lines in vitro . It may be defined as a profile of chemoresistance depending on the drug used for induction . This may have a potential implication for monitoring chemoresistance to modulate or to prevent its amplification . Several questions are always open to discussion, including the method of detection, the true prognostic impact of chemoresistance, the dynamic expression of such mechanisms, depending on the cell status, the host response and the mechanism of induction . In MM, the over-expression of MDR1/P-gp is usually less than 10% at diagnosis, leading to 59-80% at relapse, depending on the clinical status . The percentage of positivity depends on the cumulative dose of vincristine and/or doxorubicin . GST pi is (over)expressed in 10-70% of patients at diagnosis, and in 30% at relapse, but in small series, as well as for topoisomerases I and II which are concerned in 53% and 6%, respectively, at diagnosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Br J Haematol, 1995 Aug, 90(4), 876 - 83
Transduction of MDR1 into human and mouse haemopoietic progenitor cells: use of rhodamine (Rh123) to determine transduction frequency and in vivo selection; Hegewisch-Becker S et al.; The MDR1 gene product P-glycoprotein (P-gp) extrudes several anticancer drugs including taxol and fluorescent dyes such as rhodamine (Rh123) . Modulation of the level of P-gp expression has the potential of overcoming multidrug resistance . One possible approach is the retroviral transfer of the human MDR1 gene into murine and human bone marrow (BM) progenitor cells . The rationale for this approach is increased chemoprotection, which allows chemotherapy of a greater level of intensity to be delivered . In this study, flow cytometric measurement of Rh123 extrusion was used to test P-gp function in human and mouse haemopoietic progenitor cells, which had been transduced with a virus containing the human MDR1 transcription unit . Human CD34+ selected cells were analysed immediately following transduction . In two successive experiments MDR1 cDNA transduction resulted in a 7% and 11% increase of P-gp expressing Rh123 dull cells . To monitor transduction efficiency over time as well as the possibility of in vivo selection of drug-resistant BM cells in mice treated with increasing numbers of taxol cycles, the assay was also successfully applied to peripheral blood lymphocytes of mice transplanted with MDR1 transduced BM cells, demonstrating increased Rh123 efflux in transduced cells . Analysis of another fluorescence assay using fluorescein di-beta galactopyranoside as a substrate for beta-galactosidase in cells transduced with a MDR1: beta-gal activity . We conclude that the Rh123 efflux assay is a sensitive method to monitor P-gp function in MDR1 cDNA transduced cells, and may be used to enrich transduced cells via flow cytometric cell sorting for Rh123 dull cells.

Jpn J Clin Oncol, 1995 Aug, 25(4), 124 - 30
Influence of systemic chemotherapy on anti-P-glycoprotein antibody-dependent cell-mediated cytotoxicity in patients with small cell lung cancer; Nabioullin R et al.; Anti-P-glycoprotein antibody (MRK-16)-dependent cell-mediated cytotoxicity (ADCC) by blood mononuclear cells (MNC) was examined in patients with small cell lung cancer (SCLC) before and after systemic chemotherapy . The effect of in vitro treatment of MNC with interleukin (IL)-2 and macrophage-colony-stimulating factor (M-CSF) was also examined . The ADCC reaction was assessed by a 6 h 51Cr-release assay using a multidrug-resistant (MDR) SCLC cell line (H69/VP cells) . The MRK-16 monoclonal antibody was able to augment spontaneous cytotoxicity by MNC, even in SCLC patients . Pretreatment of MNC with IL-2 significantly augmented their ADCC ability in SCLC patients, while M-CSF had no effect on ADCC activity . After the first cycle of systemic chemotherapy, the ADCC activity tended to decline, but ADCC of MNC pretreated with IL-2 was not affected . The results suggest that anti-P-glycoprotein antibody, in combination with a cytokine such as IL-2, may be therapeutically useful against human SCLC resistant to chemotherapeutic drugs.

Leuk Res, 1995 Aug, 19(8), 543 - 8
Comparison of cyclosporin A, verapamil, PSC-833 and cremophor EL as enhancing agents of VP-16 in murine lymphoid leukemias; Slater L et al.; Although verapamil, cyclosporin A . cremophor EL and PSC-833 are active as multidrug resistance modulators, there has been limited study of these compounds as possible chemotherapy enhancing agents against drug-sensitive tumors . We compared these agents as modifiers of VP-16 cytotoxicity in vitro and modifiers of VP-16 efficacy in vivo against drug-sensitive P388 and L1210 leukemias . Our study indicates that cyclosporin A enhances VP-16 cytotoxicity to a significantly greater extent than equimolar concentrations of verapamil or PSC-833 . Although cremophor EL shows significantly greater activity than verapamil in VP-16 cytotoxicity enhancement in vitro, it is ineffective when added to VP-16 therapy of mice bearing L1210 leukemia.

Am J Physiol, 1995 Aug, 269(2 Pt 2), R370 - 9
Daunomycin secretion by killfish renal proximal tubules; Miller DS; Epifluorescence microscopy and video-image analysis were used to measure the uptake of the fluorescent anthracycline daunomycin by intact killifish renal proximal tubules . When tubules were incubated in medium containing 2-5 microM daunomycin, the drug accumulated in the cells and the tubular lumen . At steady state, luminal fluorescence was two to three times greater than cellular fluorescence . Luminal accumulation of daunomycin was reduced when tubules were exposed to the multidrug-resistance (MDR) transporter modifiers verapamil and cyclosporin A (CSA), but not tetraethylammonium (TEA), a model substrate for the renal organic cation transport system . NaCN and vanadate reduced luminal drug accumulation . In contrast, cellular daunomycin accumulation was not affected by verapamil, CSA, TEA, or vanadate and was only slightly reduced by NaCN . When the pH of the buffer solution was decreased from 8.25 to 7.25, luminal, but not cellular, accumulation of daunomycin was again reduced by CSA; however, TEA now reduced cellular and luminal accumulation . These findings are consistent with daunomycin being actively secreted in killifish proximal tubule by two mechanisms . At pH 8.25, daunomycin crossed the basolateral membrane by simple diffusion and was secreted into the tubular lumen by the MDR transporter . At pH 7.25, daunomycin was transported across the basolateral membrane by simple diffusion and carrier-mediated uptake on the organic cation transporter and was secreted into the lumen by the MDR transporter and the organic cation/H+ exchanger.

Am J Physiol, 1995 Aug, 269(2 Pt 1), C323 - 33
Expression and function of P-glycoprotein in a mouse kidney cell line; Ernest S et al.; P-glycoprotein (PGP), a transporter conferring multidrug resistance to cancer cells, is expressed in the kidney . C219 monoclonal antibody binding revealed PGP in proximal tubules and mesangium of mouse kidneys . A cell line (TKPTS) expressing PGP was developed from proximal tubules of the 8Tg(SV40E)Bri7 mouse . Northern blot analysis demonstrated a 5.0-kb message identified as mdr1 by ribonuclease protection assay . Cyclosporin A (CSA) at 0.15 and 10 microM increased cellular accumulation of verapamil (VRP) by 32 and 121%, respectively (P < 0.001) . VRP at 5 microM increased steady-state cellular accumulation of CSA by 46% (P = 0.02) . Basal-to-apical transport of the PGP substrate vinblastine was inhibited by VRP . Rhodamine-123 (R-123) influx was rapid and independent of PGP . R-123 efflux was inhibited by VRP and CSA . Inhibition of PGP transport by VRP, CSA, and PSC-833 decreased the 50% effective dose of adriamycin . The concomitant administration of VRP and CSA was not deleterious and coincided with preferential accumulation of VRP over CSA . Inhibition of PGP-mediated transport is demonstrated as a mechanism of renal cell toxicity.

Toxicol Appl Pharmacol, 1995 Aug, 133(2), 269 - 76
In vivo induction of liver P-glycoprotein expression by xenobiotics in monkeys; Gant TW et al.; P-glycoprotein (pgp), the protein product of the multidrug resistance (mdr) gene family, can confer a multidrug resistance (mdr) phenotype to cells in which it is expressed . One member of the pgp family, pgp2, is located on the hepatocyte biliary pole where it may have a role in biliary excretion . Using primates we sought to determine if mdr gene expression and pgp levels were affected by xenobiotics excreted via the bile in man . Five drugs were studied in male and female rhesus monkeys: erythromycin, rifampicin, tamoxifen, diethylstilbesterol (DES), and probenecid . For each xenobiotic, with the exception of DES, an increase in mdr2 mRNA was observed . The results suggest that expression of mdr2 is responsive to xenobiotics, or their metabolites, that require biliary excretion . We speculate that the mdr2 gene may be a member of a class of xenobiotic responsive genes coding for proteins that actively excrete xenobiotics and/or their metabolites into the bile.

Leukemia, 1995 Aug, 9(8), 1398 - 406
Detection of P-glycoprotein with a rapid flow cytometric functional assay using Fluo-3: evaluation of sensitivity, specificity and feasibility in multiparametric analysis; Van Acker KL et al.; The specificity and sensitivity of a flow cytometric assay simultaneously measuring expression and transport function of the multidrug resistance associated P-glycoprotein (Pgp) was evaluated . The monoclonal antibody (mAb), MRK16 was used to detect phenotypic Pgp expression while Fluo-3-AM was used as a fluorescent substrate in a Pgp functional transport assay . The specificity of the functional assay was examined in two vinblastine selected human leukemic cell lines (K562/VLB2.5 and CCRF-CEM/VLB50) with acquired Pgp overexpression . Downmodulation of Pgp function in these cell lines could be demonstrated with different substances (verapamil, vinblastine, trifluoperazine, cyclosporin A, progesterone and quinidine) and was proven to be consistently higher in the vinblastine selected cells than in their non-selected drug sensitive counterparts . Unexpectedly, modulator activity was also observed in drug sensitive K562 and CCRF-CEM cell lines despite the inability to detect Pgp in those cells by MRK16 flow cytometrically . Low level expression of the MDR1 gene encoding Pgp in sensitive K562 cells was however demonstrated with a sensitive RT-PCR procedure . The small effect of Pgp modulators in non-drug selected cells could therefore be attributed to low level basal expression of Pgp and illustrates the sensitivity of the functional assay . Also, the effect of various Pgp modulators on Pgp function was more pronounced in a subpopulation of Pgp expressing lymphocytes than in lymphocytes which did not express Pgp . Finally, a correlation was found between discrete variations in Pgp expression and Pgp function of CD4+ lymphocytes, underscoring the feasibility of the functional assay in a triple parametric procedure . The triple parametric assay holds promise to detect Pgp expression and function in clinical samples containing mixtures of malignant and non-malignant cells.

Br J Cancer, 1995 Aug, 72(2), 418 - 23
Chemosensitisation of spontaneous multidrug resistance by a 1,4-dihydropyridine analogue and verapamil in human glioma cell lines overexpressing MRP or MDR1; Abe T et al.; Multidrug resistance phenotypes in human tumours are associated with the overexpression of the 170 kDa P-glycoprotein encoded by the multidrug resistance 1 (MDR1) gene, and also with that of the non-P-glycoprotein-mediated multidrug resistance gene, MRP, which encodes a 190 kDa membrane ATP-binding protein . We have previously reported that overexpression of MRP appears to be responsible for spontaneous multidrug resistance in some human glioma cell lines (Abe et al., Int . J . Cancer, 58, 860-864, 1994) . In this study, we investigated whether chemosensitising agents of P-glycoprotein-mediated multidrug resistance such as verapamil, a biscoclaurine alkaloid (cepharanthine), and a dihydropyridine analogue (NIK250) could also reverse multidrug resistance in human glioma cells . The glioma cell lines were the two MRP-expressing cell lines, T98G and IN500, an MDR1-expressing cell line, CCF-STTG1, and the MRP1 MDR1-non-expressing cell line, IN157 . Verapamil and NIK250 almost completely reversed drug resistance to vincristine, etoposide and doxorubicin in T98G cells, while they also reversed drug resistance to vincristine and etoposide, but only partially to doxorubicin in IN500 cells . Cepharanthine as well as verapamil and NIK250 reversed vincristine resistance in CCF-STTG1 cells, but cepharanthine only partially reversed drug resistance in T98G and IN500 cells . The cellular accumulation of {3H}etoposide increased about 2- and 3-fold compared with control in T98G cells in the presence of verapamil and NIK250 respectively . Furthermore, the release of doxorubicin from the nuclei of T98G cells was blocked by NIK250 . However, NIK250 and verapamil caused no apparent increase in vincristine accumulation in T98G cells . NIK250 or verapamil might exert inhibitory effects upon MRP function, resulting in a reversal of MRP-mediated spontaneous multidrug resistance in cultured human glioma cells.

Br J Cancer, 1995 Aug, 72(2), 307 - 11
P-glycoprotein is not expressed in a majority of colorectal carcinomas and is not regulated by mutant p53 in vivo; De Angelis P et al.; Overexpression of the MDR1 product, P-glycoprotein (Pgp), has been shown to be one of the mechanisms underlying the development of multidrug resistance (MDR) . Recently, one mutant p53 has been shown to stimulate the MDR1 gene promoter in vitro, whereas wild-type p53 repressed this activity . We measured Pgp and p53 expression by immunoblotting in 34 colorectal tumours, and performed mutation analyses on the p53-positive tumours to confirm the presence of mutant p53 protein . Tumour DNA indices (DIs) were also measured using flow cytometry . Pgp was detected in 44% (15/34) of the tumours and in 100% (13/13) of the normal mucosas (P = 0.0005), with highest levels of expression seen in normal mucosa, suggesting that initial drug resistance in colorectal tumours is not caused by Pgp . Highly DNA aneuploid tumours demonstrated the lowest levels of Pgp expression relative to moderately aneuploid and diploid colorectal tumours . p53 protein was detected in 53% (18/34) of the tumours, and 12 of 14 p53-positive tumours had p53 gene mutations, p53-negative tumours had approximately twice the level of Pgp expression of p53-positive tumours . Pgp expression was not associated with either p53 expression (P = 0.73) or incidence of p53 gene mutation (P = 0.70), suggesting that mutant p53 does not induce Pgp overexpression in colorectal carcinomas.

Br J Cancer, 1995 Aug, 72(2), 298 - 306
Altered MRP is associated with multidrug resistance and reduced drug accumulation in human SW-1573 cells; Eijdems EW et al.; We have analysed the contribution of several parameters, e.g . drug accumulation, MDR1 P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and topoisomerase (topo) II, to drug resistance in a large set of drug-resistant variants of the human non-small-cell lung cancer cell line SW-1573 derived by selection with low concentrations of doxorubicin or vincristine . Selection with either drug nearly always resulted in MDR clones . The resistance of these clones could be explained by reduced drug accumulation and was associated with a decrease rather than an increase in the low MDR1 mRNA level . To test whether a decrease in MDR1 mRNA indirectly affected resistance in these cells, we introduced a MDR1-specific hammerhead ribozyme into wild-type SW-1573 cells . Although this led to a substantial reduction in MDR1 mRNA, it did not result in resistance . In all resistant clones we found an altered form of the multidrug resistance-associated protein (MRP), migrating slightly slower during SDS-polyacrylamide gel electrophoresis than MRP in parental cells . This altered MRP was also present in non-P-gp MDR somatic cell hybrids of the SW-1573 cells, demonstrating a clear linkage with the MDR phenotype . Treatment of crude cellular membrane fractions with N-glycanase, endoglycosidase H or neuraminidase showed that the altered migration of MRP on SDS-PAGE is due to a post-translational modification . There was no detectable difference in sialic acid content . In most but not all doxorubicin-selected clones, this MDR phenotype was accompanied by a reduction in topo II alpha mRNA level . No reduction was found in the clones selected with vincristine . We conclude from these results that selection of the SW-1573 cell line for low levels of doxorubicin or vincristine resistance, predominantly results in MDR with reduced drug accumulation associated with the presence of an altered MRP protein . This mechanism can be accompanied by other resistance mechanisms, such as reduced topo II alpha mRNA in case of doxorubicin selection.

J Clin Oncol, 1995 Aug, 13(8), 2039 - 42
Effect of R-verapamil on the pharmacokinetics of paclitaxel in women with breast cancer; Berg SL et al.; PURPOSE: To study the effect of the multidrug-resistance reversal agent R-verapamil on the pharmacokinetic behavior of paclitaxel . METHODS: Six women with breast cancer who received paclitaxel as a 3-hour infusion with and without R-verapamil were monitored with frequent plasma sampling up to 24 hours postinfusion . Paclitaxel concentrations were measured using a reverse-phase high-pressure liquid chromatography assay . RESULTS: Concomitant administration of R-verapamil resulted in a decrease in mean (+/- SD) paclitaxel clearance from 179 +/- 67 mL/min/m2 to 90 +/- 34 mL/min/m2 (P < .03) and in a twofold increase in paclitaxel exposure (area under the curve {AUC}) . The mean end-infusion paclitaxel concentration was also twofold higher: 5.1 +/- 1.8 mumol/L versus 11.3 +/- 4.1 mumol/L (P < .03) . CONCLUSION: The alteration in paclitaxel pharmacokinetics when paclitaxel and R-verapamil are coadministered complicates the interpretation of response and toxicity data from clinical trials of this drug combination.

J Clin Oncol, 1995 Aug, 13(8), 1995 - 2004
Controlled trial of dexverapamil, a modulator of multidrug resistance, in lymphomas refractory to EPOCH chemotherapy; Wilson WH et al.; PURPOSE: Overexpression of the multidrug resistance gene (mdr-1) is present in up to 60% of relapsed lymphomas . To study its role in lymphomas, we conducted a controlled trial of dexverapamil, an inhibitor of the mdr-1 gene product, P-glycoprotein (Pgp), in lymphomas refractory to etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (EPOCH) chemotherapy . PATIENTS AND METHODS: Eligible patients had recurrent Hodgkin's (HD) or non-Hodgkin's lymphomas (NHL) and measurable disease . Patients initially received EPOCH alone and those with stable tumor over two cycles or progressive disease crossed over to receive dexverapamil and EPOCH on subsequent cycles . Dexverapamil was escalated eight dose levels, from 240 to 1,200 mg/m2/d . When possible, serial biopsies were obtained to measure mdr-1 expression by quantitative polymerase chain reaction (PCR) . RESULTS: Of 154 patients entered onto the trial, 109 had NHL and 45 had HD . The median age was 44 years, 67% had stage IV disease, and the median number of prior regimens was two (range, one to 12) in NHL and one (range, one to four) in HD . Sixty-four patients (42%) crossed over, of which eight were not assessable . The maximum-tolerated dose of dexverapamil was 900 mg/m2/d . Among 41 NHL patients (excluding mycosis fungoides), there were three complete responses (CRs) and two partial responses (PRs) (12%) and five minor responses (MRs); two of 10 HD patients achieved PRs . The mdr-1 level was measured in 44 biopsies from 19 patients . Pretherapy, mdr-1 was low (median, 2.5 U) but increased (median, 12.2 U) at crossover . Of six patients with mdr-1 levels greater than 15 U, three responded to dexverapamil, while only one of eight patients with mdr-1 levels less than 15 U responded . EPOCH and dexverapamil were well tolerated, but compared with EPOCH alone, produced more hematologic toxicity . CONCLUSION: These results suggest that Pgp plays a role in clinical drug resistance of lymphomas . However, they also suggest that mechanisms other than Pgp are prominent in heavily pretreated patients and that, although Pgp inhibition may be necessary, it is probably insufficient . Earlier intervention with dexverapamil may be more effective and warrants further study.

J Clin Oncol, 1995 Aug, 13(8), 1985 - 94
Phase I and pharmacokinetic study of the multidrug resistance modulator dexverapamil with EPOCH chemotherapy; Wilson WH et al.; PURPOSE: Dexverapamil is a competitive inhibitor of the P-glycoprotein (Pgp) efflux pump, a potent mechanism of multidrug resistance (mdr-1) in vitro . We performed a phase I study to determine the maximum-tolerated dose (MTD) and pharmacokinetics of dexverapamil with etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (EPOCH) chemotherapy . PATIENTS AND METHODS: Eligible patients had relapsed or refractory lymphoma or sarcoma . Patients initially received EPOCH alone, and those with stable or progressive disease were crossed-over to received dexverapamil on subsequent cycles of EPOCH . Dexverapamil was administered orally for 6 days and escalated over eight dose levels ranging from 240 to 1,200 mg/m2/d . Pharmacokinetics of dexverapamil and its active metabolite, nor-dexverapamil, were obtained in most patients . In seven patients, pharmacokinetics of doxorubicin, doxorubicinol, and etoposide were determined on paired cycles of EPOCH with or without dexverapamil . RESULTS: Sixty-five patients received 130 cycles of dexverapamil/EPOCH chemotherapy . The MTD of dexverapamil was 150 mg/m2 every 4 hours (900 mg/m2/d), and hypotension was the principal dose-limiting toxicity . The dexverapamil area under the curve (AUC) increased proportionally with dexverapamil dose, but significant interpatient variation occurred . At the MTD, the median plasma average concentrations of dexverapamil and nor-dexverapamil were 1.2 and 1.4 mumol/L, respectively . Dexverapamil did not affect the steady-state concentration (Css) of etoposide, but increased the Css of doxorubicin and doxorubicinol nearly twofold . The absolute neutrophil and platelet nadirs were significantly lower on the dexverapamil cycles compared with cycles of EPOCH alone, but other chemotherapy-related toxicities did not change . CONCLUSION: The phase II recommended dose of dexverapamil with EPOCH is 150 mg/m2 every 4 hours . This dose was well tolerated on an outpatient basis and achieved plasma concentrations of dexverapamil and nor-dexverapamil within the effective range for Pgp inhibition in vitro . Although dexverapamil increased the hematopoietic toxicity of EPOCH, it was mild, readily reversible, and offset by EPOCH dose reductions . Dexverapamil should be considered for further study.

J Clin Oncol, 1995 Aug, 13(8), 1958 - 65
Phase I/II trial of dexverapamil plus vinblastine for patients with advanced renal cell carcinoma; Motzer RJ et al.; PURPOSE: The reduced cardiac toxicity of the dextro-(d-) stereoisomer of verapamil (dexverapamil; Knoll Pharmaceuticals, Whippany, NJ) warrants its study as a potential multidrug-resistance (MDR) reversal agent . PATIENTS AND METHODS: Twenty-three patients with advanced renal cell carcinoma (RCC) were treated with vinblastine at a dose of 0.11 mg/kg intravenous (IV) bolus injection on days 1 and 2 every 21 days . Dexverapamil was added to subsequent cycles after resistance had been demonstrated . Dexverapamil treatment was begun 18 hours before day 1 of vinblastine administration and was given orally every 6 hours for 12 doses . Patients in group A were treated with a dose of 120 mg/m2, and those in group B were treated with 180 mg/m2 plus dexamethasone; plasma concentrations achieved in patients were correlated with in vitro effects . RESULTS: Toxicities included hypotension, asymptomatic bradycardia, and mild atrioventricular conduction delays, although one patient had dexverapamil discontinued for grade IV congestive heart failure . There were no partial or complete responses . The mean day-1 serum dexverapamil plus norverapamil plasma concentrations were 2,575 ng/mL (range, 697 to 6,015 ng/mL) for group A and 1,654 ng/mL (range, 710 to 4,132 ng/mL) for group B at the time of vinblastine administration . These concentrations were in the range of those that reversed vinblastine resistance in vitro . CONCLUSION: The advantage of dexverapamil as an MDR reversal agent is its potential for achieving desired blood levels with substantially less toxicity than the racemic mixture of verapamil . Based on tolerability, it is a suitable drug for further study in clinical trials of malignancies other than RCC that attempt to achieve MDR reversal . The dose of 120 mg/m2 given orally every 6 hours, with dose escalation based on individual tolerance, represents a feasible schedule to be considered for such studies.

Exp Parasitol, 1995 Aug, 81(1), 1 - 8
Plasmodium falciparum: detection of P-glycoprotein in chloroquine-susceptible and chloroquine-resistant clones and isolates; Cremer G et al.; Recent studies have suggested the potential involvement of multidrug resistance (mdr) genes in resistance to quinoline-containing compounds in Plasmodium falciparum parasites . Three different antibodies were used to detect the malarial mdr 1 protein product by indirect immunofluorescence and/or immunoblot in fresh clinical isolates and clones of P . falciparum from different geographic origins . A 160-kDa protein was detected in all five parasite clones by immunoblot and around the food vacuole by IFA, regardless of the level of sensitivity to chloroquine and the modulation by desipramine of chloroquine accumulation . Our results suggest that chloroquine resistance is not correlated with the presence of the Pfmdr1 product.

Cytometry, 1995 Aug 1, 20(4), 315 - 23
Influence of S9788, a new modulator of multidrug resistance, on the cellular accumulation and subcellular distribution of daunorubicin in P-glycoprotein-expressing MCF7 human breast adenocarcinoma cells; Merlin JL et al.; A triazinoaminopiperidine derivative synthesized as a modulator of multidrug resistance, S9788, was investigated in the human breast adenocarcinoma MCF7DXR cell line expressing P-glycoprotein . In addition to being less sensitive to daunorubicin, the resistant cell line showed dramatic alterations in the subcellular distribution of daunorubicin, as observed via fluorescence microscopy and quantified via tritiated daunorubicin nuclear distribution analysis . Compared to verapamil and cyclosporin A at 2 and 5 mumol/liter, S9788 proved to be more potent in restoring the cellular accumulation and the subcellular distribution of daunorubicin in the resistant cells . Significant activity of S9788 was observed at 2 mumol/liter, which is clinically achievable, and S9788 restored the nuclear distribution of the drug to the level observed in the parental sensitive cell line . Consequently, the restoration of the cytotoxicity of daunorubicin by S9788 was nearly complete (> 90%) at 2 mumol/liter, wheras cyclosporin A reached this level of activity at 5 mumol/liter, and verapamil was always less active at both concentrations . These results suggest that the modulation of multidrug resistance by S9788 is not only related to the enhancement of the cellular accumulation but also especially by the restoration of the subcellular distribution of the drugs to their nuclear sites of action.

Cytometry, 1995 Aug 1, 20(4), 307 - 14
Flow cytometric assessment of the cellular pharmacokinetics of fluorescent drugs; Dordal MS et al.; Development of multidrug resistance (MDR) in cancer cells decrease net doxorubicin uptake as a result of either increased efflux, or decreased intracellular sequestration, or decreased membrane permeability . Kinetic parameters of drug uptake can distinguish among these forms of altered transport . Cellular uptake of fluorescent drugs was monitored by a flow cytometric assay using a rapid-injection system and analyzed with a three-compartment model in which rapid diffusion from extracellular fluid into the cell was followed by uptake into a nonexchangeable pool . In agreement with our recent studies of 14C-doxorubicin distribution (Dordal et al.: J Pharmacol Exp Ther 271:1286-1290, 1994), sequestration of doxorubicin was decreased 2.7-fold in P-glycoprotein-expressing SU-4R lymphoma cells compared to drug-sensitive SU-4 cells (14.0 +/- 4.8 vs . 5.0 +/- 0.9 nl s-1) without a change in membrane permeability or evidence of active efflux . In contrast, sequestration of the highly fluorescent dye rhodamine 123 was decreased 20-fold (17.1 +/- 8.3 vs . 0.9 +/- 0.8 nl s-1) . Resistant cells were significantly less permeable to rhodamine than sensitive cells (3.8 +/- 1.2 vs . 10.2 +/- 2.6 x 10(5) cm2 s-1), and rhodamine efflux was increased by 24% . Thus, SU-4R cells exhibit multiple alterations that cause decreased intracellular drug concentrations, of which decreased sequestration is quantitatively the most significant.

