Eur J Biochem, 1992 Jul 15, 207(2), 567 - 79 Kinetics of daunorubicin transport by P-glycoprotein of intact cancer cells; Spoelstra EC et al.; Drug permeation across the plasma membrane of multidrug-resistant cells depends on the kinetics of the P-glycoprotein-mediated pump activity as well as on the passive permeation of the drug . We here demonstrate a method to characterize kinetically the pump in intact cells . To this purpose, we examined the membrane-transport properties of daunorubicin in various sensitive cancer cell lines and in their multidrug resistant (MDR) counterparts . First, we determined the passive permeability coefficient for daunorubicin . Then, using a flow-through system, the drug flux into the cell was measured after inhibition of the P-glycoprotein-mediated efflux pump . Combining the two results allowed us to calculate the intracellular free concentration of the drug . In the steady-state, the pump rate must equal the net rate of passive diffusion of the drug and, therefore, the same experiments gave us the pumping rate of daunorubicin . These experiments were then repeated at various extracellular drug concentrations . By plotting the pumping rate versus the intracellular drug concentration, we then characterized the P-glycoprotein kinetically . Four independent methods were used to measure the passive permeability coefficient for the cell line A2780 . Similar values were obtained . Maximal pump rates (Vmax) showed a good correlation with the amount of P-glycoprotein in the cell lines used . We obtained saturation curves for the variation of the pump rates with the intracellular daunorubicin concentrations . These curves were typical for positive cooperativity, which provides evidence that at least two binding sites for daunorubicin are present on the active transport system of daunorubicin . The apparent Km values for P-glycoprotein-mediated transport, the intracellular free cytosolic daunorubicin concentrations at half-maximal velocity for the cell lines used, were approximately 1.5 microM . Except for the cell lines with the highest amount of P-glycoprotein, the passive efflux rate of daunorubicin proved to be a substantial part of the total daunorubicin efflux rate for the cell lines used . In cell lines with relatively low levels of P-glycoprotein, passive daunorubicin efflux was even the main route of daunorubicin transport from the cells, determining the intracellular steady-state concentrations of daunorubicin. Cancer Res, 1992 Jul 1, 52(13), 3539 - 46 Uptake of the noncytotoxic transport probe procainamide in the Chinese hamster ovary model of multidrug resistance; Speeg KV Jr et al.; Many of the cytotoxic substrates of the multidrug transporter are organic cations . Cimetidine, procainamide, and tetraethylammonium bromide were used in a Chinese hamster ovary model of multidrug resistance, to study handling of noncytotoxic cationic transport probes . Cimetidine and procainamide, but not tetraethylammonium, accumulated to a greater extent (5-fold) in the sensitive CHOAUXB1 (AB) cell line than in the resistant CHRC5 (C5) cell line . Accumulation of both cimetidine and procainamide was significantly increased by verapamil in C5 but not AB . Procainamide accumulation in both AB and C5 was temperature dependent and occurred by passive diffusion . Diltiazem, nifedipine, rifampin, tamoxifen, rhodamine, and ethidium also increased procainamide accumulation in C5 but not AB . Azide in glucose-free medium increased procainamide accumulation in C5, and this was reversed when glucose, but not 3-O-methylglucose, was added . Procainamide efflux rates were similar in AB and C5 and not affected by verapamil or azide . The initial rate of procainamide uptake was higher in AB than in C5, and both verapamil and azide increased the initial rate of procainamide uptake in C5 . Thus, differences in accumulation of the noncytotoxic transport probe procainamide in the colchicine-sensitive and colchicine-resistant components of the Chinese hamster ovary cell line mimic the accumulation of known cytotoxic substrates for the multidrug transporter, such as colchicine, vinblastine, and doxorubicin . The differential accumulation of procainamide is due to differences in rates of drug influx, rather than efflux . Since procainamide influx is passive and decreased accumulation in the resistant line appears to parallel M(r) 170,000 glycoprotein presence and activity, we would speculate that decreased procainamide accumulation may be due to an indirect effect of the M(r) 170,000 glycoprotein, such as its effect on intracellular pH. Biochim Biophys Acta, 1992 Jul 7, 1139(3), 169 - 83 P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines; Nielsen D et al.; MDR has been studied extensively in mammalian cell lines . According to usual practice, the MDR phenotype is characterized by the following features: cross resistance to multiple chemotherapeutic agents (lipophilic cations), defective intracellular drug accumulation and retention, overexpression of P-gp (often accompanied by gene amplification), and reversal of the phenotype by addition of calcium channel blockers . An hypothesis for the function of P-gp has been proposed in which P-gp acts as a carrier protein that actively extrudes MDR compounds out of the cells . However, basic questions, such as what defines the specificity of the pump and how is energy for active efflux transduced, remain to be answered . Furthermore, assuming that P-gp acts as a drug transporter, one will expect a relationship between P-gp expression and accumulation defects in MDR cell lines . A review of papers reporting 97 cell lines selected for resistance to the classical MDR compounds has revealed that a connection exists in most of the reported cell lines . However, several exceptions can be pointed out . Furthermore, only a limited number of well characterized series of sublines with different degrees of resistance to a single agent have been reported . In many of these, a correlation between P-gp expression and transport properties can not be established . Co-amplification of genes adjacent to the mdr1 gene, mutations {122}, splicing of mdr1 RNA {123}, modulation of P-gp by phosphorylation {124} or glycosylation {127}, or experimental conditions {26,78} could account for some of the complexity of the phenotype and the absence of correlation in some of the cell lines . However, both cell lines with overexpression of P-gp without increased efflux {i.e., 67,75} and cell lines without P-gp expression and accumulation defects/increased efflux {i.e., 25,107} have been reported . Thus, current results from MDR cell lines contradict--but do not exclude--that P-gp acts as multidrug transporter . Other models for the mechanism of resistance have been proposed: (1) An energy-dependent permeability barrier working with greater efficacy in resistant cells . This hypothesis is supported by studies of influx which, although few, all except one demonstrate decreased influx in resistant cells; (2) Resistant cells have a greater endosomal volume, and a greater exocytotic activity accounts for the efflux.(ABSTRACT TRUNCATED AT 400 WORDS) Science, 1992 Jul 3, 257(5066), 99 - 103 Selection of drug-resistant bone marrow cells in vivo after retroviral transfer of human MDR1; Sorrentino BP et al.; Experiments were performed to determine if retroviral-mediated transfer of the human multidrug resistance 1 gene (MDR1) into murine bone marrow cells would confer drug resistance to the cells and whether the MDR1 gene could be used as a dominant selectable marker in vivo . When mice transplanted with bone marrow cells containing a transferred MDR1 gene were treated with the cytotoxic drug taxol, a substantial enrichment for transduced bone marrow cells was observed . This demonstration of positive selection establishes the ability to amplify clones of transduced hematopoietic cells in vivo and suggests possible applications in human therapy. Br J Cancer, 1992 Jul, 66(1), 62 - 8 Reversal of multidrug resistance by surfactants; Woodcock DM et al.; Cremophor EL, a pharmacologically inactive solubilising agent, has been shown to reverse multidrug resistance (MDR) . Using flow cytometric evaluation of equilibrium intracellular levels of daunorubicin (DNR), we found that eight other surface active agents will also reverse MDR . All the active detergents contain polyethoxylated moieties but have no similarities in their hydrophobic components . The properties of three polyethoxylated surfactants that showed the lowest toxicities, Cremophor, Tween 80 and Solutol HS15, were examined in more detail . The concentrations of Tween 80 and Solutol required to reverse DNR exclusion were 10-fold lower than for Cremophor . However while concentrations greater than or equal to 1:10(2) of the former two surfactants resulted in breakdown of cells, even 1:10 of Cremophor did not lyse cells . Studies of the effects of Cremophor on the uptake and efflux of DNR in normal and MDR cell types showed that Cremophor increases intracellular DNR primarily by locking the rapid efflux from the cells . This blockage of drug efflux may be mediated by a substantial alteration in the fluidity of cell membranes induced by Cremophor, as shown by decreased fluorescence anisotropy of a membrane probe . Consistent with these data, coinjection of adriamycin plus Cremophor into mice carrying a multidrug resistant P388 transplantable tumour significantly increased the survival time of the mice compared with adriamycin treatment alone. Am J Trop Med Hyg, 1992 Jul, 47(1), 112 - 6 Emergence of multidrug-resistant Plasmodium falciparum in Thailand: in vitro tracking; Wongsrichanalai C et al.; Mefloquine was introduced into Thailand in 1985 for the treatment of Plasmodium falciparum infection . Recently, clinical failure of mefloquine was observed in southeastern Thailand, where an epidemic of falciparum malaria occurred . Beginning in 1984 and continuing until 1989, in vitro monitoring of P . falciparum isolates from Borai, a border district in the southeastern part of the country, showed a progressive decrease in mefloquine sensitivity until 1989; in 1990, the degree and prevalence of resistance accelerated . A similar pattern of resistance was observed for halofantrine, an antimalarial drug not yet commercially available in Thailand . In vitro sensitivity patterns of mefloquine and halofantrine elsewhere in the country remained relatively unchanged . These observations suggest a serious deterioration in available drugs for the treatment of falciparum malaria in southeastern Thailand that is predicted to spread throughout the country and Southeast Asia. Am J Trop Med Hyg, 1992 Jul, 47(1), 1 - 5 The efficacy of halofantrine in the treatment of acute malaria in nonimmune travelers; Weinke T et al.; A multicenter prospective trial was performed to investigate the efficacy and the tolerability of halofantrine in nonimmune patients with malaria imported from areas with drug-resistant falciparum parasites (mainly Africa) . Forty-five of the 74 subjects were treated with a one-day regimen (3 x 500 mg) of halofantrine, and the other 29 received the same regimen with an additional treatment on day 7 . In the second group, a 100% efficacy rate was demonstrated, but in the group receiving the one-day regimen, four recrudescences were observed in patients with falciparum malaria . Only five mild adverse reactions were seen, which disappeared spontaneously after the end of the treatment . We conclude that halofantrine is highly effective in curing malaria in nonimmune subjects . The treatment scheme for such persons should include an additional treatment on day 7 for nonimmune individuals . This drug was well tolerated in our patients, indicating that halofantrine will be useful in the treatment of multidrug-resistant malaria in nonimmune persons. Cancer Res, 1992 Jul 1, 52(13), 3750 - 9 Elevated level of nuclear protein kinase C in multidrug-resistant MCF-7 human breast carcinoma cells; Lee SA et al.; Previous studies have demonstrated elevated levels of protein kinase C (PKC) activity in multidrug-resistant human breast carcinoma MCF-7/ADR cells compared to control drug-sensitive MCF-7/WT cells (R.L . Fine, J . Patel, and B.A . Chabner, Proc . Natl . Acad . Sci . USA, 85:582-586, 1988) . In our present studies, immunohistochemical localization analysis using a polyclonal PKC antibody recognizing the alpha, beta, and gamma subtypes of PKC demonstrates that immunoreactivity is enhanced in MCF-7/ADR cells, with pronounced staining noted in the nuclear region . Other studies with purified nuclei isolated from MCF-7/ADR cells also show a marked increase in the intensity of immunostaining for PKC when compared to nuclei prepared from control MCF-7/WT cells . Western blot analysis of proteins extracted from purified nuclear preparations further establishes an increase in PKC enzyme protein associated with the nuclear fraction of MCF-7/ADR cells . Subcellular fractionation studies also indicate that MCF-7/ADR cells have 4-8 times higher nuclear PKC activity compared to that of control MCF-7/WT cells . MCF-7/ADR cells also possess 3-5-fold elevated cytosolic PKC activity, while a less than 2-fold increase is found in PKC activity associated with the plasma membrane fraction of MCF-7/ADR cells . Examination of these extracts with PKC isotype-specific antisera, as well as by DEAE-cellulose chromatography, reveals that nuclei prepared from MCF-7/ADR cells contain markedly elevated amounts of a slightly altered form of PKC alpha . These results suggest that elevated levels of a modified form of PKC alpha at the nucleus may play a role in modulating nuclear events to promote the development of multidrug resistance in MCF-7 cells. Cancer Res, 1992 Jul 1, 52(13), 3655 - 60 Effect of a dihydropyridine analogue, 2-{benzyl(phenyl)amino}ethyl 1,4-dihydro-2,6-dimethyl-5-(5,5-dimethyl-2-oxo- 1,3,2-dioxaphosphorinan-2-yl)-1-(2-morpholinoethyl)-4-(3-nitrophenyl)-3 -pyridinecarboxylate on reversing in vivo resistance of tumor cells to adriamycin; Niwa K et al.; A newly synthesized dihydropyridine analogue, 2-{benzyl(phenyl)-amino}ethyl 