Multidrug resistant

Cancer Res, 1991 Oct 1, 51(19), 5417 - 24
Chemotherapy and chemosensitization of transgenic mice which express the human multidrug resistance gene in bone marrow: efficacy, potency, and toxicity; Mickisch GH et al.; A common form of multidrug resistance in human cancer results from expression of the MDR1 gene which encodes a plasma membrane energy-dependent multidrug efflux pump . We have engineered transgenic mice which express this multidrug transporter in their bone marrow cells and demonstrated that peripheral WBC of these animals provide a rapid and reliable system for assessing the bioactivity of agents that reverse multidrug resistance . Immunocytochemical analysis of bone marrow smears suggests that the activation of the MDR1 transgene has probably occurred at a very early stage of bone marrow differentiation since most bone marrow cells express the transporter . Expression of this transgene in bone marrow produces about 10-fold resistance to leukopenia induced by taxol compared to normal bone marrow . Chemosensitization of MDR1 mice to daunomycin and taxol, measured by a fall in WBC, is detectable at a dose as low as 0.01 mg/kg R-verapamil . A dose of 0.5 mg/kg R-verapamil reduces the WBC by nearly 50% . Chemosensitization of MDR-transgenic mice with 5 mg/kg R-verapamil, which is highly effective in reversing MDR and readily tolerated by mice, necessitates a reduction of the maximum tolerated dose of most chemotherapeutic agents by only 20% . In addition, detailed histopathological examination shows that treatment of mice with chemotherapeutic drugs and R-verapamil does not change the organ-related toxicity pattern but only moderately accentuates inherent toxic side effects of the chemotherapeutic agents . We conclude that MDR1-transgenic mice represent a valid model for evaluating efficacy, potency, and toxicity associated with chemotherapy and chemosensitization of multidrug-resistant cells in animals.

Br J Cancer, 1991 Oct, 64(4), 705 - 9
Modulation of vinblastine sensitivity by dipyridamole in multidrug resistant fibrosarcoma cells lacking mdr1 expression; Shalinsky DR et al.; We examined the ability of dipyridamole (DPM) to act synergistically with vinblastine (VBL) in HT1080 fibrosarcoma cells and a drug-resistant variant, HT1080/DR4, which lacks mdr1 expression, in order to determine whether DPM requires P-glycoprotein to modulate drug sensitivity . Median effect analysis of clonogenic assay was used to produce the combination index (CI50, values less than 1 indicate synergy) . DPM was mildly synergistic with VBL producing a CI50 of 0.83 +/- 0.13 for HT1080 cells and 0.80 +/- 0.16 for HT1080/DR4 cells . HT1080 and HT1080/DR4 cells accumulated 6.7 +/- 0.7 and 5.6 +/- 0.9 pmol 3H-VBL mg cells-1 at steady state (Css) and 20 microM DPM elevated the Css by 1.8 and 2.9-fold, respectively . In comparison, the CI50 was 1.1 +/- 0.2 in parental KB-3-1 cells and 0.1 +/- 0.1 in mdr1-expressing KB-GRC1 cells . The KB-3-1 and KB-GRC1 cells had a Css of 3.8 +/- 0.8 and 0.7 +/- 0.2 pmol 3H-VBL mg cells-1, respectively, and DPM elevated the Css by 9.2-fold in KB-GRC1 cells . These studies demonstrate that DPM can produce synergy independently of mdr1 expression but that much greater levels of synergy are achievable in mdr1-expressing tumour cells.

J Urol, 1991 Oct, 146(4), 982 - 6; discussion 986-7
Flow cytometric determination of the multidrug resistant phenotype in transitional cell cancer of the bladder: implications and applications; Benson MC et al.; We detail our experience with a monoclonal antibody to detect the cell surface P-glycoprotein product of the multidrug resistance gene (MDR-1) in the human bladder . A total of 32 patients had 44 different specimens analyzed . The samples consisted of 8 normal bladders, 21 transitional cell carcinomas, 1 mucinous adenocarcinoma, 3 P-0 bladder wall specimens and 10 nonmalignant urothelial samples from cystectomies . P-glycoprotein was not detected in the normal adult or pediatric bladder . Bladder specimens from 3 children with a neurogenic bladder revealed enhanced expression (21%, 14% and 4% positivity) . Transitional cell carcinoma usually demonstrates low expression at diagnosis (less than 6%), although 3 patients had enhanced initial expression (11%, 12% and 31%) . Three patients treated with chemotherapy demonstrated 56%, 76% and 50% expression of MDR-1 . Nonmalignant tissue from cystectomy specimens had low expression of MDR-1 . The specificity of this system was confirmed with human bladder cell lines . The ability of flow cytometry to detect and quantify the expression of MDR-1 may allow for the early detection of chemotherapy resistance in patients with transitional cell carcinoma treated with systemic and intravesical therapy.

Exp Cell Res, 1991 Oct, 196(2), 323 - 9
Laser scanning and confocal microscopy of daunorubicin, doxorubicin, and rhodamine 123 in multidrug-resistant cells; Weaver JL et al.; The multidrug-resistant gene (MDR1) encodes an energy-dependent drug efflux pump (P-glycoprotein) for many anti-cancer drugs . We have studied the intracellular distribution of rhodamine 123 (R123), daunorubicin (DN), and doxorubicin (DOX) in cells expressing a human MDR1 gene . The distribution of these fluorescent drugs was measured by laser scanning microscopy and confocal microscopy . We devised a new method for analysis of fluorescence line scan data to determine the intracellular distribution of fluorescent probes . This method and confocal microscopy showed that R123, DN, and DOX are localized to both plasma membrane and intracellular compartments in multidrug-resistant cells . When the cells are treated with verapamil, an inhibitor of the multidrug transporter, the amount of DOX, DN, and R123 associated with the cell rises . After inhibition, the relative distribution of DOX and DN between the cell surface and intracellular structures does not change dramatically . However, R123 tends to relocalize to intracellular sites from predominantly plasma membrane sites, indicating that this dye behaves differently than the anti-cancer drugs . These results show the subcellular distributions of R123, DN, and DOX in plasma membrane, cytoplasm, and intracellular membrane systems, but do not allow definitive distinctions among existing models of how P-glycoprotein affects the distribution of drugs.

Cancer Res, 1991 Oct 1, 51(19), 5093 - 9
Antagonistic effect of aclarubicin on daunorubicin-induced cytotoxicity in human small cell lung cancer cells: relationship to DNA integrity and topoisomerase II; Jensen PB et al.; The effect of combinations of the anthracyclines aclarubicin and daunorubicin was investigated in a clonogenic assay using the human small cell lung cancer cell line OC-NYH and a multidrug-resistant (MDR) murine subline of Ehrlich ascites tumor (EHR2/DNR+) . It was found that the cytotoxicity of daunorubicin in OC-NYH cells was antagonized by simultaneous exposure to nontoxic concentrations of aclarubicin . Coordinately, aclarubicin inhibited the formation of daunorubicin-induced protein-concealed DNA single-strand breaks and DNA-protein cross-links in OC-NYH cells when assayed by the alkaline elution technique . Aclarubicin had no influence on the accumulation of daunorubicin in these cells . In contrast, the accumulation of daunorubicin in EHR2/DNR+ cells was enhanced by more than 300% when the cells were simultaneously incubated with the MDR modulator verapamil, aclarubicin, or the two agents combined . Yet the cytotoxicity of daunorubicin was potentiated significantly only by verapamil . The increased cytotoxicity of daunorubicin in the presence of verapamil was completely antagonized when aclarubicin was used together with the MDR modulator . Finally, the effect of daunorubicin on the DNA cleavage activity of purified topoisomerase II in the presence and absence of aclarubicin was examined . It was found that daunorubicin stimulated DNA cleavage by topoisomerase II at specific DNA sites . The addition of aclarubicin completely inhibited the daunorubicin-induced stimulation of DNA cleavage . Taken together, these data indicate that aclarubicin-mediated inhibition of daunorubicin-induced cytotoxicity is due mainly to a drug interaction with the nuclear enzyme topoisomerase II . This antagonism at the nuclear level explains why aclarubicin is a poor modulator of daunorubicin resistance even though aclarubicin is able to increase the intracellular accumulation of daunorubicin in a MDR cell line.

Cancer Res, 1991 Sep 15, 51(18), 4955 - 63
Subcellular distribution of daunorubicin in P-glycoprotein-positive and -negative drug-resistant cell lines using laser-assisted confocal microscopy; Gervasoni JE Jr et al.; Four well defined multidrug-resistant cell lines and their drug-sensitive counterparts were examined for intracellular distribution of daunorubicin (DNR) by laser-assisted confocal fluorescence microscopy: P-glycoprotein-negative HL-60/AR cells, and P-glycoprotein-positive P388/ADR, KBV-1, and MCF-7/ADR cells . Both drug sensitive cell lines (HL-60/S, P388/S, KB3-1, and MCF-7/S) and drug-resistant cell lines (HL-60/AR, P388/ADR, KBV-1, and MCF-7/ADR) exposed to DNR showed a similar rapid distribution of drug from the plasma membrane to the perinuclear region within the first 2 min . From 2-10 min, the drug sensitive HL-60/S, P388/S, and MCF-7/S cells redistributed drug to the nucleus and to the cytoplasm in a diffuse pattern . In contrast, drug-resistant HL-60/AR, P388/ADR, and MCF-7/ADR redistributed DNR from the perinuclear region into vesicles distinct from nuclear structures, thereby assuming a "punctate" pattern . This latter redistribution could be inhibited by glucose deprivation (indicating energy dependence), or by lowering the temperature of the medium below 18 degrees C . The differences in distribution between sensitive and resistant cells did not appear to be a function of intracellular DNR content, nor the result of drug cytotoxicity . Drug-sensitive KB3-1 and -resistant KBV-1 cells did not fully follow this pattern in that they demonstrated an intracellular DNR distribution intermediate between HL-60/S and HL-60/AR cells with both "punctate" and nuclear/cytoplasmic uptake sometimes in the same cell . These data indicate that the intracellular distribution of DNR is an important determinant of drug resistance regardless of the overexpression of P-glycoprotein . The intracellular movement of drug requires the presence of glucose and a temperature above 18 degrees C, implicating energy-dependent processes and vesicle fusion in the distribution process . This intracellular transport of DNR away from the nucleus in multidrug-resistant cells may protect putative cell targets such as DNA against drug toxicity.

J Biol Chem, 1991 Sep 25, 266(27), 18224 - 32
Study of membrane orientation and glycosylated extracellular loops of mouse P-glycoprotein by in vitro translation; Zhang JT et al.; Increased expression of P-glycoprotein (Pgp) has been demonstrated to cause multidrug resistance (MDR) in vitro, and it may be responsible for chemotherapy failure in a number of human cancers . Pgp is a plasma membrane protein thought to function as an energy-dependent drug transporter . From its deduced protein sequence the topology of Pgp was proposed to contain 12 transmembrane domains with six extracellular loops and two cytoplasmic ATP-binding sites . To investigate further the membrane orientation of Pgp, we have expressed a full length cDNA of mouse mdr1, as well as its truncated forms, in a cell-free system supplemented with dog pancreatic microsomal membranes (RM) . We determined which domains of the in vitro-synthesized Pgp had transversed the RM membranes by analyzing their resistance to protease digestion and their glycosylation state . To our surprise, this system revealed that a significant portion of in vitro-synthesized Pgp molecules has an additional glycosylated domain in the C-terminal half . Previously, only the first predicted extracellular loop near the N terminus had been thought to be glycosylated . Furthermore, we discovered that Pgp has at least two functional signal recognition particle/docking protein dependent signal sequences, one at the N-terminal half and the other at the C-terminal half . These findings suggest a new topological model for in vitro synthesized P-glycoprotein which may be relevant to its in vivo topology.

Biochim Biophys Acta, 1991 Sep 23, 1097(2), 111 - 6
Etoposide-induced DNA damage in human tumor cells: requirement for cellular activating factors; Sinha BK et al.; Previous studies with the multidrug-resistant human HL60 cell line have shown a 3-4-fold decrease in VP-16 accumulation compared to the sensitive cell line, while the degree of resistance to VP-16 was 300-fold, indicating that other mechanisms of resistance are also operative . Since VP-16 has been shown to interfere with topoisomerase II activity, we have evaluated VP-16-dependent DNA strand break formation in the drug-sensitive and -resistant HL60 cells . Studies reported here show that the drug-resistant HL60 cells are extremely resistant to VP-16-dependent DNA cleavage compared to the sensitive cells . This decrease in DNA cleavage activity in the presence of VP-16 was, in part, related to a 2-3-fold decrease in both the amount and activity of topoisomerase II in the resistant cell line compared to the sensitive cells . Nuclei from the resistant cell line were markedly more resistant to VP-16-dependent DNA cleavage than the WT cell nuclei . Interestingly, WT nuclei were found to be relatively more resistant to VP-16-induced DNA cleavage than the intact WT cells . Addition of WT cytosolic proteins to WT nuclei, however, significantly stimulated VP-16-dependent DNA cleavage and slightly increased DNA cleavage in resistant cell nuclei . In contrast, cytosolic proteins from the resistant cells had no effect on DNA cleavage in nuclei isolated from either cell line . These observations indicate that a decrease in the amount and activity of topoisomerase II in resistant HL60 cells translates into a decrease in VP-16-dependent DNA breakage and contributes to the resistance to VP-16 . Furthermore, the cytosolic fraction from WT cells contains some factor, not present in the resistant cells, which is necessary for the maximal drug-induced DNA cleavage.

J Natl Cancer Inst, 1991 Sep 18, 83(18), 1307 - 15
Effect of recombinant fibroblast interferon and recombinant immune interferon on growth and the antigenic phenotype of multidrug-resistant human glioblastoma multiforme cells; Reddy PG et al.; To study the effect of drug resistance on the response of stage IV astrocytomas to interferon, a human glioblastoma multiforme cell line, GBM-18, was transfected with an expression-vector plasmid containing a human multidrug resistance (MDR) gene (pHaMDR1/A), and clones surviving in colchicine were isolated . GBM-18 multidrug-resistant subclones displayed cross-resistance to other chemotherapeutic agents, including vincristine, doxorubicin, and dactinomycin . The multidrug-resistant phenotype was reversible when GBM-18 multidrug-resistant cells were cultured in colchicine and the calcium-channel blocker verapamil . The level of the MDR1 gene (also known as PGY1) message was increased in GBM-18 multidrug-resistant cells selected for increased resistance to colchicine, and this effect was not correlated with an amplification of the MDR1 gene . In both parental GBM-18 and GBM-18 multidrug-resistant cells, growth was suppressed to a greater degree when cultures were treated with the combination of fibroblast interferon (IFN-beta) and immune interferon (IFN-gamma) . Parental cells and multidrug-resistant subclones varied in their de novo and/or interferon-modulated expression of HLA class I and class II antigens, a high-molecular-weight melanoma-associated antigen, and intercellular adhesion molecule 1 (ICAM-1) . Of the antigens tested, ICAM-1 and HLA class I antigens were the most sensitive to enhanced expression induced by IFN-beta and IFN-gamma when used alone or in combination . The results of the present study indicate that multidrug-resistant human glioblastoma multiforme cells retain their increased sensitivity to the antiproliferative activity of the combination of IFN-beta plus IFN-gamma, and differences in antigenic phenotype are apparent in independent multidrug-resistant glioblastoma multiforme clones.