Anticancer Drugs, 1995 Aug, 6(4), 578 - 85
Cytotoxic effect of calcein acetoxymethyl ester on human tumor cell lines: drug delivery by intracellular trapping; Liminga G et al.; Calcein acetoxymethyl ester (calcein/AM) and some related cellular dyes with a cytoplasmic distribution were investigated with respect to cellular hydrolysis, accumulation, efflux and cytotoxicity in a panel of established human cell lines, including multidrug resistant (MDR) phenotypes . At 0.1-1 micrograms/ml, calcein/AM was highly cytotoxic against several cell lines, even after short-term exposure (30 min) . Calcein/AM induced no immediate loss (3 h) of membrane integrity and the drug was more active against low compared with high density plated cells . In cell lines with the MDR phenotype and in the renal carcinoma cell line ACHN, the drug was considerably less active . Non-esterified calcein had no effect and calcein/AM was significantly more potent than other structurally related fluorescein analogs and AM esters tested . Although MDR cell lines showed a decreased cellular hydrolysis and accumulation of the dye, there was no strict relationship between cytoplasmic calcein exposure and cytotoxic activity . The rate of efflux was low in the two most sensitive cell lines, the human lymphoma U-937-GTB and its vincristine (vcr) resistant subline U-937/vcr10, while the remaining cell lines showed similar biphasic efflux patterns, including cell lines of the MDR phenotype . The results show that calcein/AM has cytotoxic activity against human tumor cell lines at low concentrations . The effect appears dependent on the intracellular trapping of the drug, although the specific cellular target remains unknown . Due to its cytotoxic efficacy and unique principle of cellular drug delivery, further investigation of calcein/AM and related compounds as potentially new anticancer agents seems warranted.

J Formos Med Assoc, 1995 Aug, 94(8), 449 - 57
Gestational trophoblastic diseases: current trends and perspectives; Wang TH et al.; Gestational trophoblastic diseases (GTD) include a spectrum of diseases from the potentially premalignant hydatidiform mole to the highly aggressive choriocarcinoma . Most complete moles have diploid chromosomes, nearly always of pure paternal origin, whereas most partial moles have triploid chromosomes, containing one haploid maternal set and two paternal sets . The first-line treatment of molar pregnancies is suction evacuation . In patients with persistent trophoblastic diseases or choriocarcinoma, single agent or multiagent chemotherapy is indicated, depending on the prognostic score of the individual patient . With careful follow-up and appropriate treatment, nearly all patients with gestational trophoblastic diseases can be cured . Although many advances have been made in the cytogenetics, molecular biology and immunobiology of GTD, the reasons for its unique curability remain unclear . Studies comparing induction of apoptosis and multidrug resistance gene expression, in normal trophoblasts and GTD, may elucidate the mechanism behind the good response of GTD to chemotherapy . This may give some innovative insight into chemoresistance.

J Clin Invest, 1995 Aug, 96(2), 1026 - 34
Downregulation of mdr-1 expression by 8-Cl-cAMP in multidrug resistant MCF-7 human breast cancer cells; Scala S et al.; 8-Cl-cAMP, a site-selective analogue of cAMP, decreased mdr-1 expression in multidrug-resistant human breast cancer cells . A sixfold reduction of mdr-1 mRNA expression by 8-Cl-cAMP began within 8 h of treatment and was associated with a decrease in the synthesis of P-glycoprotein and with an increase in vinblastine accumulation . A reduction in mdr-1 expression after 8-Cl-cAMP treatment was also observed in multidrug-resistant human ovarian cancer cell lines . 8-Cl-cAMP is known to change the ratio between the two regulatory subunits, RI and RII, of protein kinase A (PKA) . We observed that RI alpha decreased within 24 h of 8-Cl-cAMP treatment, that RII beta increased after as few as 3 h of treatment, and that PKA catalytic activity remained unchanged during 48 h of 8-Cl-cAMP treatment . The results are consistent with the hypothesis that mdr-1 expression is regulated in part by changes in PKA isoenzyme levels . Although 8-Cl-cAMP has been used to differentiate cells in other model systems, the only differentiating effect that could be detected after 8-Cl-cAMP treatment in the MCF-7TH cells was an increase in cytokeratin expression . Evidence that the reduction of mdr-1 mRNA occurred at the level of gene transcription was obtained by measuring chloramphenicol acetyltransferase (CAT) mRNA in MCF-7TH cells transfected with an mdr-1 promoter-CAT construct prior to 8-Cl-cAMP treatment . Thus, 8-Cl-cAMP is able to downregulate mdr-1 expression and suggests a new approach to reversal of drug resistance in human breast cancer.

Clin Chem, 1995 Aug, 41(8 Pt 1), 1087 - 93
Nonradioactive quantification of mdr1 mRNA by polymerase chain reaction amplification coupled with HPLC; van Hille B et al.; We describe a new strategy for quantifying mRNA by using polymerase chain reaction (PCR) coupled with HPLC . PCR-amplified products are directly analyzed with a specific HPLC column and are quantified by standardization against a housekeeping gene, beta-actin . To evaluate the experimental conditions, we examined the multidrug-resistance-associated mdr1 gene expression in two drug-sensitive cell lines (RPMI-8226 and KB-3-1) and in their drug-resistant sublines . This revealed a significant overexpression of the mdr1 gene in chemoresistant sublines that paralleled the degree of in vitro drug resistance and the amounts of mRNA determined by other methods . Results were consistently reproducible, with imprecision (CV) < 25% . The reverse transcription option PCR/HPLC protocol described here is a sensitive, nonradioactive method for quantifying mdr1 mRNA in cell lines or clinical tumor samples, and can be applied to the simultaneous quantification of several mRNA species.

AIDS Res Hum Retroviruses, 1995 Aug, 11(8), 893 - 901
Transmembrane P-glycoprotein (P-gp/P-170) in HIV infection: analysis of lymphocyte surface expression and drug-unrelated function; Lucia MB et al.; P-glycoprotein (P-gp/P-170), a transmembrane efflux pump known to be one of the mechanisms responsible for multidrug resistance in cancer therapy, is constitutively expressed in several solid human tissues as well as in normal peripheral blood lymphocytes and bone marrow cells . In particular, this molecule has been associated with the transport of perforin and other cytolysins in natural killer (NK) and T cytotoxic lymphocytes . In the present study, we analyzed peripheral blood lymphocytes (PBLs) from controls and HIV+ patients for phenotypic expression and function of the P-gp/P-170 molecule . We found that 90% of all PBL subsets (i.e., CD4+, CD8+, CD56+, and CD19+ cells) expressed surface P-gp/P-170 both in controls and HIV+ patients . However, a significant decrease in CD4+/P-170+ and CD19+/P-170+ cells was observed in HIV+ individuals with respect to controls . PHA and IL-2 stimulation of PBLs was unable to increase the expression of P-gp/P-170 both in controls and HIV+ patients, despite the increased detection of the CD25 molecule . On the other hand, stimulation with anti-CD3 determined a significant increase in lymphocyte P-gp/P-170 . The function of P-gp/P-170, assessed by a flow cytometric assay for rhodamine-123 (Rh123) efflux, was significantly reduced in CD16+ NK cells and CD19+ B cells from HIV+ patients . The Rh123 efflux by NK cells correlated (p < 0.01) with the NK cytotoxicity against the 51Cr-labeled K562 cell line . Last, the effect of the antiretroviral drugs AZT, ddI, and ddC on P-gp expression and function was evaluated . The dideoxynucleoside compounds did not inhibit P-gp/P-170 function of normal mononuclear cells in vitro, and did not increase P-gp/P-170 expression in vivo, in patients undergoing antiretroviral therapy with AZT . These findings provide further evidence of a possible involvement of the P-gp/P-170 system in specific immunological lymphocyte functions, and especially in cytotoxic-type functions . In addition, it is possible to suggest, on the basis of our experimental data, that the dideoxynucleoside class of antiretroviral agents does not contribute to the phenotypic and functional alterations related to P-glycoprotein during HIV infection.

Hematol Oncol Clin North Am, 1995 Aug, 9(4), 889 - 908
Multidrug drug resistance in pediatric sarcomas; Chan HS et al.; PURPOSE . Although P-glycoprotein overexpression is an important cause of multidrug resistance in vitro, whether this resistance mechanism is equally applicable to the clinic has still to be fully established . This review has examined the immunohistochemical and molecular biologic tools used to evaluate soft tissue sarcomas of children and adults, in order to determine whether P-glycoprotein can limit the efficacy of chemotherapy . DESIGN . Because soft tissue sarcomas are successfully treated by cytotoxic substrates of P-glycoprotein in many, but not all children, these tumors may be useful models for determining whether this drug efflux transporter is a clinically relevant cause of chemoresistance . RESULTS . Certain studies of acute myelogenous leukemia, lymphoma, and myeloma in adults and rhabdomyosarcoma, neuroblastoma, and acute lymphoblastic leukemia in children have provided the best current evidence for a strong corelation between the expression of P-glycoprotein and outcome of chemotherapy . Based on these observations, several clinical trials have been initiated to determine whether pharmacologic chemosensitization improves chemotherapy responses and cure rates in P-glycoprotein-expressing malignancies . Thus, early identification of lower levels of P-glycoprotein in tumors that still might respond to chemosensitizer-modulated chemotherapy may be highly pertinent to conducting more informative multidrug resistance reversal trials . CONCLUSION . The question of whether the multidrug resistance P-glycoprotein is a clinically relevant cause of chemoresistance may ultimately be answered by the successful prevention of chemotherapy failure by chemosensitizers that specifically reverse this drug efflux mechanism.

Semin Oncol, 1995 Aug, 22(4 Suppl 11), 35 - 41
Induction of resistance to 2',2'-difluorodeoxycytidine in the human ovarian cancer cell line A2780; Ruiz van Haperen VW et al.; 2',2'-Difluorodeoxycytidine (gemcitabine; dFdC) is a nucleoside analog with promising antitumor activity . To be active it must be phosphorylated by deoxycytidine kinase (dCK) . We induced resistance to gemcitabine in the human ovarian carcinoma cell line A2780 by exposure to increasing concentrations of gemcitabine . At 72 hours' exposure the IC50, defined as the concentration of gemcitabine causing 50% growth inhibition, increased from 0.6 nmol/L gemcitabine in A2780 to 92 mumol/L in the resistant variant, AG6000 . AG6000 is cross-resistant to other drugs that require activation by dCK, such as I-beta-D-arabinofuranosylcytosine, 5-aza-2'-deoxycytidine, and 2-chlorodeoxyadenosine . AG6000 was also cross-resistant to 2',2'-difluorodeoxyuridine (dFdU), the deamination product of gemcitabine . In addition, cross-resistance to the multidrug-resistance drugs doxorubicin and vincristine was observed . This was not associated with induction of P-glycoprotein . No accumulation of gemcitabine triphosphate could be detected in AG6000 cells, in contrast to the parental A2780 cells . There was no specific dCK activity in extracts from AG6000 cells . Western blot analysis using a polyclonal anti-dCK antibody did not reveal any dCK protein in AG6000 cell extracts . Reverse transcribed and polymerase chain reaction amplified mRNA, using specific dCK primers, demonstrated that AG6000 expressed a normal length amplicon of 701 base pairs, besides an aberrant amplicon of 500 base pairs . Although the resistant cell line is routinely cultured in 6 mumol/L gemcitabine, the resistant phenotype can be maintained for at least 10 passages without gemcitabine . These results indicate that the gemcitabine resistance phenotype is stable and mainly due to dCK deficiency.

J Physiol, 1995 Aug 1, 486 ( Pt 3), 707 - 14
Failure of P-glycoprotein (MDR1) expressed in Xenopus oocytes to produce swelling-activated chloride channel activity; Morin XK et al.; 1 . P-glycoprotein, the protein product of the multidrug resistance (MDR1) gene, has ATP-dependent transporter activity . It has been suggested that P-glycoprotein may also function as a volume-regulated chloride channel or chloride channel regulator . To assess the chloride channel function of P-glycoprotein, we examined swelling-activated chloride conductances in Xenopus oocytes injected with human MDR1 cRNA . 2 . Functional expression of P-glycoprotein in Xenopus oocytes was confirmed using Western blot analysis and by assessing transport of the P-glycoprotein substrate, calcein AM . 3 . Endogenous, swelling-activated chloride conductances were virtually absent by the time P-glycoprotein expression was confirmed . Thus, this expression system afforded the advantage of assessing putative MDR1-associated chloride currents in the absence of background currents . 4 . The currents activated by hypotonic shock (50%) in both MDR1-injected and control (water-injected) oocytes were not significantly different . The swelling response was due in part to the activation of a potassium-selective conductance which could be inhibited by barium . No chloride-selective currents were activated by hypotonic shock in the presence or absence of barium . Therefore, we conclude that P-glycoprotein expression does not produce a swelling-activated chloride conductance in the Xenopus oocyte expression system.

Int J Cancer, 1995 Jul 28, 62(3), 283 - 90
Differential effects of verapamil and quinine on the reversal of doxorubicin resistance in a human leukemia cell line; Bennis S et al.; We studied the restoration of doxorubicin accumulation and sensitivity by verapamil and quinine in a variant of the human erythroleukemia cell line K562 selected for resistance to doxorubicin and presenting a multidrug-resistance (MDR) phenotype . Verapamil was able to completely restore doxorubicin accumulation in the resistant cells to the level obtained in sensitive cells, but only partially reversed doxorubicin resistance . Quinine, in contrast, had a relatively weak effect on doxorubicin accumulation but was able to completely restore doxorubicin sensitivity in the resistant cells . In addition, verapamil was able to decrease azidopine binding to P-glycoprotein, whereas quinine was not . Quinine also modified the intracellular tolerance to doxorubicin, which suggests that it is able to modify drug distribution within the cells . Confocal microscopy revealed that verapamil and quinine were able to restore nuclear fluorescence staining of doxorubicin in resistant cells; since this was obtained for quinine without significant increase of doxorubicin accumulation, this observation confirms that quinine acts principally on doxorubicin redistribution within the cells, allowing the drug to reach its nuclear targets . When used in association, verapamil and quinine reversed doxorubicin resistance in a synergistic fashion . We conclude that verapamil and quinine do not share the same targets for reversal of MDR in this cell line; whereas verapamil directly interferes with P-glycoprotein and mainly governs drug accumulation, quinine has essentially intracellular targets involved in drug redistribution from sequestration compartments.

N Engl J Med, 1995 Jul 27, 333(4), 229 - 33
Tuberculosis in New York City--turning the tide; Frieden TR et al.; BACKGROUND . From 1978 through 1992, the number of patients with tuberculosis in New York City nearly tripled, and the proportion of such patients who had drug-resistant isolates of Mycobacterium tuberculosis more than doubled . METHODS . We reviewed, confirmed, and analyzed data obtained during the surveillance of patients with tuberculosis . RESULTS . From 1992 through 1994, there was a 21 percent decrease in reported cases of tuberculosis in New York City . An evaluation of the surveillance system revealed very few unreported cases . The number of cases decreased by more than 20 percent among blacks and Hispanics, persons with documented human immunodeficiency virus infection, homeless persons, and patients with multidrug-resistant tuberculosis; in all these groups, tuberculosis is likely to result from recent transmission . In contrast, the number of cases of tuberculosis increased among elderly and foreign-born persons, in whom the disease is likely to result from the reactivation of an infection acquired many years earlier . Enrollment in a program of directly observed therapy, in which health workers watch patients take their medications, increased from fewer than 100 patients to nearly 1300, with more than 32,000 patient-months of observation from 1992 through 1994 . CONCLUSIONS . Epidemiologic patterns strongly suggest that the decrease in cases resulted from an interruption in the ongoing spread of M . tuberculosis infection, primarily because of better rates of completion of treatment and expanded use of directly observed therapy . Another contributing factor may have been efforts to reduce the spread of tuberculosis in institutional settings, such as hospitals, shelters, and jails . Expansion of measures to prevent and control tuberculosis and support of international control efforts are needed to ensure continued progress.

J Med Chem, 1995 Jul 21, 38(15), 2955 - 63
Novel hexakis(areneisonitrile)technetium(I) complexes as radioligands targeted to the multidrug resistance P-glycoprotein; Herman LW et al.; Transport substrates and modulators of the human multidrug resistance (MDR1) P-glycoprotein (Pgp) are generally lipophilic cationic compounds, many with substituted aryl moieties . We sought to synthesize aromatic technetium-isonitrile complexes to enable functional detection in vivo of Pgp expression in tissues . A series of substituted aromatic isonitrile analogs were synthesized from their corresponding amines by reaction with dichlorocarbene under phase transfer-catalyzed conditions, and the non-carrier-added hexakis(areneisonitrile)Tc-99m(I) complexes were produced by reaction with pertechnetate in the presence of sodium dithionite . Cellular accumulation in vitro, whole body biodistribution, and the imaging properties of these lipophilic, monocationic organometallic complexes were determined in Chinese hamster lung fibroblasts expressing MDR Pgp, in normal rats, and in rabbits, respectively . For this initial series, verapamil (50 microM), the classical Pgp modulator, significantly enhanced cellular accumulation or displaced binding of Tc complexes of 1b, 1d, 1h, 2a, 2d, 3a, and 3b, indicative of targeted interactions with Pgp . Most complexes, despite their modestly high lipophilicity, were excluded by the blood/brain barrier, and several complexes displayed simultaneously high hepatobiliary and renal excretion in vivo, consistent with the physiological expression pattern of Pgp in these tissues . Selected Tc- and Re-areneisonitrile complexes of this class have potential applicability to the functional imaging and modulation, respectively, of MDR Pgp in human tissues.

Proc Natl Acad Sci U S A, 1995 Jul 18, 92(15), 6996 - 7000
The bacterial colicin active against tumor cells in vitro and in vivo is verotoxin 1; Farkas-Himsley H et al.; We have identified verotoxin 1 (VT1) as the active component within an antineoplastic bacteriocin preparation from Escherichia coli HSC10 studied over two decades . Recombinant VT1 can simulate the toxicity of anticancer proteins (ACP), and the antineoplastic activity of ACP (and VT1) was abrogated by treatment with anti-VT1 antibody . Similarly, VT1 mimics the protective effect of ACP in a murine metastatic fibrosarcoma model . Prior immunization with VT1 B subunit prevents the effect of VT1 or ACP in this model . The activity of ACP against a variety of human ovarian cell lines was mimicked by VT1, and multidrug-resistant variants were significantly hypersensitive . Primary ovarian tumors and metastases contain elevated levels of globotriaosylceramide compared with normal ovaries, and overlay of frozen tumor sections showed selective VT binding to tumor tissue and the lumen of invading blood vessels . Our contention that VT1 could provide an additional approach to the management of certain human neoplasms is discussed.

Biochemistry, 1995 Jul 18, 34(28), 9159 - 65
Involvement of cytoplasmic factors regulating the membrane orientation of P-glycoprotein sequences; Zhang JT et al.; Chinese hamster pgpl P-glycoprotein (Pgp) is a membrane transport protein that causes multidrug resistance (MDR) by actively extruding a wide variety of cytotoxic agents out of cells . It may also function as a peptide transporter and as a chloride channel . Previously, we have shown that hamster pgpl Pgp is expressed in more than one topological form and that the generation of these structures is modulated by charged amino acids flanking the predicted transmembrane (TM) segments 3 and 4 . Different topological structures of Pgp may be involved in different functions . In this study, we examined the role of cytoplasmic components in cell-free translation systems in modulating the topologies of Pgp . By using rabbit reticulocyte lysate (RRL) and wheat germ extract (WGE) expression systems, we showed that WGE contains a soluble, heat-labile, high molecular weight fraction that regulates the membrane topology of truncated Pgp molecules . These results and our previous findings indicate that the membrane topology of a mammalian polytopic membrane protein may be regulated both by the amino acid sequence of the protein and by soluble cytoplasmic component(s) . We speculate that Pgp expressed in various cell types may have different topological structures modulated by specific cytoplasmic factors.

Biochem Pharmacol, 1995 Jul 17, 50(2), 187 - 96
Novel dithiane analogues of tiapamil with high activity to overcome multidrug resistance in vitro; Eliason JF et al.; Dithiane analogues of tiapamil are highly active as modifiers of P-glycoprotein mediated multidrug resistance (MDR) in vitro . In an assay using the P-glycoprotein over-expressing cell line KB-8-5, the most active analogues for decreasing vincristine resistance were the racemate Ro 11-5160 and its two enantiomers, Ro 44-5911 (R) and Ro 44-5912 (S) . In the KB-8-5 assay, the resistance modification index (RMI) of Ro 11-5160 was approximately 12-fold higher than those of the most active reference compounds tested, dipyridamole, cepharanthine, reserpine and cyclosporin A, when compared at concentrations equal to one-tenth of the IC50 of each compound (RMI0.1) . The enantiomers have similar resistance modifying activities, but the (S) enantiomer Ro 44-5912 is somewhat more active, fully reverting the vincristine sensitivity of KB-8-5 cells to the level of the parental KB-3-1 cells at a concentration of 2 microM . The (R) enantiomer attained this level of modification at a concentration of 3.5 microM . These concentrations are both well below their IC50 values for KB-8-5 cells (150 microM) . The enantiomers appear to interact with P-glycoprotein because they inhibited {3H}azidopine and {3H}-vinblastine binding to plasma membrane fractions prepared from resistant K562/ADR cells . However, in addition to their resistance modifying activities with KB-8-5 cells, these compounds also decreased the IC50 values of vincristine and doxorubicin with KB-3-1 cells that do not express detectable levels of P-glycoprotein . Ro 44-5911 overcame doxorubicin and vincristine resistance in three colorectal cancer cell lines (DLD-1, WiDr and COLO 201) that express P-glycoprotein . No effect was seen with the 3 colorectal cell lines on the IC50 values of three drugs not related to the MDR phenotype, 5-fluorouracil, 5'-deoxy-5-fluorouridine and cis-diaminodichloroplatinum (II) . The in vitro vasodilatory activity of these dithianes, measured with strips of rat aorta contracted with KCl, was about 5% of that of verapamil . These results suggest that diathianes could be useful agents for MDR modification in vivo.

Biochem Pharmacol, 1995 Jul 17, 50(2), 169 - 75
Azelastine and flezelastine as reversing agents of multidrug resistance: pharmacological and molecular studies; Hu YP et al.; The effects of two new phthalazinone derivatives, azelastine (AZ) and flezelastine (FZ), on the reversal of resistance to doxorubicin (dox) were studied using two variants of the rat C6 glioblastoma cell line, selected with dox (C6 0.5) or with vincristine (C6 1V) . Both lines presented a multidrug-resistant phenotype which was, in the case of C6 0.5 cells, likely to be accompanied by an additional mechanism leading to intracellular tolerance of the drug . Both AZ and FZ reversed dox resistance in a concentration-dependent manner, and FZ was shown to be at least three times more potent than AZ . FZ was able, at a relatively high concentration (30 microM), to completely restore dox sensitivity in both cell lines . Both drugs were able to virtually restore dox accumulation to the level reached in sensitive cells, and, interestingly, this complete restoration occurred at lower concentrations of modulator than required for complete reversal of resistance . FZ was able to reverse dox intracellular tolerance of C6 0.5 cells and to restore dox accumulation at the IC50 to the level observed in sensitive cells . AZ and FZ both inhibited azidopine binding to membrane preparations of C6 0.5 and C6 1V cells, although FZ was more potent . Both drugs more successfully inhibited azidopine binding to membranes prepared from C6 1V cells (which express the mdr1b gene product) than to membranes from C6 0.5 cells (which express the mdr1a gene product) . In view of its potent activity on MDR, further preclinical evaluation of FZ is warranted.

FEBS Lett, 1995 Jul 17, 368(2), 385 - 8
ATP-dependent efflux of calcein by the multidrug resistance protein (MRP): no inhibition by intracellular glutathione depletion; Feller N et al.; In this study we report that the multidrug resistance protein (MRP) transports calcein from the cytoplasmic compartment of tumor cells, in contrast to P-glycoprotein which transports calcein acetoxymethyl ester from the plasmamembrane . The transport of calcein by MRP is ATP-dependent and is inhibited by probenecid and vincristine . Intracellular glutathione (GSH) depletion which occurred when cells were exposed to buthionine sulfoximine had no effect on the efflux of calcein, whereas it reversed the daunorubicin accumulation deficit in MRP overexpressing tumor cells . In conclusion, ATP-dependent transport of calcein and possibly other organic anions by MRP is not inhibited by a large decrease of the intracellular GSH concentration, that inhibits daunorubicin efflux by MRP.

Biochem Biophys Res Commun, 1995 Jul 17, 212(2), 494 - 500
Immune response against the murine mdri protein induced by vaccination with synthetic lipopeptides in liposomes; Tosi PF et al.; Intrinsically, or after exposure to chemotherapeutic drugs, many cancer cells overexpress a class of high molecular weight membrane glycoproteins associated with the multidrug resistance (mdr) of these cells . This report describes an immunization protocol eliciting autoantibodies specific to extracellular epitopes of the murine mdr 1 P-glycoprotein (Pgp) . Synthetic peptides with the sequences of extracellular loops of murine Pgp were covalently coupled with four palmitic acid moieties per peptide molecule . These "lipopeptides" were reconstituted in the bilayer of liposomes containing lipid A and used to immunize mice . Antibodies against the lipopeptides corresponding to loop 2 and 4 were elicited in sera of immunized mice . They reacted specifically with extracellular epitopes of the naturally occurring murine Pgp . After interaction with resistant cancer cells, the antibodies induced an average 50% increase in cellular accumulation of doxorubicin and Bodipy-verapamil . In the presence of these antibodies the resistance of L1210 mdr cells was reduced from an LD50 of 4 x 10(-5) M to 5 x 10(-7) M doxorubicin.

Blood, 1995 Jul 15, 86(2), 491 - 501
Expression of retroviral vectors containing the human multidrug resistance 1 cDNA in hematopoietic cells of transplanted mice; Sorrentino BP et al.; Transfer of the human multidrug resistance 1 (MDR1) gene to hematopoietic stem cells offers an approach to overcome the myelosuppression caused by a number of antineoplastic drugs . This study was designed to determine the effect of MDR1 gene transfer on overall P-glycoprotein (P-gp) expression in murine hematopoietic cells . Mice were transplanted with bone marrow cells infected with either of two different MDR1 retroviral vectors . A reverse-transcriptase polymerase chain reaction-based assay was used to quantify expression levels of both endogenous and vector-derived P-gp encoding transcripts in hematopoietic cells of transplanted mice . Expression of both a truncated and full-length MDR1 mRNA species was noted in bone marrow and spleen colony cells . The truncated message resulted from cryptic mRNA splice sites within the MDR1 cDNA and was detected with both vectors . Full-length message levels exceeded those from the endogenous genes in all but one case and roughly approximated that seen in the modestly drug-resistant cell line SW620 . We conclude that transfer of MDR1 retroviral vectors resulted in a significant increase in P-gp expression in most cases; however, aberrant splicing of MDR1 transcripts can result in reduced expression of vector-derived P-gp.

J Med Chem, 1995 Jul 7, 38(14), 2789 - 93
Synthesis, pharmacologic activity, and structure-activity relationships of a series of propafenone-related modulators of multidrug resistance; Chiba P et al.; A series of {(o-acylaryl)oxy}propanolamines have been prepared and evaluated for multidrug resistance-reverting activity in a human tumor cell model . Structure-activity relationship studies indicate that the phenylpropiophenone moiety as well as the substitution pattern at the nitrogen atom is crucial for activity of the compounds . Incorporation of the ether oxygen into a benzofuran substructure, which renders the compound an arylethanolamine, decreased biologic activity . Highest activity could be observed with the arylpiparazines 4f-h, which not only completely restored daunomycin sensitivity but also showed moderate activity in restoring etoposide toxicity.

J Biol Chem, 1995 Jul 7, 270(27), 16167 - 75
Reconstitution of drug transport by purified P-glycoprotein; Shapiro AB et al.; P-glycoprotein confers multidrug resistance upon cells in which it is highly expressed, reducing the effectiveness of numerous cytotoxic drugs, including many of those used for chemotherapy of cancer . Although P-glycoprotein is widely believed to function as an ATP-dependent drug efflux pump, the unusually broad substrate specificity of P-glycoprotein has engendered the proposal of other, less direct mechanisms . None of the hypothetical mechanisms has been definitively tested, however, in a purified system where other cellular components and processes are absent . We have used a fluorescent substrate of P-glycoprotein, Hoechst 33342, to measure transport activity in real-time of highly purified P-glycoprotein in a reconstituted liposome system in which the P-glycoprotein has a uniformly inside-out orientation . Using this system, we demonstrated MgATP-dependent, chemosensitizer-inhibitable transport of Hoechst 33342 . Transport was prevented by omission of Mg2+, by substitution of nonhydrolyzable adenylyl-beta,gamma-imidodiphosphate for ATP, by inhibition of the ATPase activity of P-glycoprotein with vanadate and N-ethylmaleimide, and by the chemosensitizers verapamil and amiodarone . Measurements of intraliposomal pH during Hoechst 33342 transport detected no large pH changes in P-glycoprotein-containing liposomes . These results are inconsistent with a mechanism in which P-glycoprotein affects drug accumulation by directly altering intracellular pH . The Hoechst 33342 transport assay results are consistent with mechanisms in which P-glycoprotein alone is sufficient to transport drugs out of the membrane bilayer.