1,4-dihydro-2,6-dimethyl-5-(5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorina n-2-yl)-1- (2-morpholinoethyl)-4-(3-nitrophenyl)-3-pyridinecarboxylate (PAK-200), at 5 microM inhibited the efflux of {3H}vincristine from KB-C2 cells and increased the accumulation of {3H}vincristine in KB-C2 cells to a level similar to that in KB-3-1 cells . PAK-200 inhibited the photoaffinity labeling of P-glycoprotein in KB-C2 membranes by {3H}azidopine . At 5 microM, PAK-200 enhanced the cytotoxic effect of Adriamycin on drug-sensitive KB-3-1 cells, multidrug-resistant KB-8-5 cells, and two human colorectal carcinoma tumor lines, COK-28LN and COK-36LN, by factors of 2, 5, 2, and 3 times, respectively . The calcium antagonistic activity of PAK-200 was about 1000 and 5 times lower than that of another dihydropyridine analogue, nicardipine, and of verapamil, respectively . PAK-200 in combination with Adriamycin completely suppressed the growth of KB-3-1 and COK-36LN and partially suppressed the growth of KB-8-5 but had no significant effect on COK-28LN cells xenografted in nude mice . The level of MDR1 expression of COK-36LN was about 3 times higher than that of COK-28LN, but lower than that of KB-8-5 cells . These results suggest that the interaction of PAK-200 with P-glycoprotein may be partly correlated with the enhancement of the antitumor effect of Adriamycin on xenografted KB-8-5 and COK-36LN cells in nude mice. Cancer Res, 1992 Jul 1, 52(13), 3648 - 54 Response of human breast cancer cells to heat shock and chemotherapeutic drugs; Ciocca DR et al.; Previous studies have shown that certain chemotherapeutic drugs are less effective on tumor cells when cells have been previously exposed to hyperthermia . In the present study, we have evaluated whether specific modifications in heat shock protein (hsp) expression are associated with resistance to anticancer drugs . RNA levels for hsp90, hsp70, and hsp27 were studied by Northern and slot blots, while proteins were studied by two-dimensional gel electrophoresis, in MCF-7/BK and MDA-MB-231 breast cancer cells . The sensitivities of these cells to doxorubicin, colchicine, 5-fluorouracil, cisplatin, actinomycin D, and methotrexate were tested by clonogenic assays . These techniques were applied to both cell lines before (control) and after heat shock . The study revealed that elevated hsp70 and hsp27 levels were associated with doxorubicin resistance . In addition, the presence of phosphorylated hsp27 isoforms was also associated with doxorubicin resistance . The study showed that elevated hsps were not associated with multidrug resistance . Heat shock did not induce P170 glycoprotein mRNA overexpression or resistance to the other drugs tested . We also found that the level of doxorubicin protection conferred by the overexpression of hsp was lower than that obtained in cells expressing a multidrug resistance phenotype (MDA-A1R cells) . In these cells, heat shock did not confer additional doxorubicin resistance and hsp27 phosphorylation was deficient . Our studies suggest that specific hsps are associated with doxorubicin resistance in certain human breast cancer cells and that this mechanism seems to be independent of the multidrug resistance system. J Nucl Med, 1992 Jul, 33(7), 1373 - 7 In-vivo identification of tumor multidrug resistance with 3H-colchicine {corrected}; Mehta BM et al.; Multidrug resistance (MDR) is a major obstacle in the clinical treatment of cancer with natural-product anticancer agents . Identification of MDR in vivo could be important in the design of chemotherapeutic regimens . As a first step in developing radiolabeled drugs to detect MDR, we measured the in vivo distribution of radiolabel from {ring C, methoxy-3H}-colchicine ({3H}-CHC) in immunosuppressed mice bearing xenografts of colchicine-resistant and sensitive tumor cell lines . Experiments were done at trace (1 microgram/kg) and LD50 (4 mg/kg) dose levels . Activity concentration/injected dose was more than twice as great in sensitive as in resistant tumors (p less than 0.01) at 60 min following retroorbital injection of {3H}-CHC . There was no significant difference in activity distribution between trace- and high-dose injections for any of the tissues sampled . Chromatographic analysis of plasma and tumor extracts demonstrated extensive extravascular metabolic degradation of {3H}-CHC . The ratio of {3H}-CHC concentration of injected dose between sensitive and resistant tumors was 3:1 (p less than 0.05), due primarily to protein-bound {3H}-CHC . This preliminary study demonstrates that it is possible to distinguish multidrug resistant from sensitive tumors in vivo on the basis of radiolabel uptake from an injected MDR drug . Colchicine, labeled with 11C at the {ring C}-methoxy group, may be useful as a radiopharmaceutical for quantitative identification of MDR in human tumors using PET. J Neurooncol, 1992 Jul, 13(3), 217 - 22 Fluorescent dye rhodamine 6G as a molecular probe to study drug resistance of C6 rat glioma cells; Matsumoto Y et al.; A study was made of the membrane transport of cytoplasm and mitochondria stained fluorescence dye Rhodamine 6G (R6G) . In rat glioma C6 cells and 1-(4-amino-2-methyl-5-pyrymidinyl)-methyl-3-(2-chloroethyl) -3-nitrosourea hydrochloride (ACNU) and vincristine (VCR) resistant cell lines (C6/ACNU, C6/VCR), the rate of uptake of R6G decreased in C6/VCR cells, but verapamil increased the intracellular accumulation of R6G in C6/VCR . The intracellular accumulation of R6G of C6/ACNU cells was essentially the same as that of wild-type cells . C6/ACNU cells did not show cross resistance and were sensitive to VCR and cisplatin . C6/VCR cells showed cross resistance to ACNU and CDDP, but C6/VCR cells in the presence of verapamil were more sensitive to drugs than C6/VCR cells in the absence of verapamil . We conclude that the reduction of R6G fluorescence staining intensity in C6/VCR cells compared to wild-type cells may be associated with the mechanism of multidrug resistance (MDR) but does not reflect the mechanism of resistance to ACNU . Verapamil increased the accumulation of R6G in C6/VCR cells and overcame MDR, suggesting that there is a correlation between the MDR overcoming effect and enhancement of R6G accumulation, and that this correlation validates the use of the R6G staining test for clinical and laboratory investigation of MDR. Invest New Drugs, 1992 Jul, 10(2), 63 - 71 Toremifene and its metabolites enhance doxorubicin accumulation in estrogen receptor negative multidrug resistant human breast cancer cells; Wiebe V et al.; The enhanced accumulation of doxorubicin by agents known to reverse multidrug resistance provides a good functional test for evaluating modulating activity . In the present study, the non-steroidal triphenylethylene toremifene selectively increased doxorubicin accumulation in multidrug resistant estrogen receptor negative MDA A-1 human breast cells compared to the MDA 231 wild type cells . MDA A-1 cells were noted to be 1,000 fold resistant to doxorubicin (IC 50 = less than 0.1 microgram/ml MDA 231; IC 50 = 100 micrograms/ml MDA A-1) . Total accumulation of doxorubicin, expressed as area under the time concentration curve (AUC), was increased significantly in doxorubicin resistant cells (156% increase) versus wild type MDA 231 cells (6% increase) . Correction of the accumulation defect to doxorubicin in drug resistant cells required a 18-20 hour pre-incubation with toremifene . The effects of toremifene on cell cycle in MDA A-1 cells was analyzed by flow cytometric techniques . Toremifene had a dose response relationship in blocking cells in G0-G1 reducing the number of cells entering S phase of the cell cycle . This effect was maximal at concentrations which increased the accumulation of doxorubicin in MDA A-1 cells . Several metabolites of toremifene were also noted to increase doxorubicin accumulation in MDA A-1 doxorubicin resistant cells . Tore XVIII (deaminocarboxytoremifene), Tore IV (4-hydroxy-N-desmethyltoremifene) and N-desmethyltoremifene all increased the accumulation of doxorubicin significantly (114%, 128% and 42% respectively) . Finally, we show evidence that toremifene and its active metabolites are present in high concentrations in human plasma following a single 200 mg oral dose.(ABSTRACT TRUNCATED AT 250 WORDS) Nat Immun, 1992 Jul-Aug, 11(4), 177 - 92 P-glycoprotein-mediated multidrug resistance and cytotoxic effector cells; Savas B et al.; Multidrug resistance (MDR) is one of the major obstacles to successful cancer chemotherapy . MDR is a complex and multifactorial phenomenon . One important and common mechanism used by cancer cells as a defense against cytotoxic drugs is a 170-kD plasma membrane glycoprotein, P-glycoprotein (P-gp) . P-gp confers resistance by actively pumping cytotoxic drugs out of cancer cells . Paradoxically, P-gp overexpression on tumor cells is frequently associated with enhanced susceptibility to lymphokine-activated killer cell activity . This enhanced susceptibility is not observed with P-gp- MDR cells, nor is susceptibility to natural killer cells increased . The physiologic, evolutionary and immunologic concepts with regard to the P-gp and the possible intervention of the function of the P-gp in cancer therapy are reviewed. Biochem Int, 1992 Jul, 27(2), 301 - 10 Amino acid transport in multidrug-resistant Chinese hamster ovary cells; Daly SE et al.; In the process of assessing the effect of anthracycline drugs on cellular membrane function in cultured multidrug resistant (MDR) and its parental cells, experiments were undertaken to investigate the kinetics of neutral amino acid membrane transport (the sodium dependent A and ASC systems) . P-glycoprotein, a high molecular weight energy requiring integral membrane protein responsible for actively pumping drugs out of cells, has been shown to be overexpressed in MDR cells . It was our hypothesis that its presence might affect other membrane energy requiring systems such as amino acid transport . On establishing the concentrations of P-glycoprotein by western blotting in the two cell lines to be studied, the kinetics of membrane transport of the neutral amino acids alpha-aminoisobutyric acid (AIB) and serine (SER) were investigated using the CHRC5 multidrug resistant and AUX B1 parental Chinese hamster ovary (CHO) cells . In CHRC5 cells, the amount and rate (Vmax) of accumulated amino acids, was significantly depressed when compared to AUX B1 cells, however, there was no difference in the rates of amino acid efflux between these two cell lines . Using 1,6-diphenyl 1,3,5-hexatriene (DPH) polarization to evaluate the state of membrane fluidity in the two cell lines studied, it was seen that CHRC5 cells showed a slightly lower degree of polarization than that observed in AUX B1 cells . These results suggest, that the P-glycoprotein does not alter amino acid transport directly but may modify the activity or numbers of functional transport carriers. Mol Pharmacol, 1992 Jul, 42(1), 69 - 74 Expression of c-fos in human and murine multidrug-resistant cells; Bhushan A et al.; In both mouse sarcoma 180 and human KB cells selected for the multiple drug resistance (MDR) phenotype, there is an elevation in the steady state mRNA level of c-fos . There is no detectable gene amplification for c-fos, nor is there any significant change in the rate of mRNA transcription or degradation, suggesting that other factors are responsible for the increased expression level in resistance . Cells selected for resistance to methotrexate, a drug not in the MDR group, do not have an increase in c-fos mRNA expression . When drug-sensitive cells are exposed for 30 min to an ED50 concentration of vinblastine, Adriamycin, colchicine, or VP-16, but not to methotrexate or cisplatin, there is a 3-6-fold induction in the level of c-fos message . Because the former drugs are members of the MDR class and the latter are not, the results are consistent with the hypothesis that induction of c-fos by low levels of cytotoxic drugs may be an early event in the acquisition of the MDR phenotype . If this were the case, then c-fos would be expected to act in concert with c-jun to control transcription by binding to a specific DNA regulatory site . Consistent with this explanation is the existence of an AP-1 sequence in the promotor region for the P-glycoprotein gene (mdr1), as well as the fact that c-jun is also overexpressed in MDR cells. Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 5824 - 8 Efficient inhibition of P-glycoprotein-mediated multidrug resistance with a monoclonal antibody; Mechetner EB et al.; P-glycoprotein (Pgp), encoded by the MDR1 gene, is an active efflux pump for many structurally diverse lipophilic compounds . Cellular expression of Pgp results in multidrug resistance (MDR) in vitro and is believed to be a clinically relevant mechanism for tumor resistance to chemotherapy . We have developed a mouse monoclonal antibody, UIC2, that recognizes an extracellular epitope of human Pgp . UIC2 inhibited the efflux of Pgp substrates from MDR cells and significantly increased the cytotoxicity of Pgp-transported drugs, under the conditions where no effect was detectable with other anti-Pgp antibodies . Potentiation of cytotoxicity by UIC2 was observed with all the tested drugs associated with MDR (vinblastine, vincristine, colchicine, taxol, doxorubicin, etoposide, actinomycin D, puromycin, and gramicidin D) but not with any of the drugs to which MDR cells are not cross-resistant (methotrexate, 5-fluorouracil, cis-platinum, G418, and gentamicin) . The inhibitory effect of UIC2 in vitro was as strong as that of verapamil (a widely used Pgp inhibitor) at its highest clinically achievable concentrations . Our results suggest that UIC2 or its derivatives provide an alternative or supplement to chemical agents for the reversal of MDR in clinical cancer. J Neuroimmunol, 1992 Jul, 39(1-2), 123 - 32 Characterization of a rat retinal endothelial cell culture and the expression of P-glycoprotein in brain and retinal endothelium in vitro; Greenwood J; Retinal vascular endothelia form one aspect of the blood-retinal barrier and, like the blood-brain barrier, control the passage of molecules and cells into the parenchyma . To facilitate comparative in vitro studies, rat retinal endothelial cells have been cultured and characterised . Using immunocytochemical techniques, retinal endothelium was positive for von Willebrand's factor, tight junction-associated polypeptide (ZO-1) and the transferrin receptor . The cells also expressed high-affinity uptake of acetylated low-density lipoprotein . Using the monoclonal antibodies JSB-1 and C219, the product of the multidrug resistance gene, P-glycoprotein, was found to be expressed on primary cultures of both brain and retinal endothelium. J Cell Physiol, 1992 Jul, 152(1), 87 - 94 Reduced mRNA levels for the multidrug-resistance genes in cAMP-dependent protein kinase mutant cell lines; Chin KV et al.; We have previously shown that in Chinese hamster ovary (CHO) cells, a mutant cell line with a defective regulatory subunit (RI) for the cAMP-dependent protein kinase (Abraham et al: Mol . Cell . Biol., 7:3098-3106, 1987), and a transfectant cell line expressing the same mutant kinase, showed increased sensitivity to a number of drugs that are known to be substrates for the multidrug transporter (P-glycoprotein) . In the current study we have investigated the mechanism by which cAMP-dependent protein kinase controls drug resistance . We report here that the sensitivity of the kinase defective CHO cell lines to multiple drugs results from decreased RNA levels for the multidrug-resistance gene . Similar results were obtained with mouse Y1 adrenal cells . Wild-type Y1 cells had high levels of P-glycoprotein due to expression of both the mdr1b and mdr2 genes, whereas the cAMP-dependent protein kinase mutant Kin 8 cells had decreased RNA levels for these genes . A Kin 8 transfectant with restored cAMP-dependent protein kinase activity recovered mdr expression, indicating a cause and effect relationship between the protein kinase mutations and mdr expression . No changes in nuclear run-off assays could be detected, suggesting a non-transcriptional mechanism of regulation . Wild-type Y1 cells are more drug sensitive despite having higher levels of P-glycoprotein than the mutant cells . This paradoxical result may be explained by the higher rate of synthesis of steroids by the wild-type Y1 cells, which appear to be inhibitors of P-glycoprotein transport activity. Cancer Res, 1992 Jul 1, 52(13), 3768 - 75 Immunohistochemical identification of P-glycoprotein in previously untreated, diffuse large cell and immunoblastic lymphomas; Niehans GA et al.; Expression of P-glycoprotein has been linked to multidrug resistance in cancer cell lines and human tumors . We investigated the frequency and clinical significance of P-glycoprotein immunoreactivity in 57 previously untreated diffuse large cell and immunoblastic lymphomas . Banked frozen tissue, which had been obtained prior to chemotherapy, was tested for reactivity with 2 monoclonal antibodies (MRK16 and C219) that recognize different domains of P-glycoprotein, using an immunoperoxidase technique . Thirteen of 57 lymphomas (23%) showed strong staining of greater than 50% of neoplastic cells; 15 of 57 (26%) showed labeling of a minority (11-50%) of neoplastic lymphocytes; 14 of 57 (25%) yielded equivocal results (reactivity in less than 10% of cells); and 15 of 57 (26%) were negative for P-glycoprotein . The 2 monoclonal antibodies were comparable in reactivity . Expression of MDR-1 mRNA was determined in 6 cases with sufficient available tissue, and did not correlate well with the percentages of cells reactive for P-glycoprotein by immunohistochemistry . Thirty-nine of our 57 patients completed multiagent chemotherapy . Contrary to our expectations, we found that P-glycoprotein immunoreactivity did not decrease the likelihood of response to induction chemotherapy . Median survival also was not adversely affected. Korean J Intern Med, 1992 Jul, 7(2), 111 - 7 Effects of buthionine sulfoximine treatment on cellular glutathione levels and cytotoxicities of cisplatin, carboplatin and radiation in human stomach and ovarian cancer cell lines; Lee KS et al.; Chemotherapy failure remains a significant medical problem in the treatment of neoplastic disease and is thought to be due to many different factors including membrane transport, p-glycoprotein in multidrug resistance, glutathione and its related enzymes, topoisomerase II and DNA repair . Glutathione is a major constituent of non-protein thiol and participates in detoxification of chemotherapy and radiation . Thus, glutathione concentration is correlated with sensitivity to alkylating agents and radiation, and increased in resistant cell lines . Buthionine sulfoximine (BSO) is an inhibitor of glutathione biosynthesis and may increase cytotoxicities of alkylating agents, including melphalan and cisplatin, and radiation in sensitive and resistant cell lines . We studied effects on cellular glutathione levels and cytotoxicities of cisplatin, carboplatin and radiation by BSO treatment in human stomach cancer cell line (SNU-1) and ovarian cancer cell line (OVCAR-3) . The results were as follow: 1) After BSO treatment of 1 mM and 2 mM for 2 days, the intracellular thiol concentration was depleted to 75.7% and 76.2% in SNU-1, and 74.1% and 63.0% in OVCAR-3, respectively . 2) The intracellular thiol concentration in SNU-1 was depleted to 33.4% after BSO 2 mM for only 2 hours incubation and 71.5% after small amount of BSO (0.02 mM) for 2 days . 3) The recovery of intracellular thiol concentration required more than 3 days after BSO removal . 4) BSO inhibited partially the growth of SNU-1 and OVCAR-3 . 5) The cytotoxicities of cisplatin and carboplatin were markedly enhanced both in SNU-1 and OVCAR-3 by BSO treatment . 6) The cytotoxicities of radiation was increased in OVCAR-3 and SNU-1 by BSO treatment . Therefore, it is concluded that BSO can deplete effectively the intracellular thiol concentration and enhance the cytotoxicities of cisplatin, carboplatin and radiation. Tumori, 1992 Jun 30, 78(3), 159 - 66 P-glycoprotein but not topoisomerase II and glutathione-S-transferase-pi accounts for enhanced intracellular drug-resistance in LoVo MDR human cell lines; Boiocchi M et al.; The biochemical bases of the multidrug-resistant (MDR) phenotype were investigated in drug-resistant sublines derived from LoVo human colon carcinoma cell lines by doxorubicin (DOX) and teniposide (VM26) selection . In addition to P-glycoprotein-mediated drug extrusion through the plasma-membrane, LoVo MDR cells display a further drug-resistance mechanism . That is, to achieve equitoxic effects, LoVo MDR sublines require much higher intracellular drug concentrations than those required by LoVo drug-sensitive parent cell line . Involvement of mdr1, topoisomerase II and glutathione-S-transferase-pi (GST-pi) drug-resistance systems in intracellular drug resistance was investigated . Pharmacologic and biochemical data indicated that intracellular drug resistance in LoVo MDR sublines is uniquely consequent to the drug-transporting property of intracytoplasmic membrane-bound P-glycoprotein molecules which compartment drugs in vacuole-like structures. J Med Chem, 1992 Jun 26, 35(13), 2481 - 96 New triazine derivatives as potent modulators of multidrug resistance; Dhainaut A et al.; A series of 70 triazine derivatives have been synthesized and tested for their capacity to modulate multidrug resistance (MDR) in DC-3F/AD and KB-A1 tumor cells in vitro, in comparison with verapamil (VRP), a calcium channel antagonist currently used in therapy as an antihypertensive drug, which also shows MDR modulating activity . Among the 12 selected compounds, 16 (S9788) showed high MDR reversing properties in vitro (300- and 6-fold VRP at 5 microM in DC-3F/AD and KB-A1 cells, respectively) and induced a strong accumulation of adriamycin . The relationship between the increase of ADR accumulation and the fold reversal induced by these compounds and their lack of effects on the sensitive DC-3F cells suggest that they act mainly by inhibiting the P-glycoprotein (Pgp) catalyzed efflux of cytotoxic agents, as already described for a majority of MDR modulators . In vivo, in association with the antitumor drug vincristine (0.25 mg/kg), 16 (100 mg/kg) increased the T/C by 39% in mice bearing the resistant tumor cell line P388/VCR . According to these interesting properties, 16 was selected for a clinical development because it was more bioavailable than 34, even though it was less active. Biochem Pharmacol, 1992 Jun 23, 43(12), 2601 - 8 Stereoisomers of calcium antagonists which differ markedly in their potencies as calcium blockers are equally effective in modulating drug transport by P-glycoprotein; Hollt V et al.; The (-)-isomer of verapamil is 10-fold more potent as a calcium antagonist than the (+)-isomer . However, both enantiomers are equally effective in increasing cellular accumulation of anticancer drugs {Gruber et al., Int J Cancer 41: 224-226, 1988} . In addition to verapamil, there exists a wide variety of stereoisomers with phenylalkylamines and dihydropyridine structures which markedly differ in their potency as calcium antagonists . We have tested these drugs for their ability to increase intracellular accumulation of {3H}vinblastine ({3H}VBL) in a doxorubicin-resistant cell line (F4-6RADR) derived from the Friend mouse leukemia cell line (F4-6P) and in COS-7 monkey kidney cells . Both cell types express substantial amounts of multidrug resistance gene 1 mRNA and P-glycoprotein as revealed by RNA and immuno blot analysis . The enantiomers with phenylalkylamine structures {(+/-)-verapamil; (+/-)-devapamil; (+/-)-emopamil)} and with dihydropyridine structures {(+/-)-isradipine; (+/-)-nimodipine; (+/-)-felodipine; (+/-)-nitrendipine; (+/-)-niguldipine} increased {3H}VBL accumulation in both cell lines at micromolar concentrations . Although the stereoisomers of these drugs differ markedly in their potency as calcium channel blockers they were about equally effective in increasing VBL levels in the cells . There was no substantial difference in the potencies of the phenylalkylamine drugs in affecting cellular {3H}VBL transport . Major potency differences, however, were observed in the dihydropyridine drug series with the niguldipine isomers as the most effective drugs . Moreover, the niguldipine enantiomers were equally as effective in reversing VBL resistance in F4-6RADR cells as were the verapamil enantiomers . Since (-)-niguldipine (B859-35) displays a 45-fold lower affinity for calcium channel binding sites than (+)-niguldipine, but is equally potent in inhibiting drug transport by P-glycoprotein and in reversing drug resistance, it may be, in addition to (+)-verapamil, another useful candidate drug for the treatment of multidrug resistance in cancer patients. MMWR Recomm Rep, 1992 Jun 19, 41(RR-11), 61 - 71 Management of persons exposed to multidrug-resistant tuberculosis; National action plan to combat multidrug-resistant tuberculosis; At no time in recent history has tuberculosis (TB) been as great a concern as it is today . TB cases are on the increase, and the most serious aspect of the problem is the recent occurrence of outbreaks of multidrug-resistant (MDR) TB, which pose an urgent public health problem and require rapid intervention . A Task Force composed of representatives of many federal agencies has developed a National Action Plan for addressing this problem . The Task Force identified a number of objectives to be met if MDR-TB is to be successfully combatted . These objectives fall under the categories of a) surveillance and epidemiology--determining the magnitude and nature of the problem; b) laboratory diagnosis--improving the rapidity, sensitivity, and reliability of diagnostic methods for MDR-TB; c) patient management--effectively managing patients who have MDR-TB and preventing patients with drug-susceptible TB from developing drug-resistant disease; d) screening and preventive therapy--identifying persons who are infected with or at risk of developing MDR-TB and preventing them from developing clinically active TB; e) infection control--minimizing the risk of transmission of MDR-TB to patients, workers, and others in institutional settings; f) outbreak control; g) program evaluation--ensuring that TB programs are effective in managing patients and preventing MDR-TB; h) information dissemination/training and education; and i) research to provide new, more effective tools with which to combat MDR-TB . The Action Plan lays out a series of activities to be undertaken at the national level . For each category, the Plan presents statements of problems to be overcome, followed by a summary of the objective to be achieved and steps to be carried out . For each implementation step, responsibility is assigned to the appropriate organization and start-up dates are listed. MMWR Recomm Rep, 1992 Jun 19, 41(RR-11), 51 - 7 Meeting the challenge of multidrug-resistant tuberculosis: summary of a conference; Comparative cellular pharmacology of daunorubicin and idarubicin in human multidrug-resistant leukemia cells; Leukemia Service, Memorial Sloan Kettering Cancer Center, New York, NY 10021We examined the effect of daunorubicin (DNR), the new anthracycline derivative idarubicin (IDR), and verapamil on two leukemia cell lines that displayed the multidrug resistant (MDR) phenotype and used laser flow cytometry to quantitate intracellular anthracycline content . The vinblastine-resistant human lymphoblastic leukemia cell line CEM-VBL demonstrated minimal DNR uptake; simultaneous incubation with verapamil and DNR increased intracellular DNR uptake fourfold . IDR uptake was 10 times more rapid in these cells and simultaneous incubation with IDR and verapamil resulted in only a 1.2-fold increase of intracellular IDR . Similar results were observed in the vincristine-resistant human myeloid leukemia cell line HL-60/RV+ . Intracellular retention of DNR and IDR was also measured in each cell line . In CEM-BVL cells, 38% of the original DNR concentration remained after a 2-hour resuspension in fresh medium compared with 71% of the original IDR concentration . In HL-60/RV+ cells, 36% of the DNR concentration remained compared with 51% of