Cancer Res, 1991 Sep 15, 51(18), 4898 - 902
Reversal of adriamycin resistance by recombinant alpha-interferon in multidrug-resistant human colon carcinoma LoVo-doxorubicin cells; Scala S et al.; Reversal of the drug resistance phenotype by the use of agents which induce cell differentiation offers an experimental approach to the study of chemoresistance . In numerous in vitro models, alpha-interferon (alpha-IFN) has been shown to induce phenotypical changes and to modulate the growth of cancer cells . The aim of the present study was to define the effect of alpha-IFN on the Adriamycin sensitivity of the human colon adenocarcinoma cell line, LoVo, and its Adriamycin-resistant variant, LoVo/DX . Pretreatment of LoVo/DX cells with 500 units/ml of alpha-IFN increased sensitivity to low doses of Adriamycin . Similar treatment conditions did not change the sensitivity of the parental cell line . Following treatment of the LoVo/DX cells with alpha-IFN plus 100 ng/ml Adriamycin for 1 h, 30% of the cells survived compared to 100% of untreated cells . This effect was not related to changes in cell cycle kinetics induced by alpha-IFN treatment and did not result from variations in the expression of P-glycoprotein at the cell surface, as assessed by flow cytometric analysis using monoclonal antibody MRK16 . Adriamycin accumulation was increased by alpha-IFN as assessed by spectrofluorometric analysis . Thus, the data suggest that in LoVo/DX cells, alpha-IFN increased Adriamycin cytotoxicity through modulation of the multidrug resistance phenotype.

Biochem Pharmacol, 1991 Sep 12, 42(7), 1427 - 32
The role of protein kinase C and the phosphatidylinositol cycle in multidrug resistance in human ovarian cancer cells; Anderson L et al.; The present study aimed to investigate the role of protein kinase C (PKC), the phosphatidylinositol pathway (PI) and cytosolic calcium in multidrug resistance (MDR) in human ovarian carcinoma cells . Binding of the phorbol ester 13,14-dibutyrate (PDBu) was 3-fold higher in resistant A2780AD versus sensitive A2780 cells indicating increased PKC activity . However, when inositol phosphate production (IP) was measured in quiescent cells similar total IP release was seen in both lines suggesting no difference in the basal turnover of PI . Non-specific stimulation of the PI pathway was achieved with the calcium ionophore A23187 which increased IP production in a time- and dose-dependent fashion in both cell lines but was significantly less effective in A2780AD . The PI pathway was investigated further using the agonists aluminium fluoride, serum and bombesin but these agents failed to elicit a response . The effect of a wide range of Adriamycin concentrations on the PI cycle and cell growth was also studied . Intracellular calcium was measured with the fluorescent dye fura-2-pentaacetoxymethylester (Fura-2) . A23187 produced a rise in cytosolic calcium in A2780 and A2780AD but from a level 3-fold lower in the unstimulated resistant cell line . The dose responsiveness of this effect was greater but irreversible in A2780AD cells . Collectively these results imply that alterations in PI turnover appear not to be responsible for the differences in PDBu binding and calcium handling observed between A2780 and A2780AD and suggests only a minor role for the PI cycle in the maintenance of MDR in human ovarian cancer cell lines.

J Biol Chem, 1991 Sep 5, 266(25), 16796 - 800
Azidopine noncompetitively interacts with vinblastine and cyclosporin A binding to P-glycoprotein in multidrug resistant cells; Tamai I et al.; It is believed that P-glycoprotein (P-gp) is an energy-dependent drug efflux pump responsible for decreased drug accumulation in multidrug resistant (MDR) cells . In this study, we investigated whether azidopine, a photoactive dihydropyridine calcium channel blocker, is transported by P-gp in MDR Chinese hamster lung cells, DC-3F/VCRd-5L, and whether its binding site(s) on P-gp are distinct from those of Vinca alkaloids and cyclosporins . The efflux of azidopine from MDR cells was energy-dependent and inhibited by the cytotoxic agent vinblastine (VBL) . Cyclosporin A (CsA), a modulator of MDR, also increased azidopine accumulation in MDR cells by decreasing the energy-dependent efflux of azidopine . P-gp in these cells was the only protein specifically bound to {3H}azidopine in photoaffinity experiments . The specific photoaffinity labeling of P-gp by {3H}azidopine was inhibited by CsA, SDZ 33-243, nonradioactive azidopine, and VBL with median concentrations (IC50) of 0.5, 0.62, 1.7, and 25 microM, respectively . The equilibrium binding of azidopine to plasma membranes of MDR variant DC-3F/VCRd-5L cells showed a single class of specific binding sites having a dissociation constant of 1.20 microM and a maximum binding capacity of 4.47 nmol/mg of protein . Kinetic analysis indicated that the inhibitory effect of VBL and CsA on azidopine binding to plasma membranes of MDR cells was noncompetitive, indicating that azidopine binds to P-gp at a binding site(s) different from the binding site(s) of these drugs.

Br J Haematol, 1991 Sep, 79(1), 50 - 6
Analysis of multidrug resistance (MDR-1) gene expression in chronic lymphocytic leukaemia (CLL); Shustik C et al.; Multidrug resistance (MDR) in cultured human cells is caused by the overexpression of the MDR-1 gene . This gene codes for P-glycoprotein, a proposed ATP-dependent drug efflux pump, which reduces the net intracellular accumulation of a large group of chemotherapeutic agents in resistant cells . We have measured the level of expression of the human MDR-1 gene in a series of patients with chronic lymphocytic leukaemia (CLL) . Forty-eight patients included in the study were at different stages of disease and were either untreated or had been treated with alkylating agents or alkylating agents in combination with drugs of the MDR spectrum, and were tested over a period of 3 years . The level of MDR-1 expression was monitored by Northern blotting analysis using a specific cDNA hybridization probe and also after polymerase chain reaction (PCR) amplification of MDR-1 complementary DNA (cDNA) . Four of 28 previously untreated patients showed intrinsically high levels of MDR-1 mRNA while 5/19 treated patients had elevated MDR-1 expression . Elevated MDR-1 expression in treated patients was unrelated to the type of chemotherapy and was independent of previous exposure to drugs of the MDR spectrum . Intrinsic MDR-1 gene expression in positive patients did not appear to influence their response to chemotherapy with non-MDR drugs such as alkylating agents.

Exp Cell Res, 1991 Sep, 196(1), 26 - 32
Restoration of daunomycin retention in multidrug-resistant P388 cells by submicromolar concentrations of SDZ PSC 833, a nonimmunosuppressive cyclosporin derivative; Boesch D et al.; Overexpression of P-glycoprotein may cause increased efflux of a variety of anticancer drugs (ACD) leading to multidrug resistance (MDR) of tumor cells . Two sublines of murine monocytic leukemia P388 cells were used, one parental (Par-P388) and one multidrug resistant (MDR-P388) . In cell growth inhibition assays in vitro, the Par-P388 cells showed a normal sensitivity to daunomycin (DAU) while the MDR-P388 cells were 200-fold resistant . In cellular fluorescence assays, DAU retention in MDR-P388 cells reached only 5% of the level achieved in Par-P388 cells . This cell line pair was used to compare the nonimmunosuppressive cyclosporin analog PSC 833 with several resistance-modifying agents (RMAs) for their in vitro chemosensitizing activity and for their restoration of DAU retention . PSC 833 sensitized the MDR-P388 cells 60- and 140-fold when used at 0.1 and 0.3 micrograms/ml (0.08 and 0.25 microM), respectively, a complete restoration of sensitivity being obtained at 1.0 micrograms/ml PSC 833 . Similarly as little as 0.1 micrograms/ml (0.08 microM) PSC 833 was sufficient to restore intracellular DAU retention to 60% of the level found in Par-P388 cells, a 3-fold higher concentration restoring virtually the whole DAU retention . For both these activities, PSC 833 was at least one order of magnitude more active than CsA, which was itself an order of magnitude stronger than verapamil, another RMA already used in clinic . Since PSC 833 had no effect on the PAR-P388 cells, neither on chemosensitization nor on drug retention, it is assumed that it acts on the P-glycoprotein, which is highly expressed on the membrane of the MDR-P388 cells, by inhibiting the function of the P-glycoprotein pump and thus restoring a normal ACD-sensitivity of the MDR-P388 cells.

Cancer Res, 1991 Sep 1, 51(17), 4665 - 70
Characterization of multidrug resistance by fluorescent dyes; Kessel D et al.; Fluorimetric techniques were used to examine accumulation of fluorescent probes by the P388 murine leukemia and an anthracycline-resistant subline, P388/Adriamycin(ADR), which expresses the multidrug-resistant phenotype . P388 could be differentiated from P388/ADR on the basis of fluorescence intensity measurements using 3 classes of cationic dyes that are sensitive to membrane potential differences: rhodamine esters, cyanines, and styrylpyridinium dyes . But fluorescence intensity differences were also observed with potential-insensitive dyes: zwitterionic rhodamines and an acridine orange derivative . In all cases, fluorescence intensity differences were caused by impaired dye accumulation, and could be eliminated by treatment of P388/ADR cells with verapamil . Moreover, fluorescence signals from 2 anionic potential-sensitive dyes, merocyanine 540 and a bis-oxonol, were identical in P388 and P388/ADR . None of these dyes could be used to delineate CCRF-CEM, a lymphoblastic leukemia of human origin from the CEM/VM-1 subline that exhibits a markedly atypical drug resistance pattern not based on an enhanced outward transport . But accumulation of both neutral and cationic dyes was impaired in CEM/VLB100, a subline of CCRF-CEM expressing mdr . These studies show that many cationic and neutral fluorescent probes are substrates for the enhanced outward drug transport system associated with P388/ADR cells, and cannot be used to probe membrane-potential differences in cells expressing the mdr phenotype . With several dyes, differences in fluorescence intensity were sufficient so that flow cytometry could be used to delineate P388 from P388/ADR and CCRF-CEM from CEM-VLB100 . The latter technique may be useful for identifying malignant cell populations expressing multidrug resistance in patients with neoplastic disease.

Southeast Asian J Trop Med Public Health, 1991 Sep, 22(3), 380 - 5
Clinical trial of artesunate and artemether on multidrug resistant falciparum malaria in Thailand . A preliminary report; Bunnag D et al.; A 5-day course of oral artesunate at total doses of 1200, 600, 650 mg and intramuscular artemether 480 mg proved effective (90-100% cured) in the treatment of multidrug resistant falciparum malaria in Thailand . Shorter courses yielded high recrudescence rates . The fever clearance and parasite clearance times were short . The side effects were mild and transient including occasional abnormal electrocardiograms and pain at the injection site . Slight reduction of neutrophil leucocytes and reticulocytes was observed . Further studies of artesunate and artemether should be carried out to find the optimum dosage regimen and to clarify the hematological effects.

Trans R Soc Trop Med Hyg, 1991 Sep-Oct, 85(5), 578 - 83
Genetic diversity of Plasmodium falciparum in a village in eastern Sudan . 2 . Drug resistance, molecular karyotypes and the mdr1 genotype of recent isolates; Babiker HA et al.; Isolates of Plasmodium falciparum from a Sudanese village have been collected as part of a study of parasite genetic diversity during seasonal malaria epidemics . The sensitivity in vitro to chloroquine, pyrimethamine and mefloquine of these isolates has been determined . To assess the utility of pulse field gel chromosome separations in isolate characterization, 18 samples from individual patients in a single village were studied using this technique . Extensive variation in chromosome size was detected, no 2 isolates having identical molecular karyotypes . No multidrug resistance (mdr) gene amplification polymorphisms were detected in either chloroquine-resistant or chloroquine-sensitive isolates in this sample.

Pathol Res Pract, 1991 Sep, 187(7), 892 - 905
What's new in cytostatic drug resistance and pathology; Dietel M; A major problem in cytostatic treatment of malignant tumors is the development of chemoresistant cell clones . An increased understanding of chemoresistance related mechanisms, improved methods for the detection and localization of resistant cell populations including predictive conclusions on the effectiveness of cytostatic drugs would contribute to the advancement of anti-tumor strategies . This paper reviews current concepts suggested for the development of cellular resistance to natural product drugs (anthracyclines, Vinca alkaloids, epipodophyllotoxines, antibiotics; so-called multidrug resistance substances), alkylating agents (nitrosureas, busulfan and mitomycin C), heavy metal compounds (cisplatin) and antifolates (5-fluorouracil, methotrexate) and describes the role of drug transporting and binding proteins (P-170-glycoprotein), detoxifying enzymes (glutathion-S-transferase, dihydrofolate reductase), DNA repair enzymes (topoisomerase I and II, polymerase alpha and beta), and genomic alterations (amplification, double minutes and homogeneous staining regions) due to resistance . It is focussed on the employment of morphological methods (light microscopy, immunocytochemistry, electron microscopy, fluorescence analysis, in situ hybridization, computer aided morphometric analysis) which will help to detect resistant cell clones in tumor biopsies . First correlations between histological data and clinical course will be reported . In the future, the morphological determination of chemoresistance may play an important role in applied functional tumor pathology.