Anal Biochem, 1995 Jul 1, 228(2), 226 - 31
Facile preparation of inside-out plasma membrane vesicles from tumor cells for functional studies of pharmacologically relevant translocating ATPases; Schlemmer SR et al.; A reasonably facile and effective procedure is described for the preparation of inside-out plasma membrane vesicles from tumor cells . The method incorporates nitrogen cavitation, optimized with respect to the applied N2 pressure, in the absence of added divalent cations followed by differential centrifugation and discontinuous, sucrose gradient centrifugation . With the three tumor cell types utilized, multidrug-resistant (MEL/VCR-6) and parental (MEL/O) murine erythroleukemia cells and methotrexate-resistant (L1210/R24) L1210 leukemia cells, yields were in the range of 8-12 mg of plasma membrane vesicles/10(10) cells at a purity of 87-94% with average inside-out sidedness among preparations varying from 65 to 93% depending upon the cell type . Inside-out plasma membrane vesicles so derived were capable of sustaining ATP-dependent transport inward of two common antitumor cytotoxic agents, vinblastine and methotrexate . The former was demonstrated with inside-out vesicles from only P-glycoprotein-overexpressing, multidrug-resistant MEL/VCR-6 cells, while the latter was readily demonstrated in inside-out vesicles from all three cell types.

Cell Signal, 1995 Jul, 7(5), 431 - 43
Regulation of multidrug resistance through the cAMP and EGF signalling pathways; Rohlff C et al.; The development of cross-resistance to many natural product anticancer drugs, termed multidrug resistance (MDR), is a serious limitation to cancer chemotherapy . MDR is often associated with overexpression of the MDR1 gene product, P-glycoprotein, a multifunctional drug transporter . Understanding the mechanisms that regulate the transcriptional activation of MDR1 may afford a means of reducing or eliminating MDR . We have found that MDR1 expression can be modulated by type I cAMP-dependent protein kinase (PKA) . This suggests that MDR may be modulated by selectively downregulating PKA activity to effect inhibition of PKA-dependent trans-activating factors which may be involved in MDR1 transcription . High levels of type I PKA occur in primary breast carcinomas and patients exhibiting this phenotype show decreased survival . The selective type I PKA inhibitors, 8-Cl-cAMP and Rp8-Cl-cAMP{S}, may be particularly useful for downregulating PKA, and inhibit transient expression of a reporter gene under the control of MDR1 promoter elements . Thus, investigations of the signalling pathways involved in transcriptional regulation of MDR1 may lead to a greater understanding of the mechanisms governing the expression of MDR and provide a focus for pharmacological intervention.

Zhonghua Nei Ke Za Zhi, 1995 Jul, 34(7), 463 - 7
{Analysis of the multidrug resistance MDR1 gene expression in clinical leukemia with RT-PCR}; Zhang H et al.; The method of reverse transcription coupled with polymerase chain reaction (RT-PCR) has been used to detect multidrug resistance gene MDR1 expression in 73 leukemia patients . Specificity of MDR1 PCR product was identified by a probe labelled with biotin . In this paper, beta 2M mRNA (a 120bp of PCR product) was selected as an internal control for semi-quantitative analysis of MDR1 mRNA (a 157bp of PCR product) . Detection of MDR1 expression was positive in 31 of the 73 patients with leukemia . The percentage of MDR1+ and the value of MDR1/beta 2M in the relapsed acute leukemia and CML in blast crisis were 76.00% and 0.798 +/- 0.266, they were significantly higher than those in newly diagnosed acute leukemia (25.00% and 0.386 +/- 0.128) and complete remission leukemia (25.00% and 0.151 +/- 0.059), MDR1/beta 2M in newly diagnosed was significantly higher than that in complete remission . We found that MDR1 gene expression correlated well with response to chemotherapy in 53 cases of acute leukemia . The refractory rate in patients with MDR1+ was 75.00%, while it was 15.00% in MDR1- (P < 0.01) . We thought that the determination of MDR1 expression level in acute leukemia could provide a valuble information for designing chemotherapeutic regimen in individual patient, and a high level of MDR1 expression correlated closely with drug resistance in clinical leukemia chemotherapy.

J Cell Physiol, 1995 Jul, 164(1), 132 - 41
Regulation of multidrug resistance gene mdr1b/mdr1 expression in isolated mouse uterine epithelial cells; Kuo MT et al.; The mammalian uterine epithelium (UE) undergoes drastic physiological and morphological changes during pregnancy . Steady-state levels of murine mdr1b mRNA, transcribed from a multidrug resistance gene encoding a membrane protein which functions as a transporter of lipophilic cytotoxic agents, are low in nonpregnant, cycling UE, but drastically increase (about 1,500- to 2,000-fold) at day 8 of gestation . At day 16 of gestation, levels of mdr1b mRNA are 2,500- to 3,000-fold higher than those in the cycling UE cells . Levels of mdr1b mRNA were elevated to levels comparable to those observed during pregnancy, in the UE of ovariectomized mice following 5-8 days of estrogen and progesterone administration . Withdrawal of these hormones resulted in a drastic reduction of mdr1b mRNA within 36 hr . These results suggested that steroid hormones alone can account for increased mdr1b mRNA expression and do not require the presence of other placenta/embryo-derived factors . Moreover, the hormonal effect on uterine mdr1b mRNA biosynthesis during pregnancy apparently is a delayed phenomenon . Nuclear run-on assays demonstrated that the rate of mdr1b transcription in UE cells prepared from 15-day pregnant mice (d-15 UE cells) was about two- to three-fold higher than that in nonpregnant UE cells . This increased transcription rate alone cannot account for mdr1b mRNA accumulation during pregnancy . mdr1b mRNA expression was investigated in primary cultures of d-15 UE cells . mdr1b mRNA levels decayed by 50% within 3-4 hr of culture and reached a steady-state 0.5-2% of initial levels by 24 hr . The rate of mdr1b mRNA decay in primary d-15 UE cells was decreased by treatment with alpha-amanitin or cycloheximide, suggesting that the decay pathway requires both transcription and de novo protein synthesis . Our results suggest that multiple mechanisms are involved in the maintenance of the high levels of mdr1b mRNA in pregnant UE cells . Furthermore, these data suggest that increased mRNA stability may contribute to the accumulation of mdr1b transcript during pregnancy.

Zhongguo Yao Li Xue Bao, 1995 Jul, 16(4), 333 - 7
Multidrug resistance in leukemic cell line K562/A02 induced by doxorubicin; Yang CZ et al.; AIM: To study the mechanism of the development of multidrug resistance in leukemic cells . METHODS: A human leukemic cell line K562/A02 was established by stepwise increase of concentrations of doxorubicin (Dox) in medium . P-glycoprotein was detected by immunohistochemistry assay . The mdr1 gene expression was measured by RT-PCR . The amplification of mdr1 gene in its genome, and DNA topisomerase II (Top II) gene expression were determined by dot-blot hybridization . RESULTS: K562/A02 was highly cross-resistant to vincristine (VCR), homoharringtonin (HHT), amsacrine (m-AMSA), daunorubicin (Dau) and etoposide (VP-16), slightly to cytosine arabinoside (Ara-C), but not cisplatin (Cis), methotrexate (MTX) and fluorouracil (5-FU), showing a typical phenotype of MDR . Intracellular accumulation of Dau in K562/A02 was 33% as high as that in K562 . P-glycoprotein P-170 was positive . In K562/A02, the mdr1 gene did not amplify, the mdr1 mRNA level was markedly higher, the Top II mRNA level was lower, and glutathione-S-transferase (GST) activity was higher than in K562 . CONCLUSION: mdr1 mRNA was overexpression and thus the encoded P-170 was responsible for MDR in K562/A02 while Top II or GST may play a role in MDR.

Anticancer Res, 1995 Jul-Aug, 15(4), 1279 - 84
Structurally modified anthracyclines retain activity in a cell line with simultaneous typical and atypical multidrug resistance; Bielack SS et al.; Resistance to the classical anthracyclines may be due to one or several mechanisms, most notably p-glycoprotein (pGP) associated multidrug resistance (mdr1, "typical mdr") and altered activity of topoisomerase II (topo II) ("atypical mdr") . Modulators of mdr1 will be of limited value in case of combined forms of resistance . A Friend murine erythroleukemia cell line (F4-6R) carrying both mdrl and topo II mediated anthracycline resistance was used to determine the efficacy of structurally altered anthracyclines against such extended multidrug resistance . Proliferation assays showed that 3'N-morpholinyl substituted anthracyclines were able to retain much of their activity even in this setting . MX2 (KRN8602; 3'-deamino-3'-{4-morpholinyl}-13-deoxo-10-hydroxycarminomycin+ ++), which is 9-alkylated in addition to carrying a 3'N-morpholinyl group, was the most promising agent tested.

Mol Pharmacol, 1995 Jul, 48(1), 21 - 9
B9209-005, an azido derivative of the chemosensitizer dexniguldipine-HCl, photolabels P-glycoprotein; Borchers C et al.; P-glycoprotein is an energy-dependent drug extrusion pump for a variety of anticancer drugs and is involved in the development of multidrug resistance in cancer . Dexniguldipine-HCl is a potent chemosensitizer for P-glycoprotein-mediated multidrug resistance in vitro, and clinical phase I/II trials are underway . To investigate the mechanisms of chemosensitization and to identify the binding sites for dexniguldipine-HCl on target proteins involved in chemosensitization, {3H}B9209-005, an azido derivative of dexniguldipine-HCl, was synthesized and used as a photoaffinity ligand . In two models of multidrug resistance reversal, i.e., sensitization to vincristine and modulation of rhodamine-123 uptake, B9209-005 and dexniguldipine-HCl showed identical biological activities . Photoaffinity labeling experiments with {3H}B9209-005 in cell membranes from multidrug-resistant CCRF ADR-5000 cells, in comparison with labeling experiments with {3H}azidopine (an established photoaffinity ligand for P-glycoprotein), showed that {3H}B9209-005 labeled two proteins, with apparent molecular masses of 170 and 95 kDa . The pharmacological specificity of labeling was demonstrated by inhibition of photoincorporation by several cytostatic drugs transported by P-glycoprotein, as well as by chemosensitizers . Immunoprecipitation of the labeled proteins with the P-glycoprotein-specific monoclonal antibody C 219 and with a site-directed polyclonal antibody to the amino-terminal sequence of P-glycoprotein (amino acids 389-406) identified these proteins as intact P-glycoprotein and the amino-terminal fragment thereof . No specific labeling was obtained in the drug-sensitive parent cell line CCRF-CEM, which is devoid of significant P-glycoprotein expression . Maximal labeling of 17 pmol of the 170-kDa protein/mg of crude membrane protein was obtained . The affinity of {3H}B9209-005 for binding to and photoincorporation into P-glycoprotein was 5-fold greater than that of {3H}azidopine, and photoincorporation of {3H}B9209-005 showed a different photoincorporation pattern, compared with {3H}azidopine, in that the latter compound was incorporated specifically into the carboxyl-terminal 55-kDa fragment of P-glycoprotein . In contrast to {3H}azidopine, no specific labeling of this fragment was obtained with {3H}B9209-005, indicating different binding sites for or different photoincorporation of the two dihydropyridine ligands . Because B9209-005 carries the photoreactive azido group in the dihydropyridine moiety, whereas the azido group of azidopine is located in the side chain, these results suggest that the dihydropyridine moiety of the two compounds probably interacts with the amino-terminal part of P-glycoprotein, whereas the side chains react preferentially with the carboxyl-terminal 55-kDa fragment.(ABSTRACT TRUNCATED AT 400 WORDS)

J Invest Dermatol, 1995 Jul, 105(1), 109 - 12
Glutathione and related enzymes in tumor progression and metastases of human melanoma; Schadendorf D et al.; We have shown previously that overexpression of p-170 glycoprotein-mediated multidrug resistance plays only a minor role in conferring chemoresistance to human melanoma cells . In addition to membrane transporters like p-170, metabolizing enzyme systems have been implicated in altered drug sensitivity . Recently, glutathione and associated enzymes have been associated with resistance to alkylating substances, particularly in gastrointestinal and gynecologic cancers . In this study, we investigated whether increased levels of glutathione and related enzymes may play a role in chemoresistance in melanoma . Levels of glutathione, glutathione S-transferase (GST), glutathione reductase, and gamma-glutamyl transpeptidase were analyzed in melanoma and non-melanoma cell lines . In addition, 18 melanoma metastases derived from skin and lymph nodes were examined . Levels of gamma-glutamyl transpeptidase were statistically different in cells derived from melanocytic tumors compared with non-melanoma cell lines and normal cells . In addition, GST levels in metastases derived from skin or lymph nodes were significantly lower than those in permanent cell lines . However, levels of glutathione and related enzymes in metastases and cell lines fluctuated over a wide range, up to 40-fold, regardless of treatment status or origin of metastases . In a second part of the study, the expression of GST isoenzymes alpha, mu, and pi was studied by immunohistology in 10 benign nevi, 29 primary melanomas, and 39 melanoma metastases before and during chemotherapy . Expression of GST isoenzymes was increased with tumor progression, and GST pi was the strongest isoform expressed . However, no correlation was found between GST levels by immunohistochemistry and the course of tumor progression, between GST levels in metastases obtained before or during chemotherapy, or between GST levels and clinical response . These data suggest that alterations in glutathione metabolism and the expression of GST do not play a major role in resistance to chemotherapeutic drugs in melanoma.

Radiol Clin North Am, 1995 Jul, 33(4), 707 - 17
Tuberculosis and AIDS; Goodman PC; In the mid-1980s, the incidence of tuberculosis (TB) in the United States increased . Recently, TB became an index diagnosis for AIDS . Chest radiographs in patients with AIDS and TB may be atypical; various features are addressed in this article . Radiologists should be aware of the possibility of TB because this disease is not only communicable to health care workers and other patients but more recently has become difficult to treat because of multidrug-resistant Mycobacterium tuberculosis.

Am J Hematol, 1995 Jul, 49(3), 183 - 93
Comparative study of multidrug resistance evaluated by means of the quantitative immunohistochemical detection of P-glycoprotein and the functional release of rhodamine 123; Delville JP et al.; The immunological detection of P-Glycoprotein (P-GP) and the functional release of Rhodamine 123 (R123) have been compared in a number of human and murine cancer cell lines, in chemo- and/or radiotherapy-resistant subclones, and in clinical specimens from patients . The chemoresistance level was established from the viability index (IC50) in the presence of doxorubicin . Cytocentrifuge preparations were immunostained with JSB-1 monoclonal antibody followed by the alkaline phosphatase anti-alkaline phosphatase technique . The strength of the reaction was quantified by a digital image analyser . The kinetic incorporation and release of Rhodamine 123 were evaluated by flow cytometry . The parent cell lines and radiotherapy resistant subclones showed a low IC50, were JSB-1 negative and retained R123 during the whole experiment, while the chemoresistant and radio-chemoresistant cell line mutants had a high IC50, were JSB-1 positive, and actively pumped the R123 out of the cells . Good correlations were obtained between the IC50, the digital image analysis, and flow cytometry . The kinetic profile of the R123 release allowed the distinction between typical and atypical multidrug resistance phenotypes . These findings were confirmed in clinical specimens from patients . We conclude that antigenic and functional studies are complementary and are useful in experimental and clinical approaches to multidrug resistance.

Am J Respir Crit Care Med, 1995 Jul, 152(1), 355 - 9
Transmission of multidrug-resistant tuberculosis in a large urban setting; Friedman CR et al.; Multidrug resistance has become an increasingly important problem in the control and prevention of tuberculosis in large urban centers . Although several small outbreaks of multidrug-resistant (MDR) tuberculosis in New York City have been reported, the increase in the number of cases is not fully explained by these recognized outbreaks, and the modes of transmission have not been clearly delineated . Transmission patterns of MDR tuberculosis in New York City, therefore, were studied by stratifying Mycobacterium tuberculosis isolates from 167 newly diagnosed tuberculosis patients according to their DNA restriction fragment length polymorphisms (RFLP) . Forty-three (34%) of 127 drug-susceptible isolates and 19 (79%) of 24 multidrug-resistant isolates had RFLP patterns representing possible recent exogenous infection (primary tuberculosis) . Patients who had such isolates were more likely to be seropositive for human immunodeficiency virus (58%; p < 0.05), non-Hispanic black (56%; p < 0.005), U.S.-born (57%; p < 0.001), and have MDR tuberculosis (79%; p < 0.0005) . In a logistic regression model, primary tuberculosis remained significantly associated with MDR tuberculosis and black race . In contrast to previous reports, in New York City recent exogenous transmission accounts for most new cases of multidrug-resistant tuberculosis.

Br J Cancer, 1995 Jul, 72(1), 82 - 9
Regulation by glutathione of drug transport in multidrug-resistant human lung tumour cell lines overexpressing multidrug resistance-associated protein; Versantvoort CH et al.; Previous studies have shown that multidrug resistance (MDR) in the doxorubicin-selected lung tumour cell lines COR-L23/R, GLC4/ADR and MOR/R is associated with overexpression of the MRP gene . In this study we report that resistance to daunorubicin, vincristine and rhodamine 123 can be partially reversed in these cell lines by exposing the cells to buthionine sulphoximine (BSO), an inhibitor of glutathione (GSH) synthesis . This effect of BSO on drug resistance was associated with an increased intracellular accumulation of daunorubicin and rhodamine 123, owing to inhibition of the enhanced drug efflux . In contrast, the accumulation of daunorubicin was not increased by BSO treatment in a P-glycoprotein (P-gp)-mediated MDR cell line . BSO treatment (25 microM, 20 h) of the cell lines resulted in 60-80% depletion of cellular GSH levels . The effects of BSO on daunorubicin accumulation in the COR-L23/R and GLC4/ADR cells were associated with cellular GSH depletion . In addition, increase of cellular GSH levels in BSO-treated COR-L23/R and GLC4/ADR cells as a result of incubation with 5 mM GSH ethyl ester restored the accumulation deficit of daunorubicin . However, the transport of daunorubicin did not increase the GSH release in any of the cell lines . These results demonstrate that drug transport in MRP- but not in P-gp-overexpressing MDR tumour cell lines can be regulated by intracellular GSH levels.

Radiat Res, 1995 Jul, 143(1), 17 - 25
The effect of hyperthermia and verapamil on melphalan cytotoxicity and transport in multidrug-resistant Chinese hamster ovary cells; Averill-Bates DA et al.; The effect of both hyperthermia and verapamil on cytotoxicity and transport of melphalan was studied in a pleiotropic drug-resistant Chinese hamster ovary cell line (CHRC5) and in the drug-sensitive parent line (AuxB1) . The CHRC5 cell line was selected for resistance to colchicine but is also cross-resistant to other drugs including melphalan . Verapamil (10 microM) increased melphalan cytotoxicity in drug-resistant cells but not in drug-sensitive cells . Hyperthermia (40 to 45 degrees C) increased melphalan cytotoxicity in both cell lines . In drug-resistant but not drug-sensitive cells, melphalan cytotoxicity was increased further when verapamil was combined with hyperthermia (40 to 45 degrees C) . The increased cytotoxicity caused by verapamil in drug-resistant cells was accompanied by alterations in membrane permeability to melphalan . The cellular uptake of melphalan after 15 min increased in the presence of verapamil (7 to 30 microM) at 37 and 42 degrees C . When verapamil (10 microM) was present, the rate of efflux of melphalan from CHRC5 cells decreased by almost 40% at 37 degrees C . The rate of efflux was increased at 42 degrees C relative to 37 degrees C, but with verapamil the rate decreased to that obtained at 37 degrees C in CHRC5 cells . In drug-sensitive cells, verapamil (< or = 50 microM) did not affect either uptake or efflux of melphalan . These findings suggest that verapamil could be beneficial by increasing the effectiveness of melphalan in the elimination of multidrug-resistant cells . The combination of hyperthermia and verapamil could be especially advantageous by increasing melphalan cytotoxicity in a localized target region.

Hokkaido Igaku Zasshi, 1995 Jul, 70(4), 573 - 89
{Studies on the microtubules assembly of multidrug-resistant human leukemic cells}; Hara N; The development of drug resistance in cancer cells is a significant clinical problem for the successful cancer chemotherapy . Since the cytoskeleton, including microtubules, may be involved in modulating cellular signal transduction, morphological and structural changes, the microtubules assembly of multidrug resistant cells was examined using Confocal Laser Microscope MRC500 system (Bio Rad) . In this study, multidrug resistant cells were established by the continuous exposure to ADR(adriamycin) starting with 20 nM up to 1 microM . The expression of MDR-1 (multidrug resistance) gene was detected in K562 leukemia cells and to more extent in the multidrug resistant K562/ADR cells, but not in HL-60 leukemia cells and multidrug resistant HL-60/ADR cells by RT-PCR method . The chronological features of microtubules assembly in the parent cell lines were lost on day 3, after incubation with 20nM of ADR . In accordance with development of drug resistance, the microtubules assembly appeared to be more dense and stronger than that of parent cells . During the development of drug resistant cells, the ADR-accumulation in the nucleus was decreased according to the increase of microtubules assembly . In the case of incubation with 0.5 microM colcemid, an inhibitor of microtubules polymerization, for 3 hours, the stainings of microtubules were lost their fine network and appeared to be diffuse and dot-like pattern . At the same time, both untreated HL-60/ADR and K562/ADR showed the decrease of ADR-accumulation, but the accumulations both colcemid treated resistant cells were increased the same level of their parent cells at the point of 120 min . These results suggested that the resistance to ADR in human leukemia cells correlated with microtubules assembly, and the microtubules assembly played an important role of drug resistance with or without MDR-1 gene overexpression.

Eur Respir J, 1995 Jul, 8(7), 1184 - 92
Drug-resistant tuberculosis in the 1990s; Yew WW et al.; There has been an upsurge of tuberculosis in many parts of the world in the past decade . The high rates of drug-resistant tuberculosis currently reported in many countries are alarming . The most catastrophic phenomenon is the emergence of multidrug-resistant strains of Mycobacterium tuberculosis . These organisms have caused epidemic outbreaks in nosocomial and health-care settings in the USA and some European countries . In addition to immigration, poverty, alcoholism and intravenous substance abuse, human immunodeficiency virus (HIV) infection has also had a significant impact on the prevalence of drug resistance, since amongst these patient groups a common factor giving rise to drug resistance is noncompliance . Rapid drug susceptibility tests are needed, and effective chemotherapy regimens with newly developed drugs in combination with traditional second-line antituberculosis agents for established multidrug-resistant tuberculosis are urgently being sought . There is also a quest for other novel modalities of therapy . Measures should be actively adopted to prevent the development of drug resistance . Well formulated short-course chemotherapy as initial treatment and ensurance of compliance are the most important components . The organization of a national tuberculosis control programme with a sound and adequately functioning infrastructure remains the most effective strategy to combat the resurgence of tuberculosis and to curtail drug resistance.

Cytometry, 1995 Jul 1, 20(3), 228 - 37
Binding diversity of antibodies against external and internal epitopes of the multidrug resistance gene product P-glycoprotein; Lehne G et al.; P-glycoprotein (Pgp) is a trans-membraneous protein that is associated with multidrug resistance (MDR) in human cancer, including hepatocellular carcinomas and leukemias . There is no consensus regarding methods of choice for analysis of Pgp expression, and development of reliable analytical methods is now essential . We have studied the the Pgp expression in human hepatoma and leukemia cell lines using flow cytometry . The aim of the study was to compare binding properties of anti-Pgp antibodies reacting with surface (MRK16, UIC2) and cytoplasmic (C219, JSB-1) epitopes to assess which antibody performed best with respect to fluorescence discrimination . By histogram subtraction the fractions of resistant human hepatoma cells positive for Pgp were 99% (MRK16), 97% (UIC2), 77% (JSB-1), and 51% (C219), demonstrating variations in antibody reactivity . The resolution in detecting decreasing levels of Pgp in hepatoma cells was superior for the externally binding antibodies, showing that there is a correlation between antibody reactivity and fluorescence discrimination . Similar results were obtained for parental and resistant KG1a human leukemia cell lines . The Pgp epitopes remained reactive to the anti-Pgp MAbs after methanol fixation and cryopreservation . By dual parameter flow cytometry it was shown that Pgp expression in viable cells may be assessed together with uptake of epirubicin, which was low in cells expressing high levels of Pgp and vice versa . In conclusion, all tested antibodies proved useful for flow cytometric detection of high levels of Pgp, but the externally binding ones were superior in detection of low and variable levels of Pgp.

Cytometry, 1995 Jul 1, 20(3), 218 - 27
Dyes providing increased sensitivity in flow-cytometric dye-efflux assays for multidrug resistance; Frey T et al.; In an effort to improve detection of P-glycoprotein-mediated multidrug resistance (mdr), several dyes were compared to rhodamine 123 (R123) in efflux assays . Two dyes (SY-38 and SY-3150) were found that provided better sensitivity . These dyes were effluxed by a cell line known to be mdr-positive (P388/R84) but not by an mdr-negative cell line (P388) . Efflux was blocked by both verapamil and cyclosporine A . Efflux from KG1a cells was less than from P388/R84, just as has been seen with R123 and daunomycin . In further experiments, a model system was used to demonstrate two-color immunofluorescence plus efflux measurements . This was done using a sequential staining method . A procedure was devised that, at least for this model system, allowed single-step staining with both dye and antibody . The sensitivity of the efflux measurement was slightly compromised by using this one-step staining method.

Drugs Aging, 1995 Jul, 7(1), 19 - 29
P-glycoprotein expression and regulation . Age-related changes and potential effects on drug therapy; Gupta S; P-Glycoprotein is a member of a superfamily of adenosine triphosphate-binding cassette transporter proteins and plays an important role in multidrug resistance in cancer cells . P-Glycoprotein is known to transport a wide variety of substances ranging from ions to peptides . P-Glycoprotein is expressed on a variety of normal cells, however its physiological function is unclear . The apical and polar distribution on secretory cells suggests a secretory role for P-glycoprotein . More recently, cells of the immune system have been shown to express P-glycoprotein . There is evidence to suggest that P-glycoprotein may play a role in the secretion of certain cytokines (especially those lacking signal sequence) and cytotoxic molecules . In this article, the basic structure, gene regulation and expression of P-glycoprotein are reviewed . Furthermore, age-related changes in the expression of P-glycoprotein and potential effects on drug therapy in the elderly are discussed.

Hum Gene Ther, 1995 Jul, 6(7), 905 - 15
Retroviral coexpression of a multidrug resistance gene (MDR1) and human alpha-galactosidase A for gene therapy of Fabry disease; Sugimoto Y et al.; Human alpha-galactosidase A (alpha-Gal A; EC.3.2.1.22) is a lysosomal exoglycosidase encoded by a gene on Xq22 . Deficiencies of this enzyme result in Fabry disease, an X-chromosome-linked recessive disorder that leads to premature death in affected males . For treatment of genetic diseases, we have developed a retroviral vector system, pSXLC/pHa, that enables coexpression of drug-selectable markers with a second nonselectable gene as part of a bicistronic message using the promoter from the Harvey murine sarcoma virus and an internal ribosomal entry site (IRES) from encephalomyocarditis virus . Retroviral vectors based on this system that carry the human alpha-Gal A cDNA either upstream (pHa-alpha Gal-IRES-MDR) or downstream (pHa-MDR-IRES-alpha Gal) from the IRES relative to the drug-selectable MDR1 (P-glycoprotein) cDNA were constructed . Each of eight independent vincristine-resistant, pHa-alpha Gal-IRES-MDR-transfected clones and all four vincristine-resistant, pHa-alpha Gal-IRES-MDR retrovirus-transduced clones showed significantly higher activity of alpha-Gal A than the parental cells . More than 50% of the vincristine-resistant, pHa-MDR-IRES-alpha Gal-transfected clones and all four vincristine-resistant, pHa-MDR-IRES-alpha Gal retrovirus-transduced clones showed significantly higher activity of alpha-Gal A than the parental cells . In these bicistronic vectors, the cDNA whose translation was cap-dependent (upstream) was expressed at higher levels than when the same cDNA was translated in an IRES-dependent manner (downstream) . These vectors may prove useful in the gene therapy of Fabry disease.