the IDR concentration . After incubation of CEM-VBL and HL-60/RV+ cells with DNR for 1 hour followed by resuspension in fresh medium plus verapamil, intracellular DNA retention increased 5- and 5.2-fold, respectively . However, incubation of these cells for 1 hour with IDR followed by resuspension in fresh medium plus verapamil resulted in only a 1.6- and 2.4-fold increase in intracellular IDR retention . Lastly, clonogenic experiments were performed to correlate intracellular anthracycline content with cytotoxicity . DNR alone had a minimal effect on the clonogenic growth of CEM-VBL cells, whereas the combination of DNR plus verapamil resulted in approximately 80% growth inhibition . However, incubation of these cells with IDR alone resulted in greater than 95% growth inhibition . These results suggest that IDR may be more effective than DNR in leukemia cells that display the MDR phenotype. FEBS Lett, 1992 Jun 15, 304(2-3), 256 - 60 ATP and GTP as alternative energy sources for vinblastine transport by P-170 in KB-V1 plasma membrane vesicles; Lelong IH et al.; Purified plasma membrane vesicles isolated from multidrug-resistant human KB-V1 cells accumulate {3H}vinblastine in an energy-dependent manner . The accumulation of {3H}vinblastine in the presence of ATP is a saturable process . ATP can be replaced by other purine nucleotide triphosphates, of which GTP is the most efficient. Cancer Lett, 1992 Jun 15, 64(2), 177 - 83 Reversal of vinblastine resistance by a new staurosporine derivative, NA-382, in P388/ADR cells; Miyamoto K et al.; Activities of a newly synthesized compound, N-ethoxycarbonyl-7-oxo-staurosporine (NA-382), on cyclic AMP-dependent protein kinase (A-kinase), Ca2+/phospholipid dependent protein kinase (C-kinase), and drug resistance were investigated and compared with those of staurosporine . Protein kinase-inhibitory activity of NA-382 was lower but more selective to C-kinase than that of staurosporine . NA-382 was less toxic to P388 cells and at a non-cytotoxic concentration completely reversed the vinblastine (VBL) resistance of Adriamycin-resistant P388 (P388/ADR) cells without influence on the effect of VBL on the parental P388/S cells . However, the cytotoxicity of staurosporine was too high to give the combination effect with VBL . NA-382 dose-dependently increased VBL-accumulation and inhibited VBL-efflux in P388/ADR with higher potency than staurosporine . Both compounds inhibited the photolabeling of {3H}azidopine on 140-kDa P-glycoprotein in the plasma membrane from the resistant cells . These results suggest that a staurosporine analog, NA-382, reverses multidrug resistance by inhibiting the drug-efflux system or P-glycoprotein. Cancer Res, 1992 Jun 15, 52(12), 3409 - 17 Resistance to N-benzyladriamycin-14-valerate in mouse J774.2 cells: P-glycoprotein expression without reduced N-benzyladriamycin-14-valerate accumulation; Lothstein L et al.; N-Benzyladriamycin-14-valerate (AD 198) is a highly lipophilic analogue of Adriamycin with novel cytotoxic mechanisms, greater in vivo antitumor activity, and the ability to circumvent multidrug resistance due to P-glycoprotein-mediated drug efflux or decreased topoisomerase II activity . To identify the mechanism(s) which may confer AD 198 resistance, J774.2 mouse macrophage-like cells were selected for growth in cytotoxic levels of AD 198 (AD 198R) . AD 198R cells exhibited over-expression of the mdr1b (P-glycoprotein) gene, cross-resistance to Adriamycin and vinblastine, and potentiation of drug cytotoxicity by verapamil . However, net intracellular accumulation of AD 198 in AD 198R cells was unchanged compared to parental cells, while Adriamycin and vinblastine accumulations were reduced 40% and 95%, respectively . AD 198 was localized in the perinuclear region of the cytoplasm in both parental and AD 198R cells, with additional vesicular compartmentalization in AD 198R cells . Verapamil-induced reversal of AD 198 resistance coincided with some drug redistribution from cytoplasmic vesicles, but without redistribution of AD 198 into the nucleus . These results suggest that AD 198 resistance was not conferred through a P-glycoprotein-mediated reduction in intracellular drug accumulation but through other cytoplasmic mechanisms, including, but not limited to, drug compartmentalization. Presse Med, 1992 Jun 13, 21(22), 1033 - 7 {Drug resistance genes}; Marie JP; Among the different mechanisms of multidrug resistance, the overexpression of the mdr1 gene has been actively investigated during the last 5 years . This gene encodes a 170 kDa protein, named P-gp, a member of a transporter superfamily, the ABC (ATP Binding Cassette) proteins . P-gp actively expels out of the tumoral cell different drugs like anthracyclins, vinca alkaloids, epipodophyllotoxins . The involvement of mdr1 gene in clinical drug resistance is now demonstrated, and several trials using P-gp modulators and chemotherapy are going on in resisting tumors . Other intrinsic drug resistance mechanisms, such as increase of intracellular glutathione content or decrease of topoisomerase activity, could be involved in clinical drug resistance. Biochim Biophys Acta, 1992 Jun 11, 1107(1), 105 - 10 Specific drug binding by purified lipid-reconstituted P-glycoprotein: dependence on the lipid composition; Saeki T et al.; We fused P-glycoprotein with beta-galactosidase at the C-terminus aiming to study the mechanism of drug binding of P-glycoprotein in reconstitution experiments . Expression of the fusion protein in NIH 3T3 cells conferred a multidrug-resistant phenotype, suggesting that beta-galactosidase fusion at the C-terminus does not affect the functions of P-glycoprotein . The fusion protein was partially purified by simple immunoprecipitation with anti-beta-galactosidase polyclonal antibody, and its {3H}azidopine binding property was investigated in the presence of various compositions of liposomes . The purified P-glycoprotein, after reconstitution into liposomes, was capable of binding {3H}azidopine . When the cholesterol content of liposomes was increased to a weight ratio of 20%, the specific binding activity of the partially purified fusion protein was stimulated, and when the cholesterol content was increased higher, the binding activity decreased . The binding was specifically decreased by competition with vinblastine . Stigmasterol was less effective, and ergosterol was the least effective in stimulating the specific binding. Nucleic Acids Res, 1992 Jun 11, 20(11), 2841 - 6 Regulation of P-glycoprotein gene expression in hepatocyte cultures and liver cell lines by a trans-acting transcriptional repressor; Gant TW et al.; Previously we have demonstrated that expression of the multidrug resistance (mdr) genes in rat liver and primary rat hepatocyte cultures is induced by exposure to 2-acetylaminofluorene and 3-methylcholanthrene . The mdr expression induced by both of these compounds occurs primarily via increased gene transcription . To determine the nature of possible regulatory proteins involved in mdr gene regulation we inhibited protein synthesis using cycloheximide or emetine in primary rat hepatocyte cultures, mouse (HePa 1), human (Hep G2) and rat (H4-II-E) cell lines . Each cell type responded by strongly increasing its steady state mdr1 mRNA levels . In hepatocytes increased mdr expression was observed after greater than 50% inhibition of protein synthesis, and was first detected after 2h of protein synthesis inhibition with maximal induction occurring by 24h . Nuclear run-on analysis showed that the increased steady state mRNA level was due to increased gene transcription without alteration of the transcription start site . Combined these data indicate that one regulatory mechanism by which mdr gene expression is controlled is via a trans-acting transcriptional repressor. N Engl J Med, 1992 Jun 4, 326(23), 1514 - 21 An outbreak of multidrug-resistant tuberculosis among hospitalized patients with the acquired immunodeficiency syndrome; Edlin BR et al.; BACKGROUND . Since 1990 several clusters of multidrug-resistant tuberculosis have been identified among hospitalized patients with the acquired immunodeficiency syndrome (AIDS) . We investigated one such cluster in a voluntary hospital in New York . METHODS . We compared exposures among 18 patients with AIDS in whom tuberculosis resistant to isoniazid and streptomycin was diagnosed from January 1989 through April 1990 (the case patients) with exposures among 30 control patients who had AIDS and tuberculosis susceptible to isoniazid, streptomycin, or both . We also compared exposures among the 14 case patients hospitalized during the six months before the diagnosis of tuberculosis (the exposure period) with those among 44 control patients with AIDS matched for duration of hospitalization . Mycobacterium tuberculosis isolates were typed with analysis of restriction-fragment-length polymorphism (RFLP) . RESULTS . Case patients with drug-resistant tuberculosis were significantly more likely than controls with drug-susceptible tuberculosis to have been hospitalized during their exposure periods (14 of 18 vs . 10 of 30) (odds ratio, 7.0; 95 percent confidence interval, 1.6 to 36; P = 0.006) . Case patients hospitalized during their exposure periods were significantly more likely to have been hospitalized on the same ward as a patient with infectious drug-resistant tuberculosis than were either controls with drug-susceptible tuberculosis hospitalized during their exposure periods or controls matched for duration of hospitalization (13 of 14 vs . 2 of 10 and 23 of 44) (odds ratio, 52; 95 percent confidence interval, 3.1 to 2474; P less than 0.001; and odds ratio, infinity; 95 percent confidence interval, 2.4 to infinity; P = 0.005, respectively) . Among those hospitalized on the same ward, the rooms of case patients were closer to that of the nearest patient with infectious tuberculosis than were the rooms of controls matched for duration of hospitalization . M . tuberculosis isolates from 15 of 16 case patients had identical patterns on RFLP analysis . Of 16 patients' rooms tested with air-flow studies, only 1 had the recommended negative-pressure ventilation . CONCLUSIONS . Multidrug-resistant tuberculosis is readily transmitted among hospitalized patients with AIDS . Physicians must be alert to this danger and must enforce adherence to the measures recommended to prevent nosocomial transmission of tuberculosis. Leuk Res, 1992 Jun-Jul, 16(6-7), 631 - 7 The cytotoxic effects of 5-OH-1,4-naphthoquinone and 5,8-diOH-1,4-naphthoquinone on doxorubicin-resistant human leukemia cells (HL-60); Segura-Aguilar J et al.; The effect of 5-OH-1,4-naphthoquinone and 5,8-diOH-1,4-naphthoquinone, two quinones highly reactive with oxygen, was studied on HL-60 and HL-60R cells . The multidrug resistance developed by the doxorubicin-resistant HL-60 cell line did not prevent the cytotoxic effect of these compounds, at clinically relevant concentrations . An increase in cellular defenses against oxygen radicals seemed to be one of the features developed by HL-60R, since the homogenate from this cell line had only 65% of the ability of the original cell line to form oxygen radicals during doxorubicin reduction . This result may be explained in part by the slight increase in superoxide dismutase and DT-diaphorase enzymatic activities. Mol Pharmacol, 1992 Jun, 41(6), 1034 - 8 Overcoming of vinblastine resistance by isoquinolinesulfonamide compounds in adriamycin-resistant leukemia cells; Wakusawa S et al.; We investigated the effects of seven isoquinoline derivatives in overcoming resistance to vinblastine in Adriamycin-resistant mouse leukemia P388/ADR cells and human myelogeneous leukemia K562/ADR cells . N-(2-Methylpiperazyl)-5-isoquinoline-sulfonamide (H-7), N-{2-(methylamino)ethyl}-5-isoquinolinesulfonamide (H-8), and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) did not reverse resistance to vinblastine in these resistant cells . N-{2-{N-{3-(4-Chlorophenyl)-2-propenyl}amino}ethyl}-5- isoquinolinesulfonamide (H-86) and N-{2-{N-{3-(4-chlorophenyl)-1-methyl-2-propenyl}- amino}ethyl}-5-isoquinolinesulfonamide (H-87) caused significant accumulation of intracellular vinblastine and marked reversal of the resistance to vinblastine in both resistant cell lines . Addition of a formyl group at the terminal amino group of H-86 (H-85) or addition of an aminoethyl group to the nitrogen atom at the sulfonamide group of H-86 (W-66) reduced those activities . The activity on vinblastine accumulation seems to correlated with the hydrophobicity of the compounds . The compounds that effectively reversed resistance to vinblastine inhibited {3H}vinblastine efflux and photoaffinity labeling of P-glycoprotein with a photosensitive analogue of vinblastine, N-(p-azido-(3-{125I}iodo)-salicyl)-N'-beta-aminoethylvindesine . Although these isoquinoline derivatives inhibited protein kinase A and protein kinase C with various potencies, these inhibitory activities did not correlate with the reversal of drug resistance . These results indicate that hydrophobic isoquinoline derivatives reverse multidrug resistance due to the suppression of drug binding to P-glycoprotein, without involvement of their activities on protein kinase A and protein kinase C. Cancer Res, 1992 Jun 1, 52(11), 3241 - 5 Increased accumulation of drugs in multidrug-resistant cells induced by liposomes; Warren L et al.; A multidrug-resistant cell such as the human lymphoblastic leukemic cell CEM/VLB100 accumulates far less vinblastine (VLB) than its drug-sensitive parent, CEM . When CEM/VLB100 cells are exposed to liposomes consisting of the phospholipids cardiolipin, dioleoylphosphatidic acid, or phosphatidylinositol bearing unsaturated fatty acids and then tested for uptake of VLB, accumulation of drug rapidly rises to levels approaching those of CEM cells, which are relatively unaffected by the liposome treatment . The liposomes are not carriers of entrapped drug . Phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine are inactive, and the addition of cholesterol to liposomes inhibits uptake . Exposure of cells to liposomes does not appear to alter the efflux of drugs . We suggest