J Pediatr Surg, 1991 Sep, 26(9), 1107 - 12
Multidrug resistance in human neuroblastoma cells; LaQuaglia MP et al.; Neuroblastoma remains a significant problem in pediatric oncology . Recently a "multidrug-resistance" gene that may cause cells to become resistant to various chemotherapeutic agents has been cloned . The gene encodes the high-molecular-weight plasma membrane protein known as P-glycoprotein . To study the expression of this gene in cells exhibiting the multidrug-resistant phenotype, a panel of sublines selected with several different natural product drugs was established . The drug-sensitive parental BE(2)-C cells were clonally isolated from the human neuroblastoma SK-N-BE(2) line and exhibit a 150-fold increase in the copy number of the N-myc protooncogene . Sublines were selected by stepwise increases in the concentration of actinomycin-D, doxorubicin, vincristine, or colchicine . Gene amplification was assessed using Southern analysis, and RNA levels were determined by Northern and dot-blot analysis . Western blotting was used to determine protein levels . N-myc amplification and expression were simultaneously determined to assess possible alterations associated with development of multidrug resistance . Amplified P-glycoprotein-encoding genes were not seen in control lines but were clearly present in those that had undergone exposure to each of the chemical agents . Similarly, steady-state messenger RNA and protein levels were greatly increased in the drug-resistant sublines . We conclude that human neuroblastoma cells can acquire the multidrug-resistant phenotype after exposure to various chemotherapeutic agents.

Ophthalmology, 1991 Sep, 98(9), 1425 - 31
Multidrug-resistant phenotype in retinoblastoma correlates with P-glycoprotein expression; Chan HS et al.; Chemotherapy plays an important role in therapy for patients with extraocular and metastatic retinoblastoma . The authors used chemotherapy for management of selected patients with uncontrolled intraocular tumors or tumors larger and more posteriorly located than those conventionally treated with local cryotherapy or photocoagulation . Rapid regrowth of some tumors after an initial excellent chemotherapy response led us to investigate the hypothesis that failure of treatment is caused by P-glycoprotein-related multidrug resistance . By using a sensitive immunoperoxidase method, increased P-glycoprotein was detected in five multidrug-resistant and two selectively plant alkaloid-resistant retinoblastoma cell lines and in the intraocular and metastatic tumors from which they were derived . In four chemotherapy-treated cases, increased P-glycoprotein in the tumor samples correlated with clinically relevant drug resistance . None of the four chemosensitive tumor cell lines had increased P-glycoprotein expression . Continuous surveillance of P-glycoprotein levels in metastatic retinoblastoma may be a useful guide to drug therapy.

J Cell Physiol, 1991 Sep, 148(3), 479 - 84
Modulation of ATP and drug binding by monoclonal antibodies against P-glycoprotein; Georges E et al.; The role of P-glycoprotein in mediating the drug-resistance phenotype in multidrug resistant cells is now well documented . It is thought to function as an energy-dependent drug-efflux pump of broad specificity . Structurally, P-glycoprotein is an internally duplicated molecule containing two large multi-spanning transmembrane domains and two cytoplasmic ATP binding domains . In this report we demonstrate that monoclonal antibodies C219, C494, and C32 directed against short linear regions of the P-glycoprotein molecule inhibit ATP binding to P-glycoprotein in vitro . We also provide direct evidence that both predicted ATP-binding domains bind ATP and that there is co-operativity between the two sites . In addition, the capacity of P-glycoprotein to bind the calcium channel blocker, azidopine, is inhibited differentially by the antibodies . These observations are the first evidence linking specific perturbations of the P-glycoprotein molecule with ATP and drug binding.

Schweiz Med Wochenschr, 1991 Aug 31, 121(35), 1249 - 53
{Interactions of cytostatic agents with other drugs}; Sauter C; With the degree of polypharmacy currently practiced in the field of oncology, there are undoubtedly many drug interactions . In the present study the influence of "non-cytotoxic" drugs on anticancer drugs is discussed, but not the reverse . Not only is the augmentation (reversal of multidrug resistance) or the reduction of antitumor properties of cytotoxic drugs observed, but also cytostatic activities of "non-cytotoxic" drugs themselves . Examples are calmodulin inhibitors such as phenothiazines and tricyclic antidepressants . Interactions may also increase side effects of cytostatic drugs or even neutralize the antitumoral activity . To ensure that interactions are not overlooked, all medicaments being administered should be listed . It is, however, not feasible yet to determine serum concentrations of all the drugs given to the patient . The antitumor activity of supportive care could be evaluated in randomized studies (e.g . cytostatic drugs +/- antidepressants).

MMWR Morb Mortal Wkly Rep, 1991 Aug 30, 40(34), 585 - 91
Nosocomial transmission of multidrug-resistant tuberculosis among HIV-infected persons--Florida and New York, 1988-1991; Interaction of forskolin with the P-glycoprotein multidrug transporter; Molecular Pharmacology Laboratory, Food and Drug Administration, Bethesda, Maryland 20892Forskolin and 1,9-dideoxyforskolin, an analogue that does not activate adenylyl cyclase, were tested for their ability to enhance the cytotoxic effects of adriamycin in human ovarian carcinoma cells, SKOV3, which are sensitive to adriamycin and express low levels of P-glycoprotein, and a variant cell line, SKVLB, which overexpresses the P-glycoprotein and has the multidrug resistance (MDR) phenotype . Forskolin and 1,9-dideoxyforskolin both increased the cytotoxic effects of adriamycin in SKVLB cells, yet had no effect on SKOV3 cells . Two photoactive derivatives of forskolin have been synthesized, 7-O-{{2-{3-(4-azido-3- {125I}iodophenyl)propionamido}ethyl} carbamyl}-7-deacetylforskolin, 125I-7-AIPP-Fsk, and 6-O-{{2-{3-(4-azido-3- {125I}iodophenyl)propionamido}ethyl}carbamyl}forskolin, 125I-6-AIPP-Fsk, which exhibit specificity for labeling the glucose transporter and adenylyl cyclase, respectively (Morris et al., 1991) . Both photolabels identified a 140-kDa protein in membranes from SKVLB cells whose labeling was inhibited by forskolin and 1,9-dideoxyforskolin . There was no specific labeling of proteins in membranes from the SKOV3 cells . The overexpressed 140-kDa protein in SKVLB membranes was identified as the P-glycoprotein by immunoblot analysis and immunoprecipitation using anti-P-glycoprotein antiserum . Total inhibition of photolabeling of the P-glycoprotein was observed with verapamil, nifedipine, diltiazem, and vinbalastine, and partial inhibition was observed with colchicine and cytochalasin B . Forskolin was less effective at inhibiting the photolabeling of the P-glycoprotein than 1,9-dideoxyforskolin or a lipophilic derivative of forskolin . The data are consistent with forskolin binding to the P-glycoprotein analogous to that of other chemosensitizing drugs that have been shown to partially reverse MDR.(ABSTRACT TRUNCATED AT 250 WORDS)

Tidsskr Nor Laegeforen, 1991 Aug 20, 111(19), 2423 - 7
{Resistance to chemotherapeutic agents in the treatment of cancer . Emphasizing the multiresistant phenotype}; Holte H; Despite considerable efforts, rather limited progress has been achieved in the treatment of disseminated cancer with chemotherapeutic agents during the last decade . Most tumours, especially those of epithelial origin, are more or less resistant to cytostatics right from initiation of therapy . Other tumours, on the other hand, like leukemias and lymphomas, are highly responsive initially but may become resistant during the course of therapy (secondary resistance) . This type of resistance is due to random mutations in the malignant cells . Several mechanisms of cellular resistance have been described, the most important being characterized by simultaneous resistance to several unrelated chemotherapeutic agents (pleiotropic drug resistance or multidrug resistance) . This mechanism is due to a membrane-associated efflux pump which transports the cytostatic agents out of the cells . The clinical importance of multidrug resistance and the possibility of inhibiting the function of the pump by pharmacological agents are currently being examined.

Cancer Res, 1991 Aug 15, 51(16), 4243 - 9
Isolation and initial characterization of mouse tumor cells resistant to porphyrin-mediated photodynamic therapy; Luna MC et al.; Photodynamic therapy (PDT)-resistant variants of the RIF-1 mouse tumor cell line have been isolated following a protocol of repeated porphyrin incubation and light treatments . Two porphyrin incubation procedures, employing either an extended (16 h) or a short (1 h) incubation, were used in order to obtain cell strains exposed to conditions with differing intracellular photosensitizer localization . Two clones from each PDT porphyrin incubation protocol were selected for in vitro and in vivo analyses based on degree of resistance and plating efficiency . Resistant variants had increased protein content and were larger than the parental RIF-1 cells . In vitro growth rates were similar for all cell strains . Both 16-h PDT-resistant variants exhibited modest resistance to ionizing radiation and one of the 16-h PDT-resistant variants demonstrated increased sensitivity to hyperthermia . The PDT-resistant variants did not exhibit a multidrug resistance phenotype nor did they have altered porphyrin uptake properties . The parental and resistant RIF cells had comparable basal levels of antioxidant enzymes, reduced glutathione and stress proteins, but the number of cells required to produce in vivo tumor growth in 50% of inoculated animals was increased for all PDT-resistant variants . The resistant cells exhibit a stable phenotype and should be useful in studies designed to define PDT mechanisms of action.

Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 7386 - 90
Multidrug resistance after retroviral transfer of the human MDR1 gene correlates with P-glycoprotein density in the plasma membrane and is not affected by cytotoxic selection; Choi K et al.; Multidrug resistance (MDR) in mammalian cells is associated with the expression of the MDR1 gene encoding P-glycoprotein (P-gp), an and active efflux pump for various lipophilic compounds . MDR transfectants can be isolated after MDR1 gene transfer and selection with cytotoxic drugs; low levels of drug resistance have also been observed in unselected NIH 3T3 mouse cells after retrovirus-mediated transfer of mouse mdr1 cDNA . MDR cell lines possess multiple phenotypic changes, suggesting that P-gp function could be complemented by some additional mechanisms associated with cytotoxic selection . To determine whether cytotoxic selection contributes to the MDR phenotype of MDR1-expressing cells, NIH 3T3 cells infected with a recombinant retrovirus carrying the human MDR1 gene were selected by two different procedures: (i) noncytotoxic selection for increased P-gp expression on the cell surface by multiple rounds of immunofluorescence labeling and flow sorting or (ii) one or more steps of selection with a cytotoxic drug . The levels of MDR in both types of infectants showed an excellent correlation with the P-gp density in the plasma membrane, expressed as immunoreactivity with a P-gp-specific antibody normalized by reactivity with an antibody against an unrelated antigen . Cytotoxic selection conferred no additional increase in resistance relative to P-gp density . These results indicate that P-gp density in the plasma membrane may be sufficient to determine the level of MDR.

Cancer Res, 1991 Aug 15, 51(16), 4226 - 33
In vivo circumvention of P-glycoprotein-mediated multidrug resistance of tumor cells with SDZ PSC 833; Boesch D et al.; The new nonimmunosuppressive cyclosporin analogue, SDZ PSC 833, is a very potent multidrug-resistance modifier . In vitro, it was shown to be at least 10-fold more active than cyclosporin A (Sandimmune), itself more active than verapamil, on most P-glycoprotein-expressing multidrug-resistant (MDR) tumor cell lines . In vivo, SDZ PSC 833 was tested in a few protocols of combined therapy with either Vinca alkaloids or doxorubicin as anticancer drugs, using the homologous tumor-host system (P388 cells of DBA/2 origin grafted into DBA/2 or B6D2F1 mice) . Although these MDR-P388 tumor cells belong to a highly resistant variant that in vitro required about 150-fold more anticancer drug for 50% cell growth inhibition than the parental P388 cells, significant prolongation of survival times of the MDR-P388 tumor-bearing mice was obtained when treated with a combination of SDZ PSC 833 p.o . were otherwise ineffective doses of anticancer drugs given i.p . This chemosensitizing effect of SDZ PSC 833 was dose-dependent and was most effective in a protocol combining administration of SDZ PSC 833 p.o . 4 h before a doxorubicin i.p . injection: in comparison with the survival of MDR-P388 tumor-bearing mice treated with the anticancer drug alone, the pretreatment with SDZ PSC 833 at 25 and 50 mg/kg gave 2- to 3-fold increases of survival times . Since the MDR-P388 tumor cells used in our studies belong to a highly resistant variant, with a much higher degree of drug resistance than the one known to occur in cancer patients, SDZ PSC 833 appears to be a very promising chemosensitizer.

Cancer Res, 1991 Aug 15, 51(16), 4213 - 8
Decreased DNA topoisomerase II in daunorubicin-resistant Ehrlich ascites tumor cells; Friche E et al.; We have previously shown that the multidrug-resistant EHR2/DNR+ cells, which overexpress P-glycoprotein, accumulate only about 20-30% of daunorubicin at steady state compared to the sensitive cells . These cells have been thought to be a "pure" P-glycoprotein cell line . We now report that the EHR2/DNR+ cells exhibit decreased DNA topoisomerase II catalytic activity . We also found that the amount of immunoreactive DNA topoisomerase II from these cells is about one-third that seen in the drug-sensitive cell line . In agreement with the decreased activity and amount of topoisomerase II, the number of DNA-protein complexes stabilized by teniposide (VM-26) was reduced by about 50% in nuclear extracts from EHR2/DNR+ cells . Furthermore, using an intact cell assay for DNA protein complexes, we found that the VM-26-stimulated complexes formed in the drug-resistant cells never reached the level seen in the drug-sensitive cells . Verapamil and Cremophor EL block P-glycoprotein-mediated efflux of "natural product" drugs and increase their accumulation in resistant cells . Coincubation of the EHR2/DNR+ cells with VM-26 and either of these modulators increased the number of complexes formed in the resistant cells . However, neither modulator increased the number of topoisomerase II-DNA complexes in the drug-resistant cells to the level seen in the EHR2 cells . We conclude that the resistance of EHR2/DNR+ cells is due in part to reduced amounts of DNA topoisomerase II . Furthermore, we note that a single cell line can express features of both P-glycoprotein-associated multidrug resistance and altered topoisomerase II-associated multidrug resistance.

J Natl Cancer Inst, 1991 Aug 7, 83(15), 1098 - 102
Involvement of vacuolar H(+)-adenosine triphosphatase activity in multidrug resistance in HL60 cells; Marquardt D et al.; HL60 cells isolated for resistance to vincristine (HL60/Vinc cells) or doxorubicin (HL60/Adr cells) contain enhanced levels of an energy-dependent drug efflux pump . HL60/Vinc cells contain the drug transporter P-glycoprotein, whereas the HL60/Adr isolate does not . In the present study, we examined the possible involvement of vacuolar H(+)-adenosine triphosphatase (H(+)-ATPase) activity in drug resistance in HL60 cells . We utilized bafilomycin A1, an agent which selectively inhibits vacuolar H(+)-ATPase activity at low concentrations . The results showed that bafilomycin A1 induced a major increase in drug accumulation and inhibited drug efflux in both HL60/Adr cells and HL60/Vinc cells . Similar results were obtained with 7-chloro-4-nitrobenz-2-oxa 1,3 diazole, an agent which is also capable of inhibiting vacuolar H(+)-ATPase . Azide, an inhibitor of F1F0 mitochondrial ATPase, and vanadate and ouabain, which are inhibitors of E1E2-type ATPase, did not affect drug levels in resistant cells . We also observed that bafilomycin A1 did not compete with {3H}azidopine binding to P-glycoprotein . Thus, bafilomycin A1 does not appear to function as a substrate for P-glycoprotein . These results suggest an involvement of vacuolar H(+)-ATPase activity in the pathway of drug efflux from HL60/Adr cells and HL60/Vinc cells . The mechanism of this action remains to be determined.