Eur J Cancer, 1995 Jul-Aug, 31A(7-8), 1295 - 8
Multidrug resistance and the role of P-glycoprotein knockout mice; Schinkel AH et al.; Drug resistance, be it intrinsic or acquired, is a major problem in cancer chemotherapy . In vitro, one well characterised form of resistance against many different cytotoxic drugs is caused by the MDR1 P-glycoprotein, a large plasma membrane protein that protects the cell by actively pumping substrate drugs out . Available evidence suggests that this protein may cause drug resistance in at least some clinical tumours . Drugs inhibiting the MDR1 P-glycoprotein activity are, therefore, co-administered during chemotherapy of these tumours . To predict the biological and pharmacological effects of the blocking of this protein, we have generated mice with a genetic disruption of the drug-transporting mdr1a P-glycoprotein . These mice are overall healthy, but they accumulate much higher levels of substrate drugs in the brain, and have markedly slower elimination of these drugs from the circulation . For some drugs, this leads to dramatically increased toxicity, indicating that P-glycoprotein inhibitors should be used with caution in patients.

Eur J Cancer, 1995 Jul-Aug, 31A(7-8), 1291 - 4
MDR1/P-glycoprotein expression in colorectal cancer; Linn SC et al.; Drug resistance to multiple chemotherapeutic agents is considered a major cause of chemotherapy failure . An extensively studied and relatively well understood type of cellular drug resistance is P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) . Pgp acts as an energy-dependent drug efflux pump, thereby decreasing the intracellular drug concentration and causing drug resistance, in in vitro experiments . Colorectal cancer and colorectal mucosa generally express high levels of Pgp, and this may contribute to the general unresponsiveness of colorectal cancer to natural product, anticancer drugs . The controversies concerning the prognostic role of Pgp expression and its contribution to tumour aggressiveness, and possible reasons for the disappointing results of clinical MDR reversal trials in colorectal cancer are discussed.

Eur J Cancer, 1995 Jul-Aug, 31A(7-8), 1283 - 7
CPT-11 (irinotecan) in the treatment of colorectal cancer; Armand JP et al.; Colorectal cancer is one of the most common cancers in the Western World . Although 50% of patients are cured by surgery alone, the outcome is poor in high-risk patients (Dukes stages B2 and C) despite adjuvant chemotherapy with 5-fluorouracil (5-FU)-based regimens . CPT-11 (irinotecan) is a promising new agent for the treatment of colorectal cancer with a unique mechanism of action . CPT-11 is a DNA topoisomerase I inhibitor, which has not demonstrated susceptibility to the P-glycoprotein-mediated multidrug-resistant phenotype . Phase II studies with CPT-11 have demonstrated definite activity against colorectal cancer in both chemotherapy-naive and pretreated patients (response rates of 15-32% observed) even with clinical evidence of resistance to 5-FU . The response rate appears to be consistent, reproducible and equivalent to that achieved with 5-FU plus folinic acid in chemotherapy-naive patients.

Yakugaku Zasshi, 1995 Jul, 115(7), 513 - 22
{Mechanism of resistance to antitumor agents--its involvement in blood-brain barrier}; 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 the blood-brain barrier . The physiological function of the P-glycoprotein in relation to therapy is discussed.

J Clin Pathol, 1995 Jul, 48(7), 679 - 81
P-glycoprotein positive, drug resistant invasive lymphoepithelial thymoma: treatment response to chemotherapy with cyclosporin and quinine; Gala JL et al.; A case of invasive drug resistant thymoma, expressing P-glycoprotein, which showed noticeable clinical response to chemotherapy and the multidrug resistance modulating agents cyclosporin and quinine is reported . A 46 year old man presented with severe left shoulder pain and a diagnosis of invasive lymphoepithelial thymoma was made following chest x ray and a computed tomography scan . The patient underwent extensive chemotherapy without resolution of the tumour . More than 90% of the malignant epithelial cells were strongly positive for P-glycoprotein and based on this observation, cyclosporin and quinine were added to the chemotherapy regimen . The mediastinal mass completely resolved and the size of the pleural metastasis decreased substantially . The patient, however, died of an intercurrent infection . This case report highlights the feasibility and efficacy of using cyclosporin and quinine in combination with VAD chemotherapy in the treatment of invasive thymoma.

Chemotherapy, 1995 Jul-Aug, 41(4), 296 - 305
Does P-glycoprotein play a pivotal role in the drug resistance of an MDR variant, K562/Dox?
Hu X, Yang H, Pan QR, Zheng S.
A multidrug-resistant (MDR) variant, K562/Dox, was selected from repeated exposure of human erythroleukemia cell line K562 to doxorubicin (Dox) . K562/Dox displayed typical MDR features with respect to its cross-resistance to a variety of functionally and structurally unrelated compounds: vincristine (Vin), Dox, mitomycin C, reduced steady-state intracellular anthracycline accumulation, and elevated P-glycoprotein expression/mdr1 mRNA transcription/mdr1 gene amplification . Nevertheless, by incubation of cells with Dox/epirubicin (Epi)/daunorubicin (Dau) (5-80 micrograms/ml), the initial drug uptake was similar (p > 0.05) in K562/Dox and K562 cells, suggesting P-glycoprotein-mediated drug efflux would not occur unless a relatively high cellular drug concentration was reached . After 8 h incubation of cells with 50 ng/ml Dox (5 times higher than its IC50 to K562 cells), there were only slight differences (p > 0.05) in intracellular drug levels between K562/Dox and K562 cells, clearly indicating that K562/Dox, circumventing drug toxicity in this case, was irrelevant to reduced drug accumulation caused by P-glycoprotein . Similar results were obtained when Epi or Dau was applied . Despite complete restoration of anthracycline accumulation in K562/Dox cells in the presence of 6 mumol/l verapamil, the reversal of their drug resistance was incomplete . These results suggest that P-glycoprotein-mediated drug efflux possibly did not play a primary role in the drug resistance of K562/Dox cells.

Z Naturforsch {C}, 1995 Jul-Aug, 50(7-8), 565 - 70
Pharmacokinetic interaction between 4'-epidoxorubicin and the multidrug resistance reverting agent quinine; Czejka M et al.; The serum and red blood cell (RBCs) disposition of epirubicin (EPR) after intravenous bolus injection without and with coadministered quinine ( QUI) was investigated in patients undergoing a cyclic chemotherapy with EPR . QUI possesses a statistical significant influence on the EPR serum concentrations and, as a consequence, on the pharmacokinetic parameters for the initial distribution phase of EPR . Within the first 15 min after administration, EPR was distributed from the central compartment distinctly faster in compare to the control, when QUI was preadministered (t(1/2) = 6 min for the control group and t(1/2) = 3 min with QUI; -46%, p < 0.05) . Yet, in the beta-phase when drug-elimination predominates, no statistical significant influence of QUI in regard to EPR serum and RBC concentrations could be observed . Half-life of elimination was 0.5 h for the control group and 8.6 h for the QUI group (-10%) . The mean initial serum concentration (co) was reduced significantly by QUI from 7359 +/- 506 ng/ml to 4351 +/- 1682 ng/ml (-42%, p < 0.005) . Furthermore, QUI caused a reduction of the serum bioavailability of EPR (expressed as AUC(o-24)-values) from 3404 +/- 1008 ng/ml x h to 2359 +/- 1073 ng/ml x h (-31%, p < 0.05) . Vd and Vdbeta were increased at about 90% and the mean total body clearance was accelerated from 45.3 to 1487 ml/min, but due to the large standard deviations the calculated difference for these parameters was not statistically significant . In the observed time interval of 24 h, the red blood cell coefficient of distribution k(rbc) of EPR was lower if QUI was coadministered (k(rbc) = 1.25 +/- 0.12 for the control group k(rbc) = 1.15 +/- 0.13 under QUI; p < 0.04) . The results point out that QUI induces an accelerated distribution of EPR from the blood into the tissue and that QUI additionally may have influence on the red-blood cell partitioning of EPR.

Biotechniques, 1995 Jul, 19(1), 84, 86 - 8, 90
Construction of RNA standards for high-resolution automatic product analysis in quantitative competitive RT-PCR; Repp R et al.; The exponential character of PCR amplification may compromise quantitative assays because it multiplies minor sample-to-sample variations . To overcome these problems, several authors have used recombinant standard DNA or RNA molecules to be spiked into the samples in a dilution series of known copy numbers before co-amplification by PCR . To obtain an equal efficacy of reverse transcription and PCR amplification, standard and template molecules should be highly homologous . However, the limited resolution of commonly used agarose gel electrophoresis requires rather large differences in size and nucleotide sequence to separate both molecules from each other after PCR . Due to a much higher resolution, automatic post-PCR analyzing systems based on laser-induced fluorescence may help to overcome these difficulties . For using the capabilities of these systems in quantitative competitive RT-PCR, we developed a protocol to construct recombinant RNA standard molecules that only differ from the target sequence by a small deletion of 8 nucleotides . It is based on PCR-induced mutagenesis and solid-phase in vitro transcription . This protocol was applied to quantify multidrug resistance gene (MDRI) mRNA in malignant cells, but it can easily be adapted to any gene of interest.

J Infect Dis, 1995 Jul, 172(1), 70 - 8
Drug resistance and heterogeneous long-term virologic responses of human immunodeficiency virus type 1-infected subjects to zidovudine and didanosine combination therapy . The AIDS Clinical Trials Group 143 Virology Team; Shafer RW et al.; Plasma human immunodeficiency virus (HIV) type 1 RNA levels, CD4 lymphocyte changes, and drug resistance were studied in HIV-infected patients with 200-500 CD4 lymphocytes/microL who received zidovudine and didanosine combination therapy for 2 years . Among 35 patients, 10 had sustained and 16 had transient > 10-fold reductions in HIV RNA: 9 did not have 10-fold HIV RNA reductions . Only patients with sustained HIV suppression maintained increased CD4 cell counts for 2 years (370 to 501 cells/microL; P = .006) . Patients with transient HIV suppression were more likely to develop drug-resistant HIV strains (12/16 vs . 5/19, P = .01) and reverse transcriptase (RT) mutations (4.5 vs . 2.5/strain; P = .02) than were patients with sustained or no HIV suppression . Zidovudine resistance occurred with RT mutations at codons 41, 67, 70, 215, and 219 . Multidrug resistance occurred with mutations at codons 62, 75, 77, 116, and 151 . Mutations occurred at codons 60, 68, 118, 210, and 228 in > or = 4 patients each . Heterogeneity exists among individual virologic responses to zidovudine and didanosine combination therapy . HIV resistance mechanisms during combination therapy appear more complex than reported with monotherapy.

Antimicrob Agents Chemother, 1995 Jul, 39(7), 1609 - 11
Efficacy of the herbicide trifluralin against four P-glycoprotein-expressing strains of Leishmania; Chan MM et al.; Drug resistance has emerged as a major obstacle to chemotherapy for many infectious diseases . Trifluralin, an antimicrotubule herbicide, is a new experimental drug for treatment of leishmaniasis . Here, we found that it was effective against two strains of Leishmania that express the multidrug-resistant genes ldmdr1 and lmpgpA and two strains that express proteins that are immunologically cross-reactive with mammalian P glycoproteins . These results suggest that trifluralin is not subject to counteractions of these multidrug resistance mechanisms of Leishmania species.

Antimicrob Agents Chemother, 1995 Jul, 39(7), 1542 - 5
Synergistic activities of clarithromycin and antituberculous drugs against multidrug-resistant Mycobacterium tuberculosis; Cavalieri SJ et al.; The rise of multidrug-resistant Mycobacterium tuberculosis has complicated therapy for tuberculosis and led us to search for a potentially active combination of drugs against these strains . The susceptibilities of 12 strains of multidrug-resistant M . tuberculosis to standard antituberculous drugs (isoniazid, rifampin, ethambutol, and pyrazinamide), clarithromycin, and its metabolite, 14-hydroxyclarithromycin, were determined by use of the BACTEC radiometric method . All strains were resistant to at least two of the antituberculous drugs . Clarithromycin and 14-hydroxyclarithromycin MICs were in the range indicating resistance at > or = 8.0 micrograms/ml for all strains . Combination testing by the BACTEC method was performed with various concentrations of isoniazid, rifampin, and ethambutol, and with clarithromycin/14-hydroxyclarithromycin at fixed concentrations of 2.0/0.5 micrograms/ml, respectively . Addition of clarithromycin/14-hydroxyclarithromycin to these antituberculous drug mixtures resulted in a 4- to 32-fold reduction in MICs of isoniazid, rifampin, and ethambutol and made resistant strains susceptible . Fractional inhibitory concentrations ranged from 0.23 to 0.50 for all strains, suggesting a synergistic interaction between standard antituberculous drugs and clarithromycin/14-hydroxyclarithromycin . The ability of clarithromycin/14-hydroxyclarithromycin to enhance the activities of isoniazid, ethambutol, and rifampin in vitro suggests that this combination may be efficacious in the treatment of multidrug-resistant M . tuberculosis infections.

Pathol Res Pract, 1995 Jul, 191(6), 563 - 70
New aspects of cell biology in osteosarcoma; Grundmann E et al.; Among the solid tumors of childhood and adolescence, osteosarcoma (OS) represents the most prominent example of efficient aggressive chemotherapy with secondary surgical therapy . A specific subclassification of the tumor is indispensable and must include recent results of cell biology . The co-distribution of different collagen types I-VI reflects the diverse differentiation of osteosarcoma cells, supporting the concept of a pluripotent mesenchymal cell to be the stem cell of the tumor . In contrast, osteonectin (SPARC) may not be considered as a reliable marker for osteosarcoma . The experience of special proteins being secreted by osteosarcoma cells is rather limited . Detailed molecular biological studies are still lacking . A loss of alleles on chromosome 17, particularly in the defined region 17p 13, can be observed in more than 75% of all OS, suggesting the contribution of a tumor suppressor gene, p53, located in that region . It is a 53 kd nucleophosphoprotein binding the major transforming protein, the large T antigen of Simian Virus 40 . Immunohistological results showed positive staining with the antibody Pab 240 in 13 of 18 cases . In one osteoblastic OS, a novel splice mutation resulting in a fusing of exon 5 directly to exon 7 was detected . RB1 gene is also of major importance for the tumorigenesis of OS . The multidrug resistance (mdr) is associated with a membrane-bound channel-forming transport protein, the P-glycoprotein . It is a conserved plasma membrane component of about 170 kd . Both the human isoforms mdr 1 and mdr 3 are localised in the long arm of chromosome 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Pharmacol, 1995 Jun 29, 50(1), 69 - 74
Effects of KS-501, KS-502 and their enantiomers on calmodulin-sensitive enzyme activity and cellular proliferation; Hait WN et al.; Calmodulin plays an important role in cellular proliferation as part of a signal transduction pathway activated by phospholipase C . Drugs that block the ability of calmodulin to bind to and activate its target enzymes inhibit the growth of a wide variety of malignant cells . To identify more potent and selective inhibitors of this potential target for new drug development, we studied two recently synthesized compounds, KS-501 and KS-502, for their activity against calmodulin-sensitive enzymes and for their ability to block the growth of parental and multidrug-resistant leukemic cells . KS-501 and KS-502 inhibited the activation of a calmodulin-sensitive cyclic nucleotide phosphodiesterase . The mechanism of enzyme inhibition was through interfering with calmodulin activation rather than through a direct effect on the enzyme . KS-501 was more potent than KS-502 and was studied in greater detail . This compound inhibited the activation of calmodulin kinase I and II, but had less effect against cyclic adenosine 3',5'-monophosphate (cyclic AMP)-sensitive kinase . KS-501 was also more effective than KS-502 in inhibiting the growth of sensitive L1210 leukemic lymphocytes . Both compounds were less effective inhibitors of multidrug-resistant L1210 leukemia than of the parental line . These studies identify a new class of calmodulin inhibitor, with selectivity for calmodulin-dependent kinases over cyclic AMP-dependent protein kinase . Since the total synthesis of the KS-compounds has been accomplished, it should now be possible to develop derivatives with greater activity and selectivity.

Biochem Pharmacol, 1995 Jun 29, 50(1), 61 - 8
Isolation and characterization of a cell line resistant to 5-{3-(2-nitro-1-imidazoyl)-propyl}-phenanthridinium bromide (2-NLP-3), a DNA-intercalating hypoxic cell radiosensitizer and cytotoxin; Cowan DS et al.; A DNA-targeted hypoxic cell radiosensitizer and cytotoxin, 5-{3-(2-nitro-1-imidazoyl)-propyl}-phenanthridinium bromide (2-NLP-3), has been shown previously to have increased efficacy over untargeted analogues in vitro . To further study the mechanism of action of this compound, a cell line, CHO-1000, derived from Chinese hamster ovary (CHO) AA8-4 cells was isolated . This cell line is capable of continuously growing in a concentration of 2-NLP-3 approximately 10-fold greater than that tolerated by wild-type CHO cells . The resistance of CHO-1000 to 2-NLP-3 was compared with that of the P-glycoprotein overexpressing, multidrug resistant Chinese hamster cell line CHR-C5 (C5) . The resistance of CHO-1000 cells to the acute toxic effects of 2-NLP-3 under both hypoxic and aerobic exposure conditions was intermediate to that of the sensitive CHO wild-type cells and the resistant C5 cells . A similar pattern was seen for the hypoxic cell radiosensitizing ability of 2-NLP-3 . 2-NLP-3 produced significant depletion of glutathione under both hypoxic and aerobic conditions in all three cell lines studied, and the degree of depletion was correlated with drug toxicity . CHO-1000 and C5 cells were significantly more resistant to colchicine and doxorubicin compared with wild-type cells . The toxicity pattern of 2-NLP-3 and its comparison phenanthridinium ion, P3, was not the same for CHO-1000 cells compared with C5 cells . Verapamil was an effective agent for reversing the hypoxic resistance to 2-NLP-3 in both CHO-1000 and C5 cells, but only a partial reversal of aerobic resistance was observed in CHO-1000 cells . These results indicate that the resistant phenotype of CHO-1000 is mediated to some degree by P-glycoprotein expression, but that other as yet unidentified factors are also involved.

J Med Chem, 1995 Jun 23, 38(13), 2418 - 26
Synthesis and activity against multidrug resistance in Chinese hamster ovary cells of new acridone-4-carboxamides; Dodic N et al.; A number of tricyclic carboxamides have been synthesized and tested to evaluate their ability to reverse multidrug resistance in the CHRC/5 cell line . Among them the acridone derivatives were the most potent . A key feature is the presence of a dimethoxybenzyl or phenethylamine cationic site, separated from the tricyclic lipophilic part by a carbamoylphenyl chain . Optimization led to compounds 2 orders of magnitude more active than the prototype inhibitors verapamil and amiodarone . On the basis of in vitro and in vivo activities, 9,10-dihydro-5-methoxy-9-oxo-N-{4-{2-(1,2,3,4-tetrahydro-6,7- dimethoxyisoquinol-2-yl)ethyl}phenyl}-4-acridinecarboxamide (84) has been selected for further development.

Int J Cancer, 1995 Jun 9, 61(6), 880 - 6
Correlation between functional and molecular analysis of mdr1 P-glycoprotein in human solid-tumor xenografts; Broxterman HJ et al.; The contribution of P-glycoprotein (Pgp) to multidrug resistance in human solid tumors is generally estimated from bulk mRNA measurements or immunohistochemistry, while direct measurement of the effect of Pgp on intracellular drug concentrations has not been pursued . We investigated the feasibility and sensitivity of a method for probing Pgp-mediated drug transport in cells isolated from solid tumors, using xenograft models . Human tumor xenografts (XG) were grown by s.c . injection of Pgp-expressing cell lines 2780AD, BRO/mdr1 and KB8-5 . Tumor uptake of doxorubicin (DOX) after administration of DOX to the mice was determined . XG from untreated mice were enzymatically dissociated . The effect of the Pgp modulator bepridil on steady-state cellular daunorubicin (DNR) and vincristine (VCR) accumulation and chemosensitivity of these XG cells was compared with its effects in the cell lines (CL) . mdr1 mRNA and Pgp (by flow cytometry) were measured . Also, the dependence on intracellular ATP concentration, {ATP}i, of the modulator effect was determined in intact KB8-5 cells . The results showed that i.v . administration of DOX to the mice led to lower DOX levels in the Pgp-expressing XG than in the "sensitive" XG, suggesting the presence of an in vivo functional Pgp in these XG tumor models . Dissociated, viable XG cells appeared to have ATP levels sufficient to sustain Pgp-ATPase-coupled drug transport . This was inferred from experiments using KB8-5 CL, which showed half-maximal inhibition of DNR transport at an {ATP}i of 1 to 2 mM . The effect of bepridil on DNR and VCR accumulation and chemosensitivity in the XG cells was in accordance with the XG expression of mdr1/Pgp . In KB8-5 XG cells, Pgp function was hardly detectable, in accordance with decreased mdr1/Pgp expression in vivo . In conclusion, Pgp activity can be determined in freshly dissociated XG human tumor cells . The results obtained with the more necrotic KB8-5 XG may represent some of the interpretation problems arising when low levels of Pgp expression occur within a heterogeneous cell population, such as may be expected in clinical human tumors . Also our results indicate that Pgp activity may be impaired in vivo at {ATP}i below 2 mM, which are realistic values for human solid tumors.

J Biol Chem, 1995 Jun 9, 270(23), 14085 - 93
Cloning, overexpression, purification, and characterization of the carboxyl-terminal nucleotide binding domain of P-glycoprotein; Sharma S et al.; Multidrug-resistant tumor cells overexpress P-glycoprotein (170 kDa), a member of the ABC (ATP Binding Cassette)-transporter superfamily . P-glycoprotein has been implicated in transport of a broad range of amphiphilic, hydrophobic drugs from tumor cells . The sequence and structural organization of P-glycoprotein, which consists of 12 transmembrane helices and two cytoplasmic nucleotide binding domains, is similar to other ABC-transporters . It is believed that the nucleotide binding domains of various ABC transporters, which have 30-50% sequence identity, play an important role in coupling ATP hydrolysis to the transport process . To allow structure-function studies of the nucleotide binding domains, the carboxyl-terminal nucleotide binding domain (NBD) of Chinese hamster P-glycoprotein has been cloned, overexpressed, and purified both by itself and as a fusion with maltose-binding protein . It has been demonstrated that the carboxyl-terminal NBD, when overexpressed in Escherichia coli in the absence of transmembrane helices, has very low ATPase activity . This suggests that the amino-terminal nucleotide binding domain and possibly interaction with the transmembrane domains may be required for full ATPase activity . It is also consistent with the idea that the ATPase activity of P-glycoprotein is stimulated in the presence of drugs . Circular dichroism spectral analysis and the ability of carboxyl-terminal NBD, both by itself and as a fusion with maltose-binding protein, to bind ATP-agarose beads and P-glycoprotein specific monoclonal antibodies suggests that the polypeptide folds into a functional domain . Gel filtration chromatography and cross-linking studies indicate that the carboxyl-terminal NBD has a tendency to self-associate to form oligomers . It is speculated that the carboxyl-terminal NBD may play a role in self-association of P-glycoprotein molecules in the plasma membrane.

Biochim Biophys Acta, 1995 Jun 9, 1262(2-3), 113 - 23
A homologue of the mammalian multidrug resistance gene (mdr) is functionally expressed in the intestine of Xenopus laevis; Castillo G et al.; P-glycoprotein is an integral membrane protein that functions in multidrug resistance (MDR) cells as a drug efflux pump to maintain intracellular concentrations of antitumor drugs below cytotoxic levels . A homologue of the mammalian mdr gene has been isolated and characterized from Xenopus laevis (Xe-mdr) . The cDNA was isolated from a tadpole cDNA library using the full length mouse mdrlb cDNA as a probe . The Xe-mdr encodes a protein that is 66% identical to the mouse mdrlb and 68% identical to the human mdrl . The predicted structure of the Xe-mdr gene product identifies twelve membrane spanning domains and two ATP binding sites both of which are the hallmark of the ABC (ATP binding cassette) transporters . Xe-mdr mRNA is expressed as a single message of 4.5 kb and is found predominantly in the intestine . Xe-mdr message is increased 3- to 4-fold in the ileum compared to the rest of the small intestine . In situ hybridization of sequential sections from the small intestine localized the expression of the Xe-mdr to the cells lining the lumenal epithelium . Brush border membrane vesicles prepared from the small intestine of Xenopus laevis effluxed vinblastine in an ATP-dependent manner . Efflux was decreased by verapamil, a known inhibitor of P-glycoprotein function . These studies indicate that the structure of Xe-mdr has been conserved and suggest that the protein has a role in maintaining the function of the normal intestine in Xenopus.

Proc Natl Acad Sci U S A, 1995 Jun 6, 92(12), 5421 - 5
Enhanced secretion of glycocholic acid in a specially adapted cell line is associated with overexpression of apparently novel ATP-binding cassette proteins; Brown RS Jr et al.; Secretion of anionic endo- and xenobiotics is essential for the survival of animal and plant cells; however, the underlying molecular mechanisms remain uncertain . To better understand one such model system--i.e., secretion of bile acids by the liver--we utilized a strategy analogous to that employed to identify the multidrug resistance (mdr) genes . We synthesized the methyl ester of glycocholic acid (GCE), which readily enters cells, where it is hydrolyzed to yield glycocholic acid, a naturally occurring bile acid . The rat hepatoma-derived HTC cell line gradually acquired resistance to GCE concentrations 20-fold higher than those which inhibited growth of naive cells, yet intracellular accumulation of radiolabel in resistant cells exposed to {14C}GCE averaged approximately 25% of that in nonresistant cells . As compared with nonresistant cells, resistant cells also exhibited (i) cross-resistance to colchicine, a known mdr substrate, but not to other noxious substances transported by hepatocytes; (ii) increased abundance on Northern blot of mRNA species up to 7-10 kb recognized by a probe for highly conserved nucleotide-binding domain (NBD) sequences of ATP-binding cassette (ABC) proteins; (iii) increased abundance, as measured by RNase protection assay, of mRNA fragments homologous to a NBD cRNA probe; and (iv) dramatic overexpression, as measured by Western blotting and immunofluorescence, of a group of 150- to 200-kDa plasma membrane proteins recognized by a monoclonal antibody against a region flanking the highly conserved NBD of mdr/P-glycoproteins . Finally, Xenopus laevis oocytes injected with mRNA from resistant cells and incubated with {14C}GCE secreted radiolabel more rapidly than did control oocytes . Enhanced secretion of glycocholic acid in this cell line is associated with overexpression of ABC/mdr-related proteins, some of which are apparently novel and are likely to include a bile acid transport protein.

Q J Nucl Med, 1995 Jun, 39(2), 122 - 8
Differences between accumulation of 99mTc-MIBI and 201Tl-thallous chloride in tumour cells: role of P-glycoprotein; Ballinger JR et al.; Both 99mTc-MIBI and 201Tl have been used for tumour imaging . It has recently been reported that 99mTc-MIBI is a substrate for P-glycoprotein (Pgp), a membrane pump which mediates multidrug resistance . We have evaluated the role of Pgp in the cellular accumulation of 201Tl by using sensitive and resistant strains of Chinese hamster ovary (CHO) fibroblasts (AuxB1 and CHRC5, respectively) grown in suspension culture . 201Tl accumulation was the same in sensitive and resistant cells, whereas 99mTc-MIBI accumulation was much lower in resistant cells than in sensitive ones . Down-modulation of Pgp with 100 microM verapamil did not alter cellular accumulation of 201Tl while it significantly increased 99mTc-MIBI accumulation in both types of cell . Similarly, 10 microM verapamil did not affect the rate of washout of 201Tl from preloaded cells, while 99mTc-MIBI washout was greatly reduced in the presence of verapamil . These results suggest that 201Tl will accumulate in both sensitive and resistant tumour cells, whereas 99mTc-MIBI will be extruded from resistant cells and therefore may be less useful for tumour detection when the tumour cells express high Pgp levels.