that the liposomal lipids, introduced into the plasma membranes of CEM/VLB100 cells, change their properties so that accumulation of drugs by cells is largely restored . The cytotoxicity of VLB in CEM/VLB100 cells is increased approximately 10-fold by cardiolipin liposomes. Cancer Res, 1992 Jun 1, 52(11), 3157 - 63 Drug transport mechanisms in HL60 cells isolated for resistance to adriamycin: evidence for nuclear drug accumulation and redistribution in resistant cells; Marquardt D et al.; HL60 cells isolated for resistance to Adriamycin are multidrug resistant and defective in the cellular accumulation of drug . These cells do not contain detectable levels of P-glycoprotein . At the present time the mechanism by which HL60/Adr cells reduce drug levels is not known . To gain insight into the molecular basis of this system we have analyzed transport pathways and the distribution of daunomycin in drug-resistant HL60 cells . Using a cell fractionation technique we find that the major portion of daunomycin accumulates in the nucleus of both sensitive and resistant cells . Further studies reveal, however, that under efflux conditions drug is retained in the nuclei of sensitive cells but rapidly removed from the nuclei of the resistant isolate . Essentially identical results are obtained when daunomycin distribution and transport are analyzed by fluorescence microscopy . A number of agents which alter transport processes have been tested for their effect on drug accumulation in resistant cells . Thus we find that brefeldin A, which disassembles Golgi, and various lysosomotropic agents such as chloroquine and methylamine do not affect drug levels . In contrast the protonophores nigericin and monensin induce an increase in drug accumulation and inhibit efflux . The results of this study thus suggest that resistance in HL60/Adr cells is related to a mechanism whereby drug is transported to the nucleus and thereafter rapidly redistributed to the extracellular space . The molecular basis of this transport pathway is not known. Am J Clin Oncol, 1992 Jun, 15(3), 216 - 21 Radiation resistance in a doxorubicin-resistant human fibrosarcoma cell line; Miller PR et al.; In clinical practice, cancers refractory to chemotherapy can appear relatively radioresistant . Recent work in multidrug-resistant cell lines has yielded conflicting results concerning the relationship between drug resistance and radiation resistance . The current study examines the radiation response of a human fibrosarcoma (HT1080) and a doxorubicin-resistant subline (HT1080/DR4) . Using soft-agar colony formation after graded doses of x-rays as an endpoint, HT1080/DR4 had an increased D0 (D0 = 2.1 Gy) and a broader initial shoulder (n = 2.7, Dq = 2.1 Gy) than the parental HT1080 line (D0 = 0.7, Gy, n = 1.2, Dq = 0.3 Gy), suggesting that HT1080/DR4 has an increased capacity to repair radiation-induced DNA damage . This possibility was tested by comparing the cell lines' ability to accumulate sublethal damage . In split-dose recovery experiments, HT1080/DR4 demonstrated increased ability to repair sublethal radiation damage following fractionated irradiation, compared with the HT1080 parental line . The mechanism for this radiation resistance is not clear, but a variety of cellular alterations seen in drug-resistant cell lines are discussed with reference to areas of further study. Mol Pharmacol, 1992 Jun, 41(6), 1008 - 15 Regulation by phorbol ester and protein kinase C inhibitors, and by a protein phosphatase inhibitor (okadaic acid), of P-glycoprotein phosphorylation and relationship to drug accumulation in multidrug-resistant human KB cells; Chambers TC et al.; Covalent modification by phosphorylation is a characteristic of the P-glycoproteins expressed in multidrug-resistant cells . This report describes analysis of P-glycoprotein phosphorylation in multidrug-resistant human KB-V1 cells and a study of the relationship of phosphorylation and drug accumulation . In isolated membranes, phosphorylation of P-glycoprotein by purified protein kinase C (PKC) was rapid, and time-dependent dephosphorylation was inhibited by okadaic acid, an inhibitor of type 1 and type 2A protein phosphatases . In 32P-labeled intact KB-V1 cells, P-glycoprotein phosphorylation was stimulated by both 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, and okadaic acid . Two-dimensional thin layer tryptic phosphopeptide maps indicated that the sites of phosphorylation were similar in control, TPA-treated, and okadaic acid-treated cells and that they corresponded to those phosphorylated by PKC in vitro . The protein kinase inhibitor staurosporine, and the PKC-selective inhibitors calphostin C and the alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, inhibited P-glycoprotein phosphorylation in vitro and in intact cells . Drug accumulation assays demonstrated that in KB-V1 cells TPA caused a decrease, whereas staurosporine and calphostin C caused an increase, in accumulation of {3H}vinblastine . These compounds did not significantly alter {3H}vinblastine levels in drug-sensitive KB-3 cells . These results suggest that PKC is chiefly responsible for P-glycoprotein phosphorylation in KB-V1 cells, that membrane-associated protein phosphatases 1 and 2A are active in dephosphorylation of P-glycoprotein, and that phosphorylation of P-glycoprotein may be an important mechanism for modulation of drug-pumping activity. Nippon Rinsho, 1992 Jun, 50(6), 1400 - 6 {Expression of multidrug resistance 1 and correlation with clinical drug resistance in acute leukemia}; Asou N et al.; Expression of multidrug resistance (mdr 1) gene, which encodes a transmembrane efflux pump referred to P-glycoprotein, leads to the decreased intracellular accumulation of various lipophilic drugs, such as vinca alkaloids, anthracyclines and epipodophyllotoxins . As these drugs are commonly used in chemotherapy for acute leukemia, it is of importance to determine whether mdr 1/P-glycoprotein expression is associated with clinical resistance . In several reports, some leukemia cells from untreated patients have expression of mdr 1/P-glycoprotein . We quantitatively detected low levels of mdr 1 expression in all cases of untreated acute leukemia and normal hematopoietic cells, using the reverse transcriptase-polymerase chain reaction . Carefully designed clinical trials including mdr 1 reversing agents may have significant consequences for the treatment of acute leukemia. Jpn J Cancer Res, 1992 Jun, 83(6), 644 - 9 Effector cell analysis of human multidrug-resistant cell killing by mouse-human chimeric antibody against P-glycoprotein; Nishioka Y et al.; A mouse-human chimeric monoclonal antibody (mAb), MH162, against P-glycoprotein was previously found to be more effective than an all-mouse mAb (MRK16) in lysis of multidrug-resistant (MDR) tumor cells by blood mononuclear cells . The present study was performed to identify the effector cells responsible for the chimeric mAb-dependent cell-mediated cytotoxicity (ADCC) against MDR cells . The ADCC reaction was assessed by a 6-h 51Cr release assay . Highly purified lymphocytes (greater than 99%), monocytes (greater than 99%) and neutrophils (greater than 96%) were obtained from peripheral blood of the same healthy donors . A comparison of these three effector cell populations showed no difference between MH162 and its all-murine counterpart MRK16 in MDR cell lysis by monocytes or neutrophils . But MH162 was more effective than MRK16 in lymphocyte-mediated lysis of the MDR cells . The lymphocytes responsible for this ADCC had CD16+ Fc receptors . Pretreatment of monocytes with colony-stimulating factors (IL-3, GM-CSF and M-CSF) caused significant increase in their MH162-mediated lysis of MDR cells . Another anti-P-glycoprotein chimeric mAb (MH171) was also more effective than its murine counterpart MRK17 in lymphocyte-mediated lysis of MDR cells . These findings suggest that mouse-human chimeric mAbs may be useful therapeutically for in vivo destruction of MDR cancer cells by the ADCC reaction. Br J Haematol, 1992 Jun, 81(2), 145 - 52 Expression of multidrug resistance gene mdr1 mRNA in a subset of normal bone marrow cells; Marie JP et al.; The multidrug resistance gene mdr1, encoding P-glycoprotein (P-gp), can be expressed at high levels in tumour cells derived from normal tissues with constitutive high expression of this gene . In myelogenous leukaemia, the incidence of increased expression of mdr1 gene contrasts with the low expression of this gene in normal bone marrow (b.m.) . To detect cells expressing mdr1 gene in normal and post-chemotherapy b.m., we used in situ RNA hybridization and RNA phenotyping by the polymerase chain reaction for mdr1 mRNA detection . The presence of P-gp was evaluated by immunocytochemistry with MRK16 . Fifteen b.m . (eight normal and seven post chemotherapy) were tested by in situ hybridization and either PCR (three b.m.) or immunocytochemistry (11 b.m.) or both (one b.m.) . With in situ mRNA hybridization, a subset (7.7% +/- 3.1%) of b.m . cells expressed mdr1 mRNA in all cases tested, with no significant differences between normal b.m . and post chemotherapy b.m . 18% of myeloid recognizable cells and 7% of the cells with lymphoid morphology expressed mdr1 mRNA . By RNA phenotyping, the four samples tested for in situ hybridization and two additional post chemotherapy b.m . expressed mdr1 . MRK16 was unable to detect a significant number of cells expressing P-gp either by immunocytochemistry in the 12 b.m . tested for in situ hybridization (0% in nine cases; 0.4%, 1% and 3% of positive cells in three cases), or by flow cytometry in six additional normal b.m . (0-1.4% positive cells). J Pharmacol Exp Ther, 1992 Jun, 261(3), 1222 - 30 Postnatal development of organic cation transport and mdr gene expression in mouse kidney; Dutt A et al.; The apical surface of the proximal tubular epithelium is the site of both P-glycoprotein localization and postulated active secretion of organic cations in the mammalian kidney . P-glycoprotein has been shown to act as a pleiotropic drug efflux pump across the cell membrane of tumor cells expressing the multidrug resistance phenotype, whereas the renal organic anion and organic cation secretory systems serve the function of pleiotropic drug transport across the proximal tubule epithelium . Because most known substrates for P-glycoprotein are organic cations, we tested the hypothesis that the physiological function of this protein in the kidney is to mediate renal organic cation secretion . In one approach, we compared the postnatal development of organic cation transport with that of kidney mdr gene expression . Cimetidine-sensitive uptake of classical substrates for renal secretion (N-methyl nicotinamide and tetraethylammonium) into kidney slices developed gradually in neonate mice, reaching adult capacity in 4 to 6 weeks . P-glycoprotein and its mRNA, as estimated by immunohistochemical methods and RNAse protection analysis, were undetectable at birth and were expressed abruptly at the adult level between 2 and 3 weeks of age . In another approach, classical inhibitors of renal organic cation secretion (cimetidine and cyanine 863) failed to reverse resistance to adriamycin in Chinese hamster ovary and P388 cell lines, which possess the phenotypic traits of multidrug resistance . These results suggest that the cimetidine-sensitive component of organic cation secretion is mediated by a protein other than the P-glycoprotein in the mammalian kidney. Endocrinology, 1992 Jun, 130(6), 3246 - 56 Characterization of multidrug-resistant pituitary tumor cells; Nelson EJ et al.; These studies were designed to investigate the role of P-glycoprotein in an endocrine cell line . Drug-resistant pituitary cells were obtained by growing GH4C1 cells in the presence of increasing concentrations of colchicine . Cells resistant to colchicine at 0.4 micrograms/ml, termed GH4C1/RC.4, exhibited the multidrug-resistance phenotype, as the LD50 values for colchicine, puromycin, actinomycin D, and doxorubicin were between 8 and 30 times greater than the corresponding values for the parental GH4C1 cells . Verapamil at 10 microM increased the sensitivity of GH4C1/RC.4 cells to colchicine, puromycin, and actinomycin D, almost completely reversing the drug resistance . Flow cytometry and fluorescence microscopy were used to demonstrate that GH4C1/RC.4 cells retained less rhodamine 123 than GH4C1 cells, and that the rate of efflux of rhodamine 123 was much faster for GH4C1/RC.4 cells . Immunocytochemical staining with a monoclonal antibody, C219, to the 170-kilodalton P-glycoprotein showed directly that GH4C1/RC.4 cells overexpress P-glycoprotein . We used drug-resistant pituitary cells to assess the possible role of P-glycoprotein in uptake and efflux of several hormones . At equilibrium, GH4C1 and GH4C1/RC.4 cells bound similar amounts of {125I}L-triiodothyronine and {125I}L-thyroxine, and verapamil did not alter either equilibrium binding or thyroid hormone efflux kinetics . Multidrug-resistant GH4C1/RC.4 cells retained less {3H}hydrocortisone than parental GH4C1 cells at equilibrium, and verapamil increased the equilibrium concentration of {3H}hydrocortisone 3.6-fold . The effect of verapamil was due to its ability to reverse multidrug resistance, since two other chemosensitizers, quinidine and vinblastine, increased {3H}hydrocortisone retention as effectively as verapamil but another calcium channel blocker, nifedipine, had no effect . The drug-resistant GH4C1/RC.4 line synthesized more GH (290%) and much less PRL (5%) than the parent . Hydrocortisone stimulated GH synthesis and inhibited PRL similarly in GH4C1 and GH4C1/RC.4 cells . The results show that the GH4C1/RC.4 line is multidrug-resistant and overexpresses the 170-kilodalton P-glycoprotein and suggest that the P-glycoprotein pump contributes to hydrocortisone kinetics. Cancer Res, 1992 Jun 1, 52(11), 3029 - 34 Multidrug-resistant phenotype of disease-oriented panels of human tumor cell lines used for anticancer drug screening; Wu L et al.; Disease-oriented panels of human tumor cell lines used by the National Cancer Institute for large-scale in vitro anticancer