Mol Cell Biol, 1991 Aug, 11(8), 3940 - 8
Isolation and characterization of Drosophila multidrug resistance gene homologs; Wu CT et al.; Mammalian multidrug-resistant cell lines, selected for resistance to a single cytotoxic agent, display cross-resistance to a broad spectrum of structurally and functionally unrelated compounds . These cell lines overproduce a membrane protein, the P-glycoprotein, which is encoded by a member(s) of a multigene family, termed mdr or pgp . The amino acid sequence of the P-glycoprotein predicts an energy-dependent transport protein with homology to a large superfamily of proteins which transport a wide variety of substances . This report describes the isolation and characterization of two Drosophila homologs of the mammalian mdr gene . These homologs, located in chromosomal sections 49EF and 65A, encode proteins that share over 40% amino acid identity to the human and murine mdr P-glycoproteins . Fly strains bearing disruptions in the homolog in section 49EF have been constructed and implicate this gene in conferring colchicine resistance to the organism . This work sets the foundation for the molecular and genetic analysis of mdr homologs in Drosophila melanogaster.

J Neurosurg, 1991 Aug, 75(2), 277 - 83
Cross-resistance patterns in ACNU-resistant glioma sublines in culture; Saito Y et al.; Three ACNU-resistant clones (R1, R3, and R12) were isolated from 9L rat glioma cells under selection pressure of ACNU in vitro . The authors have investigated the mechanisms of resistance and characteristics of these clones at the cellular level by studying cross-resistance patterns to chemical and physical agents . Although these resistant sublines showed complete cross-resistance to methyl-chloroethylnitrosourea (MCNU), no cross-resistance was observed for other alkylating agents, while each of the resistant sublines showed partial cross-resistance to structurally dissimilar toxic agents (vinblastine, Adriamycin, and VP-16) . No difference in ACNU uptake was observed between 9L and R3 cells, and resistance patterns among alkylating agents suggested that the mechanism of ACNU resistance was specific to bifunctional nitrosoureas . Based on a transport study, this multidrug resistance could be explained by reduced intracellular uptake of these drugs, but there seemed little possibility that membrane P-glycoprotein, which usually is observed in typical multidrug-resistant cells, was expressed in these ACNU-resistant cells because enhanced drug efflux was not found in ACNU-resistant sublines . Significant collateral sensitivity to L-asparaginase indicated that ACNU might disturb the asparagine synthetic pathways by its mutagenic action . The increased level of total glutathione in the resistant sublines may be one mechanism of radiation or ACNU resistance.

Curr Opin Oncol, 1991 Aug, 3(4), 677 - 83
Rhabdomyosarcoma and other soft tissue sarcomas of childhood; Goren MP; Often treated as a single clinical entity, the pediatric soft tissue sarcomas include neoplasms of diverse histology that can originate from any anatomic site and exhibit varied patterns of local spread and metastasis . Recent developments with potential clinical impact include new diagnostic and prognostic markers stemming from advances in molecular biology, and increasing concern for the late effects of therapy in a growing population of long-term survivors . Initial clinical data indicate the value of MyoD1 protein expression in classifying previously indeterminate primitive undifferentiated tumors . The reported sensitivity and specificity of P-glycoprotein expression for the identification of chemoresistant disease in pediatric sarcomas should spawn studies to clarify its prognostic value and potential therapeutic strategies to circumvent the multidrug-resistance phenotype . Tumor cell DNA ploidy may also have prognostic and diagnostic value . An abundant interest in alleviating the late effects of adjuvant therapy and surgery is reflected in many clinical reports by oncologists, surgeons, radiotherapists, ophthalmologists, and other specialists.

Br J Cancer, 1991 Aug, 64(2), 361 - 4
A phase II study of epidoxorubicin in colorectal cancer and the use of cyclosporin-A in an attempt to reverse multidrug resistance; Verweij J et al.; We determined the ability of the multidrug resistance (MDR) reversal agent cyclosporin-A to increase anthracycline drug accumulation in colorectal tumour cells in vitro, using the technique of on-line flow cytometry . Data of four previously untreated patients showed that cyclosporin-A can increase intracellular net-uptake of daunorubicin . A phase II study was initiated in 24 colorectal cancer patients . They received cyclosporin-A at a dose of 3 mg kg-1 over 1 h as i.v . infusion, at 7 h and at 1 h preceding cytotoxic drug administration . At the end of the second cyclosporin-A administration epidoxorubicin 90 mg m-2 was administered as i.v . bolus . Cycles were repeated every 3 weeks . Median cyclosporin-A peak blood levels and levels at 18 h after cytotoxic drug administration appeared to be 6248 ng ml-1 and 1012 ng ml-1 respectively . Only one partial response was observed, despite these high cyclosporin-A levels . Cyclosporin-A did not cause major toxicity, only a 29% incidence of hot flushes was observed . Epidoxorubicin toxicities were as expected but the frequency of severe leucocytopenia was striking . This treatment schedule can not be considered active in colorectal cancer.

Br J Cancer, 1991 Aug, 64(2), 267 - 73
Resistance to the antimitotic drug estramustine is distinct from the multidrug resistant phenotype; Speicher LA et al.; Following EMS mutagenesis, three estramustine (EM) resistant DU 145 human prostatic carcinoma cell lines were clonally selected by exposure to incrementally increasing concentrations of the drug . Although only low levels of resistance (approximately 3-fold) were attainable, this resistance was stable in the absence of continuous drug exposure . These EM-resistant clones (EMR 4,9,12) did not exhibit cross resistance to vinblastine, taxol, or adriamycin, and had collateral sensitivity to cytochalasin B . None of the lines had elevated expression of P-glycoprotein mRNA or glutathione S-transferase activity, suggesting a phenotype distinct from the classic multi-drug resistance phenotype . This conclusion was supported further by the observation that two MDR cell lines (FLC mouse erythroleukaemic and SKOV3 human ovarian carcinoma cells) showed sensitivity to EM . Fluorescent activated cell sorting analysis of the effects of EM on cell cycle traverse revealed that at EM concentrations up to 20 microM an increasing percentage of wild type cells were blocked in G2/M; no such effect occurred in EMR lines . Differential interference contrast microscopy was employed to study EM's effect on mitosis . EMR lines were able to form functional, albeit smaller, spindles at EM concentrations that resulted in chromosomal disorganisation and inhibition of mitotic progression in wild type cells . EMR lines were able to progress through mitosis and cytokinesis at the same rate as untreated cells . Tritiated EM was used to evaluate potential drug uptake/efflux mutations in ERM clones . EMR 4 and 9 incorporate less EM than wild type cells; however, they have significantly decreased cellular volumes . The initial efflux rate constants for EMR clones were greater than for wild type cells . Within 5 min greater than 70% of the drug was lost from resistant cells compared to a 50% loss by the wild type . Although the specific mechanisms of resistance have yet to be defined, the lack of collateral resistance to other MDR/anti-microtubule agents could serve as the basis for the clinical use of EM in combination chemotherapy.

FASEB J, 1991 Aug, 5(11), 2523 - 8
Molecular manipulations of the multidrug transporter: a new role for transgenic mice; Pastan I et al.; Multidrug resistance in human cancer is associated with overexpression of the MDR1 gene which encodes a 170,000 molecular weight membrane glycoprotein that transports cytotoxic drugs out of cancer cells . The MDR1 gene is normally expressed in intestine, kidney, liver, and adrenal glands, and in tumors derived from these tissues, but it is not expressed in normal bone marrow . Transgenic mice that express the MDR1 gene in their bone marrow have been developed, and because of this expression these mice are resistant to the bone marrow-suppressive effects of daunomycin, doxorubicin, taxol, and several other anticancer drugs . These mice can be used in several different ways to develop new types of drugs to treat human cancer.--Pastan, I.; Willingham, M . C.; Gottesman, M . Molecular manipulations of the multidrug transporter: a new role for transgenic mice.

Proc Natl Acad Sci U S A, 1991 Aug 1, 88(15), 6585 - 9
Reversed siderophores act as antimalarial agents; Shanzer A et al.; We describe here a family of biomimetic iron carriers that display high binding efficiency for ferric ions and favorable permeation properties across erythrocytic membranes . These carriers inhibit in vitro growth of Plasmodium falciparum by scavenging intracellular iron . The chemical features were realized by reproducing the iron-binding cavities of natural iron carriers (siderophores) and by systematic substitutions of their hydrophilic envelopes for more hydrophobic ones . In contrast to natural carriers, which participate in receptor-mediated iron uptake in cells and act as growth promoters, our synthetic carriers were designed to penetrate cellular membranes by diffusion, scavenge intracellular iron, and thereby act as growth inhibitors . Based on these properties we designate the compounds reversed siderophores and refer to the specific analogs of the natural ferrichrome as synthetic ferrichromes . The antimalarial activity of the synthetic ferrichromes correlated with their lipophilicity, and this antimalarial activity was averted when the chelators were applied as iron (III) complexes . The sites of synthetic ferrichrome action reside in the intraerythrocytic parasite and not in serum or on normal erythrocyte components . The agents were effective against all stages of parasite growth and against a variety of multidrug-resistant strains of P . falciparum . The most potent agent of this synthetic ferrichrome series, SF1-ileu, was not toxic to mammalian cells in culture and was 15-fold more potent and 20-fold faster acting than desferrioxamine . Taken in toto, these agents constitute a series of promising candidates for future use in malaria chemotherapy.

Blood, 1991 Aug 1, 78(3), 586 - 92
Multidrug resistance (mdr1) gene expression in adult acute leukemias: correlations with treatment outcome and in vitro drug sensitivity; Marie JP et al.; Resistance to multiple chemotherapeutic agents has been related to the production of P-glycoprotein, a trans-membrane drug efflux pump that is encoded by the multidrug resistance (MDR) gene mdr1 . To investigate whether mdr1 could be involved in clinical resistance to chemotherapy in acute leukemias, we have analyzed retrospectively the RNA from adult acute leukemia cells by slot-blot hybridization with a human mdr1 probe . Units of mdr1 expression were defined by reference to drug-sensitive human sarcoma and K562 leukemia cell lines (1 U) and the highly resistant doxorubicin selected leukemia cells K562/R7 (50 U) . We studied 41 adult patients with acute leukemias: 5 acute lymphoblastic leukemias, 23 acute myeloid leukemias, and 13 secondary leukemias or blast crisis of chronic myelogenous leukemia . Expression of 10 U or more of mdr1 was found in 6 of 31 (19%) leukemias at diagnosis, versus 5 of 10 (50%) after relapse from therapy, P = .06 . The complete remission rate and in vitro sensitivity to daunorubicin were both correlated with low expression (1 U, v 2 U or more) of mdr1 . Among 36 evaluable attempts to induce remission, the complete remission rate was 67% (8 of 12) for patients with undetectable or minimal mdr1 expression (1 U), versus 29% (7 of 24) in patients with 2 U or more of expression, P = .03 . In vitro resistance to daunorubicin or other MDR-related drugs was associated with expression of 2 U or more of mdr1 in 11 of 11 cases, while specimens that were sensitive to these agents were negative for mdr1 expression in 5 of 11 cases, P = .03 . These data suggest that mdr1 expression contributes to chemoresistance in acute leukemia . Determination of mdr1 gene expression may be useful in designing therapy for patients with leukemia.

Invest New Drugs, 1991 Aug, 9(3), 219 - 25
Differences in N-acetylation of the experimental antitumor agent batracylin in the mouse and the rat; Ames MM et al.; Batracylin (NSC-320846) is a quinalzolineone recently evaluated as a potential antitumor agent by the National Cancer Institute . The analog was active against a number of murine tumors, including colon adenocarcinoma 38 and multidrug resistant sublines of P-388 leukemia . Preclinical toxicity studies revealed that batracylin was much more toxic when administered orally to rats than to mice . The combined sex LD10 in mice was 5,655 mg/m2 while 576 mg/m2 was lethal to all rats treated at that dose . We determined that following oral administration of batracylin, systemic exposure of parent drug to the rat was only 14.9% of that to the mouse . It was subsequently noted that systemic exposure of a relatively non-polar metabolite was approximately 9 times greater in the rat than in the mouse . The metabolite was identified as N-acetylbatracylin by TLC, HPLC and mass spectral analyses . Observations by the National Cancer Institute that N-acetylbatracylin was not toxic following oral administration to mice or rats prompted evaluation of systemic exposure following oral administration to rats . Following oral administration of N-acetylbatracylin to rats, systemic exposure was almost nil . Indeed, exposure of rats to N-acetylbatracylin was several orders of magnitude greater following oral administration of six-fold lower doses of the parent drug, batracylin . Thus, N-acetylation may play a role in the toxicity of batracylin despite the lack of toxicity observed following oral administration of N-acetylbatracylin . In addition, further metabolism of the N-acetyl conjugate, analogous to that of other aromatic amines, may be involved in the pharmacology of batracylin and similar analogs.

Arzneimittelforschung, 1991 Aug, 41(8), 855 - 8
{Reversal of multidrug resistance with (R)-verapamil in vitro and in vivo}; Pommerenke EW et al.; The clinical use of racemic (R/S)-verapamil (CAS 52-53-9) as resistance modifier is limited because of the cardiovascular activity of the substance . The stereoisomer (R)-verapamil shows significant less cardiovascular activity . Therefore we tested the resistance modifying abilities of (R)-verapamil and (R/S)-verapamil in murine multidrug-resistant L 1210 ascites-tumor-cells in vitro and in vivo . The present results demonstrate, that the (R)-isomer has the same resistance modifying effects as racemic verapamil . Both modifying substances have no effects in the parental (sensitive) and cytosine-arabinoside resistant L 1210 ascites-tumor-cells . Thus, (R/S)-verapamil and (R)-verapamil show their resistance modifying abilities only in multidrug-resistant tumor-cells.