Med Oncol, 1995 Jun, 12(2), 79 - 86
Multidrug resistance gene (mdr1) RNA levels in relation to P-glycoprotein content of leukemic cells from patients with acute leukemia; Albertioni F et al.; The clinical relevance of multidrug resistance gene (mdr1) expression in tumor cells remains largely unclear . Conflicting results regarding mdr1 gene expression and clinical outcome have been obtained . Little is known about regulation of mdr1 gene expression, and the conflicting results might be explained by the fact that mdr1 RNA levels do not reflect expression at the protein level . The aim of the present study was to investigate the relationship between mdr1 RNA levels and P-glycoprotein (Pgp) content of leukemic cells from patients with acute myelogenous or lymphocytic leukemia . Mdr1 RNA levels were determined by a quantitative RNA-RNA solution hybridization method, and Pgp by Western blot technique with enhanced chemiluminescence for immunodetection . Pgp was detected in 14/14 leukemic cell samples while mdr1 RNA was detectable (> 0.15 copies/cell) in cells from only six out of the 14 patients . Mdr1 RNA levels did not correlate with the Pgp content of leukemic cells (r = 0.284, p = 0.306) . Relapsed leukemias had significantly (p = 0.016) higher levels of Pgp than de novo untreated leukemias (the mean and SD optical density units were 0.56 +/- 0.18 and 0.25 +/- 0.17 respectively) while no difference was found in RNA levels . The findings support post-transcriptional level regulation of mdr1 gene expression and stress the importance of accurate determinations of the Pgp content of tumor cells in studies of the relationship between mdr1 gene expression and clinical outcome.

Trop Med Parasitol, 1995 Jun, 46(2), 83 - 7
Evaluation of resistant-reversal, CDRI compound 87/209 and its possible mode of action in rodent experimental malaria; Srivastava R et al.; The resurgence of malaria in form of resistance against chloroquine (CQ) has decreased the importance of the drug as a chemotherapeutic agent . If an agent in combination with CQ can make CQ resistant plasmodia susceptible to CQ, the problem of drug resistance may then be solved . Use of conventional drugs like verapamil, desipramine along with CQ suggested the feasibility of this approach . This report is concerned with a new class of compound, CDRI compound 87/209 (15 mg/kg b . wt.) which is given in combination with chloroquine (10 mg/kg b . wt.) for 10 consecutive days to chloroquine resistant P . berghei/P . yoelii nigeriensis (multidrug resistant) infected Mastomys coucha/Swiss albino mice respectively, displayed a potential in curing the animals . A tentative mode of action of the CDRI compound 87/209 based upon its unique property of inhibiting heme-oxygenase (a heme degrading enzyme) has been presented . It is likely that CDRI compound 87/209 in combination with chloroquine may reverse the resistance acquired by the malarial parasites and in combination with CQ is capable of clearing the parasite from the animals.

Br J Haematol, 1995 Jun, 90(2), 375 - 83
Circumvention of P-glycoprotein-mediated drug resistance in human leukaemic cells by non-immunosuppressive cyclosporin D analogue, SDZ PSC 833; Jiang XR et al.; Cyclosporin A (CSA) exhibits greater multidrug resistance (MDR) modulating activity in vitro than other MDR modulators such as verapamil and quinidine . However, the immunosuppressive and nephrotoxic effects of CSA may limit its clinical use . PSC 833, a new cyclosporin D derivative, exerts a higher MDR reversal activity but lacks toxic or immunosuppressive effects . The drug-resistant sublines K/DAU100, K/DAU200, K/DAU300, K/DAU400, K/DAU500 and K/DAU600 have been derived from the drug-sensitive parental cell line, K562 cl.6 and CEM/VLB100 is a drug-resistant derivative of CCRF-CEM . We report a comparison of the effects of PSC 833 and CSA on daunorubicin (DAU) transport kinetics and chemosensitivity in these cell lines . Both CEM/VBL100 and K562 cl.6 DAU-resistant cells displayed high levels of P-glycoprotein (PGP), decreased DAU accumulation and increased DAU efflux when compared to their parental cells . PSC 833 was 1.6-, 3.4-, 4.9- and 4.6-fold more effective than CSA in reversing DAU resistance in higher resistance CEM/VLB100, K/DAU400, K/DAU500 and K/DAU600 cells respectively . DAU transport kinetics showed that PSC 833 was more effective than CSA in increasing cellular DAU accumulation and decreasing DAU efflux in higher resistant leukaemia subclones . PSC 833 could restore DAU retention at lower doses and was more active than CSA in all the resistant cells . A 89-100% restoration of intracellular DAU retention were gained by PSC 833 at 1.0 microM in K562 cl.6 DAU-resistant sublines, whereas a 73-100% restoration of DAU retention was obtained by CSA only at 30.0 microM in the same resistant sublines . PSC 833 at 3.0 microM is sufficient to restore full DAU retention in all resistant cells . CSA, however, even at 30.0 microM, cannot confer full restoration of DAU retention in higher resistance K562 cl.6/DAU sublines . By measuring MDR modulator-mediated short-term inhibition of PGP function, PSC 833 was found to be at least 10-30 times more active than CSA . As no effect on DAU retention and sensitivity has been found in sensitive parental cells with PSC 833, it is suggested that PSC 833 may act by blocking the effluxing function of PGP in the resistant leukaemia cells.

Anticancer Drug Des, 1995 Jun, 10(4), 333 - 46
Binding to DNA and cytotoxic evaluation of ascididemin, the major alkaloid from the Mediterranean ascidian Cystodytes dellechiajei; Bonnard I et al.; The isolation of ascididemin from the Mediterranean ascidian Cystodytes dellechiajei is described . This alkaloid consists of a planar pentacyclic chromophore which was investigated for its DNA-binding and cytotoxic properties . Spectroscopic measurements provided evidence that the drug intercalates into DNA . DNase I footprinting assays indicated that the binding of ascididemin to GC-rich sequences is favoured over binding to AT-rich and mixed sequences . Chemical probes were used to detect ligand-induced structural changes in DNA . The alkaloid induces a hyper-reactivity of the DNA towards potassium permanganate, but not towards diethylpyrocarbonate, just as is the case with ethidium bromide; it has little effect on the catalytic activities of topoisomerases I and II . Ascididemin exhibits marked cytotoxicity towards human leukaemic cells in vitro and appears to be practically equally toxic for drug-sensitive and multidrug-resistant cell lines . The results suggest that DNA, but not topoisomerases, may represent the critical cellular target at which this marine alkaloid exhibits its potent cytotoxic properties in vitro.

Anticancer Drug Des, 1995 Jun, 10(4), 277 - 97
Synthesis and biological evaluation of amino-substituted benzo{f}pyrido{4,3-b} and pyrido{3,4-b}quinoxalines: a new class of antineoplastic agents; Nguyen CH et al.; In order to study the structure-activity relationships in the series of new intercalating polycyclic agents, 1-amino-substituted pyrido{3,4-b}quinoxalines, benzo{f}pyrido{4,3-b}quinoxaline derivatives bearing a dibasic side chain and their benzo{f}pyrido{3,4-b} isomers have been synthesized . Biological evaluation was carried out for topoisomerase I and II inhibition, and for in vitro and in vivo antitumor properties in several models . Results demonstrate that appropriately substituted benzo{f}pyrido{4,3-b}quinoxaline derivatives are inhibitors of topoisomerase I and II, and have significant antitumor properties in various experimental models . In addition, the most active compounds appear to be minimally recognized by tumor cells expressing the multidrug resistance phenotype.

Arch Biochem Biophys, 1995 Jun 1, 319(2), 498 - 503
Purification and characterization of tubulin from parental and vincristine-resistant HOB1 lymphoma cells; Lee WP; A multidrug-resistant lymphoma cell line resistant to 1.0 microM vincristine (designated HOB1/VCR1.0) was established . The tubulins of parental and resistant cell lines were purified by ion-exchange chromatography . Two-dimensional polyacrylamide gel electrophoresis of tubulins showed a decrease in the basic component of beta-tubulin in the HOB1/VCR1.0 cell line; native isoelectric focusing of tubulins showed decreased expression of two more basic tubulin dimers in the same cell line . The {3H}vincristine-tubulin binding studies were performed by filtration and HPLC and displayed the tubulin of HOB1/VCR1.0 cells having a weaker binding affinity to vincristine than those of parental HOB1 and HOB1/VCR0.5 cells . The binding constant Ka of purified tubulin to vincristine, calculated from the slope of the Scatchard curve, for parental HOB1 cells was 5.6 x 10(6), and that for HOB1/VCR1.0 cells was 3.1 x 10(6) . The Scatchard kinetics was also used to determine the binding ability of the purified tubulins to {3H}colcemid: the Kas for parental and HOB1/VCR1.0 cells were 3.9 x 10(5) and 2.0 x 10(5), respectively . The current study suggests that high-level resistant cells, HOB1/VCR1.0, tend to express fewer tubulin isoforms of stronger binding affinities to antimitotic agents; that is, they preserve weak drug-binding forms rather than produce additional species . This may be a mechanism for the cells to protect themselves from drug injury when the P-glycoprotein cannot efficiently pump out the agent of high concentration within the cells.

J Cell Physiol, 1995 Jun, 163(3), 538 - 44
P-glycoprotein stability is affected by serum deprivation and high cell density in multidrug-resistant cells; Muller C et al.; The control of P-glycoprotein (Pgp) expression in multidrug-resistant cells (MDR) is complex and may be regulated at different levels . We have investigated Pgp stability in four different human and hamster MDR cell lines . Using a pulse-chase procedure we show that Pgp half-life is between 14 and 17 h in all these cell lines when they are growing exponentially . However, in the presence of a low level of serum, Pgp half-life is increased four to sixfold . A similar effect is observed when the cell cultures are maintained in high cell density . The increased Pgp stability appears to be differently regulated as serum deprivation results in a general enhanced degradation of total cytoplasmic and membrane proteins . Moreover, the observed serum effect suggests the involvement of growth factors in the control of Pgp stability . These findings suggest that protein stability may be an important factor in the regulation of Pgp expression.

Arch Fam Med, 1995 Jun, 4(6), 541 - 6
Tuberculosis in the homeless; Barclay DM 3rd et al.; The prevalence of tuberculosis in the homeless is on the rise . The presence of human immunodeficiency virus and multidrug-resistant tuberculosis in the homeless has contributed to this high prevalence . Several factors, including alcoholism, substance abuse, and psychiatric illness, combine to make it difficult to diagnose and treat tuberculosis in the homeless . Medical providers are likely to encounter homeless individuals in a number of settings, including emergency departments, community and free clinics, public hospitals, and health maintenance organizations . Appropriate screening, prevention, and treatment should be undertaken in collaboration with local health departments . The use of directly observed therapy and of the treatment regimens published by the Centers for Disease Control and Prevention improves treatment outcomes among the homeless.

Biochem J, 1995 Jun 1, 308 ( Pt 2), 381 - 90
Characterization of the ATPase activity of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells; Sharom FJ et al.; P-Glycoprotein (Pgp) was isolated from CHRC5 membranes by selective detergent extraction and further purified by lentil lectin affinity chromatography . The purified product displayed a very high basal ATPase activity (1.65 mumol/min per mg protein in the absence of added drugs or lipids) with an apparent Km for ATP of 0.4 mM . There was no evidence of cooperativity, suggesting that the two ATP sites operate independently of each other . Pgp ATPase activity was stimulated by verapamil, trifluoperazine and colchicine, and inhibited by daunomycin and vinblastine . All drugs and chemosensitizers acted as mixed activators or inhibitors, producing changes in both the Vmax of the ATPase and the Km for ATP . ADP competitively inhibited Pgp ATPase, with a Ki of 0.2 mM . The macrolide antibiotics bafilomycin A1, concanamycin A and concanamycin B, inhibited Pgp ATPase at concentrations of 0.1-10 microM, and at an inhibitor:protein stoichiometry of 0.65-1.0 mumol/mg protein, which is at the low end of the range characteristic of P-type ATPases . Pgp ATPase was relatively selective for adenine nucleotides . Several phospholipids stimulated Pgp ATPase activity in a dose-dependent manner, whereas others produced inhibition . Metabolic labelling showed that the endogenous phospholipids associated with purified Pgp consisted largely of phosphatidylethanolamine and phosphatidylserine, with only a small amount of phosphatidylcholine . 32P-Labelling studies indicated that purified Pgp was partially phosphorylated . It can be concluded that Pgp is a constitutively active, adenine nucleotide-specific ATPase whose catalytic activity can be modulated by both drugs and phospholipids.

Ann Thorac Surg, 1995 Jun, 59(6), 1405 - 7; discussion 1408-9
Current role of surgery in Mycobacterium tuberculosis; Treasure RL et al.; From January 1986 through December 1993, we operated on 59 patients with documented Mycobacterium tuberculosis infection . Indications for operation were as follows: multidrug-resistant tuberculosis (MDRTB) in 19 patients; bronchopleural fistula secondary to Mycobacterium tuberculosis infection in 12; massive hemoptysis in 5; destroyed lung in 7; solitary nodule in 7; trapped lung in 3; complicated cavity in 4; and empyema in 2 . Sixty-five operative procedures were performed: pneumonectomy with latissimus muscle flap in 15 patients; pneumonectomy in 3; lobectomy in 16; segmental or wedge resection in 11; decortication in 5; window thoracostomy in 3; thoracoplasty with myoplasty in 4; tube thoracostomy in 4; return to operating room for bleeding in 2; Clagett procedure in 1; and drainage of a cold abscess in 1 . There were no operative deaths . Major postoperative complications occurred in 5 patients . The two late deaths were in patients with MDRTB: 1 with progressive disease and massive hemoptysis and the other with a relapse of MDRTB . Of the patients operated on as part of their therapeutic regimen for MDRTB, 17 (89%) of 19 have remained culture negative . We conclude that (1) surgery still plays an important role in the management of patients with Mycobacterium tuberculosis infection; (2) surgical intervention can be performed with acceptable mortality and morbidity; (3) a variety of procedures are needed to effect cure; and (4) encouraging results in patients with MDRTB support surgical therapy in this difficult group of patients.

Am J Hematol, 1995 Jun, 49(2), 143 - 8
Pretreatment fibrinogen levels are associated with response to chemotherapy in patients with small cell carcinoma of the lung: Department of Veterans Affairs Cooperative Study 188; Meehan KR et al.; Small cell carcinoma of the lung (SCCL) responds commonly to combination chemotherapy but resistance to therapy follows . Prior reports have suggested that a relationship may exist between plasma fibrinogen levels and response to therapy in SCCL . This study was designed to determine the possible predictive value of the fibrinogen level for tumor response (chemoresistance) in SCCL . Pretreatment fibrinogen levels were correlated with outcome and response to therapy in a cohort of 119 previously untreated patients with SCCL who were admitted to VA Cooperative Study 188 . Higher pretreatment fibrinogen levels at diagnosis correlated significantly with more advanced stage of disease at entry (P < 0.001) and with reduced overall survival (P = 0.030) . In addition, higher pretreatment fibrinogen levels were correlated significantly with a reduced likelihood of achieving subsequent disease regression with combination chemotherapy (P = 0.005) . Because several clinical trials have shown that anticoagulant therapy improves tumor response rates and survival of SCCL, we postulate that tumor cell thrombin generation not only promotes SCCL growth but may also be primarily responsible for both increased fibrinogen levels and for resistance to chemotherapy . These findings provide incentive for studies of thrombin effects on the development of multidrug resistance, and for new clinical trials of more potent and specific inhibitors of thrombin that may further improve tumor response and survival in SCCL.

Am Fam Physician, 1995 Jun, 51(8), 1929 - 34, 1937-8
Positive PPD and chemoprophylaxis for tuberculosis infection; Pickwell SM; The appearance of an indurated area of 5 mm or more 48 to 72 hours after administration of purified protein derivative (PPD) is considered a positive reaction in persons who have recently had close contact with an individual with active tuberculosis, in persons with radiographic findings consistent with a past history of tuberculosis or in persons with known or suspected human immunodeficiency virus infection . Ten or more millimeters of induration is considered a positive reaction in persons at increased risk of tuberculosis . Induration of 15 mm or more is considered a positive result in all other persons . Candidates for a six-month course of isoniazid include persons under age 35 who are recent converters and have induration of 10 mm or more and persons over age 35 with 15 mm or more of induration . Patients with HIV infection and those with radiographic evidence of previous tuberculosis should receive 12 months of therapy . A regimen of pyrazinamide and either ethambutol, ofloxacin or ciprofloxacin is recommended for contacts of patients with multidrug-resistant tuberculosis.

Pediatr Clin North Am, 1995 Jun, 42(3), 649 - 64
Drug-resistant malaria in children and in travelers; Longworth DL; Malaria remains a significant cause of childhood morbidity and mortality worldwide . Drug resistance in Plasmodium falciparum has become widespread in the past 30 years, and in some parts of the world multidrug resistance is common . Chloroquine resistance in Plasmodium vivax has recently been recognized in Indonesia . The mechanisms of drug resistance have been defined for the antifolate antimalarial agents but remain incompletely understood for the quinolines . Judicious use of antimalarial compounds will be essential to prevent the emergence and spread of further drug resistance . The history, geographic distribution, and mechanisms of drug resistance are reviewed, together with current recommendations regarding prophylaxis and therapy.

J Pharm Pharmacol, 1995 Jun, 47(6), 524 - 9
Antitumour effects and pharmacokinetics of combination of vinblastine with a staurosporine derivative, NA-382, in P388/ADR-bearing mice; Miyamoto K et al.; The effects of a staurosporine derivative, N-ethoxycarbonyl-7-oxostaurosporine (NA-382), on the pharmacokinetics of vinblastine were evaluated, compared with those of verapamil, in multidrug-resistant P388/ADR-bearing mice . At first, the in-vitro experiments indicated that NA-382 permeated into the cells better and were more effective in combined cytotoxicity with vinblastine and on accumulation of vinblastine than with verapamil in P388/ADR cells . In combined intraperitoneal injection with vinblastine (200 micrograms kg-1) into P388/ADR-bearing mice, NA-382 in a suspension form (10 mg kg-1) prolonged the life-span of the mice near to that of P388/S-bearing mice treated with vinblastine alone, but verapamil even at the maximum tolerated dosage (30 mg kg-1) barely affected the in-vivo antitumour effect of vinblastine . When simultaneously administered with vinblastine to P388/ADR-bearing mice, NA-382 maintained significantly higher vinblastine levels in the tumour cells for 24 h and gave a larger area under the time-intracellular vinblastine concentration curve (0 to 24 h) than those receiving vinblastine alone, with long retention of the agent in ascitic fluid . Verapamil increased the cellular vinblastine content for only 6 h, accompanying a rapid elimination of the agent from the ascitic fluid . This study indicates that NA-382 is more effective against multidrug-resistance than verapamil, and its suspension is also advantageous for cancer chemotherapy of multidrug-resistant tumours.

Anticancer Drugs, 1995 Jun, 6(3), 432 - 7
Development of drug resistance is reduced with idarubicin relative to other anthracyclines; Hargrave RM et al.; Multidrug resistance (MDR) is associated with poor prognosis in leukemia, and anthracyclines, which are used in the treatment of leukemia, are associated with the expression of P-glycoprotein and the development of MDR . We report here that idarubicin, a new anthracycline approved for use in the treatment of acute myelogenous leukemia (AML), did not induce P-glycoprotein expression in the K562 human leukemia cell line under conditions where daunorubicin, doxorubicin and epirubicin did induce expression of P-glycoprotein . The P-glycoprotein expressing, multidrug resistant sublines developed to daunorubicin (K/DNR), doxorubicin (K/DOX) and epirubicin (K/EPR) were cross-resistant to the other anthracyclines and to vinblastine, taxol, colchicine and actinomycin D, but were not resistant to idarubicin or etoposide . The idarubicin treated subline, K/IDA, was only resistant to taxol but was 12-fold sensitized to etoposide, suggesting that idarubicin had affected topoisomerase II in this subline.

Farmaco, 1995 Jun, 50(6), 365 - 8
Azolophenanthridines as antineoplastic agents; Aiello E et al.; Pyrrolo-, pyrazolo- and triazolo-phenanthridines were synthetized by using a Pschorrtype cyclization reaction or an intramolecular cyclization of arylnitrenium ions . By using these synthetic methods several azolo-phenanthridines, variously functionalized either in the azolo ring and in the phenanthridine moiety, were prepared . The title compounds, tested against murine leukemia cell lines, sensible and multidrug resistant, showed moderate activity with IC50 in the range 5-50 microM.

Nurse Pract, 1995 Jun, 20(6), 34 - 6, 39-40
A tuberculosis control plan for ambulatory care centers; Wolf L; Tuberculosis and a multidrug resistant form of TB have reemerged in epidemic proportions . Tuberculosis is more prevalent among HIV-infected individuals, the homeless, foreign-born individuals from countries with high rates of TB, prison inmates, individuals in long-term care facilities, individuals from low-socioeconomic backgrounds, and intravenous drug users . Urgent care centers, which have gained popularity as an easy access to medical care, are at risk for seeing undetected infectious patients . By developing a TB control plan for ambulatory care centers, specific triage criteria can be instituted to promote early identification of the potentially infectious patient and provide guidelines for identification, prevention, environmental controls, education, and follow-up of exposed health care workers . This article discusses the mandatory OSHA policy for occupational exposure to tuberculosis based on the 1993 CDC guidelines for TB transmission and how a TB control plan for ambulatory care centers can be developed from these guidelines.

S Afr Med J, 1995 Jun, 85(6), 499 - 504
Tuberculosis drug resistance in the Western Cape; Weyer K et al.; OBJECTIVES . Drug resistance is a serious problem in the treatment of tuberculosis and a threat to successful tuberculosis control programmes . Local health workers have expressed concern that the increasing tuberculosis epidemic in the Western Cape is partly attributable to drug resistance . The aim of this study was to determine the prevalence of tuberculosis drug resistance (including multidrug resistance) and to investigate possible relationships between drug resistance and patient demographic characteristics . DESIGN, SETTING, SUBJECTS, OUTCOME MEASURES . During a defined period, all adult (> or = 15 years) patients with pulmonary tuberculosis (confirmed by culture) from all tuberculosis clinics in the Western Cape were included . Previous tuberculosis treatment history was obtained by interviews, utilising a standardised questionnaire . Acquired drug resistance was determined on cultures from patients with a prior history of tuberculosis treatment, while initial resistance was determined from tuberculosis cases with no history of previous treatment . RESULTS . Data from 7,266 patients were analysed . After adjusting for missing information by way of a random sample validation study, 32% of patients were found to have a history of previous treatment, 63% indicated no previous treatment, and in 5% the treatment history was unknown . Rates for initial resistance were found to be low at 3,9% for isoniazid, 1,1% for rifampicin and 0,2% for ethambutol . Combined resistance to isoniazid and rifampicin (multidrug resistance) was found to be 1,1% in patients not treated before . Acquired resistance rates were higher at 10,8% for isoniazid, 4,2% for rifampicin, 0,3% for ethambutol and 4,0% for multidrug resistance . Logistic regression analysis of the data indicated that drug resistance was not influenced by population group, gender or age . Patients with a history of tuberculosis treatment were found to be at an increased risk of developing drug resistance (relative risk 2,6) . Some regions in the Western Cape had higher proportions of previously treated patients with consequent higher acquired resistance rates . CONCLUSIONS . Results from this study indicated that drug resistance is currently not a major problem in the Western Cape, rates comparing favourably with those reported from developed countries and being much lower than those for developing countries . Every effort should therefore be made to maintain the status quo and to prevent the emergence of further resistance . The priority for tuberculosis control in the Western Cape should remain to limit transmission of the disease by reducing the infectious pool through improved cure of (especially) smear-positive cases.

J Clin Microbiol, 1995 Jun, 33(6), 1617 - 23
Genotypic detection of Mycobacterium tuberculosis rifampin resistance: comparison of single-strand conformation polymorphism and dideoxy fingerprinting; Felmlee TA et al.; Detection of mutations in the rpoB gene of Mycobacterium tuberculosis can be used as an accurate predictor of rifampin resistance in the majority of strains tested . Simple but highly accurate screening methods must be developed for the detection of these mutations . Either DNA sequence analysis or single-strand conformation polymorphism (SSCP) screening can be used to detect rpoB mutations, but these techniques either are expensive or yield results that may prove difficult to interpret when used in a clinical setting . This report describes the use of dideoxy fingerprinting (ddF) as a postamplification screening method to identify rifampin-resistant genotypes . The ddF protocol was performed on the amplified rpoB fragment with no preparatory steps, thus making ddF practical for laboratories equipped for polyacrylamide gel electrophoresis . When compared with the results of SSCP analysis, ddF results were more easily interpreted and contained more sequence-dependent information that facilitated differentiation of functionally significant and silent mutations . The ddF method was used for genotypic determination of rifampin susceptibility of 20 multidrug-resistant strains of M . tuberculosis . The results of this analysis were concordant with DNA sequence analysis and conventional clinical laboratory methods.

Free Radic Biol Med, 1995 Jun, 18(6), 963 - 76
Antioxidant and cytotoxic tocopheryl quinones in normal and cancer cells; Thornton DE et al.; We found previously that {d}-alpha-tocopherol (alpha-T) and {d}-gamma-tocopherol (gamma-T) are lipid antioxidants (thiobarbituric acid test) in model systems containing arachidonic acid (AA), cumene hydroperoxide, and Fe3+ and in smooth muscle cell (SMC) cultures challenged with AA . We now show that {d}-alpha-tocopherylquinone (alpha-TQ), {d}-delta-tocopherylquinone (delta-TQ), and {d}-gamma-tocopherylquinone (gamma-TQ) are antioxidants at low concentrations and prooxidants at high concentrations in the model system . Prooxidant activity is greater with gamma-TQ than either alpha-TQ or delta-TQ . Low concentrations of alpha-TQ, delta-TQ, and gamma-TQ are also antioxidants in SMC cultures challenged with AA . Unlike alpha-TQ, partially substituted gamma-TQ and glutathione (GSH) form a Michael adduct which has been purified and characterized . We found previously that alpha-T, gamma-T, and alpha-TQ are mitogenic in SMC . We now report that both delta-TQ and gamma-TQ but not alpha-TQ show concentration-dependent cytotoxicity (changes in morphology, propidium iodide stain) in SMC cultures . Cytotoxicity is greater with gamma-TQ than delta-TQ . An acute lymphoblastic leukemia (ALL) cell line shows greater chemosensitivity (MTT and Neutral Red assays) to gamma-TQ than to either doxorubicin (DOX) or vinblastine (VLB) . An ALL cell line resistant to both DOX and VLB retains the same chemosensitivity to gamma-TQ as the drug-sensitive ALL cell line . ALL cell lines are unaffected by either alpha-TQ or the GSH Michael adduct of gamma-TQ . These data show that partially substituted tocopheryl quinones capable of forming Michael adducts are potential chemotherapeutic agents for multidrug-resistant cancer cells.