drug screening were evaluated for multidrug-resistant phenotype at the functional (in vitro drug sensitivity) and molecular levels . The cell line panels manifested a broad range of sensitivities to drugs typically associated with multidrug resistance (MDR) as well as to drugs not associated with MDR . Individual cell lines displayed unique and characteristic profiles of response . Patterns of correlated response were observed among, but not between, MDR and non-MDR drugs . Strong evidence of correlated response was limited to drugs sharing an intracellular mechanism of action . Several tumor cell lines exhibited a high degree of resistance to MDR drugs and relative sensitivity to non-MDR drugs, contained high levels of MDR-1 mRNA, and expressed cell surface P-glycoprotein detectable with one or more monoclonal antibodies . Parallel expression of all of these features representing the classic MDR phenotype was observed among members of the colon and renal tumor panels . Certain individual cell lines among other panels (lung, ovarian, melanoma, and central nervous system) also manifested some aspects of the MDR phenotype to various extents . Identification of MDR cell lines used for large-scale in vitro anticancer drug screening will facilitate interpretation of data in a way which may allow identification of new drug leads of potential value in treatment of MDR tumor cell populations. Mol Cell Biol, 1992 Jun, 12(6), 2855 - 65 Multidrug resistance in Leishmania donovani is conferred by amplification of a gene homologous to the mammalian mdr1 gene; Henderson DM et al.; Drug resistance is a major impediment to the effective treatment of parasitic diseases . The role of multidrug resistance (mdr) genes and their products in this drug resistance phenomenon, however, remains controversial . In order to determine whether mdr gene amplification and overexpression can be connected to a multidrug resistance phenotype in parasitic protozoa, a mutant strain of Leishmania donovani was generated by virtue of its ability to proliferate in medium containing increasing concentrations of vinblastine . The vinblastine-resistant strain, VINB1000, displayed a cross-resistance to puromycin and the anthracyclines, a growth phenotype that could be attributed to an impaired ability to accumulate the toxic drugs . By using the polymerase chain reaction, two different DNA fragments, LEMDR06 and LEMDRF2, were amplified from leishmanial genomic DNA, and each amplified fragment encoded a product that was significantly homologous to parts of the mammalian P-glycoprotein . In the VINB1000 strain, the mdr gene recognized by the LEMDR06 probe was amplified approximately 50-fold in copy number, whereas the mdr genes that hybridized to LEMDRF2 or to a fragment of the previously characterized ltpgpA gene were not amplified . Moreover, the VINB1000 cell line expressed a LEMDR06 gene transcript of 12.5 kb in size that was not detected in the parental wild-type strain . To furnish a functional test for mdr gene amplification and expression in L . donovani, the L . donovani gene recognized by the LEMDR06 polymerase chain reaction product, ldmdr1, was isolated from a genomic library, transfected into wild-type cells, and amplified over 500-fold by selection in 0.5 mg of G418 per ml . The resulting transfectants were resistant to all drugs to which VINB1000 cells were resistant and sensitive to all drugs to which VINB1000 cells were sensitive . These studies demonstrate that amplification of the ldmdr1 gene either by direct selection or subsequent to transfection can confer a drug-resistant phenotype in parasitic protozoa similar to that observed for MDR mammalian cells. Exp Hematol, 1992 Jun, 20(5), 565 - 8 In vitro effect of P-glycoprotein (P-gp) modulators on drug sensitivity of leukemic progenitors (CFU-L) in acute myelogenous leukemia (AML); Marie JP et al.; Cyclosporine A (CyA), a potent reversant of multidrug resistance (mdr), was studied for its effects on the sensitivity of leukemic progenitors (leukemia colony-forming units, CFU-L) to daunorubicin (DNR) and mitoxantrone . CyA was first compared to verapamil and cefoperazone for reversion of mdr in the mdr1+ cell line K562/DOX . A dramatic increase of sensitivity to 10(-6) M DNR was noted with CyA (0.5 and 1 microgram/ml) and verapamil (1 and 5 micrograms/ml), but not for cefoperazone (0.3 and 0.6 mg/ml) . The sensitivity of K562/DOX to 10(-6) M mitoxantrone was also slightly enhanced by CyA . The change in CFU-L drug sensitivity in the presence of CyA was then tested in 12 relapsing/resisting patients and in 3 untreated patients with acute myelogenous leukemia (AML) . No change in CFU-L sensitivity to DNR was noted, despite the presence of a subset of P-glycoprotein positive (P-gp+) cells in three out of the ten evaluable cases . Among the six evaluable cases tested with mitoxantrone and CyA, an increase of 50% in CFU-L sensitivity to mitoxantrone was noted in one (of three) P-gp+ patient . These data suggest that the CFU-L in AML rarely expressed the P-gp and that other mechanisms of drug resistance could be involved in AML. Biochem Biophys Res Commun, 1992 May 29, 185(1), 284 - 90 Functionally active homodimer of P-glycoprotein in multidrug-resistant tumor cells; Naito M et al.; P-glycoprotein plays a key role in multidrug resistance of tumor cells . In order to elucidate the possible quarternary structure/function relationship of P-glycoprotein, we treated multidrug-resistant human leukemia K562/ADM cells with the crosslinking reagent, disuccinimidyl suberate . In addition to 180K P-glycoprotein, a 340K protein was immunoprecipitated with an anti-P-glycoprotein monoclonal antibody, MRK-16 . The 340K protein is most probably a dimeric P-glycoprotein, since only the 180K P-glycoprotein was immunoprecipitated with MRK-16 when K562/ADM cells were treated with the cleavable crosslinking reagent, dithiobis(succinimidylpropionate), and analysed under reduced conditions . The dimeric P-glycoprotein was photolabeled with {3H}azidopine like the 180K monomeric P-glycoprotein and the photolabeling was inhibited by excess amount of vincristine and verapamil . The dimeric P-glycoprotein could be a functionally active form of the protein involved in the transport of antitumor agents. Int J Cancer, 1992 May 28, 51(3), 433 - 8 Pharmacologic interactions between the resistance-modifying cyclosporine SDZ PSC 833 and etoposide (VP 16-213) enhance in vivo cytostatic activity and toxicity; Keller RP et al.; Cyclosporin A reverses multidrug resistance (MDR) and increases the in vivo cytostatic activity and toxicity of the anticancer agent etoposide (VP 16-213) . SDZ PSC 833 (PSC 833), a non-immunosuppressive, non-toxic cyclosporin and very active modifier of P-gp 170-mediated MDR, elicits similar effects when administered with adriamycin . The underlying mechanisms, however, are not yet understood . The present pharmacological interaction study with PSC 833 and VP 16-213 was carried out to reveal the nature of this enhancement of cytostatic activity and toxicity . Rats pre-treated with either PSC-833 or solvent received a single dose of VP 16-213 . Plasma levels of VP 16-213 were measured by high-performance liquid chromatography (HPLC) . The resulting increase in cytostatic activity and toxicity of VP 16-213 mediated by PSC 833 was paralleled by marked changes in the pharmacokinetic parameters of VP 16-213 in vivo . Bioavailability and blood levels of VP 16-213 were significantly increased 30 min after administration if PSC 833 had been given before . The disappearance rate of VP 16-213 from the intravascular compartment was considerably slowed down by PSC 833 . In drug-sensitive xenografts of human colon carcinoma, the PSC-833-induced pharmacologic changes in vivo could be counteracted by dose reduction of VP 16-213 while a full therapeutic potential was maintained . Doses of VP 16-213, 1.5 to 2 times smaller, combined with PSC 833, were as effective in terms of tumor-growth inhibition as the maximum tolerated dose of VP 16-213 alone . Thus, pharmacologic interactions between PSC 833 or other resistance modifiers and VP 16-213 and other cytostatic agents require careful attention if they are to be used in humans to overcome MDR. Biochem Pharmacol, 1992 May 28, 43(10), 2091 - 102 High cell density-dependent resistance and P-glycoprotein-mediated multidrug resistance in mitoxantrone-selected Chinese hamster cells; Muller C et al.; Mitoxantrone (MIT) resistance has been studied in a colony selected from the CHO AA8 parental line in one step under a low degree of selective pressure (9 nM) . The cells of the clonal isolate AA8/MIT C1(0) were sensitive to 9 nM MIT at low cell density but able to grow at high density . Parental AA8 cells were not able to grow under the latter condition . Decreased MIT accumulation (-20%) was observed at this step (step 0) in the absence of overexpression of mdr RNA coding for the drug efflux pump P-glycoprotein . Furthermore, AA8/MIT C1(0) did not exhibit cross resistance to vincristine, Adriamycin and etoposide at low cell density . During subsequent controlled growth for 2 months at high cell density in the presence of 9 nM drug, an additional selection occurred leading to a 4-fold MIT-resistant subline AA8/MIT C1(+) . This subline was characterized at this step (step I) and after an additional 4 months of culture in the presence of 9 nM MIT (step II) . Analysis of mdr gene expression and gene copy number showed an increase in mdr RNA and a pattern of mdr gene amplification which changed between step I and II . AA8/MIT C1(+)II exhibited a classical multidrug resistance phenotype with decreased accumulation of {14C}MIT and cross-resistance to vincristine, Adriamycin and etoposide . The ability to form the cleavable complex in the presence of etoposide in DNA topoisomerase II-containing nuclear extracts was identical in AA8/MIT C1(+)II and AA8 cell lines . These results demonstrate a new sequence of events in MIT resistance: low level of drug resistance at high cell density followed by mdr gene amplification. J Biol Chem, 1992 May 25, 267(15), 10638 - 44 Energy-dependent accumulation of daunorubicin into subcellular compartments of human leukemia cells and cytoplasts; Slapak CA et al.; Anthracycline accumulation was evaluated by flow cytometry or radiolabeled drug assays in cells and cytoplasts (enucleated cells) prepared from parental and multidrug-resistant human K562 leukemia cells . Treatment with energy inhibitors, such as dinitrophenol (DNP) or sodium azide/deoxyglucose, led to a marked decrease in daunorubicin accumulation in parental cells and cytoplasts . Another ionophore, monensin, also caused a significant decrease in daunorubicin accumulation; however, ATPase inhibitors ouabain, vanadate, and N-ethylamaleimide had little or no effect . The lysosomatropic agents chloroquine and methylamine caused a moderate decrease in anthracycline accumulation . Fluorescence microscopy showed that the DNP-sensitive daunorubicin uptake occurred in a nonnuclear subcellular compartment . Studies using increasing daunorubicin concentrations demonstrated fluorescence quenching that occurred in the nonnuclear, DNP-sensitive compartment . The effect of inhibitors on the accumulation of rhodamine 123 and acridine orange strongly implicated lysosomes as the principal compartment of this inhibitable daunorubicin accumulation . Cytoplasts from P-glycoprotein containing multidrug-resistant K562 cells demonstrated a verapamil-reversible, decreased daunorubicin accumulation that was observed in resistant whole cells . Verapamil pretreatment of cytoplasts from resistant cells revealed the subcellular DNP-sensitive uptake present in parental cytoplasts . These studies demonstrate that cytoplasts are an effective means to study drug transport in mammalian cells without nuclear drug binding . Parental K562 cells and cytoplasts exhibit an energy-dependent accumulation of daunorubicin into cytoplasmic organelles that is also present in resistant cells and cytoplasts when P-glycoprotein mediated efflux is inhibited. Cancer Res, 1992 May 15, 52(10), 2874 - 9 Relationship of VP-16 to the classical multidrug resistance phenotype; Sehested M et al.; The classical multidrug resistance (MDR) phenotype is characterized by cross-resistance between a number of chemically unrelated drugs due to an increased efflux across the plasma membrane via a P-glycoprotein-mediated mechanism . The epipodophyllotoxin derivatives etoposide (VP-16) and teniposide (VM-26) are usually included among the drugs recognized by this MDR phenotype, and the MDR EHR2/DNR cell line is greater than 50-fold cross-resistant to VP-16 . The steady-state accumulation of VP-16 in EHR2/DNR cells is only half that of wild-type EHR2 cells, and deprivation of energy by sodium azide surprisingly increased accumulation to a similar extent in both sublines . Efflux was rapid (halflife of 32-35 s) and similar in both sublines, while initial influx was markedly lower in the resistant cells . The temperature coefficients over 10 degrees C for VP-16 in- and efflux indicated passive transport in both sublines . In agreement with this finding, up to 10-fold molar excess (50 microM) VM-26 had no effect on VP-16 accumulation in MDR cells . VP-16 at a 100-fold molar excess inhibited azidopine photoaffinity labeling of P-glycoprotein by only 30% and vincristine binding to plasma membrane vesicles from EHR/DNR cells by 45% . However, VP-16 itself did not differentially bind to plasma membrane vesicles from EHR2 and EHR2/DNR cells . Finally, neither VP-16 accumulation nor cytotoxicity in EHR2/DNR cells were increased to the same degree as for daunorubicin and vincristine by verapamil, and the modulation was similar in wild-type and resistant cells . Thus, although VP-16 may be a substrate for P-glycoprotein, its other transport characteristics such as rapid diffusion and sensitivity to membrane perturbation in wild-type cells lessen any effect of P-glycoprotein-mediated efflux, resulting in a lack of differential modulation by verapamil . These results may be considered when planning clinical trials involving MDR modulators and epipodophyllotoxin