Planta Med, 1991 Aug, 57(4), 341 - 3
Antimalarial activity of Tanzanian plants and their active constituents: the genus Uvaria; Nkunya MH et al.; Petroleum ether, dichloromethane, and methanol extracts of leaves, stem, and root bark of nine Uvaria species: U . dependens, U . faulknerae, U . kirkii, U . leptocladon, U . lucida ssp . lucida, Uvaria sp . (Pande)k U scheffleri, and U . tanzaniae were tested for their in vitro activity against the multidrug resistant K1 strain of Plasmodium falciparum . The IC50 values of the extracts varied between 5 and 500 micrograms/ml . The most active extracts were obtained from the stem and root bark of U . lucida ssp . lucida and Uvaria sp . (Pande) and the root bark of U . scheffleri, all of which had IC50 values between 5 and 9 micrograms/ml . Among the compounds isolated, uvaretin, diuvaretin, and (8',9'-dihydroxy)-3-farnesylindole were the most active (IC50 = 3.49, 4.20, and 2.86 micrograms/ml, respectively).

Protein Expr Purif, 1991 Aug, 2(4), 256 - 65
Strategies for the purification of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells; Doige CA et al.; P-glycoprotein, a hydrophobic 170-kDa integral protein overexpressed in the plasma membrane of multidrug-resistant cells, is proposed to function as an ATP-dependent drug efflux pump . Plasma membrane preparations highly enriched in P-glycoprotein were isolated from multidrug-resistant cells by discontinuous sucrose gradient and Ca2+ precipitation methods . Several strategies were used for P-glycoprotein purification, with the goal being to achieve both good yields and purity, while keeping experimental manipulation to a minimum . P-glycoprotein was solubilized from the plasma membrane using 3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulfonate . Immunoaffinity chromatography using C219 monoclonal antibody produced low yields of moderately pure protein . Sequential lectin affinity chromatography on RCA-120 followed by lentil lectin resulted in a P-glycoprotein preparation that showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . A fraction of P-glycoprotein did not bind to RCA-120, most likely as a result of heterogeneous glycosylation . A combination of chromatography on RCA-120 followed by immunoaffinity chromatography on C219 resulted in low yields of very pure P-glycoprotein.

Acta Trop, 1991 Aug, 49(3), 215 - 25
The effect of verapamil alone and in combination with trypanocides on multidrug-resistant Trypanosoma brucei brucei; Kaminsky R et al.; Following previous studies of verapamil reversal of multidrug resistance in cancer cells and chloroquine resistance in malaria, the effect of the calcium channel blocker verapamil was investigated on multidrug-resistant and susceptible Trypanosoma brucei brucei . Resistance of cloned parasites to diminazene aceturate (Berenil) and isometamidium chloride (Samorin) was expressed in a cell-free in vitro culture system . Verapamil showed antitrypanosomal activity against both, multidrug-resistant and susceptible trypanosomes at concentrations above 1 micrograms/ml . Verapamil did not reverse multidrug resistance when used at concentrations of 0.1 or 1.0 micrograms/ml in combination with diminazene aceturate or isometamidium chloride . Results obtained in vitro correlate with observations in mice . It is suggested that multidrug resistance in African trypanosomes is due to mechanisms other than those occurring in cancer cells, malaria or South-American trypanosomiasis.

Histopathology, 1991 Aug, 19(2), 131 - 40
Immunohistochemical detection of the multidrug transport protein P170 in human normal tissues and malignant lymphomas; Pileri SA et al.; Two monoclonal antibodies, MRK16 and C219, both directed at the 170 kDa P-glycoprotein multidrug resistance agent, were applied to frozen sections or cytospin preparations from normal human tissues and 60 non-Hodgkin's malignant lymphomas . Adrenal gland, kidney, liver and pancreas were always stained by the reagents, albeit with slightly different patterns . Brain capillaries as well as macrophages and some elements of the bone marrow, peripheral blood, ovarian stroma and colonic, gastric and jejunal mucosa were positive in all examined preparations . There were differences in the staining patterns with the two antibodies . Among the 60 non-Hodgkin's lymphomas, 25 contained a number of positive cells, which ranged from 2% to 100% . No correlation was seen between the expression of P170 and histological type, stage, clinical symptoms or growth fraction . A close relationship was shown between the presence of P170 positive elements and the clinical course of the disease (P less than 0.001).

Bioessays, 1991 Aug, 13(8), 381 - 7
Multidrug resistant transgenic mice as a novel pharmacologic tool; Mickisch GH et al.; Multidrug resistance resulting from expression of an energy-dependent drug efflux pump encoded by the human MDR1 gene is a major impediment to effective cancer therapy . Pharmacologic intervention aimed at inhibiting this multidrug transporter should improve existing chemotherapy of human cancer, but drug development has been delayed by the difficulty and expense of developing valid animal models . Using recombinant DNA technology, a transgenic mouse has been engineered whose bone marrow is protected from the toxic effects of chemotherapy by expression of the MDR1 gene . This animal system allows the rapid screening of drugs which inhibit the multidrug transporter and heralds a new era of using transgenic animals for pharmacologic screening.

Semin Cancer Biol, 1991 Aug, 2(4), 227 - 33
On the origins of clinical drug resistance; O'Brien JP et al.; Recent progress in understanding the clinical importance of certain laboratory models of drug resistance is cause for clinical oncologists to rethink some of the current concepts used in developing chemotherapy programs . The following discussion will reexamine a popular model of resistance development and highlight certain insights gained from clinical and laboratory studies of multidrug resistance . Such insights both challenge traditional concepts of cancer treatment and suggest new therapeutic approaches that would otherwise not have been apparent.

Semin Cancer Biol, 1991 Aug, 2(4), 213 - 26
Multidrug resistance mediated by P-glycoproteins; Schinkel AH et al.; Overexpression of P-glycoprotein genes is a well-established cause of one form of multidrug resistance . P-glycoproteins are plasma membrane proteins containing two ATP-binding sites and twelve putative transmembrane segments . P-glycoproteins are thought to act as ATP-dependent drug efflux pumps, actively extruding a range of structurally different, hydrophobic drugs from the cell . This simple model can account for the properties of multidrug resistant cells, even those that seem to require more complex explanations . The structure and function of P-glycoprotein genes has been studied in mammals and in several lower eukaryotes . These studies are helping to delineate the range of drugs that can be transported by P-glycoproteins; the genetic mechanisms that can lead to elevated cellular P-glycoproteins levels; and the evolution of the versatile and prolific P-glycoprotein gene family . The physiological function of the human P-glycoproteins encoded by the MDR1 and MDR3 (or MDR2) genes remains a matter of speculation.

Eur J Haematol, 1991 Aug, 47(2), 146 - 51
Absence of correlation between cytotoxicity and drug transport by P-glycoprotein in clinical leukemic cells; Kato S et al.; Development of resistance to cytotoxic agents is a common problem in the treatment of acute leukemia . In cell lines having multidrug resistance (MDR) phenotype, a decrease in the intracellular accumulation of drugs has been closely related to the overexpression of P-glycoprotein/mdr1 genes . We analyzed the relationship between the cytotoxicity of adriamycin (ADR) in vitro, intracellular accumulation of ADR, and the expression of P-glycoprotein on fresh leukemic cells from 19 patients at their initial presentation and from 9 relapsed patients . Pretreatment patients showed significantly higher ratio of complete remission than relapsed patients, and mean value of IC50 for adriamycin in initial presentation was higher than at relapse . But we found no significant relationship between in vitro cytotoxicity and drug transport . In addition, only 2 of the 5 relapsed patients examined by monoclonal antibody C219 expressed the P-glycoprotein . These results suggest that the acquisition of clinical drug resistance may involve various mechanisms other than the reduction of drug accumulation with P-glycoprotein expression.

J Urol, 1991 Aug, 146(2), 447 - 53
New potent verapamil derivatives that reverse multidrug resistance in human renal carcinoma cells and in transgenic mice expressing the human MDR1 gene; Mickisch GH et al.; Multidrug resistance in human renal cell carcinoma is mainly caused by expression of the MDR1 gene and is characterized by a broad spectrum cross resistance to many natural product chemotherapeutic agents . This resistance can be overcome by applying chemosensitizers which inhibit the function of the MDR1 gene product P-glycoprotein . The development of new reversing agents with fewer side effects and a higher potency in modifying resistance is a high priority of research on drug resistance . We have evaluated four new verapamil derivatives on 21 primary human renal cell carcinomas in vitro, and also tested them in an MDR-transgenic mice model . These mice express the human MDR1 gene in their bone marrow cells and measurement of their white blood counts provides a simple, rapid and reliable system to screen for the potency of MDR-reversing agents in vivo . We demonstrate here that all four drugs are effective in reversing multidrug resistance in primary cultures of human renal cell carcinomas when used in combination with vinblastine chemotherapy, and to a lesser extent with doxorubicin or daunomycin chemotherapy . Our in vivo data indicate that two of these reversing agents display low toxicity at high concentrations and are more effective at low, clinically achievable concentrations, than the other two drugs and R-verapamil . These results make the two new drugs attractive candidates to be taken into clinical trials.

Cancer Commun, 1991 Aug, 3(8), 265 - 70
Selenium-dependent glutathione peroxidase expression is inversely related to estrogen receptor content of human breast cancer cells; Townsend AJ et al.; The absence of estrogen receptors (ER) in human breast tumors has been associated with a poorer prognosis compared to patients with ER positive breast cancer . Previous studies from our laboratory have shown that a multidrug resistant human breast cancer cell line selected for resistance to Adriamycin (ADR) exhibited markedly increased expression of both the pi class glutathione S-transferase (GST-pi) and the selenium-dependent glutathione peroxidase . These studies also revealed that the ER status was inversely related to the expression of GST-pi in six human breast cancer cell lines and primary tumor specimens . In the present study, we have examined the relationship between ER status and several biological properties of these cells, including their levels of glutathione peroxidase (GSH-Px) and catalase expression, their capacity to generate toxic hydroxyl radicals (degrees OH) by redox cycling of ADR, and their sensitivities to the cytotoxic effects of ADR and the oxidant, H2O2 . Our results show that expression of GSH-Px, but not catalase, is inversely related to the ER status in these cell lines . Formation of the degree OH induced by treatment of cells with ADR was inversely proportional to the GSH-Px activity in these cell lines, and thus directly related to the ER status . Sensitivity of these cells to ADR or to H2O2, however, was not consistently related to ER status, GSH-Px, or catalase activity, or to ADR induced degree OH radical formation . These results indicate that these parameters are not predictive of cellular susceptibility to oxidative damage in these cell lines under the conditions studied.

Cell, 1991 Jul 12, 66(1), 85 - 94
Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells; Chaudhary PM et al.; Hematopoietic stem cells show reduced staining with a mitochondrial fluorescent dye, rhodamine 123 (Rh-123), which was supposed to indicate decreased mitochondrial activity in these cells . Rh123 and several other fluorescent dyes are substrates for transport mediated by P-glycoprotein (P-gp), an efflux pump responsible for multidrug resistance in tumor cells . We have found that staining of human bone marrow cells with fluorescent dyes is potentiated by P-gp inhibitors and inversely correlated with P-gp expression . P-gp is expressed in practically all hematopoietic progenitor cells, including long-term culture-initiating cells . The highest levels of P-gp among the progenitors are associated with cells displaying characteristics of pluripotent stem cells . These results have implications for stem cell purification and bone marrow resistance to cancer chemotherapy.

Biochim Biophys Acta, 1991 Jul 10, 1093(2-3), 147 - 52
Cellular daunomycin fluorescence in multidrug resistant 2780AD cells and its relation to cellular drug localisation; Lankelma J et al.; Multidrug resistant (MDR) 2780AD human ovarian carcinoma cells were loaded with the fluorescent anticancer agent daunomycin (DN) . Fluorescence anisotropy was lower than for corresponding A2780 wild-type cells, indicating that DN was less rigidly bound than in the wild-type cells . Average fluorescence quenching of DN was lower for 2780AD cells . Data were fitted into a model with a highly quenched fraction (fraction A), corresponding to DN intercalated in DNA, and an unquenched fraction (fraction B) . The ratio A/B was one order of magnitude lower for the MDR cells than for the wild-type cells . Two other MDR cell lines were investigated and low A/B ratios were found in both cases . Thus, evidence has been provided that in MDR cells the DNA-bound fraction is relatively low and that more free DN is present, for example in acidic vesicles.

Biochem Pharmacol, 1991 Jul 5, 42(2), 391 - 402
Biochemical and pharmacological characterization of MCF-7 drug-sensitive and AdrR multidrug-resistant human breast tumor xenografts in athymic nude mice; Mimnaugh EG et al.; The phenotypic expression of multidrug resistance by the doxorubicin-selected AdrR human breast tumor cell line is associated with overexpression of plasma membrane P-170 glycoprotein and increased cytosolic selenium-dependent GSH-peroxidase activity relative to the parental MCF-7 wild-type line (WT) . To determine whether doxorubicin resistance by AdrR cells persists in vivo, and to further investigate the possibility of biochemical differences between WT and AdrR solid tumors, both tumor cell lines were grown as subcutaneous xenografts in athymic nude mice . Tumorigenicity depended upon cell inoculation burden, and tumor incidence was similar for both cell lines (greater than 80% tumor takes at 10(7) cells/mouse) at 14 days, provided 17 beta-estradiol was supplied to the animals bearing the WT tumors . However, the growth rate for the AdrR xenografts was only about half that of WT xenografts . Doxorubicin (2-8 mg/kg, i.p., injected weekly) significantly diminished the growth of the WT tumors, but AdrR solid tumors failed to respond to doxorubicin . The accumulation of 14C-labeled doxorubicin was 2-fold greater in WT xenografts that in AdrR, although there were no differences in host organ drug levels in mice bearing either type of tumors . Membrane P-170 glycoprotein mRNA was detected by slot-blot analysis in the AdrR tumors, but not in WT . Electron spin resonance 5,5-dimethylpyrroline-N-oxide-spin-trapping experiments with microsomes and mitochondria from WT and AdrR xenographs demonstrated a 2-fold greater oxygen radical (superoxide and hydroxyl) formation from activated doxorubicin with WT xenographs compared to AdrR . Selenium-dependent glutathione (GSH)-peroxidase, superoxide dismutase and GSH-S-aryltransferase activities in AdrR xenografts were elevated relative to WT . Although the activities of the latter two enzymes were similar to those measured in both tumor cell lines, GSH-peroxidase activities were elevated 70-fold (WT) and 10-fold (AdrR) in xenografts compared to tumor cells . In contrast, in both WT and AdrR solid tumors in vivo, catalase, NAD(P)H-oxidoreductases, and glutathione disulfide (GSSG)-reductase activities, and GSH and GSSG levels were not markedly different, and were essentially the same as in cells in vitro . Like the MDR cells in culture, AdrR tumor xenografts were extremely resistant to doxorubicin and retained most of the characteristics of the altered phenotype . These results suggest that WT and AdrR breast tumor xenografts provide a useful model for the study of biochemical and pharmacological mechanisms of drug resistance by solid tumors in vivo.