Jpn J Cancer Res, 1995 Jun, 86(6), 607 - 15
New flow cytometric method for detection of minimally expressed multidrug resistance P-glycoprotein on normal and acute leukemia cells using biotinylated MRK16 and streptavidin-RED670 conjugate; Takeshita A et al.; To evaluate the expression of multidrug resistance (MDR) on normal and leukemia cells, we examined P-glycoprotein (P-gp) by a newly devised flow cytometric method, utilizing a biotinylated monoclonal antibody (mAb) against P-gp (MRK16), a streptavidin-RED670 conjugate (SA-RED670) and appropriate emission filters . The combination of biotinylated MRK16 (b-MRK16) and SA-RED670 resulted in higher sensitivity as compared with standard methods such as the use of streptavidin-phycoerythrin (SA-PE) conjugate . The sensitivity was examined in K562, K562/ADR, NOMO-1, NOMO-1/ADR and HL60 cells, and compared with the data obtained from reverse transcription polymerase chain reaction (RT-PCR) of mdr-1 gene . P-gp positivity on flow cytometry was 10.4%, 99.9%, 1.4%, 90.4% and 0%, respectively . Mdr-1 mRNA was well expressed in K562/ADR and NOMO-1/ADR cells, but not in NOMO-1 and HL60 cells . In K562 cells, mdr-1 was found after 40 cycles of PCR, but not 25 cycles . These data are well correlated with those from the flow cytometry . We then studied the P-gp expression on normal peripheral blood cells and acute leukemia cells . P-gp was little expressed on peripheral lymphocytes, monocytes and granulocytes . It was also little expressed on blast cells from 5 patients with acute promyelocytic leukemia (AML) and 5 acute lymphocytic leukemia (ALL) expressed P-gp at diagnosis, ranging from 8.5% to 34.5% (16.9 +/- 11.8%) and from 2.3% to 45.6% (24.0 +/- 17.8%), respectively . All 9 relapsed or refractory cases expressed P-gp, ranging from 21.1% to 99.8% (52.2 +/- 29.9%) . Significant differences were found in APL, CD34-positive and relapse and refractory cases (P = 0.0006, 0.0007 and 0.0088, respectively) . These results indicate that this flow cytometric analysis is useful for the evaluation of clinical MDR status and can identify a group of patients with resistant leukemia.

J Can Dent Assoc, 1995 Jun, 61(6), 492, 495 - 8
Dentistry and tuberculosis in the 1900s; Riben PD et al.; The number of new cases--or incidence--of tuberculosis is increasing in nearly every region of the world . A number of forces have resulted in the increased incidence of TB in developed countries, including the HIV epidemic, homelessness, and emigration from highly endemic regions . Although the number of new cases in Canada is relatively constant, the TB experience in the United States serves as a reminder that this situation could change rapidly . The appearance of multidrug-resistant tuberculosis has added to the urgency of situation . The basic methods of preventing TB transmission include preventing the release of the organism into the air, removing the organism from the air, and preventing the inhalation of the organism . Identifying and appropriately treating every person with active tuberculosis is an extremely important component of the control strategy; adequate ventilation, filtering air, and ultraviolet germicidal irradiation are methods used to remove the organism from the air; and masks and other personal protective devices, such as high-efficiency particulate air filters (HEPA), have been suggested as a means of preventing inhalation of the organism . In addition, identifying new TB infections and using chemoprophylaxis often prevents infection from progressing to active disease . Given the route by which tuberculosis is transmitted, it is necessary for both dentists and allied dental personnel to be aware of the risks they may face in day-to-day practice, and the means by which they can protect themselves and their patients.

Am J Trop Med Hyg, 1995 Jun, 52(6), 536 - 8
Use of amphotericin B in drug-resistant cases of visceral leishmaniasis in north Bihar, India; Jha TK et al.; Thirty-four multidrug-resistant cases of Indian visceral leishmaniasis (kala-azar) were treated with amphotericin B . A complete hemogram, liver and renal function tests, determination of serum electrolyte levels, a chest radiograph, and an electrocardiogram were done before, during, and after completion of therapy . Assessment for clinical and parasitologic cure was done weekly . Thirty-one patients who completed treatment had full cure after receiving 10-15 injections at six-months follow up . One patient died of myocarditis . A febrile reaction was observed in all cases, while thrombophlebitis was found in six cases (18.75%) . Anorexia, nausea, and vomiting were found in seven cases (21.88%) . No significant nephrotoxicity or electrolyte disturbances were observed . It is concluded that amphotericin B is an effective second-line drug for Indian visceral leishmaniasis, but unpredictable drug-induced myocarditis remains a problem.

Nat Med, 1995 Jun, 1(6), 578 - 82
The drug resistance-related protein LRP is the human major vault protein; Scheffer GL et al.; Multidrug-resistant cancer cells frequently overexpress the 110-kD LRP protein (originally named Lung Resistance-related Protein) . LRP overexpression has been found to predict a poor response to chemotherapy in acute myeloid leukaemia and ovarian carcinoma . We describe the cloning and chromosome localization of the gene coding for this novel protein . The deduced LRP amino acid sequence shows 87.7% identity with the 104-kD rat major vault protein . Vaults are multi-subunit structures that may be involved in nucleo-cytoplasmic transport . The LRP gene is located on chromosome 16, close to the genes coding for multidrug resistance-associated protein and protein kinase C-beta, and may mediate drug resistance, perhaps via a transport process.

J Hosp Infect, 1995 Jun, 30 Suppl, 322 - 8
Treatment of multidrug-resistant tuberculosis; Cohn DL; Recent outbreaks of multi-drug-resistant tuberculosis (MDR-TB) have resulted in significant morbidity and mortality in patients with AIDS . The poor outcomes are attributable to delayed diagnoses, slow reporting of antimycobacterial susceptibility results, inadequate treatment regimens and profound immunosuppression . There are no prospective clinical trials which have evaluated the optimal treatment of MDR-TB . A retrospective study has shown that in immunocompetent patients with secondary MDR-TB, only 56% responded to prolonged courses of multiple drug regimens, and 22% died of TB . In patients with AIDS, even fewer patients respond, with median survivals of 2-4 months . In general, better responses have been associated with in vitro susceptibility of patients' isolates . If possible, patients with MDR-TB should receive at least three drugs to which their isolates are susceptible for at least 24 months; these regimens are likely to include ethambutol, pyrazinamide, a quinolone, and an aminoglycoside . Selected patients benefit from surgical intervention combined with aggressive chemotherapy . MDR-TB is best prevented by directly observed therapy of patients with susceptible organisms and rigorous infection control practices in areas of high incidence of MDR-TB . Effective treatment regimens for MDR-TB await the development of novel compounds which have better in vitro activity against MDR-TB than currently available drugs.

Tuber Lung Dis, 1995 Jun, 76(3), 185 - 9
Mycobacteria in HIV-infected patients in Buenos Aires; Di Lonardo M et al.; SETTING: F . J . Muniz Hospital and Department of Phthisiopneumonology, in Buenos Aires . OBJECTIVE: To analyze bacteriological findings concerning tuberculosis and other mycobacteriosis, in association with HIV infection and AIDS . DESIGN: From June 1985 to December 1991, 2521 samples from 1259 HIV-seropositive and AIDS patients were analyzed: 1133 samples were of bronchopulmonary origin and the remaining 1388 of extrapulmonary origin . Drug susceptibility tests were performed using the proportions method . RESULTS: Mycobacterial disease was confirmed by culture in 240 of the 1259 HIV/AIDS patients (19%) . Mycobacterium tuberculosis was isolated in 223 of these cases (92.9%) and M . bovis in two, while M . avium-complex (MAC) strains were identified as the cause of disease in 14 patients (5.8%) . In only one case was disease due to M . kansasii . Blood cultures were positive in 21.2% of these 240 cases . Resistance of M . tuberculosis to antituberculosis drugs was found in 9.4% of the 223 isolates . In only one case was multidrug resistance detected, in a patient who had received previous treatment . CONCLUSION: Smear examination, although less sensitive than in HIV-negative patients, was still a simple and reliable tool for the rapid diagnosis of mycobacterial disease . Blood culture aided in the successful diagnosis of about half of the cases of disseminated tuberculosis and of all cases of MAC disease . An alarming spread of tuberculosis was detected among a group of HIV-positive prisoners, and the possible emergence of multidrug resistance should be anticipatedPIP: An increase in human immunodeficiency virus (HIV)-associated mycobacterial tuberculosis has led to a reversal of an earlier trend in Argentina toward a decline in the incidence of tuberculosis . A bacteriologic study conducted at the Muniz Hospital in Buenos Aires June 1985-December 1991 confirmed the reliability of smear examination for the rapid diagnosis of mycobacterial disease . 2521 samples were obtained from 1259 HIV-infected individuals during the study period and mycobacterial disease was confirmed by culture in 240 cases (19%) . The smears were positive in 59.0% of the 122 pulmonary cases, 22.0% of the 72 extrapulmonary cases, and 56.5% of the 46 pulmonary-extrapulmonary cases . Blood cultures were positive in 21.2% of the 240 cases . In patients with pulmonary localization, acquired immunodeficiency syndrome (AIDS) was diagnosed between a few months to two years after the onset of tuberculosis; those with the two other localizations had already advanced to AIDS at the time of blood smear . Resistance to one or more antitubercular drugs was found in 9.4% of cases .

Leukemia, 1995 Jun, 9(6), 1025 - 31
Multidrug resistant cells with high proliferative capacity determine response to therapy in acute myeloid leukemia; te Boekhorst PA et al.; High spontaneous proliferation of acute myeloid leukemia (AML) in vitro is an unfavorable, tumor-specific prognostic factor . We investigated the frequency of drug-resistant tumor cells with high proliferating capacity in de novo AML and analyzed the expression of multiple resistance parameters in relation to the response to chemotherapy and overall survival . Thirty-eight patients were included in this study . P-glycoprotein (P-gp) expression was found in 28/38 patients and was associated with lower intracellular accumulation of DNR (P = 0.0001) . Thirty-five out of 38 patients were treated with 1-2 regimens of daunorubicin (DNR)/cytarabine (Ara-C), and 57% attained a complete remission (CR) . Failure to achieve a CR correlated with autonomous growth (P = 0.0064), CD34 and P-gp expression alone (P = 0.0005 and P = 0.048 respectively), and with simultaneous expression of P-gp and CD34 (P = 0.0001), but not with expression of the non-P-gp drug resistance associated-protein (p110), the multidrug resistance-associated protein (MRP), Ara-CTP formation or Ara-C incorporation, respectively . AML cells with CD34/P-gp double expression were more frequently observed in samples with high autonomous growth (P = 0.003) . The median survival was 6 months in CD34+/P-gp+ patients as compared with 15 months in other AML patients (P = 0.003) . In patients with de novo AML who fail on chemotherapy, a population of autonomously proliferating, immature AML cells with a multidrug resistant phenotype can be recognized . These cells thus show primary resistance to chemotherapy and have the potential for rapid regrowth, leading to resistant disease.

Lab Invest, 1995 Jun, 72(6), 760 - 4
Immunomagnetic purification of human breast carcinoma cells allows tumor-specific detection of multidrug resistance gene 1-mRNA by reverse transcriptase polymerase chain reaction in fine-needle aspirates; Maas RA et al.; BACKGROUND: The heterogenous composition of tumors is a major obstacle for the measurement of mRNA levels in cancer cells . We report here a combination of immunomagnetic purification of cancer cells and reverse transcriptase polymerase chain reaction (RT-PCR) that enables highly sensitive detection of multidrug resistance gene 1 (MDR1)-mRNA levels in human breast carcinoma cells obtained from fine needle aspirates (FNA) . EXPERIMENTAL DESIGN: Murine mAb 115D8 directed against episialin (MUC1/MAM6, epithelial membrane Ag) was used in combination with goat anti-mouse-coated magnetic microbeads to purify human T47D breast carcinoma cells (115D8+, MDR1-) from different mixtures with COLO320 human colon carcinoma cells (115D8-, MDR1+) and to purify carcinoma cells from FNA taken from axillary lymph node metastases in breast cancer patients . The efficacy of the purification was determined by FACS-analysis and by measurement of MDR1-mRNA levels by semiquantitative RT-PCR . RESULTS: FACS-analysis demonstrated that T47D cells could be purified up to 99.8% from mixtures with COLO320 cells ranging from 3:1 to 1:3 . The MDR1-mRNA level in these enriched mixtures, as detected by RT-PCR, was reduced 250-fold . It was demonstrated that MDR1 expression present in an FNA from a lymph node metastasis of breast carcinoma could be attributed completely to the leukocytes present in this FNA, because MDR1 expression was no longer detectable after purification of the tumor cells . CONCLUSION: The combination of immunomagnetic purification of breast carcinoma cells and RT-PCR enables the measurement of cancer-specific MDR1 mRNA levels in small cell samples obtained by FNA.

J Biol Chem, 1995 May 26, 270(21), 12351 - 4
Multidrug resistance protein (Mdr)-alkaline phosphatase hybrids in Escherichia coli suggest a major revision in the topology of the C-terminal half of Mdr; Beja O et al.; Recent studies reveal that the organization of the multidrug resistance protein (Mdr) in the membrane is probably not exactly as predicted from hydropathy profiling . When expressed in Escherichia coli, phoA gene fusions can be utilized to study the membrane topology of Mdr . Using this approach, it was proposed recently that the N-terminal hydrophobic domain of Mdr spans the membrane six times, in a different fashion from that predicted by hydropathy analysis (Bibi, E . and Beja, O . (1994) J . Biol . Chem . 269, 19910-19915) . In this study, we analyze mdr-phoA fusions constructed in the C-terminal half of Mdr . Overall, the results presented here lead to a significant revision in the membrane topology model of the C-terminal half of Mdr . The new topology is discussed with regard to the hydropathy profiles of the well characterized ABC proteins MalG and MalF, which are strikingly similar to those of the N- and C-terminal halves of Mdr, respectively.

Biochim Biophys Acta, 1995 May 24, 1236(1), 155 - 62
Collateral sensitivity of multidrug resistant cells to narcotic analgesics is due to effects on the plasma membrane; Callaghan R et al.; It has previously been demonstrated that opiates interact directly with P-glycoprotein in drug resistant Chinese hamster ovary (CHO) cells (Callaghan, R . and Riordan, J.R . (1993) J . Biol . Chem . 268, 16059-16064) . In this study we have examined the effects of several opiates on the growth of drug sensitive and resistant CHO and human MCF7 cell lines . The growth of P-glycoprotein expressing cells was inhibited by the opiates pentazocine, pethidine and naloxone to a greater extent than in drug sensitive cells . Since P-glycoprotein is localised at the plasma membrane the effects of opiates on membrane biophysical properties were investigated . The opiates caused a fluidizing effect in membranes from P-glycoprotein expressing cells and decreased the basal level of P-glycoprotein phosphorylation . In addition, they were able to increase the leakage of the membrane impermeant compound 6-carboxyfluorescein entrapped in model membrane vesicles . The ability to alter membrane biophysical properties correlated with the inhibitory effects on growth of drug resistant cells . These results suggest that the collateral sensitivity of P-glycoprotein expressing cell lines to opiates is mediated by the drugs' effects on the plasma membrane.

Toxicology, 1995 May 23, 99(3), 207 - 17
Lymphotoxicity and myelotoxicity of doxorubicin and SDZ PSC 833 combined chemotherapies for normal mice; Pourtier-Manzanedo A et al.; In the mouse, the P-glycoprotein-directed chemosensitizer SDZ PSC 833 could both restore a therapeutic window for doxorubicin against multidrug-resistant tumors, by inhibiting P-glycoprotein function, and increase the anti-cancer drug efficacy against drug-sensitive tumors, by increasing doxorubicin bioavailability . Since the success of such combined chemotherapy treatments might have been limited by the myelotoxicity of doxorubicin and the P-glycoprotein expression on some blood cells, their lymphotoxicity and myelotoxicity was studied on normal B6D2F1 mice, and whenever possible, the persistence of blood cell alterations was also searched for in scid recipients of lymphohaematopoietic grafts from the donor mice . Analyzed parameters were blood, lymphoid and myeloid cell numbers, proliferative responses to T- and B-cell mitogens, and serum immunoglobulin levels . Cell alterations caused by doxorubicin alone were potentiated by SDZ PSC 833, but did not persist in scid recipients . Chemotherapy regimens combining SDZ PSC 833 and doxorubicin, and known for their therapeutic benefit for multidrug-resistant tumor-bearing mice, only caused a rather mild toxicity for the lympho-myeloid system of normal mice.

Biochem Pharmacol, 1995 May 17, 49(10), 1541 - 4
In vivo and in vitro evidence for ATP-dependency of P-glycoprotein-mediated efflux of doxorubicin at the blood-brain barrier; Ohnishi T et al.; We investigated the role of ATP in the active efflux of doxorubicin (DOX) mediated by P-glycoprotein (P-gp), the multidrug-resistance (MDR) gene product, at the blood-brain barrier . In transient brain ischemic rats prepared with 4-vessel occlusion of vertebral and common carotid arteries for 20 min, a procedure that depleted their brain ATP content to 3% that of normal rats, the estimated permeability coefficient of DOX was increased 17-fold (to 243 +/- 2.5 microL/min/g brain) . When the ATP content recovered to a normal level by means of 30-min and 24-hr cerebral recirculation of blood, the permeability coefficient recovered to 14.0 +/- 5.0 and 18.4 +/- 2.3 microL/min/g brain (mean +/- SEM, N = 3-6), respectively, very close to the control permeability (14.3 +/- 1.5 microL/min/g brain) . The uptake of DOX by primary cultured brain capillary endothelial cells expressing P-gp at the luminal membrane was increased significantly (up to 2-fold), which correlated well with the decrease of cellular ATP contents caused by treating the cells with metabolic inhibitors . Evidence for the ATP-dependent transport of DOX obtained from the present in vivo and in vitro studies strongly indicates that P-gp in the brain capillaries functions actively as an efflux pump in the physiological state, providing a major mechanism to restrict the transfer of DOX into the brain.

Cancer Res, 1995 May 15, 55(10), 2090 - 6
Phase I clinical and pharmacokinetic study of 3'-deamino-3'-(2-methoxy-4-morpholinyl)doxorubicin (FCE 23762); Vasey PA et al.; Methoxymorpholinyldoxorubicin (FCE 23762) is a novel, highly lipophilic doxorubicin analogue . It possesses potent in vitro and in vivo antitumor activity including efficacy in multidrug-resistant tumor cell lines . It is also metabolically activated in vivo resulting in an 80-fold increase in potency over the parent drug . In this phase I study the drug was administered by i.v . bolus injection at 3-week intervals . Fifty-three patients with refractory solid tumors were treated; 133 courses of FCE 23762 were administered at doses ranging from 30 to 2250 micrograms/m2 . The dose limiting toxicity was reversible myelo-suppression (granulocytopenia and thrombocytopenia), demonstrating a delayed nadir and recovery in comparison to doxorubicin . Other toxicities included transient elevation of hepatic transaminases, delayed and prolonged nausea and vomiting, mucositis, anorexia, fatigue, and diarrhea . Heavily pretreated patients demonstrated more myelosuppression than previously untreated patients at 1250 micrograms/m2 . No cardiotoxicity was observed . Four objective tumor responses were seen: one complete response in a patient with pelvic recurrence of cervical cancer; one partial response in a patient with cutaneous and lymph gland metastases from head and neck cancer; and two minor responses in patients with liver metastases from colorectal cancer . Plasma concentrations of FCE 23762 and its 13-dihydro metabolite, FCE 26176, were measured in 20 patients at doses > or = 675 micrograms/m2, using HPLC with fluorescence detection . The area under the plasma concentration-time curve ranged from 30 to 80 ng/h/ml; plasma data suggested linear kinetics in the range of tested doses (although there was considerable interpatient variability) . The maximum tolerated dose defined in this study using this schedule is 1500 micrograms/m2 . A safe phase II dose for previously untreated patients using this schedule is 1250 micrograms/m2; however, this may actually be below the optimal dose for this patient population.

Cancer Res, 1995 May 15, 55(10), 2029 - 34
Identification of a sister gene to P-glycoprotein; Childs S et al.; The P-glycoproteins (Pgps) are a small family of transport proteins associated with the multidrug resistance phenotype of cell lines selected for growth in cytotoxic drugs . Utilizing low stringency screening, we have identified a novel gene closely related to the Pgps expressed in the pig and other mammalian liver which we have called Sister of P-glycoprotein (spgp) . Sequence of this gene shows it to be a member of the ATP-binding cassette family of transporters and the gene most closely related to Pgp identified to date . The function of spgp is not known, but it can be recognized by at least one Pgp mAb, C219 . This cross-reactivity has implications for expression studies in tissues and tumors utilizing this and other Pgp antibodies.

J Biol Chem, 1995 May 12, 270(19), 11040 - 2
Functional modulation of multidrug resistance-related P-glycoprotein by Ca(2+)-calmodulin; Schlemmer SR et al.; Studies with inside-out plasma membrane vesicles from multidrug-resistant (MDR 3) murine erythroleukemia (MEL/VCR-6) cells have provided evidence for down-modulation of P-glycoprotein (P-gp) function by Ca(2+)-calmodulin (CLM) . These studies showed that CLM in the presence or absence of Ca2+ had no effect on binding of {3H}vinblastine (VBL) by P-gp in inside-out plasma membrane vesicles . However, profound inhibition of ATP-dependent {3H}VBL efflux by these vesicles was demonstrated by the addition of subnanomolar concentrations of CLM (IC50 = 0.15 +/- 0.02 nM) . The addition of 1 microM Ca2+ reduced the inhibition of {3H}VBL efflux by CLM, shifting the concentration required for inhibition to the nM range (IC50 = 2.55 +/- 0.35 nM) . The inhibition of as 0.01 mM Ca2+, and no inhibition occurred with concentrations greater than 0.2 mM Ca2+ . Binding of CLM, itself, to P-gp was demonstrated in two ways . The P-gp content of detergent-solubilized plasma membrane from MEL/VCR-6 cells could be appreciably depleted by treating this material with CLM-Sepharose beads as shown by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting with anti-P-gp antibody (C219) before and after CLM-Sepharose treatment . Also, depletion of P-gp from solution by CLM was less in the presence of 1 mM Ca2+ . Blotting of P-gp after SDS-PAGE of plasma membrane from MEL/VCR-6 cells was also obtained using 125I-CLM as a probe . These results strongly suggest that the MDR 3 homolog of P-gp is a CLM-binding protein and that direct interaction of Ca(2+)-CLM with P-gp, while not affecting its binding of {3H}VBL, down-modulates the translocation of this agent in the presence of ATP.

Biochem Pharmacol, 1995 May 11, 49(9), 1255 - 60
Rifampicin enhances anti-cancer drug accumulation and activity in multidrug-resistant cells; Fardel O et al.; Rifampicin, a semi-synthetic antibiotic used in the treatment of tuberculosis and belonging to the chemical class of rifamycins, was examined for its effect on anti-cancer drug accumulation and activity in multidrug resistant cells overexpressing P-glycoprotein (P-gp) . Rifampicin was shown to strongly enhance vinblastine accumulation in both rat hepatoma RHC1 and human leukemia K562 R7 multidrug resistant cells, but had no effect in rat SDVI drug-sensitive liver cells . By contrast, two other rifamycins, rifamycins B and SV, had no or only minor effect on vinblastine accumulation in RHC1 cells . Efflux experiments revealed that rifampicin was able, like the well-known chemosensitizer agent verapamil, to decrease export of vinblastine out of resistant cells . Rifampicin, when used at a concentration close to plasma concentrations achievable in humans (25 microM), was able to increase sensitivity of RHC1 cells to both vinblastine and doxorubicin . Rifampicin was also demonstrated to inhibit P-gp radiolabeling by the photoactivable P-gp ligand azidopine, thereby suggesting that the antituberculosis compound can interfere directly with P-gp drug binding sites . These results thus indicate that rifampicin was able to down-modulate P-gp-associated resistance through inhibition of P-gp function.

Arch Biochem Biophys, 1995 May 10, 319(1), 309 - 15
Reversal of multidrug resistance phenotype by surfactants: relationship to membrane lipid fluidity; Dudeja PK et al.; Previous studies have suggested that multidrug resistance (MDR) reversal by polyoxyethylene surfactants involves alterations in plasma membrane lipid physical state of resistant cells as one of the possible mechanism(s) . To date, however, a detailed and critical examination of the relationship between membrane lipid fluidity and MDR reversal by these surfactants has not been performed . In the present studies, therefore, a series of experiments were conducted to critically examine the role of membrane lipid physical state in MDR reversal by employing a unique class of clinically important nontoxic lipophilic surfactants and the KB-8-5-11 drug-resistant cell line . MDR reversal was assessed by rhodamine-123 uptake . The effect of surfactants on plasma membrane lipid fluidity of these cells was assessed utilizing a fluorescence polarization technique with fluorophores DPH, TMA . DPH, 2-AS, and 12-AS . Our studies demonstrated that: (i) in vitro addition of active MDR-reversing surfactants (Solutol HS-15, Tween 40, and Cremophor EL, 10 micrograms/ml each) decreased lipid fluidity of isolated crude plasma membranes of resistant cells; (ii) the inactive surfactants (octylglucoside, hecameg) failed to influence membrane lipid fluidity; (iii) cells grown in the presence of active surfactants also exhibited a decreased plasma membrane lipid fluidity as measured with intact cells utilizing the probe TMA.DPH; and (iv) active surfactants did not influence lifetimes of the excited state of the fluorophores . These findings demonstrate that decrease of the plasma membrane lipid fluidity of KB 8-5-11 resistant cells may be one of the important mechanism(s) of MDR reversal by polyoxyethylene surfactants.

Lancet, 1995 May 6, 345(8958), 1148 - 50
Emergence of fluoroquinolone-resistant tuberculosis in New York City; Sullivan EA et al.; 22 patients infected with fluoroquinolone-resistant Mycobacterium tuberculosis in New York City were identified between January, 1991, and November, 1993 . In 16 patients resistance arose as a result of inadequate or inappropriate treatment . 6 patients had primary infection with fluoroquinolone-resistant organisms; 5 acquired the organisms nosocomially . Seven distinct patterns of restriction-fragment length polymorphism were identified in isolates from 21 patients . Fluoroquinolones should be restricted to patients with multidrug-resistant disease or intolerance to other antituberculosis drugs . All patients with multidrug-resistant tuberculosis should be on directly observed therapy.

Biochem Biophys Res Commun, 1995 May 5, 210(1), 21 - 30
Involvement of phospholipase C in heat-shock-induced phosphorylation of P-glycoprotein in multidrug resistant human breast cancer cells; Yang JM et al.; The phosphorylation of P-glycoprotein has been appreciated for many years, yet little is known about the factors that initiate this post-translational modification . To determine whether the activation of P-glycoprotein phosphorylation could occur in response to cellular stress and to investigate the possible signal pathways involved, we studied the effect of heat shock on the phosphorylation of P-glycoprotein in sensitive and resistant MCF-7 human breast cancer cells . Treatment of multidrug resistant MCF-7/AdrR cells with heat shock increased the phosphorylation of P-glycoprotein . The response was not seen in the sensitive MCF-7 line, which does not express this drug transporter . Phosphorylation of P-glycoprotein induced by heat shock was not dependent on synthesis of new proteins, since phosphorylation was not inhibited by cycloheximide and the content of P-glycoprotein, as measured by immunoblotting, did not change after heat shock . The activation of P-glycoprotein phosphorylation by heat shock may be initiated through activation of phospholipase C, since heat shock stimulated the activity of this enzyme, as evidenced by increased formation of inositol trisphosphate and diacylglycerol and by phosphorylation of phospholipase C-gamma . U-73122, an inhibitor of phospholipase C and staurosporine, an inhibitor of protein kinase C, both decreased the heat-shock-induced phosphorylation of P-glycoprotein . These results suggest that heat shock induces phosphorylation of P-glycoprotein through the activation of the phospholipase C/protein kinase C pathway.

Int J Cancer, 1995 May 4, 61(3), 375 - 80
Resistance-associated factors in human small-cell lung-carcinoma GLC4 sub-lines with increasing adriamycin resistance; Versantvoort CH et al.; Previous studies have shown that the in vitro-selected adriamycin-resistant human small-cell lung-carcinoma cell line GLC4-ADR150 displays multidrug resistance as the result of 3-fold decreased DNA-topoisomerase II (topo II) activity and a 6-fold reduction in adriamycin accumulation . Not the MDR1 gene, but the MRP gene, was over-expressed in this cell line . The aim of our study was to establish which of these drug-resistance-associated factors are already involved in drug resistance occurring at early steps of selection with adriamycin . To address this question, changes in expression of topo II alpha/topo II beta, MRP and drug accumulation were measured along with adriamycin resistance (from 2- to 10- to 150-fold) and in a partial revertant cell line (10-fold resistant) . Topo II alpha and II beta mRNA and protein levels were decreased in the resistant sub-lines, except in the 10-fold-resistant cell line . Cellular daunorubicin accumulation was decreased 1.2- to 5-fold with increasing resistance . MRP mRNA was over-expressed in all resistant sub-lines, with a marked increase in the 10-fold-resistant cells (level of expression as high as in the GLC4-ADR150 cells) . Expression of an ATP-binding 190-kDa membrane protein and Western-blot analysis with anti-MRP anti-serum ASPKE, was in accordance with the expression of MRP mRNA in all cell lines . Expression of MRP mRNA and protein, however, was not proportional with the decrease in drug accumulation in all resistant sub-lines . This study also shows that drug accumulation, topo II and MRP expression were all changed at the earliest stage of resistance development of GLC4 cells upon adriamycin selection.