derivatives. Cancer Res, 1992 May 15, 52(10), 2797 - 801 Cinchonine, a potent efflux inhibitor to circumvent anthracycline resistance in vivo; Genne P et al.; Circumvention of multidrug resistance is a new field of investigation in cancer chemotherapy, and safe and potent multidrug resistance inhibitors are needed for clinical use . We investigated several analogues of quinine for their ability to increase anthracycline uptake in resistant cancer cells . Cinchonine was the most potent inhibitor of anthracycline resistance in vitro, and its activity was little altered by serum proteins . Serum from rats treated with i.v . cinchonine produced greater uptake of doxorubicin in cancer cells (DHD/K12/PROb rat colon cells and K562/ADM human leukemic cells) than did serum from quinine-treated rats (ex vivo assay) . Cinchonine was more effective than quinine in reducing tumor mass and increasing the survival of rats inoculated i.p . with DHD/K12/PROb cells and treated i.p . with deoxydoxorubicin . Moreover, the acute toxicity of cinchonine in rats and mice was lower than that of other quinine-related compounds . The lower toxicity and greater potentiation of in vivo anthracycline activity produced by cinchonine are favorable characteristics for its use as an anti-multidrug resistance agent in future clinical trials. Eur J Biochem, 1992 May 15, 206(1), 137 - 49 Quantitative expression patterns of multidrug-resistance P-glycoprotein (MDR1) and differentially spliced cystic-fibrosis transmembrane-conductance regulator mRNA transcripts in human epithelia; Bremer S et al.; P-glycoprotein (MDR1), that confers multidrug resistance in cancer, and the cystic-fibrosis transmembrane-conductance regulator (CFTR), that is causative defective in cystic fibrosis, belong to the family of ATP-binding transport proteins . The expression of MDR1 and CFTR in human epithelial tissues and the cell lines T84 and HT29 was estimated by primer-directed reverse transcription (RT) and subsequent monitoring of the kinetics of cDNA product formation during the polymerase chain reaction (PCR) . MDR1 mRNA was found in high levels, 15-50 amol mRNA/microgram RNA, in the intestine, kidney, liver and placenta, and in low levels, 0.2 amol/microgram RNA, in respiratory epithelium . Large amounts of CFTR mRNA were measured in the gastrointestinal tract, whereas the kidney, as the phenotypically normal organ, and the lung, as the most severely affected organ in cystic fibrosis, both contained low amounts, 3 amol CFTR/microgram RNA . CFTR transcript levels of 1-5 amol/microgram RNA were determined in lymphocytes and lymphoblast cell lines, suggesting that lymphoblasts are an accessible source for the study of the molecular pathogenesis of cystic fibrosis . When transcripts were scanned by overlapping RT/PCR analyses, only transcript of expected size was detected for MDR1 mRNA, where variable in-frame deletions of either exon 4, 9 or 12 were observed in CFTR mRNA . The complete loss of single exons was seen at proportions of 1-40% in all investigated tissues and cell lines with large donor-to-donor variation . Exons 9 and 12 of the CFTR gene encode parts of the evolutionarily well-conserved first nucleotide-binding fold including the two Walker motifs . Alternative splicing may give rise to various CFTR forms of different function and localization. Proc Natl Acad Sci U S A, 1992 May 15, 89(10), 4564 - 8 Amino acid substitutions in the sixth transmembrane domain of P-glycoprotein alter multidrug resistance; Devine SE et al.; Eukaryotic cells can display resistance to a wide range of natural-product chemotheraputic agents by the expression of P-glycoprotein (pgp), a putative plasma membrane transporter that is thought to mediate the efflux of these agents from cells . We have identified, in cells selected for multidrug resistance with actinomycin D, a mutant form of pgp that contains two amino acid substitutions within the putative sixth transmembrane domain . In transfection experiments, this altered pgp confers a cross-resistance phenotype that is altered significantly from that conferred by the normal protein, displaying maximal resistance to actinomycin D . These results strongly implicate the sixth transmembrane domain in the mechanism of pgp drug recognition and efflux . Moreover, they indicate a close functional homology between pgp and the cystic fibrosis transmembrane regulator in which the sixth transmembrane domain has also been shown to influence substrate specificity. J Natl Cancer Inst, 1992 May 6, 84(9), 711 - 6 Various methods of analysis of mdr-1/P-glycoprotein in human colon cancer cell lines; Herzog CE et al.; BACKGROUND: Multidrug resistance (MDR) mediated by high levels of mdr-1 (also known as PGY1)/P-glycoprotein (Pgp) has been studied in tissue culture systems; however, most tumor samples which express mdr-1/Pgp have much lower levels . PURPOSE: We wanted to determine if levels seen clinically could be detected by commonly used methods and to determine if these levels conferred MDR reversible by Pgp antagonists . METHODS: We studied multi-drug-resistant cell lines and sublines with levels of mdr-1/Pgp expression comparable to those seen clinically . We evaluated the expression of mdr-1 RNA by Northern blot analysis, slot blot analysis, polymerase chain reaction (PCR) analysis, and in situ hybridization . We evaluated protein expression by immunofluorescence, immunohistochemistry, fluorescence-activated cell sorting, and immunoblotting analyses . Drug resistance and reversibility were determined by cell growth during continuous drug exposure . RESULTS: In most cases, the low level of mdr-1/Pgp present in these cell lines could be detected by each method, but the assays were at the limit of sensitivity for all methods except the PCR method . These low levels of mdr-1/Pgp are capable of conferring MDR, which can be antagonized by verapamil . CONCLUSIONS: Levels of mdr-1/Pgp similar to those found in clinical samples can be detected by each of these methods, but the PCR method was the most sensitive and most reliably quantitative . IMPLICATIONS: In vitro sensitization by the addition of verapamil in cell lines with these low levels of mdr-1/Pgp suggests that clinically detected levels may confer drug resistance in vivo. Anticancer Res, 1992 May-Jun, 12(3), 921 - 5 Therapeutic efficacy of interleukin-2 activated killer cells against adriamycin resistant mouse B16-BL6 melanoma; Gautam SC et al.; Development of multidrug-resistance (MDR) remains a major cause of failure in the treatment of cancer with chemotherapeutic agents . In our efforts to explore alternative treatment regimens for multidrug-resistant tumors we have examined the sensitivity of MDR tumor cell lines to lymphokine activated killer (LAK) cells . Adriamycin (ADM) resistant B16-BL6 melanoma, L1210 and P388 leukemic cell lines were tested for sensitivity to lysis by LAK cells in vitro . While ADM-resistant B16-BL6 and L1210 sublines were found to exhibit at least 2-fold greater susceptibility to lysis by LAK cells, sensitivity of ADM-resistant P388 cell was similar to that of parental cells . Since ADM-resistant B16-BL6 cells were efficiently lysed by LAK cells in vitro, the efficacy of therapy with LAK cells against the ADM-resistant B16-BL6 subline in vivo was evaluated . Compared to mice bearing parental B16-BL6 tumor cells, the adoptive transfer of LAK cells and rIL2 significantly reduced formation of experimental metastases (P less than 0.009) and extended median survival time (P less than 0.001) of mice bearing ADM-resistant B16-BL6 tumor cells . Results suggest that immunotherapy with LAK cells and rIL2 may be a useful modality in the treatment of cancers with the MDR phenotype. Anticancer Res, 1992 May-Jun, 12(3), 661 - 7 Stable expression of a cDNA encoding rat brain protein kinase C-beta I confers a multidrug-resistant phenotype on rat fibroblasts; Fan D et al.; Protein kinase C (PKC) is a Ca++- and phospholipid-dependent protein kinase that plays an important role in signal transduction pathways that regulate cell growth . Tumor cells selected for a multidrug resistant (MDR) phenotype often express elevated levels of PKC activity . To directly test whether PCK overexpression can produce an MDR phenotype, we studied rat embryo fibroblasts that were infected with the full-length cDNA clone RP58 encoding the beta I form of rat brain PKC . The PKC-beta I gene recipient R6-PKC3 cells are stable, overproduce PKC, and express an elevated level of PKC activity . R6-PKC3 cells exhibited significant resistance to adriamycin, actinomycin D, vinblastine, and vincristine but not to 5-fluorouracil . Intracellular accumulation of adriamycin, vinblastine, and vincristine was decreased in the R6-PKC3 cells, but this was not associated with an altered level of P-glycoprotein expression . Moreover, the reduction in drug accumulation appeared to be a consequence of a decreased rate of drug uptake . The data indicate that overexpression of PKC in rat fibroblasts produces an MDR phenotype without altering P-glycoprotein expression. Anticancer Res, 1992 May-Jun, 12(3), 649 - 53 Multidrug resistance in Yoshida rat ascites hepatoma cell lines; Miyamoto K et al.; Rat ascites hepatoma (AH) cell lines that were induced by dimethylaminoazobenzene and established as transplantable tumors had different sensitivities to vinblastine (VBL) . The most VBL-resistant cells, AH66, showed more cross-resistance to vincristine and anthracyclines than AH66F cells . The resistance of AH66 cells was significantly decreased by verapamil . VBL-resistance of AH66 cells was inhibited by other drugs reported as overcoming acquired multidrug resistance, while the sensitivity of AH66F cells was hardly influenced by these drugs . The lowered uptake and enhanced extrusion of the antitumor drug in AH66 cells were suppressed by verapamil . M(r) 160,000 protein in the plasma membrane from AH66 was labeled with a photoactive VBL analog and was immunopositive to a monoclonal antibody against P-glycoprotein, C219 . The sensitive cells had barely detectable levels of the surface membrane components . Specific photo-labeling with a VBL analog of P-glycoprotein of AH66 cell membrane was inhibited by reserpine and verapamil which restored the VBL resistance . These results indicate that AH66 cells are a naturally acquired multidrug-resistant cell line overexpressing a P-glycoprotein, and AH cell lines are useful to study multidrug resistance of hepatic carcinomas and development of counteracting drugs. Mol Biol Cell, 1992 May, 3(5), 507 - 20 Double minute chromosomes carrying the human multidrug resistance 1 and 2 genes are generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells; Schoenlein PV et al.; This study characterizes amplified structures carrying the human multidrug resistance (MDR) genes in colchicine-selected multidrug resistant KB cell lines and strongly supports a model of gene amplification in which small circular extrachromosomal DNA elements generated from contiguous chromosomal DNA regions multimerize to form cytologically detectable double minute chromosomes (DMs) . The human MDR1 gene encodes the 170-kDa P-glycoprotein, which is a plasma membrane pump for many structurally unrelated chemotherapeutic drugs . MDR1 and its homolog, MDR2, undergo amplification when KB cells are subjected to stepwise selection in increasing concentrations of colchicine . The structure of the amplification unit at each step of drug selection was characterized using both high-voltage gel electrophoresis and pulsed-field gel electrophoresis (PFGE) techniques . An 890-kb submicroscopic extrachromosomal circular DNA element carrying the MDR1 and MDR2 genes was detected in cell line KB-ChR-8-5-11, the earliest step in drug selection in which conventional Southern/hybridization analyses detected MDR gene amplification . When KB-ChR-8-5-11 was subjected to stepwise increases in colchicine, this circular DNA element dimerized as detected by PFGE with and without digestion with Not 1, which linearizes the 890-kb amplicon . This dimerization process, which also occurred at the next step of colchicine selection, resulted in the formation of cytologically detectable DMs revealed by analysis of Giemsa-stained metaphase spreads. Cancer Genet Cytogenet, 1992 May, 60(1), 14 - 9 A multidrug-resistant ovarian carcinoma cell line with a malignant suppressed phenotype is a CD44 gene expression defective mutant; Teyssier JR et al.; A multidrug-resistant cell subline (OV1/VCR) derived from an ovarian adenocarcinoma cell line (OV1/P) was characterized by a typical suppressed malignant phenotype and by a unique karyotypic change: del(11)(p13) . In an attempt to discern some genetic alteration of 11p genes that may be relevant to the phenotypic shift, cells were analyzed with DNA probes mapped in the deleted region and with monoclonal antibodies (MoAbs) against 11p-encoded membrane molecules . Southern blot did not detect abnormal restriction patterns of the probed sequences . OV1/VCR cells did not express the CD44 epitope (11p13 MIC4 locus) recognized by the F10-44 MoAb and did not accumulate RNAs of the CD44 (Hermes) core peptide . This defect was not detected in another OV1/P-derived drug-resistant subline that retained the malignant behavior and did not have the del(11p) marker . It may have contributed to phenotypic reversion because evidence shows that CD44 membrane molecule is involved in cell-cell interaction and growth regulation of cancer cells. Biochem J, 1992 May 1, 283 ( Pt 3), 755 - 8 Uptake of the antitrypanosomal drug 5'-({(Z)-4-amino-2-butenyl}methylamino)-5'-deoxyadenosine (MDL 73811) by the purine transport system of Trypanosoma brucei brucei; Byers TL et al.; An irreversible inhibitor of S-adenosyl-L-methionine decarboxylase (AdoMetDC), 5'-({(Z)-4-amino-2-butenyl}methylamino)-5'-deoxyadenosine (MDL 73811), was found to cure Trypanosoma brucei brucei and multidrug-resistant T . b . rhodesiense infections in mice {Bitonti, Byers, Bush, Casara, Bacchi, Clarkson, McCann & Sjoerdsma (1990) Antimicrob . Agents Chemother . 