Cancer Lett, 1991 Jul 4, 58(3), 189 - 93
Distinct patterns of phorbol ester-induced downregulation of protein kinase C activity in adriamycin-selected multidrug resistant and parental murine fibrosarcoma cells; Ward NE et al.; Specific activators of protein kinase C (PKC), including the phorbol-ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), can reduce the chemosensitivities of a variety of mammalian tumor cell lines and their cytotoxic drug-selected multidrug resistant (MDR) variants to MDR-linked drugs, thus implicating PKC in the MDR phenotype . Previously, we reported that the adriamycin-selected MDR murine fibrosarcoma cell line UV-2237M-ADRR has approximately twice as much PKC activity as the parental UV-2237M line . In this report, we show that the level of {3H}phorbol-12,13-dibutyrate specific binding activity was elevated 3.5-fold in the MDR cells, thus establishing that phorbol-ester responsive PKC is overexpressed in the MDR line . Phorbol esters mediate downregulation of PKC by stimulating proteolysis of the enzyme, without altering the rate of PKC synthesis . We report that the kinetics of TPA-induced downregulation of PKC activity differ markedly in parental and MDR UV-2237M cells, providing evidence that the overexpression of phorbol-ester responsive PKC in adriamycin-selected MDR UV-2237M-ADRR cells results, at least in part, from a reduced rate of PKC degradation in the cells.

DNA Cell Biol, 1991 Jul-Aug, 10(6), 433 - 41
Murine mdr-1, mdr-2, and mdr-3 gene expression: no coinduction with the Cyp1a-1 and Nmo-1 genes in liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin; Teeter LD et al.; Multidrug-resistance (MDR) genes are induced in the liver of rodents treated with a variety of foreign chemicals and hepatocarcinogens . It has been reported that 2,3,6,7-tetrachlorodibenzo-p-dioxin (TCDD) might increase hepatic MDR transcripts in the Fischer rat and the C57BL/6 (B6) inbred mouse strain having the high-affinity aromatic hydrocarbon (Ah) receptor, but not in the DBA/2 (D2) strain having the low-affinity Ah receptor . These intriguing results suggest that TCDD might activate MDR gene expression by way of an Ah receptor-mediated signal transduction pathway . We have attempted to confirm these data in four inbred mouse strains: two (B6 and BALB/c) having the high-affinity Ah receptor, and two (D2 and AKR) having the low-affinity Ah receptor . The RNase protection assay was used to distinguish between the MDR1, MDR2, and MDR3 mRNAs . TCDD treatment at high (100 micrograms/kg) and low (1 mu/kg) doses, a time course from 6 to 96 hr of TCDD treatment, progeny from the B6D2F1 x D2 backcross, and transcriptional run-on experiments were performed . The Cyp1a-1 (cytochrome P1450) and Nmo-1 {NAD(P)H:menadione oxidoreductase} genes, two members of the TCDD-inducible {Ah} battery, were used as positive controls . We were unable to detect significant coinduction of MDR1, MDR2, or MDR3 mRNA with CYP1A1 mRNA or with Cyp1a-1 or Nmo-1 transcription under any conditions . Therefore, we conclude that any effects that TCDD might have on MDR expression must be substantially different from TCDD effects on genes known to be induced via the Ah receptor.

J Med Chem, 1991 Jul, 34(7), 1947 - 51
Additional nucleotide derivatives of mitosenes . Synthesis and activity against parental and multidrug resistant L1210 leukemia; Iyengar BS et al.; Cytidine 5'-monophosphate and 5'-ara-CMP conjugates of 2,7-diaminomitosene, with the phosphate groups linked to C-1, were prepared by treating mitomycin C with the appropriate nucleotides . 5'-UMP conjugates were prepared from mitomycin A, 7 (M-83), and 8 (BMY-25282) by similar procedures . A conjugate could not be prepared from mitomycin C and 6-MPRP, but a sulfur-linked derivative was made with 6-MP ribonucleoside . The corresponding 1-hydroxy-2-aminomitosenes were prepared from the parent mitomycin analogues for structure-activity comparisons . All compounds were tested against L1210 murine leukemia in the MTT tetrazolium dye assay . In general, the conjugates were less potent than the parent mitomycins; however 5'-ara-CMP conjugate 14 derived from mitomycin C was more potent than the parent compound or any mitomycin tested except mitomycin A . It also was more potent than ara-C . This result establishes the value of this approach to prodrugs, at least in cell culture . Against a multi-drug-resistant L1210 cell line, all of the conjugates derived from mitomycin C were more potent than the parent compound . 6-Mercaptopurine ribonucleoside conjugate 15 was more active against the resistant cells than it was against the parental cell line.

Trans R Soc Trop Med Hyg, 1991 Jul-Aug, 85(4), 424 - 9
Malaria during pregnancy in an area of unstable endemicity; Nosten F et al.; A prospective study of malaria during pregnancy was conducted between September 1986 and December 1989 in an area of unstable (mesoendemic) malaria transmission on the Thai-Burmese border . Antenatal clinics were set up in camps for displaced persons of the Karen ethnic minority and 1358 pregnant women were enrolled at a mean estimated gestational age of 23 weeks (standard deviation 5.7 weeks) and were followed weekly until delivery . Malaria developed in 505 women (37.2%); 80.2% of infections were Plasmodium falciparum, 17.1% were P . vivax, and 2.7% were mixed . Primigravidae were infected more commonly than multigravidae: 153/322 (47.5%) compared with 318/953 (33.3%) (P less than 0.001) . The incidence of malaria declined from the 20th week of gestation (12%) towards term (4.4%) . Most infections were detected before symptoms developed, and there were no deaths associated with malaria . Despite this, malaria was associated with an overall 123 g reduction in birthweight (95% confidence interval {CI} 34-212 g) . This reduction was largely accounted for by lower birthweights of babies born to infected primigravidae (mean reduction 151 g, 95% CI 21-282 g) and women in their 2nd and 3rd pregnancies (mean reduction 185 g, 95% CI 84-286 g) . The incidence of anaemia requiring treatment was higher in women who developed malaria, 149/420 (35.4%) compared with 191/670 (28.5%), and was proportional to the number of parasitaemic episodes . Thus, despite regular antenatal clinic attendance with prompt detection and treatment of malaria (the currently employed antimalarial strategy in areas with multidrug-resistant P . falciparum), malaria still had a significant adverse effect on pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticancer Res, 1991 Jul-Aug, 11(4), 1379 - 82
Distribution, DNA damage and cytotoxic effects of etoposide in human tumor xenografts in vivo; Ullmann CA et al.; Mechanisms of resistance to VP-16 in vivo were studied using sensitive and multidrug resistant human breast tumor (MCF-7) cells, implanted bilaterally in nude mice . Administration of VP-16 (40 mg/kg, i.p.) indicated that there were no significant differences in either accumulation and retention of VP-16 or in the drug-induced bulk DNA damage in sensitive or resistant solid xenografts . Furthermore, under these conditions, no differential cytotoxic response to VP-16 was observed in solid tumors.

Anal Biochem, 1991 Jul, 196(1), 161 - 9
Quantitation of mRNA by the kinetic polymerase chain reaction assay: a tool for monitoring P-glycoprotein gene expression; Hoof T et al.; The overexpression of P-glycoprotein (mdr1) induces the phenotype of multidrug resistance to many chemically unrelated drugs . Absolute mdr1 mRNA levels in tissues, neoplasms and cell lines from various rodents and man were estimated by primer-directed reverse transcription (RT) and subsequent polymerase chain reaction (PCR) with 32P-labeled nucleotide . Aliquots taken during PCR were analyzed for product formation by two-dimensional densitometry of autoradiograms of separated cDNA on dried agarose gels . The kinetic RT/PCR assay was calibrated for the range of 10(-3) to 10(3) amol of specific mRNA by utilizing mdr1 RNA transcribed from a full-length cDNA as the external standard and aldolase mRNA as the internal standard in order to assess RNA degradation of the specimen . For various cDNA fragments differing between 200 and 600 bp in size, the yield of cDNA during logarithmic growth increased by 1.78 +/- 0.02 per cycle . Hence, the sensitive and reproducible quantitation of transcript is feasible by monitoring RT/PCR kinetics without the need for any competing cRNA or cDNA fragment provided that a high-performance thermal cycler is employed for PCR.

Anticancer Drug Des, 1991 Jul, 6(3), 189 - 93
The new potent immunosuppressant FK-506 reverses multidrug resistance in Chinese hamster ovary cells; Epand RF et al.; The macrolide FK-506, produced by Streptomyces tsukubaensis, is effective in sensitizing a multidrug resistant strain of Chinese hamster ovary cells to the cytotoxic action of vinblastine . FK-506 also promotes Adriamycin accumulation into both wild type and resistant cell lines . FK-506 may be a useful drug to enhance the effectiveness of anti-tumour cytotoxic drugs.

Anticancer Drug Des, 1991 Jul, 6(3), 179 - 88
Enhancement of activities of anti-tumor drugs by dipyridamole against multidrug-resistant human hepatoma PLC/PRF/5 cells; Tatsuta T et al.; Dipyridamole (DPM) at 10 microM enhanced the cytotoxicity of anti-tumor drugs, which were associated with multidrug resistance, more in multidrug-resistant human hepatoma PLC/PRF/5 cells (PLC/COL) than in its parental cells (PLC/S) . DPM increased, dose-dependently, the intracellular accumulation of {3H}vinblastine in PLC/COL . However, the effect was immediately diminished by its removal from the medium, indicating that DPM needed to be present together with the anti-tumor drugs to enhance the intracellular accumulation of the drugs . DPM inhibited the efflux of {3H}vinblastine from the PLC/COL cells, the binding of {3H}vinblastine to membrane vesicles of PLC/COL, and the binding of {3H}azidopine to P-glycoprotein in the plasma membrane of PLC/COL . Apparently DPM binds to P-glycoprotein and inhibits active efflux . {14C}labeled DPM was quickly incorporated into the cells and the cellular level of {14C}DPM reached a plateau after 5 min . It was slightly higher in PLC/S than in PLC/COL . The cellular {14C}DPM quickly disappeared after its removal from the medium . These results indicate that DPM binds quickly but reversibly to various kinds of cellular proteins including P-glycoprotein and inhibits active efflux of some anti-tumor drugs in multi-drug-resistant tumor cells, resulting in the enhancement of the activities of these drugs.

Am J Trop Med Hyg, 1991 Jul, 45(1), 98 - 111
Characteristics of multidrug resistance in Plasmodium and Leishmania: detection of P-glycoprotein-like components; Grogl M et al.; Multidrug-resistance (MDR) in neoplastic cells is frequently characterized by the overexpression of P-glycoprotein (PGP), a 170 kDa transmembrane glycoprotein that binds multiple cytotoxic drugs as well as calcium channel antagonists . Chloroquine resistance in Plasmodium falciparum appears to be analogous to MDR in neoplastic cells, where the induction of resistance with one drug confers resistance to other structurally and functionally unrelated drugs . To test the hypothesis that chloroquine resistance in P . falciparum and antimony resistance in Leishmania is mediated by a similar mechanism of MDR in mammalian neoplastic cells, a PGP-specific monoclonal antibody (C219) was used to determine the presence of PGP genes in resistant and sensitive Plasmodium and Leishmania parasites by indirect immunofluorescence assays and Western blotting procedures . These PGP-like components were detected in both drug-sensitive and -resistant Plasmodium and Leishmania cells . A 40-42 kDa component was observed to be greater in a chloroquine-resistant P . berghei (C line) than in a chloroquine-susceptible P line . Differences observed between Pentostam-resistant and -sensitive Leishmania promastigote clones and isolates included the increased expression of 96-106 and 23-25 kDa peptides in drug-resistant L . enrietti, and increased amounts of two different peptides in two drug-resistant L . panamensis clones (i.e., 96-106 and 43-45 kDa in WR-746-CL4, and 53 and 23-25 kDa in kDa) in amastigotes as in MDR KB carcinoma cells (KB-V1) . Comparative indirect immunofluorescent studies suggested that a correlation existed between the degree of antimony susceptibility and the concentration of the moiety recognized by C219 in two L . panamensis clones . Binding of the C219 monoclonal antibody to the PGP-like component of Leishmania was blocked by Pentostam, while the binding of C219 to multiple-drug resistant KB-V1 PGP was not inhibited by Pentostam, regardless of the PGP concentration . This suggests some degree of specificity in the binding of Pentostam to the Leishmania PGP-like components . In addition, these studies have demonstrated that drug-sensitive Leishmania accumulate two to five times more 125Sb-Pentostam than resistant clones.

Br J Cancer, 1991 Jul, 64(1), 15 - 22
Multidrug resistance-associated antigens on drug-sensitive and -resistant human tumour cell lines; Mirski SE et al.; In this paper the biochemical properties of the antigens detected by six murine monoclonal antibodies (MAbs) are described . These MAbs react selectively with the multidrug-resistant small cell lung cancer (SCLC) cell line, H69AR, compared to its sensitive parent cell line, H69 (Mirski & Cole, 1989) . Because H69AR cells do not overexpress P-glycoprotein, the antigens detected by these MAbs may be markers for non-P-glycoprotein-mediated mechanisms of resistance . We found that the 36 kDa protein precipitated by MAb 3.186 is phosphorylated and has a pI of approximately 6.7 . The 55 kDa protein precipitated by MAb 3.50 is also phosphorylated and has a pI of approximately 5.7 . Several observations suggest that MAbs 3.80, 3.177 and 3.187 recognise the same 47 kDa molecule and hence only MAb 3.187 was characterised further . This MAb precipitates an acidic protein which runs as a streak on isoelectric focusing gels . The 25 and 22.5 kDa cell surface proteins precipitated by MAb 2.54 both have a pI of approximately 7.6 . Treatment of immunoprecipitates with glycosidase F indicated that none of the proteins detected by MAbs 2.54, 3.187, 3.50 and 3.186 have large N-linked carbohydrates . The peptide nature of the epitopes detected by MAbs 2.54 and 3.186 was unequivocally demonstrated by precipitation from in vitro translation products of H69AR RNA . The antigens detected by MAbs 3.50 and 3.187 were not detectable in immunoprecipitates of translation products but the epitopes are probably peptides because they were destroyed by boiling in sodium dodecyl sulphate . When the reaction of the MAbs with a panel of 15 paired drug-sensitive and -resistant cell lines was examined in a cell enzyme-linked immunosorbent assay, only a few resistance associated reactions were observed . Most of the reactions were either negative or not resistance-associated . When tested with three SCLC cell lines, MAb 3.187 reacted in a manner consistent with the relative resistance of the cell lines . Antigens that had similar electrophoretic mobility to those from H69AR cells were precipitated from extracts of five human cell lines of various tumour types . These data indicate that the cross-reactivities of the MAbs are due to antigens shared among the cell lines and not just the expression of common epitopes on different proteins . Resistance-associated proteins with the biochemical properties of the antigens described in this paper have not been reported previously and they remain potential markers for the as yet to be determined mechanisms of drug resistance in SCLC and other human malignancies.