Rev Assoc Med Bras, 1995 May-Jun, 41(3), 255 - 6
{Infection caused by Mycobacterium tuberculosis with primary resistance to multiple drugs: a case report of a patient with AIDS}; Grinbaum RS et al.; Primary multidrug-resistant Mycobacterium tuberculosis is an important problem in the United States . There is no report in formal literature of this pathogen in Brazilian patients . CASE REPORT--We report a case of ganglionar tuberculosis diagnosed by acid-fast smears in a male, HIV positive patient . Mode of acquisition of HIV was not determined . Treatment was started, and isoniazid, rifampicin and pyrazinamide were prescribed . The patient and his family reported strict adherence to therapy, but no improvement was observed . After 75 days, the patient was admitted in our hospital because of clinical worsening . Clinical features were the presence of large submandibular and axillar lymph nodes, respiratory insufficiency and complains of abdominal pain . He died six days after admission . Culture obtained from the ganglionar aspirate disclosed M . tuberculosis susceptible to ethambutol, but resistant to isoniazid, rifampicin, pyrazinamide, ethionamide and streptomycin . DISCUSSION--Although this was a case of extrapulmonary tuberculosis, there is a concern about multidrug-resistant tuberculosis, that has been poorly evaluated in Brazil . Since high lethality and intrahospital transmission have been reported, we discuss the need of performing culture and antibiogram in suspected cases, and the prevention of the spread of M . tuberculosis to patients and health-care workers through the strict adherence to the isolation practices.

Jpn J Cancer Res, 1995 May, 86(5), 424 - 8
High-energy underwater shock wave treatment for internal iliac muscle metastasis of prostatic cancer: a first clinical trial; Hoshi S et al.; The first clinical trial of high-energy shock wave (SW) combined with chemotherapy to treat metastasis of prostate cancer in the internal iliac muscle was conducted . The patient, a 57-year-old man, diagnosed as having mucin-producing, poorly differentiated adenocarcinoma of the prostate invading the bladder wall, had been treated by total cystoprostatectomy . Five months later, metastatic tumors were found in the left axillar subcutaneous tissue and the right internal iliac muscles . For the axillar metastasis we performed radiation and left subclavicular arterial infusion of cisplatin 70 mg, THP-adriamycin (THP) 50 mg and methotrexate 50 mg . For the right internal muscular metastasis, 10,000 to 20,000 shots of SW and simultaneous intravenous injection of carboplatin 100 mg and THP 10 mg were carried out . Neither of the tumors decreased in size, but on magnetic resonance images, the SW-treated tumor exhibited a central low-intensity area . The SW-treated tumor was resected and central necrosis and a collection of mucin in the central area were observed . Hormone-resistant prostate cancer is well-known to be a multidrug-resistant tumor . It is noteworthy that SW and chemotherapy induced necrosis in such a refractory cancer without any significant side effects.

Eur J Immunol, 1995 May, 25(5), 1457 - 60
Developmentally regulated expression of P-glycoprotein (multidrug resistance) activity in mouse thymocytes; MacDonald HR et al.; P-glycoprotein (P-gly) is the transmembrane efflux pump responsible for multidrug resistance in tumor cells . The activity of P-gly in mature peripheral lymphocytes is lineage specific, with CD8+ T cells and natural killer (NK) cells expressing high levels as compared to CD4+ T cells and B cells . We have now investigated P-gly activity in immature and mature subsets of mouse thymocytes . Our data indicate that P-gly activity is undetectable in immature CD4-8- and CD4+8+ thymocyte subsets . Among mature thymocytes, P-gly activity is absent in the CD4+ subset but present in the more mature (HSAlow) fraction of CD8+ cells . Furthermore, while thymic CD4-8- T cell receptor (TCR) gamma delta cells have little P-gly activity, a minor subset of CD4-8- or CD4+ TCR alpha beta + thymocytes bearing the NK1.1 surface marker expresses high levels of P-gly activity . Collectively, our results indicate that P-gly activity arises late during thymus development and is expressed in a lineage-specific fashion.

Leukemia, 1995 May, 9(5), 808 - 14
Characterization of a multidrug resistant human erythroleukemia cell line (K562) exhibiting spontaneous resistance to 1-beta-D-arabinofuranosylcytosine; Grant S et al.; We have assessed the response of a previously characterized multidrug resistant (MDR) human erythroleukemia cell line (K562R) to the nucleoside analog antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) . This cell line has been subjected to selection pressure by intermittent exposure to daunorubicin, but not ara-C, since its initial isolation . In comparison to the parental line (K562S), K562R were approximately 15-fold more resistant to ara-C as determined by 3H-dThd incorporation, MTT dye reduction and clonogenicity . Following a 4-h exposure to 10 microM ara-C, K562S accumulated approximately seven times more ara-CTP, and incorporated approximately 250% more ara-C into DNA than their resistant counterparts . The intracellular generation of ara-CTP was not significantly influenced by the cytidine deaminase inhibitor THU or the deoxycytidylate deaminase inhibitor dTHU (1 mM each) in either cell line . Rates of dephosphorylation of ara-CTP were equivalent in sensitive and resistant cells, as were intracellular levels of both ribonucleotide and deoxyribonucleotide triphosphates . However, K562R displayed a significant (ie 70%) reduction in the level of activity of the pyrimidine salvage pathway enzyme, deoxycytidine kinase (dCK), compared to K562S cells . In contrast to U937 leukemic cells, DNA extracted from K562S and K562R cells following exposure to 10 microM ara-C for 6 h did not exhibit the characteristic internucleosomal DNA cleavage on agarose gel electrophoresis typical of drug-induced apoptosis . Lastly, Northern analysis revealed equivalent levels of dCK message in the two cell lines . K562R represents an unusual example of a classical multidrug resistant human leukemic cell line exhibiting spontaneous cross-resistance to the antimetabolite ara-C, and may prove of value in attempts to understand the mechanism(s) by which human leukemic myeloblasts survive in vivo exposure to combination chemotherapeutic regimens containing drugs that are not classically associated with the multidrug resistance phenomenon.

Leukemia, 1995 May, 9(5), 799 - 807
Lack of correlation between expression and function of P-glycoprotein in acute myeloid leukemia cell lines; Bailly JD et al.; In a panel of acute myeloblastic leukemia (AML) cell lines, representative of distinct differentiation stages, we investigated the possible correlation between drug-resistance and both expression and function of the multidrug resistance (MDR)-related P-glycoprotein (P-gp) . The AML cell lines were KG1a, KG1, TF1, HEL, ML1, and two non drug-selected P-gp positive subclones originating from HL-60 (HL-60JD) and U937 (U937AQ) . All these cells overexpressed the mdr1 gene (analyzed by RT-PCR) and displayed variable levels of P-gp expression . Flow cytometric semi-quantitative evaluation of P-gp with two P-gp specific monoclonal antibodies (MRK16 and UIC2) showed the following P-gp expression hierarchy: TF1 < KG1a < HEL < KG1 < HL-60JD < ML1 < U937AQ; the latter expressing 13 times more P-gp than TF1 . When P-gp function was assessed by Rhodamine 123 (Rh123) efflux kinetics, we found that only KG1a and KG1 cells, which have an early (immature) CD34+ CD33- CD38- phenotype, and to a lesser extent TF1, with an intermediate (CD34+ CD33+ CD38+) phenotype, displayed significant P-gp activity which could be inhibited by both verapamil and SDZ PSC 833 . In contrast, the other more mature CD33+ CD34- AML cell lines presented no Rh123 efflux capacity although they expressed higher P-gp levels . Daunorubicin (DNR) accumulation studies showed that inhibitors of P-gp increased DNR accumulation only in the immature AML cells whereas they had no impact on the mature AML cell lines . MTT drug cytotoxicity assay confirmed that the immature AML cells were 10-15-fold more resistant to DNR than the mature AML cells . Although P-gp inhibitors were able to increase the cytotoxicity of DNR in AML cells which displayed functional P-gp, they could not increase DNR cytotoxicity to levels comparable to that of the CD34- CD33+ cells, suggesting that DNR resistance of immature AML cells may not solely be related to P-gp . With drug-selection, AML subclones displayed higher levels of P-gp expression and higher extruding capacities, and therefore chemoresistance, and this independently of their initial differentiation phenotype . Finally, this study provides evidence for a lack of correlation between expression and function of P-gp in AML cells; this relationship being dependent upon leukemic cell differentiation in unselected myeloid leukemic cells.

Genes Dev, 1995 May 1, 9(9), 1111 - 22
A multidrug resistance transporter/serine protease gene is required for prestalk specialization in Dictyostelium; Shaulsky G et al.; The prestalk-specific gene, tagB, was disrupted by restriction enzyme-mediated integration (REMI) mutagenesis . Mutant aggregates exhibit a cell-autonomous defect in specialization of PST-A cells, a prestalk subpopulation that forms the tip and eventually forms the stalk of the fruiting body . Cooperative (non-cell-autonomous) defects were found in sporulation and in specialization of prestalk cells that eventually form the upper cup of the fruiting body (PST-O) . The pattern of ecmA::lacZ expression in mutant tagB- cells defines a primary prestalk population, PST-I, from which other prestalk cells differentiate . After PST-A cells differentiate, they induce remaining PST-I cells to become PST-O cells . Subsequently, prestalk cells induce encapsulation of prespore cells during culmination . tagB is homologous to serine protease and to multidrug resistance (MDR) transporter genes, implying a mechanism of action that includes proteolysis and export of peptide signals . Intercellular communication via TagB may mediate integration of cellular differentiation with morphogenesis.

J Clin Invest, 1995 May, 95(5), 2205 - 14
Generation of a drug resistance profile by quantitation of mdr-1/P-glycoprotein in the cell lines of the National Cancer Institute Anticancer Drug Screen; Alvarez M et al.; Identifying new chemotherapeutic agents and characterizing mechanisms of resistance may improve cancer treatment . The Anticancer Drug Screen of the National Cancer Institute uses 60 cell lines to identify new agents . Expression of mdr-1/P-glycoprotein was measured by quantitative PCR . Expression was detected in 39 cell lines; the highest levels were in renal and colon carcinomas . Expression was also detected in all melanomas and central nervous system tumors, but in only one ovarian carcinoma and one leukemia cell line . Using a modified version of the COMPARE program, a high correlation was found between expression of mdr-1 and cellular resistance to a large number of compounds . Evidence that these compounds are P-glycoprotein substrates includes: (a) enhancement of cytotoxicity by verapamil; (b) demonstration of cross-resistance in a multidrug-resistant cell line, (c) ability to antagonize P-glycoprotein, increasing vinblastine accumulation by decreasing efflux; and (d) inhibition of photoaffinity labeling by azidopine . Identification of many heretofore unrecognized compounds as substrates indicates that P-glycoprotein has a broader substrate specificity than previously recognized . This study confirms the validity of this novel approach and provides the basis for similar studies examining a diverse group of gene products, including other resistance mechanisms, putative drug targets, and genes involved in the cell cycle and apoptosis.

Br J Cancer, 1995 May, 71(5), 931 - 6
Rapid up-regulation of mdr1 expression by anthracyclines in a classical multidrug-resistant cell line; Hu XF et al.; Studies were carried out in a variant human multidrug-resistant (MDR) cell line CEM/A7R, which expresses very low levels of mdr1 mRNA and P-glycoprotein (P-gp) . The induction of mdr1 RNA expression by three anthracyclines, (doxorubicin, daunorubicin, epirubicin), VP-16 and two vinca alkaloids (vincristine, vinblastine) was semiquantitatively assessed by scanning Northern blots on a phosphorimager . The relative level of mdr1 expression was expressed as ratio of mdr1 to the internal RNA (actin) . A significant increase (P < 0.02) in expression of mdr1 was noted within 4 hrs of exposure to 1.5 micrograms ml-1 daunorubicin or epirubicin . Neither vinblastine nor vincristine had any effect on mdr1 levels after an 8 h exposure . With increasing concentrations of daunorubicin or epirubicin in a fixed 24 h time period, mdr1 expression increased, although a biphasic response was seen . Based on MRK 16 binding, an increase in P-gp levels was seen in the CEM/A7R line after a 24 h exposure to 1 microgram ml-1 daunorubicin or epirubicin . The rapid increase in mdr1 expression after a short period of exposure to doxorubicin, daunorubicin or epirubicin suggests that induction of mdr1 expression may have an important role in the development of drug-resistant tumours.

Br J Cancer, 1995 May, 71(5), 907 - 13
Expression of multidrug resistance-associated protein (MRP), MDR1 and DNA topoisomerase II in human multidrug-resistant bladder cancer cell lines; Hasegawa S et al.; The acquisition of the multidrug resistance phenotype in human tumours is associated with an overexpression of the 170 kDa P-glycoprotein encoded by the multidrug resistance 1 (MDR1) gene, and also with a 190 kDa membrane ATP-binding protein encoded by a multidrug resistance-associated protein (MRP) gene . Human bladder cancer is a highly malignant neoplasm which is refractory to anti-cancer chemotherapy . In order to understand the mechanism underlying multidrug resistance in bladder cancer, we established three doxorubicin-resistant cell lines, T24/ADM-1, T24/ADM-2 and KK47/ADM, and one vincristine-resistant cell line, T24/VCR, from human bladder cancer T24 and KK47 cells respectively . Both T24/ADM-1 and T24/ADM-2 cells which had elevated MRP mRNA levels showed both a cross-resistance to etoposide and a decreased intracellular accumulation of etoposide . T24/VCR cells which had elevated levels of MDR1 mRNA and P-glycoprotein but not of MRP mRNA, showed cross-resistance to doxorubicin . On the other hand, KK47/ADM cells, which had elevated levels of both MRP and MDR1 mRNA and a decreased level of topoisomerase II mRNA, were found to be cross-resistant to etoposide, vincristine and a camptothecin derivative, CPT-11 . Our present study demonstrates a concomitant induction of increased levels of MRP mRNA, decreased levels of topoisomerase II mRNA and decreased drug accumulation during development of multidrug resistance in human bladder cancer cells . The enhanced expression of the MRP gene is herein discussed in a possible correlation with the decreased expression of the topoisomerase II gene.

Trans R Soc Trop Med Hyg, 1995 May-Jun, 89(3), 296 - 8
A comparative clinical trial of two different regimens of artemether plus mefloquine in multidrug resistant falciparum malaria; Karbwang J et al.; Plasmodium falciparum in Thailand is highly resistant to available antimalarial drugs . Artemether, a derivative of artemisinin, is a promising compound currently used to cope with this situation but the course of treatment has to be at least 5 d . An effective short treatment course of this drug is possible when used in combination with mefloquine . We now report a trial of different regimens of the combination artemether/mefloquine . Fifty-seven male Thai patients, admitted to the Bangkok Hospital for Tropical Diseases, were allocated at random to receive oral artemether 300 mg as an initial dose, followed by either the standard dose of mefloquine (750 mg) at 24 h or a higher dose of mefloquine (750 mg at 24 h, then 500 mg at 30 h) . Patients were followed up in hospital for 42 d . Two patients, both in the high dose mefloquine group, were excluded as they failed to attend for follow-up . All patients had a rapid initial response to treatment with median parasite clearance times of 37 and 40 h, median fever clearance times of 33.5 and 30.5 h, and cure rates of 75 and 96% (P = 0.0248), for the standard and high doses of mefloquine respectively . No serious adverse effect was found; mild and transient dizziness, nausea, vomiting and diarrhoea were noted in half of the patients in each group . The results suggest that a 30 h short course of artemether plus mefloquine at high dose should be used in areas with documented mefloquine resistance.

Anticancer Res, 1995 May-Jun, 15(3), 911 - 6
Modulation of doxorubicin resistance in P388/ADR cells by Ro44-5912, a tiapamil derivative; De Jong G et al.; We describe here investigations into the ability of a tiapamil derivative, Ro44-5912 to overcome multidrug resistance (MDR) in doxorubicin (ADR)-resistant murine leukemic P388 cells . This compound has the formula: C27H39NO4S2.1:2C6H8O6, a M . W . of 858 and is structurally similar to verapamil, an established inhibitor of P-glycoprotein (PGP) . We have compared the MDR modulating properties of Ro44-5912 with verapamil in P388ADR cells . Doxorubicin concentration required to achieve 50% inhibition of growth (IC50) for P388ADR cells was found to be 24 microM . In contrast, treatment of P388ADR cells with Doxorubicin and 3 microM verapamil decreased the IC50 value to 2.5 microM . A further decrease was observed with 3 microM Ro44-5912 treatment, where an IC50 value of 1.1 microM was obtained . Doxorubicin accumulation was also determined by flow cytometry in order to determine whether the increased levels of chemosensitivity observed for Ro44-5912 were reflected by increased cellular drug uptake . The results revealed that Ro44-5912, at equivalent concentration, increased doxorubicin accumulation in P388ADR cells beyond that obtained with verapamil whereas no effects were seen with the parental P388 cells . The effect of Ro44-5912 on the binding of C219 monoclonal antibody to PGP in MDR cells was also studied and found not to decrease C219 expression on P388ADR cells.

Anticancer Res, 1995 May-Jun, 15(3), 873 - 8
In vitro and in vivo studies on the anticancer activity of dehydroilludin M; Kelner MJ et al.; Six first-generation illudin analogs were tested for antitumor activity using in vitro cytotoxicity and in vivo xenograft models . One analog, dehydroilludin M, inhibited xenograft growth and prolonged life span of tumor bearing animals, when administered IP or IV, whereas the parent Illudin S compound was ineffective . The efficacy of dehydroilludin M in the MV522 lung carcinoma model exceeded that of 9 known anticancer drugs, and equaled that of mitomycin C . Dehydroilludin M retained the in vitro relative selective cytotoxicity for carcinomas and myeloid leukemia cell lines noted with the parent illudin compounds . In vitro cytotoxicity data predicted response of xenografts . Dehydroilludin M also retained the in vitro activity of the parent compounds against different multidrug resistant mdr cell lines.

Anticancer Res, 1995 May-Jun, 15(3), 863 - 6
Addition of hTNF alpha potentiates cytotoxicity of taxol in human ovarian cancer lines; Berkova N et al.; Some reports indicate that the cytotoxic activity of hTNFa and taxol might be enhanced when combined with certain chemotherapeutic agents . To assess if taxol might modulate the action of hTNF alpha, we have studied the cytotoxic action of hTNF alpha in combination with taxol on two ovarian carcinoma cell lines, the SKOV3 and SKVLB . These two cell lines are resistant to the action of hTNF alpha . The SKVLB cells overexpressed the multidrug resistant gene (MDR-1) which confers a resistance to taxol . We observed that the combination of low concentration of hTNF alpha with taxol could increase the cytotoxic activity of taxol in SKOV3 cells . Furthermore, a four-hour pretreatment with taxol before adding hTNF alpha to the culture has shown a significant increase in their cytotoxic activity . This increase was not observed at the same level in SKOV3 cells pretreated with hTNF alpha . However, the phenomena was not observed in SKVLB cells . This indicates that taxol could alter the mechanism of hTNF alpha-resistance.

Anticancer Res, 1995 May-Jun, 15(3), 811 - 4
RS-33295-198: a novel, potent modulator of P-glycoprotein-mediated multidrug resistance; Slate DL et al.; A novel multidrug resistance modulator, RS-33295-198, circumvented drug resistance in human, mouse, and Chinese hamster cell lines overexpressing P-glycoprotein . It enhanced the antiproliferative activity of doxorubicin, vincristine, etoposide, and paclitaxel and increased doxorubicin retention in multidrug-resistant hamster CHRC5 cells . RS-33295-198 modulated doxorubicin resistance in a murine P388/ADR leukemia model when administered ip via continuous minipump delivery, ip by bolus injection, and orally; it also improved the efficacy of vincristine toward P388/VCR leukemia when given ip or po . RS-33295-198 showed weak activity in enhancing doxorubicin efficacy against a multidrug-resistant human sarcoma xenograft.

Anticancer Res, 1995 May-Jun, 15(3), 745 - 9
Medroxyprogesterone-acetate reverses the MDR phenotype of the CG5-doxorubicin resistant human breast cancer cell line; Zibera C et al.; In malignant cells multidrug resistance (MDR) is frequently associated with the expression of a 170 KDa P-glycoprotein (P-gp) in the plasma membrane . P-gp acts as an ATP-dependent efflux pump causing a decreased intracellular accumulation of structurally unrelated natural anticancer agents such as anthracyclines . Doxorubicin (DX) resistance is mostly related to the multidrug resistance gene product P-gp . In our experiments the revertant activity of medroxyprogesterone acetate (MPA) in comparison to that of the well known revertant agent verapamil (VRP) was investigated . In vitro tests were carried out on a DX-resistant variant (CG5/DX) obtained in our laboratory from the parental CG5 human breast cancer cell line by continuous exposure to the drug . The ability of MPA to modulate intracellular DX accumulation and to reverse MDR was evaluated . MPA appeared more active than VRP in reversing MDR, suggesting a possible role of this synthetic progestin as chemosensitizing agent in the clinical management of anthracycline-resistant breast cancer.

Anticancer Res, 1995 May-Jun, 15(3), 1117 - 21
Correlation of P-glycoprotein overexpression and cellular prognostic factors in formalin-fixed, paraffin-embedded tumor samples from breast cancer patients; Schneider J et al.; BACKGROUND: P-glycoprotein overexpression seems to play a prognostic role independent from its association with multidrug resistance . We have been able to verify this previously on fresh-frozen tumor samples, but up to now P-glycoprotein detection on formalin-fixed, paraffin-embedded material has generally been unsatisfactory . METHODS: Two distinct groups of previously untreated mammary carcinomas were studied for P-glycoprotein expression by means of immunohistochemistry, using the NCL-p-GLYp polyclonal antibody, carried out on slides from archival, paraffin-embedded material . The first group was composed of 15 tumors expressing at least three out of four factors indicative of a worse prognosis: c-erb-B2, mutant p53, cathepsin-D and PCNA (proliferating cell nuclear antigen) . The control group was composed of 16 tumors lacking expression of any of these proteins . RESULTS: There was a statistically significant difference in survival (Fisher's exact test, p = 0.0036) between both groups . All four patients who died during the follow-up period (mean 55.1 months) belonged to the first group . Three of these patients harbored tumors with the highest level of expression of P-glycoprotein . This association between P-glycoprotein overexpression and prognosis was also statistically significant (Fisher's exact test, p = 0.0059) . CONCLUSION: These results, obtained from a group of mostly operable tumors stratified for cellular risk factors, complement those previously reported by us for locally advanced, inoperable breast cancer only, using fresh-frozen tissue . P-glycoprotein again seems to play a prognostic role, independent of its involvement in multidrug-resistance . The technique used by us yielded useful results on formalin-fixed, paraffin-embedded archival tumor samples . This opens the possibility for larger studies on populations with a closed follow-up.

Anticancer Res, 1995 May-Jun, 15(3), 1023 - 32
Current aspects of the pathology of osteosarcoma; Grundmann E et al.; Since the introduction of standardized chemotherapy protocols of osteosarcoma a lot of new aspects in prognosis and curability of these have best developed . Current subclassification which divided osteosarcoma into a conventional type and eleven important recognizable varieties is one of the reason for this success . Cytological grading also serves as a good indicator for the prognosis and is an important criterion for application of adjuvant chemotherapy . Several structure proteins of the extracellular matrix have gained importance in making the diagnosis of an osteosarcoma . Immunohistochemically and biochemically evaluations could show that different collagenous-proteins can be useful for the differential diagnosis of bone tumors . The integration of molecular pathologic methods into the structural morphologic findings will be helpfull in the identification of mutated structure proteins . Oncogenes and tumor suppressor genes are of major importance for the tumorigenesis of osteosarcoma . The prognostic significance of the inactivation of p53 and RBI gene remains to be elucidated . Resistance to chemotherapy is the major mechanism responsible for the failure of osteosarcoma treatment . The main cause for this failure is multidrug resistance, which is often related to a plasma membrane protein, the P-glycoprotein . Immunohistologic investigations of P-glycoprotein are not sufficient to demonstrate the possible association between overexpression of this protein and tumor progression.

Eur J Nucl Med, 1995 May, 22(5), 434 - 42
Evaluation of tumour metabolism and multidrug resistance in patients with treated malignant lymphomas; Dimitrakopoulou-Strauss A et al.; The management of patients with treated malignant lymphomas requires functional methods to differentiate a residual soft tissue mass . Patients with treated Hodgkin's lymphoma (HL, n = 20, 68 malignant lesions, three benign lesions) or non-Hodgkin's lymphoma (NHL, n = 26, 46 malignant lesions, one benign lesion) were studied with positron emission tomography (PET) and fluorine-18 deoxyglucose (FDG) . Oxygen-15 labelled water was used (n = 14, 25 lesions) in addition to FDG in order to obtain information on the tissue perfusion . Long-term follow-up studies with PET and FDG were performed in nine patients up to 511 days after the initiation of second-line therapy . Fourteen patients underwent single-photon emission tomography (SPET) with technetium-99m sestamibi immediately prior to the first PET examination . PET with FDG displays a high sensitivity for the detection of viable tumour tissue, all the malignant lesions being correctly classified in this study . The possible limitations are inflammatory processes, which may obscure tumour detection due to increased FDG uptake, and malignant lesions with low FDG uptake due to reduced perfusion . Difficulties exist in the prognosis of long-term response, since the change in FDG uptake may be variable . Long-term therapy outcome was correlated with the slope values obtained from the standardized integral uptake (SIU) data, which provides a new approach for the evaluation of PET follow-up studies . 99mTc-sestamibi, which should reflect the multidrug resistance, was evaluated with respect to therapy outcome . A high uptake of 99mTc-sestamibi was observed in patients with stable disease or better . The data support the hypothesis that sestamibi may reflect multidrug resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Thorax, 1995 May, 50(5), 487 - 9
Prospects for global health: lessons from tuberculosis; Benatar SR; PIP: Preventable diseases continue to afflict billions of people worldwide in both rich and poor countries . With regard to tuberculosis (TB), much has been learned over the past century about Mycobacterium tuberculosis, the responsible infectious agent, and its medical treatment and cure . TB is, however, an old disease currently making a resurgence at the global level . An estimated one third of the world's population is infected with M . tuberculosis and HIV infection is increasing the proportion of those in whom infection will progress to TB disease . HIV and M . tuberculosis infections co-exist most extensively in the poorest parts of the world . TB control programs are inadequate, the degree of multidrug resistance is growing, and infections are increasingly transmitted freely across international borders . The combination of these factors suggests that scientific progress and humanitarian aid to developing countries may not be enough to avert the potential tragedy of untreatable TB . The author discusses the history of TB, the present situation, and the future .

Exp Toxicol Pathol, 1995 May, 47(2-3), 157 - 66
Alterations of melanin synthesis in human melanoma cells selected in vitro for multidrug resistance; Stromskaya TP et al.; Previous data showing the correlation of multidrug resistance (MDR) and differentiation in tumor cell populations (Melloni et al . 1988; Stavrovskaya et al . 1990) suggest that: 1) isolation of MDR cells by cytostatic drugs leads to the selection of more differentiated cell variants and 2) in more differentiated cell variants the activity of MDR-related P-glycoprotein (Pgp) is more prominent than in less differentiated cells . Here we used human melanoma cell line mS and two variants selected from mS population: a) MDR variant of mS selected by colchicine (mS-0.5) and b) mS-trRAR/2--variant obtained by introduction of expressing retinoic acid receptor RAR-alpha cDNA into mS cell . The differentiation status, expression of MDR1 gene and Pgp functioning were compared in wild-type cells and mS variants . Electron microscopic examination of melanosomes showed that the mS-0.5 subline comprised more differentiating cells in the population than parental mS cultures and that these cells were at later stages of melanogenesis . The increase in the degree of differentiation in mS-0.5 population coincided with MDR1 gene overexpression, occurrence of Pgp molecules on the cell membrane and acceleration of Pgp-mediated Rhodamine 123 (Rh123) efflux . mS-trRAR/2, proved to be more differentiated than mS cells . The MDR1 mRNA level and Rh123 efflux were not elevated in mS-trRAR/2 cells, however, retinoic acid (RA) treatment increased both the degree of differentiation and Rh123 efflux in mS-trRAR/2 to a greater extent than in mS cultures . Thus, the data obtained in this study are in favor of the suppositions mentioned above . The mechanisms of coordinated alterations of differentiation and Pgp activity in MDR cells are discussed.