34, 1485-1490} . Doses of this drug which resulted in a rapid clearance of parasites from T . b . brucei-infected rats resulted in plasma levels of 50-60 microM-MDL 73811 and an intratrypanosomal MDL 73811 concentration of 1.9 mM within 10 min of administration {Byers, Bush, McCann & Bitonti (1991) Biochem . J . 274, 527-533{ . Based on this finding we speculated that MDL 73811, which is an adenosine analogue, is a substrate for the trypanosome active purine transport system . We now report evidence that supports this hypothesis . MDL 73811 uptake by T . b . brucei in vitro was time- and temperature-dependent and was saturable over a time course in which MDL 73811 metabolism was undetectable, suggesting that MDL 73811 uptake is a transport-mediated phenomenon . Inhibition of MDL 73811 uptake by purine nucleosides is consistent with the drug being a substrate for the trypanosome purine transport system . The accumulation of MDL 73811 by cultured L1210 mouse leukaemia cells was significantly less than by trypanosomes exposed to the same pharmacologically relevant concentrations of MDL 73811 . Given that the half-life of MDL 73811 in the plasma of rats and mice is approx . 10 min, it seems likely that the existence of a highly active parasite transport system for MDL 73811 is crucial for the sensitivity of trypanosomes towards MDL 73811 in vivo, and that the absence of active transport of MDL 73811 by the host's cells may play a role in the selectivity of this drug. Hepatology, 1992 May, 15(5), 899 - 903 Effect of cyclosporine on colchicine secretion by a liver canalicular transporter studied in vivo; Speeg KV et al.; The multidrug resistance transport protein is a normal constituent of the liver canalicular membrane, although its function has not been defined in vivo . Colchicine, a multidrug resistance substrate, is eliminated mainly by the liver . Cyclosporine reverses multidrug resistance in vitro, presumably by inhibiting the multidrug resistance transporter . This study assesses biliary colchicine elimination and the effect of cyclosporine on this process . After cyclosporine administration biliary colchicine clearance decreased from 11.6 +/- 0.8 to 2.2 +/- 0.4 ml/min.kg (p less than 0.05), and the colchicine bile/plasma ratio decreased from 166 +/- 9 to 38 +/- 5 (p less than 0.05) . Cremophor EL (a cyclosporine vehicle) transiently inhibited biliary colchicine clearance and colchicine bile/plasma ratio, but to a much smaller extent than cyclosporine in vehicle . Biliary cyclosporine clearance was 0.122 and 0.024 ml/min.kg after bolus doses of 2 or 10 mg/kg intravenously, respectively . Cyclosporine bile/plasma ratio was 1.3 to 5.2 . When cyclosporine was given 16 hr before colchicine infusion, biliary colchicine clearance decreased 39% (p less than 0.05), and colchicine bile/plasma ratio decreased 51% (p less than 0.05) . Thus colchicine is actively secreted into bile and will be useful in the study of the multidrug transporter in vivo . Cyclosporine profoundly inhibits colchicine secretion into bile but is itself mainly metabolized rather than secreted . If competition for a common carrier is the basis for the interaction, then cyclosporine represents a drug that binds to but is not transported by the canalicular transporter. Cancer Res, 1992 May 1, 52(9 Suppl), 2652s - 2657s New approaches to treating early lung cancer; Roth JA; The survival from lung cancer has not changed over the past 15 years, despite the intensive application of combined modality therapy for advanced disease . Prevention and early diagnosis appear to be the most promising strategies for reduction of lung cancer mortality . A Keystone Colloquium was held April 1 to 7, 1991, on the Biology of and Novel Therapeutic Approaches for Epithelial Cancers of the Aerodigestive Tract, to discuss recent basic and clinical research in this area . This paper summarizes the presentations relevant to lung cancer, including genetic mechanisms, growth factors, neuropeptides, growth and differentiation, carcinogenesis, chemoprevention, multidrug resistance, and immunology. Cancer Biochem Biophys, 1992 May, 12(4), 241 - 52 Protein synthetic activity and adenylate energy charge in Rhein-treated cultured human glioma cells; Delpino A et al.; The effect of Rhein (RH) on the protein synthetic activity and adenylate energy charge in human glioma cells cultured in vitro has been investigated . The results demonstrate that in RH-treated cells, the protein synthesis is strongly decreased, but no modifications in the qualitative pattern occur . The extent of inhibition is a function of the drug concentration as well as of the time of exposure . Such an inhibition must be ascribed mainly to a reduction of adenylate energy charge brought about by RH because of its effect on respiration and glycolysis . The correlation between the adenylate energy charge and cell viability, as well as the possibility of using rhein as a biochemical modulator to reduce or to reverse multidrug resistance, are also discussed. Am J Pathol, 1992 May, 140(5), 1009 - 16 Comparison of an immunoperoxidase "sandwich" staining method and western blot detection of P-glycoprotein in human cell lines and sarcomas; Toth K et al.; The applicability of a multilayer immunoperoxidase "sandwich" method (IpS) developed by Chan14 for the amplified detection of P-glycoprotein (Pgp) was investigated . The authors examined 15 formalin-fixed cell lines, as well as formalin-fixed, paraffin-embedded sections from single biopsies of 46 sarcomas . The cell lines included sensitive and multidrug resistant sublines (KB, A2780, MCF-7, HeLa) with various relative degrees of resistance to doxorubicin (Dox) . The sarcoma biopsy specimens were selected on the basis of the results obtained in Western blot (WB) detection of Pgp (22 positive and 24 negative by WB) using C219 and C494 monoclonal antibodies to Pgp . The IpS method employed C219 . The least resistant cell line in which Pgp could be detected by IpS was fivefold resistant to doxorubicin, whereas Pgp was detected by WB in cells greater than twofold resistant . Cell lines having greater than fivefold resistance to Dox were positive by both IpS and WB analyses . The less resistant cell lines contained more nonreactive cells whereas the highly resistant cell lines showed more homogeneous strong membrane reactions . Among the six cell lines determined to be Pgp negative by WB analysis, no false positive immunostaining by IpS existed . One of 22 WB positive and 7 of 24 WB-negative sarcoma biopsy specimens were positive by IpS methods . Reaction varied and was always focal (a minimum of 3-5 cells, ranging up to 3-4 high power fields) indicating pronounced heterogeneous distribution of Pgp . Thus, WB can detect low average (overall) levels of Pgp in tumor samples but such low concentrations of PgP at the single cell are not detectable by IpS methods . However, IpS can discern among many Pgp-negative cells small subpopulations of immunoreactive cells, which are not detected by WB analysis due to Pgp dilution by the membrane protein of numerous Pgp negative cells . IpS and WB used together as complementary methods can provide more complete information about Pgp distribution and content, particularly in the case of heterogeneous human tumors . The IpS method is more suitable for less drastically treated (not embedded) cell line specimens than for paraffin-embedded (routine) sections . Some modification of the present IpS protocol seems necessary to increase its sensitivity and reduce the disparity with WB results. Mutat Res, 1992 May, 276(3), 285 - 90 Gene amplification in the murine SEWA system; Levan G et al.; Considerable work with DNA amplification has been carried out in the murine SEWA ascites tumor cell system . In SEWA cells there is 'spontaneous' amplification of the c-myc oncogene, and transitions between different cytogenetic expressions of gene amplification such as DM (double minutes), CM (C-bandless chromosomes) and HSR (homogeneously staining regions) of the amplified DNA have been recorded during serial in vivo transplantations . In SEWA cells it has also been shown that the c-myc-containing DM will he lost under in vitro conditions, but are rapidly recovered if the cells are reinjected into animals . Additional gene amplification has been superimposed on the c-myc amplification in SEWA cells by stepwise selection in vitro, leading to resistance to different drugs, such as methotrexate, actinomycin D, colcemid and vincristine . Cytogenetically, DNA amplification is multifaceted and, in addition to the structures mentioned, it may also take the form of CB (chromatin bodies), which have been shown to be the carriers of resistance genes in hybrids between multidrug-resistant SEWA cells and Chinese hamster CHO cells . In most instances, DM are noncentromeric and distributed by a 'hitch-hiking' mechanism at mitosis; in one colcemid-resistant SEWA line, however, we have shown that the DM carry active centromeres . The molecular mechanism behind DNA amplification appears to be complex . We have shown that in four independently derived multidrug-resistant SEWA sublines the amplicons resided on circular molecules which were about 2500 kb long and carried at least five genes, including the three mouse mdr genes . Within the circles the DNA was unrearranged compared to the organization of the DNA in sensitive cells. Mutat Res, 1992 May, 276(3), 151 - 61 From amplification to function: the case of the MDR1 gene; Roninson IB; This review describes the features of gene amplification associated with the selection of multidrug-resistant cell lines . Some of these lines carry multiple copies of the MDR1 gene that encodes P-glycoprotein, a broad specificity efflux pump . The MDR1 gene was initially identified as the common component of the amplicons found in multidrug-resistant cell lines selected with different drugs . Subsequent studies have established that increased MDR1 expression is sufficient for the multidrug-resistant phenotype . MDR1-containing amplicons may include a number of additional transcribed genes that do not appear to contribute to multidrug resistance . MDR1 amplification is associated with specific chromosomal changes and apparently non-random recombinational events . Increased expression of the MDR1 gene, however, does not necessarily require gene amplification . Although amplification of the MDR1 gene has not been found in clinical tumor samples, increased expression of this gene is commonly observed in different types of cancer and appears to be a significant marker of clinical drug resistance. Hematol Oncol, 1992 May-Aug, 10(3-4), 213 - 20 Expression of p170 protein in multiple myeloma: a clinical study; Ucci G et al.; The expression of the p170 multidrug resistance protein by bone marrow plasma cells (BMPC) was assessed at clinical presentation in 53 patients with multiple myeloma (MM) using the C219 monoclonal antibody . Twenty-two of the 53 (41 per cent) patients had variable aliquots (1-60 per cent, median = 6 per cent) of p170+ BMPC by immunocytochemistry . Five of 10 patients studied using bivariate flow cytometry had both diploid and hyperdiploid (DNA index ranged from 1.2 to 1.5) BMPC with hyperdiploid clones having significantly greater p170 expression than diploid ones . Of the 37 patients evaluated for a response, 20 (54 per cent) had responded to induction chemotherapy . The presence of p170+ BMPC was a negative indicator for achieving response . The response rate was 75 per cent for p170- and 25 per cent for p170+ cases (p < 0.01), with no difference on the basis of treatment schedule (melphalan and prednisone, 24 patients; peptichemio, vincristine and prednisone, 13 patients) . No difference in response and survival duration was found between p170+ and p170- patients . In six of nine patients studied both at diagnosis and following induction chemotherapy the p170+ BMPC% increased irrespective of the type of treatment or outcome. Horm Metab Res, 1992 May, 24(5), 210 - 3 New mechanisms of hormone secretion: MDR-like gene products as extrusion pumps for hormones? Becker KF, Allmeier H, Hollt V. P-glycoprotein, the product of the multidrug resistance (MDR1) gene, is an ATP-driven transmembrane pump that increases the resistance of cells by actively exporting toxic chemicals . In addition to transporting anticancer drugs, P-glycoprotein has been reported to extrude a variety of lipophilic drugs, such as calcium channel blockers, phenothiazines, cyclosporines etc . Interestingly, recent experiments suggest that steroid hormones may be physiologic substrates for P-glycoprotein . In addition, there exists a family of transporter genes with high structural homology to P-glycoprotein, the so-called ABC (ATP-binding casette) family . Although the physiological ligands for most of these transporters are unknown, there is increasing evidence that peptides may be transported by some of these proteins . Thus, the a-factor, a farnesylated pheromone with 13 amino acids, is exported from yeast cells by the product of the STE6 gene, a transporter protein with high homology to P-glycoprotein . Recently, we have cloned a novel member of the ABC-transporter gene family from neuroblastoma x glioma hybrid (NG-108-15) cells . This putative transporter gene ("NG-TRA") is expressed in the adrenal gland, kidney and in the brain . High amounts of NG-TRA mRNA are found in a variety of human brain tumors . Whether NG-TRA and/or other MDR-related transporters are involved in the transport of steroids, peptide hormones or growth factors remains to be established . If so, the cellular export of hormones by active pumps may represent a new mechanism of hormone secretion. Biochem Cell Biol, 1992 May, 70(5), 365 - 75 L-histidinol in experimental cancer chemotherapy: improving the selectivity and efficacy of anticancer drugs, eliminating metastatic disease and reversing the multidrug-resistant phenotype; Warrington RC; Human cancer chemotherapy is limited by two major problems: the failure of commonly used anticancer drugs to act against tumor cells in a specific manner and the ability of malignant cells to resist killing by antineoplastic agents . Experimentally, both of these problems can be solved by using L-histidinol in combination with conventional anticancer dru