Leukemia, 1991 Jul, 5(7), 592 - 7
P-glycoprotein expression and in vitro reversion of doxorubicin resistance by verapamil in clinical specimens from acute leukaemia and myeloma; Solary E et al.; The expression of the P-glycoprotein which is associated with the development of multidrug resistance in various cell lines was investigated in 87 fresh acute leukaemia and multiple myeloma samples using the specific mouse monoclonal antibody MRK16 in an indirect immunofluorescence assay . Considering a 10% positive cell cut-off value, a heterogeneous expression of P-glycoprotein was observed in 5/22 (22.7%) de novo acute leukaemias, 7/22 (31.8%) relapse or secondary acute leukaemias, 14/27 (51.8%) acute transformation of myeloproliferative or myelodysplastic syndromes and 5/16 (31.2%) multiple myelomas . This expression was not associated with specific cytogenetic abnormalities, especially alterations of chromosome 7q . Verapamil, a calcium channel blocker, has been demonstrated to circumvent the multidrug resistance in cell lines, possibly by interfering with P-glycoprotein function . Using the microculture tetrazolium assay, verapamil was demonstrated to increase the sensitivity of fresh leukaemic or myeloma cells to doxorubicin in 19/43 (43.1%) samples . The doxorubicin IC50 level and the capacity of verapamil to increase the sensitivity of blast cells to doxorubicin in vitro did not correlate with the expression of P-glycoprotein . We conclude that high non-cytotoxic concentrations of verapamil were able to increase the in vitro doxorubicin sensitivity of fresh acute leukaemia and myeloma cells without detectable expression of the P-glycoprotein.

Blood, 1991 Jul 1, 78(1), 44 - 50
Multidrug-resistant myeloma: laboratory and clinical effects of verapamil as a chemosensitizer; Salmon SE et al.; Verapamil was evaluated as a chemosensitizer for reversing multidrug resistance in multiple myeloma both in vitro and in clinical trials . Bone marrows from 59 myeloma patients in relapse were evaluated for several resistance parameters: expression of p-glycoprotein (MDR1), doxorubicin (Adriamycin) and vincristine sensitivity, and the ability of added verapamil to reduce resistance to the cytotoxic agents . We found that verapamil was capable of sensitizing myeloma cells that exhibited resistance to doxorubicin and vincristine in vitro, but did not enhance sensitivity of cells that were drug sensitive (P less than .001) . Myeloma cells expressing MDR1 immunohistochemically tended to be more doxorubicin resistant in vitro than MDR1-negative cells . In the clinical trials, 22 patients with myeloma refractory to vincristine-Adriamycin-dexamethasone (VAD) were treated with VAD plus high-dose intravenous verapamil (Ve) . Among the 22 patients treated with VAD/Ve, five achieved a partial remission (23%) . The median relapse-free survival for the VAD/Ve responders was 5.4 months and their overall survival from the start of VAD/Ve was better than that of the nonresponders . Among the subset of 10 patients whose myeloma cells were MDR1 positive, four responded clinically (40%), whereas none of five patients with MDR1-negative myeloma cells achieved remission with VAD/Ve . We also observed that myeloma cells from three of four VAD/Ve clinical responders exhibited in vitro chemosensitization with verapamil, whereas in vitro verapamil chemosensitization was seen in only one of six clinical nonresponders . Our observations demonstrate that clinical reversal of multidrug resistance can be achieved in some patients with VAD-refractory myeloma with the use of verapamil . In addition to their value in drug development, in vitro tests of MDR1 expression and of chemosensitizers plus cytotoxic drugs on the patients' bone marrow myeloma cells may identify patients who will respond clinically to chemosensitizer-containing regimens . We anticipate that chemosensitizer regimens capable of inhibiting multidrug resistance will play an increasing role in the treatment of hematologic malignancies, including B-cell neoplasms such as multiple myeloma and the non-Hodgkin's lymphomas.

Cancer Res, 1991 Jul 1, 51(13), 3345 - 52
Non-P-glycoprotein-mediated multidrug resistance in a small cell lung cancer cell line: evidence for decreased susceptibility to drug-induced DNA damage and reduced levels of topoisomerase II; Cole SP et al.; Data obtained from clinical samples suggest that non-P-glycoprotein mechanisms of multidrug resistance are likely to be important in small cell lung cancer . The H69AR cell line was derived from the H69 small cell lung cancer cell line by selection in doxorubicin (adriamycin) and does not overexpress P-glycoprotein as detected by monoclonal antibody C219 (S.E.L . Mirski et al., Cancer Res., 47:2594, 1987) . In the present study, we have used the polymerase chain reaction to verify that H69AR cells do not overexpress P-glycoprotein . Further, transport studies with radiolabeled daunomycin, VP-16, and vinblastine demonstrate that differences in net drug accumulation or efflux are not part of the resistance phenotype of H69AR cells . To determine if H69 and H69AR cells differ in their susceptibility to drug-induced DNA damage, DNA single-strand breaks (SSB) generated by VP-16 and Adriamycin were measured using the alkaline filter elution assay . Readily detectable SSB were produced in intact H69 cells by 5 microM VP-16, but 100 microM drug was required to cause similar damage in H69AR cells . H69AR cells were also resistant to SSB induction by Adriamycin . The formation of SSB by VP-16 was similarly reduced in isolated H69AR nuclei, indicating that resistance to this drug resides, at least in part, in the nucleus . No significant differences were observed in the rate or extent of repair of VP-16-induced DNA SSB in H69 and H69AR cells . The reduced susceptibility to drug-induced SSB may result from alterations in topoisomerase II, since less immunoreactive topoisomerase II was found in H69AR cells compared to H69 cells . However, changes in topoisomerase II cannot explain the resistance of H69AR cells to such drugs as the Vinca alkaloids and gramicidin D, indicating that multiple mechanisms contribute to drug resistance in this small cell lung cancer cell line.

Mol Cell Biol, 1991 Jul, 11(7), 3407 - 18
A possible role for a mammalian facilitative hexose transporter in the development of resistance to drugs; Vera JC et al.; We show that D- but not L-hexoses modulate the accumulation of radioactive vinblastine in injected Xenopus laevis oocytes expressing the murine Mdr1b P-glycoprotein . We also show that X . laevis oocytes injected with RNA encoding the rat erythroid/brain glucose transport protein (GLUT1) and expressing the corresponding functional transporter exhibit a lower accumulation of {3H}vinblastine and show a greater capacity to extrude the drug than do control oocytes not expressing the rat GLUT1 protein . Cytochalasin B and phloretin, two inhibitors of the mammalian facilitative glucose transporters, can overcome the reduced drug accumulation conferred by expression of the rat GLUT1 protein in Xenopus oocytes but have no significant effect on the accumulation of drug by Xenopus oocytes expressing the mouse Mdr1b P-glycoprotein . These drugs also increase the accumulation of {3H}vinblastine in multidrug-resistant Chinese hamster ovary cells . Cytochalasin E, an analog of cytochalasin B that does not affect the activity of the facilitative glucose transporter, has no effect on the accumulation of vinblastine by multidrug-resistant Chinese hamster cells or by oocytes expressing either the mouse Mdr1b P-glycoprotein or the GLUT1 protein . In all three cases, the drug verapamil produces a profound effect on the cellular accumulation of vinblastine . Interestingly, although immunological analysis indicated the presence of massive amounts of P-glycoprotein in the multidrug-resistant cells, immunological and functional studies revealed only a minor increase in the expression of a hexose transporter-like protein in resistant versus drug-sensitive cells . Taken together, these results suggest the participation of the mammalian facilitative glucose transporter in the development of drug resistance.

J Clin Endocrinol Metab, 1991 Jul, 73(1), 18 - 29
Mitotane enhances cytotoxicity of chemotherapy in cell lines expressing a multidrug resistance gene (mdr-1/P-glycoprotein) which is also expressed by adrenocortical carcinomas; Bates SE et al.; P-Glycoprotein (Pgp), product of the mdr-1 gene, is a 130- to 180-kDa plasma membrane phosphoglycoprotein which mediates multidrug resistance in cell culture by increasing efflux of the natural product chemotherapeutic agents . High levels of expression of mdr-1/Pgp are found in both the normal adrenal and adrenocortical cancers . By RNA in situ hybridization the expression in adrenocortical cancer is shown to be widely distributed . The present study demonstrates that decreased drug accumulation mediated by mdr-1/Pgp can be overcome by clinically achievable concentrations of mitotane (o,p'-DDD) . The increase in drug accumulation with the addition of mitotane is due at least in part to a decrease in drug efflux and results in an increase in cytotoxicity when agents of the natural product class are used . This effect is observed in cells with a broad range of mdr-1/Pgp expression, including levels comparable to those found in most adrenocortical cancers . Similar increases in drug accumulation can be demonstrated in an unselected adrenocortical cancer cell line that expresses mdr-1/Pgp . The finding that multidrug resistance mediated by mdr-1/Pgp can be reversed by mitotane provides a rational basis for exploring the use of mitotane in combination with natural product chemotherapeutic agents in adrenocortical cancer.

Res Microbiol, 1991 Jul-Aug, 142(6), 701 - 4
Site-specific recombination and shuffling of resistance genes in transposon Tn21; Avila P et al.; Many multidrug-resistant transposons found in natural isolates of Gram-negative bacteria are close relatives of Tn21 . Thus, the Tn21 subgroup of the Tn3 family of transposable elements is the most successful homogeneous group in acquiring resistance to newly introduced antibiotics . This paper summarizes the mode of acquisition of resistance genes by these elements.

Biochem Pharmacol, 1991 Jun 15, 41(12), R21 - 6
Effect of the calmodulin inhibitor trifluoperazine on phosphorylation of P-glycoprotein and topoisomerase II: relationship to modulation of subcellular distribution, DNA damage and cytotoxicity of doxorubicin in multidrug resistant L1210 mouse leukemia cells; Ganapathi R et al.; The results from the present study using the sensitive and progressively DOX resistant L1210 model system demonstrated that the effects of TFP are not due to redistribution of DOX to the nucleus, and modulation of cytotoxicity is related to effects on DOX-induced DNA strand breaks . Although TFP affects phosphorylation of PGP and TOPO II (R2 greater than R1), the comparable DNA strand breaks at lower DOX levels with TFP in the resistant sublines suggest that modulation of TOPO II function related to drug-induced DNA damage by calmodulin-mediated events may be an important mode of action.

Biochem Pharmacol, 1991 Jun 15, 41(12), 1967 - 79
Study of molecular markers of resistance to m-AMSA in a human breast cancer cell line . Decrease of topoisomerase II and increase of both topoisomerase I and acidic glutathione S transferase; Lefevre D et al.; Resistance to 0.8 microM 4'-(9-acridinylamino)methanesulphon-m-anisidide (m-AMSA) was induced by stepwise increases of drug concentration in the human tumor cell line CALc18 originating from a breast adenocarcinoma . The resistant cell line CALc18/AMSA exhibited a resistance index of 10 and a cross-resistance to other topoisomerase II inhibitors . A 3-fold decrease in the levels of topoisomerase II decatenating activity was found in CALc18/AMSA cells . By contrast, topoisomerase I activity was increased by about 3-fold in resistant cells . Interestingly this line was hypersensitive to camptothecin, a specific inhibitor of topoisomerase I . Restriction endonuclease patterns of the topoisomerase I and topoisomerase II loci were found to be identical in CALc18/AMSA and CALc18 with no evidence of gene amplification and rearrangements . Alkaline elution of m-AMSA-treated cells showed that DNA single strand breaks and DNA-protein crosslinks were decreased in CALc18/AMSA . The DNA lesions also obtained in m-AMSA-treated nuclei indicated that no drug uptake modification occurred in both cells . Moreover, the in vitro m-AMSA-induced DNA cleavage per unit of decatenating activity and the inhibitory effects of antitumoral drugs on decatenation were not found to be different with topoisomerase II from sensitive or resistant cells . However the specific cleavage induced by m-AMSA/per mg of crude protein from resistant cells was 2 to 3 times decreased . Multidrug resistance gene transcripts were not detected while levels of acidic glutathione S transferase mRNA were found to be 8 to 10-fold greater in resistant than in sensitive cell line with no amplification of the gene . In conclusion, the diminution of topoisomerase II activity and the increase of both topoisomerase I and acidic glutathione S transferase transcripts could contribute to the resistant phenotype of these breast cancer cells.

Nurs Clin North Am, 1991 Jun, 26(2), 331 - 9
Chemotherapy update; Miaskowski C; The administration of chemotherapy is one of the major forms of cancer treatment . Recent progress in the use of chemotherapeutic agents has evolved from an understanding of the basic mechanisms of tumor cell kinetics and the molecular and biochemical basis for individual and multidrug resistance patterns . These advances have led to the development of novel approaches to the administration of cancer chemotherapeutic agents . Nurses must remain knowledgeable about these concepts to understand the mechanisms underlying the administration of various combinations of cancer chemotherapy.