Ann Acad Med Singapore, 1995 May, 24(3), 442 - 6
Multidrug-resistant tuberculosis; Lee SK et al.; In 1993, the World Health Organization declared tuberculosis a global emergency . Tuberculosis is the leading cause of death attributable to a single infectious pathogen . One-third of the world's population are at risk of developing the disease . In countries confronted with the human immunodeficiency virus (HIV) epidemic, the overlap of these two populations leads to a rapid acceleration of active tuberculosis and of the emergence of multidrug-resistant tuberculosis . Multidrug-resistant tuberculosis is defined as isolates resistant to both isoniazid and rifampicin with or without resistance to other antituberculosis drugs . In the United States, outbreaks of multidrug-resistant tuberculosis have been reported in patients with HIV infection and acquired immunodeficiency syndrome (AIDS) as well as HIV sero-negative patients . These reports have caused great concern owing to the very high case-fatality rate . The treatment outcome of multidrug-resistant tuberculosis is poor . The use of second-line drugs is frequently associated with toxicity and intolerance . Patients require admission to hospitals at the beginning of treatment and adjunctive resectional surgery should be considered when the sputum does not convert after 4 months of therapy . The incidence of multidrug-resistant tuberculosis among Singapore residents remains low . Organisms resistant to one drug occurred in 3.8% of newly diagnosed tuberculosis cases with positive culture and 8.7% of relapsed tuberculosis cases with positive cultures . Organisms resistant to two or more drugs occurred in 1.6% of newly diagnosed culture positive tuberculosis cases and 4.6% of relapsed cases where cultures were positive . This is the result of vigilant surveillance, effective treatment including supervised chemotherapy and close monitoring for non-compliance.

Chem Pharm Bull (Tokyo), 1995 May, 43(5), 818 - 28
N-alkylated 1,4-dihydropyridines: new agents to overcome multidrug resistance; Ohsumi K et al.; New N-alkylated 1,4-dihydropyridine derivatives were synthesized and their ability to overcome multidrug resistance was examined in vincristine-resistant P388 cells (P388/VCR cells) . Compounds that possessed an arylalkyl substituent on the dihydropyridine ring nitrogen were more potent than verapamil in potentiating the cytotoxicity of vincristine against P388/VCR cells . However, neither drug effectively enhanced the antitumor activity of vincristine in tumor-bearing mice . Introduction of basic nitrogen-containing substituents on the side chain of 1,4-dihydropyridines gave improved activity in vitro and in vivo . The piperazine derivative 12c and 12o were more than 10 times as potent as verapamil in vitro . Four compounds selected for in vivo testing showed superior antitumor activity in P388/VCR-bearing mice in combination with vincristine . The structure-activity relationships of the compounds are discussed.

Nucl Med Biol, 1995 May, 22(4), 497 - 504
In vivo imaging and specific targeting of P-glycoprotein expression in multidrug resistant nude mice xenografts with {125I}MRK-16 monoclonal antibody; Scott AM et al.; Multidrug resistance (MDR) in tumors is associated with P-glycoprotein (Pgp) expression . In vivo quantitation of Pgp may allow MDR to be evaluated noninvasively prior to treatment planning . The purpose of this study was to radiolabel MRK-16, a monoclonal antibody that targets an external epitope of P-glycoprotein, and perform in vivo quantitation of P-glycoprotein in a MDR xenograft nude mouse model . MRK-16 was labeled with 125I by the iodogen method, with subsequent purification by size exclusion chromatography . Groups of 10 Balb c mice were each xenografted with colchicine-resistant or sensitive neuroblastoma cell lines, respectively . Whole body clearance and tumor uptake over time was quantitated by gamma camera imaging, and biodistribution studies were performed with {125I}MRK-16 and an isotype matched control antibody, A33 . Quantitative autoradiography and immunohistochemistry analysis of tumors was also evaluated to confirm specific targetting of {125I}MRK-16 . Peak tumor uptake was at 2-3 days post-injection, and was significantly greater in resistance compared to sensitive tumors (mean % injected dose/g +/- SD) (18.76 +/- 2.94 vs 10.93 +/- 0.96; p < 0.05) . Quantitative autoradiography verified these findings (19.13 +/- 0.622 vs 12.08 +/- 0.38, p < 0.05) . Specific binding of {125I}MRK-16 was confirmed by comparison to {131I}A33 in biodistribution studies, and localized to cellular components of tissue stroma by comparison of histologic and autoradiographic sections of sensitive and resistant tumors . Immunoblot analysis demonstrated a 4.5-fold difference in P-glycoprotein expression between sensitive and resistant cell lines without colchicine selective pressure . We conclude that in vivo quantitation of P-glycoprotein in MDR tumors can be performed with {125I}MRK-16.(ABSTRACT TRUNCATED AT 250 WORDS)

Leuk Lymphoma, 1995 May, 17(5-6), 367 - 74
Intrinsic expression of the multidrug transporter, P-glycoprotein 170, in multiple myeloma: implications for treatment; Pilarski LM et al.; Multidrug resistance, mediated by the P-glycoprotein 170 transport pump, is a serious problem in multiple myeloma . In this review we discuss the expression of P-gp as a differentiation antigen on normal T and B lymphocytes . In myeloma, circulating presumptively malignant B cells express P-gp prior to chemotherapy . A variety of evidence characterizes these circulating B cells as members of the malignant clone in myeloma, including the demonstration that they share immunoglobulin heavy chain (IgH) rearrangements with bone marrow plasma cells, and their extensive DNA aneuploidy . In some patients the only components of the clonal populations that express P-gp are the circulating B cells suggesting that they represent a reservoir of multidrug resistant cells that maintain malignant growth and spread in myeloma . We speculate that exposure to chemotherapy alters clonal homeostasis and exerts positive selection pressure on generative components of the myeloma clone . Thus the possibility exists that chemotherapy perpetuates rather than eradicates myeloma stem cells . P-gp is detectable on bone marrow plasma cells in myeloma but appears to be in an inactive form that is unable to mediate efflux of marker dyes . A similar phenomenon is seen for normal human monocytes which have surface P-gp but lack any functional export of P-gp substrates . P-gp appears to vary depending in a cell-type specific manner suggesting that it may be feasible to design inhibitors of P-gp which selectively block P-gp export by malignant cells and spare the function of P-gp on normal tissue, including lymphocytes and normal hematopoietic stem cells.

Oncology (Huntingt), 1995 May, 9(5), 417 - 24; discussion 429-30
Biologic and clinical advances in multiple myeloma; Varterasian ML; Recent advances in our understanding of the cellular and molecular derangements involved in multiple myeloma are beginning to be translated into novel therapeutic approaches . Growth factors, specifically interleukin-6, appear to be critical for disease progression, and interruption of autocrine and paracrine loops has been achieved, with resultant inhibition of myeloma cell growth . Oncogenes, tumor-suppressor genes, and cell-survival genes have all been found to be dysregulated in some myeloma patients . The implications of acquisition of the multidrug resistance phenotype are just beginning to be understood . High-dose therapeutic regimens with bone marrow or peripheral stem-cell rescue are being studied in an attempt to produce a cure . Autologous marrow and peripheral blood stem-cell transplantation are better suited to the older myeloma patient population than is allogeneic marrow transplantation, and have also yielded promising results.

J Biol Chem, 1995 Apr 28, 270(17), 10334 - 41
Interaction of the P-glycoprotein multidrug transporter with peptides and ionophores; Sharom FJ et al.; P-glycoprotein functions as an ATP-driven active efflux pump for many cytotoxic drugs . We now show that hydrophobic peptides and ionophores also interact with the multidrug transporter . Multidrug-resistant cells are cross-resistant to several hydrophobic peptides and ionophores, but not to some other membrane-active species . Linear peptides, cyclic peptides, and ionophores stimulated the ATPase activity of P-glycoprotein in plasma membrane vesicles by up to 2.5-fold . Drugs and chemosensitizers were able to block P-glycoprotein ATPase stimulation by verapamil, however, peptides and ionophores (with the exception of cyclosporine A) were unable to do so . Peptides and ionophores also effectively inhibited ATP-dependent drug transport by P-glycoprotein in plasma membrane vesicles . The median effect analysis was used to extract quantitative parameters from the drug transport inhibition data . Unlike drug substrates and cyclic peptides, linear peptides did not inhibit photoaffinity labeling of P-glycoprotein by {3H}azidopine . Taken together, these results indicate that certain hydrophobic peptides and ionophores are P-glycoprotein substrates, however, they affect the transporter in a different manner from drugs . Linear peptides interact with P-glycoprotein at a site distinct from those for verapamil and azidopine, whereas the interaction site for cyclic peptides and ionophores appears to be linked to these sites to varying degrees . Export of hydrophobic peptides may be an important physiological function of P-glycoprotein.

Mol Cell Endocrinol, 1995 Apr 28, 110(1-2), 205 - 11
Increased resistance to cytotoxic agents in ZR75B human breast cancer cells transfected with epidermal growth factor receptor; Dickstein BM et al.; Human breast cancer cells selected for multidrug resistance frequently overexpress ligands and receptors in the epidermal growth factor (EGF) receptor family . To determine whether this overexpression contributes to the drug resistant phenotype, EGF receptor transfected ZR75B human breast cancer cells were examined . Two EGF receptor overexpressing clones were evaluated: clone 11 with > 1 x 10(6) sites, and clone 13 with 310,000 receptor sites/cell . These were compared with clone 2-neo, which was transfected with the neomycin gene only and contained 43,000 receptor sites/cell . The EGF receptor overexpressing clones and the neo transfected control clone displayed comparable growth rates . Cytotoxicity analyses were performed with doxorubicin, vinblastine, cisplatin and 5-fluorouracil to determine the sensitivity of the clones to antineoplastic drugs . The EGF receptor overexpressing clones were found to be 1.5-5.6 times more resistant to the four drugs tested . This increase in the IC50 conferred a selective advantage when grown in the presence of 2, 3 and 6 ng/ml doxorubicin . Clone 13 cells overtook a mixed population which began with clone 2-neo comprising 95% of the cells . Clone 2-neo remained the dominant clone in the absence of drug . Finally, after long-term selection of the clones with 6 ng/ml doxorubicin, clone 2-neo became fourfold more resistant than the unselected clone 2-neo, a level which was comparable to that found in the EGF receptor overexpressing clones 11 and 13 . No additional increase in resistance was observed for these clones, suggesting that clone 2-neo had developed additional resistance mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1995 Apr 24, 156(2), 313 - 4
Analysis of a novel cDNA encoding a C219-reactive peptide isolated from methotrexate-selected multidrug-resistant human leukemic cells; Norris MD et al.; We have isolated and sequenced a novel cDNA clone (D320), from methotrexate-selected multidrug-resistant (MDR) human leukemic CCRF-CEM cells, which encodes a peptide reactive with monoclonal antibody (mAb) C219 . Despite having been cloned using this ostensibly P-glycoprotein-specific mAb, clone D320 has no significant homology with either the MDR-encoding gene or, at the amino acid (aa) level, with its product, P-glycoprotein . A putative C219-binding site has been identified in D320, which differs at two positions from the 6-aa C219 epitope previously described in P-glycoprotein.

Gene, 1995 Apr 24, 156(2), 287 - 90
Differential utilization of multiple transcription start points accompanies the overexpression of the P-glycoprotein-encoding gene in Chinese hamster lung cells; Ince TA et al.; The overproduction of P-glycoprotein (Pgp) has been associated with the development and maintenance of the multidrug resistant (MDR) phenotype, although the regulatory events responsible have not yet been elucidated . We have analyzed the overexpression of the TATA-less hamster class-I Pgp-encoding gene (Pgp1) in several MDR Chinese hamster cell lines . The MDR lung cell line DC-3F/VCRd5L, as well as the MDR ovary cell line CHRC5, express a level of Pgp1 RNA commensurate with the increase in Pgp1 dosage; in contrast, the actinomycin D (ActD)-selected sublines of DC-3F overexpress Pgp1 mRNA without a concomitant increase in Pgp1 gene-copy number . Analysis of Pgp1 transcription start point (tsp) utilization revealed that drug-sensitive DC-3F cells, as well as DC-3F/VCRd5L and CHRC5 cells, utilize one major tsp; in contrast, the ActD-resistant sublines 'switch' to a more complex pattern, using four additional Pgp1 tsp 32, 42, 52, and 67 bp downstream from the major parental tsp (+1) . This observation of a difference in the regulation of transcription of Pgp in MDR vs . drug-sensitive cells suggests that the 'switch' in tsp selection may be involved in the increased expression of Pgp1 mRNA . Interestingly, despite the existence of several hundred MDR cell lines, very few have been analyzed with respect to tsp selection; it is therefore possible that alternate tsp selection is a relatively common yet heretofore unobserved component of the MDR phenotype . Moreover, these cells provide an excellent system in which to evaluate the sequence elements and protein factors that govern the selection of tsp in TATA-less promoters.

Virology, 1995 Apr 20, 208(2), 634 - 43
Harvey murine sarcoma virus/MDR1 retroviral vectors: efficient virus production and foreign gene transduction using MDR1 as a selectable marker; Metz MZ et al.; Retroviruses are used for a variety of applications requiring the delivery of exogenous genes to cells and animals . For many of these applications, including gene therapy, safer and more efficient retroviral vectors are needed . Vectors based on Harvey murine sarcoma virus (HaMSV) are attractive because nearly all their viral sequences outside of the LTRs are derived from rat endogenous VL30 retroviruses . These sequences are not homologous to the functional viral mRNAs in commonly used retrovirus packaging cell lines, the packaging and dimerization domains of HaMSV are small and contain no splice donor sites, and the 5' sequences of HaMSV appear to confer efficient packaging and stability on genomic RNAs . HaMSV/MDR1 vectors use the human multidrug resistance gene as a dominant, selectable, amplifiable marker for gene delivery, but current versions of these vectors are large, with over 3300 nt of HaMSV sequences downstream of MDR1 . We analyzed the requirement for these downstream sequences in HaMSV vectors and found that modified HaMSV/MDR1 vectors lacking virtually all viral sequences downstream of MDR1 support the production of high-titer retroviruses and the efficient transduction, selection, and amplification of MDR1 . A reduced-size HaMSV/MDR1 vector was further modified to include a second heterologous gene under the control of an internal SV40 promoter . Using MDR1 as a selectable marker, we obtained efficient virus production, gene transduction, and expression of MDR1 plus the heterologous gene.

Biochem Biophys Res Commun, 1995 Apr 17, 209(2), 497 - 505
Selection and characterization of verapamil-resistant multidrug resistant cells; Cano-Gauci DF et al.; Multidrug resistant cells may become acutely sensitive to the calcium channel blocker verapamil, in spite of the fact that its accumulation by these cells is negligible . We selected verapamil-resistant mutants from multidrug resistant Chinese hamster ovary cells . Levels of P-glycoprotein expression and cross-resistance profiles remained unaltered in the verapamil-resistant multidrug resistant cells . As well, a photoactive verapamil analog specifically bound to P-glycoprotein in these cells . We had previously used a photoactive anthracycline to show that calcium antagonists and several anticancer drugs bind to P-glycoprotein at overlapping or interacting sites . Verapamil and its analogues no longer inhibit the binding of either anticancer drugs or calcium channel blockers to P-glycoprotein . Sequencing of P-glycoprotein revealed that no change had occurred in the coding sequence as a result of the selection procedure.

Cancer Res, 1995 Apr 15, 55(8), 1633 - 8
Relationship of multidrug resistance to rhodamine-123 selectivity between carcinoma and normal epithelial cells: taxol and vinblastine modulate drug efflux; Brouty-Boye D et al.; Preferential retention and cytotoxicity of Rhodamine-123 (Rho-123) was originally reported in a number of carcinoma cell types isolated from a variety of tissues as compared to normal epithelial cells from a limited number of other tissues . In the present study, we have examined Rho-123 selectivity in normal and tumor cell lines isolated from the same tissue source, i.e., human breast . We found that: (a) in matched pairs of normal and carcinoma breast cells, Rho-123 displays no preferential retention in either cell type; (b) there is no preferential toxicity in carcinoma as compared to normal breast cells; in fact, one of the carcinoma cell lines (MDA-MB231) shows moderate resistance to this dye; (c) all of the human breast cell lines do not express P-glycoprotein-mediated multidrug resistance; (d) the normal monkey kidney epithelial cell line CV-1, which was originally used as a model to demonstrate the relative resistance of normal epithelial cells to this drug, is found to express high levels of the mdr-1 gene, is resistant to other multidrug-resistant drugs (taxol and vinblastine), and its resistance to Rho-123 as well as decreased Rho-123 retention can be reversed by verapamil; and (e) taxol and vinblastine are found to block increased Rho-123 efflux in CV-1 cells . Thus, overall the data suggest that preferential retention and cytotoxicity of Rho-123 in carcinoma versus normal epithelial cells is related to the differential expression of the mdr-1 gene.

Blood, 1995 Apr 15, 85(8), 2147 - 53
Predictive value for treatment outcome in acute myeloid leukemia of cellular daunorubicin accumulation and P-glycoprotein expression simultaneously determined by flow cytometry; Guerci A et al.; To evaluate the clinical relevance of multidrug resistance (MDR) phenotype, the intracellular daunorubicin accumulation (IDA) and P-glycoprotein (P-gp) expression were investigated in 87 adult patients with acute leukemia: 69 patients with de novo acute myeloid leukemia (AML), 10 with AML at relapse, and eight with secondary leukemia to myelodysplastic syndromes (MDS-AML) . IDA and P-gp expression were determined by double-labeling flow cytometry analysis . Of 87 patients, 36 expressed P-gp (41%) . P-gp expression was more frequently observed in AML at relapse and MDS-AML as compared with de novo AML (P = .0001) . P-gp expression was significantly associated with CD34 expression (P = .0003) and chromosome 7 abnormalities (P = .027) . A significantly reduced IDA was observed in P-gp+ as compared with P-gp- patients (P = .0007) . Of the 87 patients, 51 achieved complete remission (CR) . A reduced IDA was observed in patients in failure as compared with patients in CR (22% +/- 17% v 42% +/- 21%; P = 10(-4) . Twelve of 36 P-gp+ patients as compared with 40 of 51 P-gp- patients achieved CR (33% v 78%; P = 10(-4) . The prognostic value of IDA and P-gp expression was confirmed in multivariate analysis . These data suggest that the determination of IDA and P-gp expression may be useful in designing therapy for patients with AML.

Biochim Biophys Acta, 1995 Apr 6, 1266(1), 1 - 8
Inhibition of lysosomal acid sphingomyelinase by agents which reverse multidrug resistance; Jaffrezou JP et al.; An increasing body of evidence appears to implicate the lipid bilayer of multidrug resistant (MDR) cells with P-glycoprotein activity . Several cationic amphiphilic drugs (CADs) have been extensively described as modulators of MDR . These same agents are also known to (1) inhibit lysosomal acid sphingomyelinase (ASmase), a phospholipid degrading enzyme, and/or (2) induce phospholipidosis in animal tissues or cultured cell lines . In this report, we randomly selected 17 CADs and evaluated their potency in modulating MDR in the murine MDR P388/ADR leukemia cell line . We compared these results with their ability to inhibit ASmase and observed a significant dose-dependent linear relationship (95% central confidence interval), between ASmase inhibition and MDR reversal . This approach permitted us to identify three new modestly potent chemosensitizers: trimipramine, desipramine, and mianserine . Modulation of MDR was not cell line specific, since CADs at 10 microM increased doxorubicin (DOX) and vinblastine (VBL) (but not methotrexate, MTX) cytotoxicity in both P388/ADR and the human MDR cell lines MES-SA/Dx5 and K562/R7, but not in the parental drug-sensitive cells . Although all chemosensitizing CADs at 10 microM significantly increased Rhodamine-123 (Rho-123) accumulation in the human leukemia MDR cell line K562/R7 and most presented significant displacement of the photoaffinity labelling probe iodoarylazidoprazosin, no correlation between these observations and the ability of CADs to sensitize MDR cells to DOX and VBL was found . In conclusion, our study strongly suggests that the chemosensitizing potency of agents such as CADs may be due to a dual mechanism of action: direct antagonism of P-gp activity and indirect modulation of P-gp activity through the disruption of cellular lipid metabolism.

Biochemistry, 1995 Apr 4, 34(13), 4402 - 11
Membrane topology of P-glycoprotein as determined by epitope insertion: transmembrane organization of the N-terminal domain of mdr3; Kast C et al.; P-Glycoproteins (P-gps) are membrane glycoproteins encoded by the mdr gene family, and their overexpression is associated with multidrug resistance (MDR) . Sequence analyses of mdr cDNAs predict a protein formed by two symmetrical halves, each composed of six transmembrane (TM) segments and one ATP-binding domain . To determine the topology of the N-terminal half of P-gp, a small antigenic peptide epitope (YPYDVPDYAIEGR) containing part of the hemagglutinin (HA) of influenza virus was inserted at six different positions of the Mdr3 protein (101, 161, 206, 244, 320, and 376) . Functional integrity of the modified proteins was tested by measuring their capacity to confer MDR in Chinese hamster ovary cells . Intracellular and extracellular localization of the tag in the full-length protein was determined in intact or permeabilized cells by immunofluorescence using a mouse monoclonal antibody (12CA5) specific for the HA epitope . While insertions at positions 101, 161, 320, and 376 did not alter P-gp function, insertions at positions 206 and 244 abrogated the capacity of P-gp to confer drug resistance . The epitope tags inserted at positions 161 and 376 were found to be located intracellularly, whereas the tags at positions 101 and 320 were located on the extracellular side of the membrane . These results indicate that the intervening segments separating predicted TM1-TM2 and TM5-TM6 correspond to extracellular regions, while the segments linking TM2-TM3 and the one located downstream of TM6 correspond to intracellular regions . These results are consistent with a six TM domain model for the N-terminal half of P-gp with an extracellular glycosylated region (TM1-TM2) and an intracellular ATP-binding site (downstream TM6) . Epitope insertion in segments linking TM3-TM4 and TM4-TM5 caused a loss of P-gp function, suggesting that the integrity of these sequences is essential either for drug transport or for proper maturation and accurate targeting of P-gp to the plasma membrane.

Gen Physiol Biophys, 1995 Apr, 14(2), 171 - 5
Effect of phorbol myristate acetate (PMA) on P-glycoprotein mediated vincristine resistance of L1210 cells; Barancik M et al.; Effect of phorbol myristate acetate (PMA) on P-glycoprotein (P-GP)-mediated vincristine resistance of the multidrug resistant mouse leukemic cell line L1210/VCR was studied by one hour lasting incubation of cells in the presence of PMA, and after three days of cultivation in the presence of the same substance . After the incubation with 100 micrograms.1-1 PMA the accumulation of {3H}-vincristine by the above cells was significantly depressed . Moreover, full reverse of verapamil-induced stimulation of {3H}-vincristine accumulation was observed in the presence of PMA . In contrary, when cells were cultivated three days in the presence of PMA, only slight but non-significant increase of {3H}-vincristine accumulation was observed . Slight increase of vincristine accumulation by cells cultivated in the presence of PMA was also supported by higher sensitivity of these cells to vincristine.

Ther Immunol, 1995 Apr, 2(2), 105 - 13
Perspectives for the control of tuberculosis; Rosen H et al.; The control of tuberculosis has become more elusive with the increased incidence of HIV, and the continued selection of multidrug-resistant organisms . The intracellular pathogenesis of, and host-response to Mycobacterium tuberculosis present challenges to both classical chemotherapeutic and vaccination approaches, with the organism able to replicate in an unrestricted manner in lung but not other tissues . Adequate control of this pathogen, that has evolved so successfully for its symbiotic exploitation of man, will require complex approaches including additional chemotherapeutics of more acceptable toxicity and efficacy, vaccination and commitment to public health measures . In this review, the worldwide scope of the disease is outlined, and its direct and indirect costs considered . The organism enjoys the protective advantages of a slow replication and of a specialized phagolysosomal intracellular niche, requiring a host-response capable of breaching these cellular barriers . The challenges of current vaccine approaches, including live vaccines, target antigen selection and the antigen delivery and adjuventation necessary to elicit adequate pulmonary responses are discussed . Our current understanding is inadequate to control TB and the rekindling of fundamental experimental approaches to the organism, and the host response it evokes, are essential to generate the preventative and therapeutic means necessary for its worldwide control.

Leuk Lymphoma, 1995 Apr, 17(3-4), 289 - 94
P170 glycoprotein expression and impaired anthracycline retention in chronic myeloid leukaemia; Damiani D et al.; Chronic myeloid leukaemia (CML) is a well known model of a disease refractory to chemotherapy, including anthracyclines and other drugs that are believed to be pumped out of the cells by a 170 Kd transmembrane glycoprotein (P170) . In 35 cases of Ph+ CML we investigated the reactivity of leukaemic cells to a P170-directed monoclonal antibody (MRK-16), by means of flow cytometry . P170 overexpression was found in 4/14 (29%) chronic phase CML cases and in 16/23 (70%) accelerated and blastic phase CML cases (P = 0.01) . The same cells were assayed for their ability to retain Daunorubicin and Idarubicin after 2-hours in vitro incubation with 1000 ng/ml of either drug . It was found that anthracycline cell concentration was negatively related with the degree of the reactivity to MRK-16 . In accelerated and blastic phase, CML cells simultaneously expressed P170 and the stem cell related marker, CD34 . These data confirm that Ph+ leukaemic cells overexpress P170, show that P170 overexpression is functionally relevant, and suggest that P170-related multidrug resistance may be an important factor for chemotherapy failure in Ph+ CML.

Anticancer Drugs, 1995 Apr, 6(2), 291 - 302
Selection of three out of 24 anti-cancer agents in poorly-differentiated gastric cancer cell lines, evaluated by the AUC/delta IC50 ratio; Nozue M et al.; The purpose of this study was to screen 24 anti-cancer drugs, either in use or in clinical study, using four cell lines, all of which originated from poorly-differentiated gastric cancers . The MTT assay was used at 1, 6, 24 or 72 h exposure times as the chemosensitivity test . We also examined P-glycoprotein expression, mdr-1 gene amplification and the modifier effect of verapamil . All four cell lines generally showed the same chemosensitivity pattern, while GCIY cells showed mdr-1 gene amplification and P-glycoprotein expression, and KATOIII cells showed the multidrug resistant pattern without P-glycoprotein expression . Both cell lines acquired higher chemosensitivity after verapamil addition . All IC50 data (with or without verapamil) were multiplied by exposure time (delta IC50) and compared with the clinical 'area under the concentration curve (AUC)' . SN-38 with/without verapamil, cisplatin with verapamil and pirarubicin with/without verapamil seemed to be the best candidates for poorly-differentiated gastric cancer chemotherapy . Plant alkaloids could also be candidates . With further experiments, we may be able to deduce commonly effective chemotherapy for poorly-differentiated gastric cancer from these drugs.

Anticancer Drugs, 1995 Apr, 6(2), 213 - 8
Multidrug resistance; Tsuruo T et al.; Since we found verapamil as a multidrug resistance (MDR) reversing agent in 1981, many MDR reversing compounds have been reported . This type of drug must have strong effects with little side effects . We recently found MS-209 and PSC-833 as reversing agents . These two compounds interacted directly with P-glycoprotein, and showed a good MDR reversing effect in vitro and in vivo . MRK16, an antibody against P-glycoprotein, also showed a good therapeutic effect against drug resistant human tumors . MS-209, PSC-833 and the antibody against P-glycoprotein are interesting candidates for clinical use in the future.

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