Southeast Asian J Trop Med Public Health, 1991 Jun, 22(2), 183 - 9
Comparison of mefloquine, chloroquine plus pyrimethamine-sulfadoxine (Fansidar), and chloroquine as malarial prophylaxis in eastern Thailand; Boudreau EF et al.; From July 1983 to March 1984 a randomized double blind prophylactic trial in Thai gem miners working across the border in Cambodia was conducted to determine the prophylactic efficacy of 3 drug regimens against P . falciparum and P . vivax malaria along the Thai-Cambodian border . Gem miners have a high incidence of malaria . Maximum duration of individual participation was 14 weeks . Of 334 participants in this study who were seen every 2 weeks, 145 received mefloquine 500 mg fortnightly, 112 received chloroquine 300 mg base weekly plus Fansidar (1000 mg sulfadoxine and 50 mg pyrimethamine) fortnightly and 77 received chloroquine as 300 mg base weekly . The significant reduction of vivax malaria in study subjects (compared to background incidence) implied good compliance with self administration of chloroquine in the intervening weeks between scheduled appointments . The attack rate in each prophylactic regimen was 2188 cases/1000/year with mefloquine, 8338 cases/1000/year with chloroquine-Fansidar and 10,207 cases/1000/year receiving chloroquine alone . There was a 79% prophylactic efficacy for mefloquine and 18% efficacy for the chloroquine plus Fansidar regimen compared to chloroquine . Using life table analysis, 56% of the mefloquine group, 6% of the chloroquine-Fansidar group and 4% of the chloroquine group were malaria free at the end of the 14 weeks study . The chloroquine plus sulfadoxine-pyrimethamine regimen prescribed for prophylaxis is no longer effective for multidrug resistant strains of P . falciparum in the study area . This study also seriously questions the efficacy of mefloquine prophylaxis.

Anticancer Drugs, 1991 Jun, 2(3), 279 - 83
FK-506 (fujimycin) reverses the multidrug resistance of tumor cells in vitro; Pourtier-Manzanedo A et al.; Cyclosporin A (CsA) and FK-506 have similar immunosuppressive activity profiles and cyclophilin-like intracellular targets . Since CsA can reverse the multidrug resistance of tumor cells showing P-glycoprotein-mediated drug efflux, the possible resistance-modulating activity of FK-506 was evaluated in vitro with multidrug-resistant P388 cells and their sensitive parental controls . Higher concentrations of FK-506 than CsA were needed to achieve a similar degree of chemosensitization, suggesting that FK-506 might interact less efficiently than CsA with the P-glycoprotein expressed in multidrug-resistant tumor cells . However, FK-506 was active on a broader range of concentrations than CsA, particularly because of direct cytostatic effects of CsA which appeared at concentrations only slightly higher than those required to show a significant resistance-modulating activity.

Curr Opin Oncol, 1991 Jun, 3(3), 537 - 44
Renal cell carcinoma; Reese JH; Renal cell carcinoma represents a significant challenge to surgeons and oncologists treating urologic malignancies . Diagnostically, it is critically important to identify the precise extent of the tumor prior to therapeutic intervention . Therapeutically, a number of controversies continue to be debated, including the role of renal-conserving surgery and the role of surgery in patients with metastatic disease . New research is beginning to identify factors involved in the multidrug-resistant properties of these tumors that may allow us, in the future, to treat these tumors more effectively with systemic chemotherapy . Utilizing immunotherapy in the form of autolymphocytes, interferon, interleukin-2, or combinations of these regimens, a number of exciting advances have been made in the treatment of metastatic renal cell carcinoma . This review examines the most recent literature on each of the above-mentioned aspects in the treatment of this difficult and challenging tumor.

Geburtshilfe Frauenheilkd, 1991 Jun, 51(6), 466 - 8
{Malignant adenoma of the cervix: a diagnostic and therapeutic dilemma}; Schneider J et al.; "Adenoma malignum" of the cervix is an extremely rare variant of cervical adenocarcinoma . Its diagnosis by means of light microscopy is rendered very difficult by its strong similarity to benign endocervical hyperplasia . During the last few years immunohistochemistry has proved to be of great help in differential diagnosis . Treatment of "adenoma malignum" also poses great problems . Most cases are diagnosed in advanced stages, although only early diagnosis offers the possibility of effective treatment . Radiotherapy has failed in all known cases in an advanced stage of the disease . Chemotherapy is also not promising as a therapeutical option, because the tumour expresses high levels of the multidrug-resistance marker P-glycoprotein.

Cancer Commun, 1991 Jun, 3(6), 181 - 9
Transfection with protein kinase C alpha confers increased multidrug resistance to MCF-7 cells expressing P-glycoprotein; Yu G et al.; Cross-resistance to anticancer drugs, termed multidrug resistance (mdr), has been functionally associated with the expression of a plasma membrane energy-dependent efflux pump, termed P-glycoprotein, the product of the mdr1 gene . When MCF-7 breast carcinoma cells were transfected with the human mdr1 gene (BC-19 cells), they expressed levels of P-glycoprotein equivalent to those of cells selected for resistance to doxorubicin (MCF-7/ADR) but exhibited 10- to 50-fold less resistance to doxorubicin and vinblastine . We have now demonstrated that when BC-19 cells were stably transfected with protein kinase C alpha (PKC alpha), resistance to doxorubicin and vinblastine was increased; wild-type MCF-7 cells transfected with PKC alpha did not exhibit any change in drug resistance . Increased resistance in PKC alpha-transfected BC-19 cells was associated with enhanced PKC activity and phosphorylation of P-glycoprotein and decreased drug accumulation . The PKC activator, phorbol dibutyrate, further increased resistance to doxorubicin and stimulated P-glycoprotein phosphorylation . These results demonstrate that transfection of P-glycoprotein-expressing cells with PKC resulted in increased mdr and that PKC may have served as an important modulator of this process.

Cancer Res, 1991 Jun 1, 51(11), 2949 - 59
A phenotype conferring selective resistance to lipophilic antifolates in Chinese hamster ovary cells; Sharma RC et al.; Trimetrexate, a lipid-soluble analogue of methotrexate, appears to enter mammalian cells by passive diffusion, thus circumventing the methotrexate transport system which is frequently a subject for alterations leading to methotrexate resistance . Using a single-step selection protocol with trimetrexate, we have isolated 45 clonal variants and found the majority of them to be selectively resistant to lipophilic antifolates while retaining their sensitivity to methotrexate and drugs involved in multidrug resistance . The majority of spontaneously induced trimetrexate-resistant clones showed a change in neither the mRNA levels of dihydrofolate reductase (24 of 30) and P-glycoprotein (26 of 30) nor their gene copy numbers, whereas a small fraction of clones (4 of 30) showed multidrug resistance gene amplification and P-glycoprotein mRNA overexpression . gamma-Irradiation prior to selection markedly enhanced the frequency of trimetrexate resistance (100-fold after 1000 rads) . None of the gamma-ray-induced trimetrexate-resistant clones (0 of 15) had evidence of dihydrofolate reductase and multidrug resistance gene amplification and/or overexpression . Flow cytometry data on trimetrexate-resistant clones showed no defect in the transport of trimetrexate . Verapamil, a modulator of the multidrug resistance phenotype, had no cytotoxic effect on parental and trimetrexate-resistant clones . However, when present with trimetrexate, verapamil (0.3-0.6 microM) reversed the lipophilic antifolate-resistant phenotype in clones that had invariant levels of P-glycoprotein and dihydrofolate reductase . This selective resistance to lipid-soluble antifolates was initially unstable but became stable after continued drug-selective growth . Two-dimensional gel electrophoresis showed some differences in protein(s) that may potentially be associated with this phenotype of selective resistance to lipophilic antifolates . We conclude that a gamma-radiation-enhanceable, verapamil-reversible, stable phenotype of selective resistance to lipid-soluble antifolates frequently emerges which requires neither the amplification nor the overexpression of dihydrofolate reductase or multidrug resistance genes.

Jpn J Cancer Res, 1991 Jun, 82(6), 747 - 51
Reversal of cisplatin resistance with amphotericin B in a non-small cell lung cancer cell line; Morikage T et al.; The potentiation of anticancer agents by non-anticancer drugs is one of the possible strategies for overcoming cellular resistance to chemotherapy . In order to overcome cis-diamminedichloroplatinum(II) (CDDP) resistance, we evaluated the sensitizing effect on CDDP-induced cytotoxicity of various non-anticancer agents which might alter membrane transport, by means of a colorimetric {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} (MTT) assay . Drugs which have previously been demonstrated to modify multidrug resistance did not show a sensitizing effect to cisplatin . Only amphotericin B (AmB) selectively conquered CDDP resistance in the CDDP-resistant cell line . A drug accumulation study done by the atomic absorption method demonstrated that the accumulation of CDDP in the resistant cell line recovered to the level of the parental cell line after treatment with AmB . Thus, AmB might overcome CDDP resistance by increasing the accumulation of CDDP.

Nature, 1991 May 23, 351(6324), 323 - 4
Restored expression of major histocompatibility class I molecules by gene transfer of a putative peptide transporter; Spies T et al.; Cytotoxic T lymphocytes recognize antigen-derived peptides bound to major histocompatibility complex (MHC) class I molecules with which they assemble in the endoplasmic reticulum or in an undefined subcompartment . There is genetic evidence that the peptides that are products of cytosolic protein degradation are transported into this compartment by a peptide supply factor (PSF), encoded in the MHC class II region . Like the corresponding genes RING4, HAM1 and mtp1, PSF is related to the multidrug-resistance family of transporters and may be a peptide pump, as translocation of peptides across membranes must occur independently of the secretory pathway . There is, however, no functional evidence for this role so far . Here we report gene transfer experiments showing that expression of PSF complementary DNA in the human lymphoblastoid cell line mutant 721.134 restores normal levels of surface HLA-A2 and -B5 . No similar effect was observed in 721.174 mutant cells, in which a homozygous deletion includes PSF among several other closely linked genes . At least one of these genes may therefore also be required for PSF function.

J Natl Cancer Inst, 1991 May 15, 83(10), 708 - 12
MDR1 gene expression and treatment outcome in acute myeloid leukemia; Pirker R et al.; To prospectively assess the role of MDR1 gene expression in patients with de novo acute myeloid leukemia (AML), levels of MDR1 RNA in blast cells were determined at diagnosis and correlated with treatment outcome in 63 patients . MDR1 RNA levels were negative in 29% and positive in 71% of the patients . The complete remission rate in response to induction chemotherapy was 89% for MDR1 RNA-negative patients and 53% for MDR1 RNA-positive patients (P = .008) . Expression of the MDR1 gene was observed in most patients who died early or had resistant disease . Kaplan-Meier curves revealed a decrease in both disease-free survival and overall survival of patients with detectable MDR1 gene expression compared with the disease-free survival and overall survival of MDR1 RNA-negative patients (P = .029 and P = .009, respectively) . These data indicate that MDR1 gene expression is an unfavorable prognostic factor and suggest that multidrug resistance is important in AML.

Biochem Pharmacol, 1991 May 15, 41(10), 1455 - 61
Circumvention of multidrug-resistance in P388 cells is associated with a rise in the cellular content of phosphatidylcholine; Ramu A et al.; In fura-2 stained drug-sensitive and multidrug-resistant P388 cells, 50 mM KCl failed to provoke an increase in the fluorescent signal, indicating that potential-dependent Ca2+ channels are not present in either cell line . Therefore the circumvention of drug-resistance by verapamil must be related to some other mechanism . In the present study, verapamil and two other circumventors of drug-resistance, tamoxifen and dipyridamole were found to induce an increase in the synthesis of phosphatidylcholine in multidrug-resistant but not in drug-sensitive cells . The relative resistance of multidrug resistance cells to permeabilization by digitonin indicates that the organization of the plasma membrane lipids in these cells must be different from the one occurring in drug-sensitive cells . Extended exposure of multidrug-resistant cells to verapamil negates the resistance to digitonin . This effect of verapamil reflects its ability to modify the lipid organization of the plasma membrane of multidrug-resistant cells . It is suggested that if the lipid composition of the cell membrane is altered by these drugs as was found for whole cells, the change could explain the increase in drug permeability.

Biochem Biophys Res Commun, 1991 May 15, 176(3), 1377 - 82
A relationship between multidrug resistance and growth-state dependent cytotoxicity of the lysosomotropic detergent N-dodecylimidazole; Wilson PD et al.; Multidrug resistance (MDR) in cultured cells and tumors is associated with overproduction of P-glycoprotein, a plasma membrane efflux pump normally present at very low levels . The cytotoxic action of N-dodecylimidazole (C12-Im), a lysosomotropic detergent, on cultured cells was previously shown to be strongly dependent on growth state, with rapidly growing cells being most sensitive and confluent cells most resistant . We show here that this may be due to a growth dependent increase in cellular P-glycoprotein activity . Both verapamil and nifedipine, structurally unrelated P-glycoprotein inhibitors, increased markedly the sensitivity of CHO fibroblasts to killing by C12-Im; the increase was greater in confluent than in growing cells . Also, verapamil inhibitable 3H-daunomycin efflux was more efficient from confluent than from subconfluent cells . The MDR cell line CH(R)C5 differed from all cell lines previously examined in that it did not show a growth-dependent decrease in C12-Im sensitivity, and sensitivity was not increased by verapamil or nifedipine . We suggest that a growth-dependent increase in MDR activity is a general property of cultured cells, except for those specifically overexpressing P-glycoprotein.

Cancer Res, 1991 May 15, 51(10), 2720 - 6
Relationship of the expression of the multidrug resistance gene product (P-glycoprotein) in human colon carcinoma to local tumor aggressiveness and lymph node metastasis; Weinstein RS et al.; P-glycoprotein mediates classic multidrug resistance by functioning as an efflux pump that excretes lipophilic chemotherapeutic drugs from cancer cells . We now report an association of P-glycoprotein in colon carcinomas with another tumor property, i.e., enhancement of local tumor aggressiveness . P-glycoprotein was detected with monoclonal antibody immunohistochemistry in 65 of 95 primary colon adenocarcinomas, which were stage B1 or greater . In all but 1 of the 95 cases, solitary invading carcinoma cells were present at the leading edge of the tumor . This subpopulation of invasive carcinoma cells expressed P-glycoprotein (P-Gp+) in 47 of the 95 surgically resected colon specimens . Cases were grouped on the basis of the presence (Group 1, 47 cases) or absence (Group 2, 48 cases) of P-Gp+ invasive carcinoma cells . There was a significantly greater incidence of vessel invasion (P less than 0.001) and lymph node metastases (P less than 0.01) in Group 1 cases . Groups 1 and 2 did not differ with respect to tumor size, depth of invasion of the bowel wall, histological grade, maximum tumor size, mitotic index, mucin production, or presence of perineural invasion (P greater than 0.1) . Our findings indicate that P-Gp+ invasive colon cancer cells may have an increased potential for dissemination, suggesting that P-glycoprotein may influence cell behavior.

Cancer Res, 1991 May 15, 51(10), 2628 - 35
Characterization of the human MDR3 P-glycoprotein and its recognition by P-glycoprotein-specific monoclonal antibodies; Schinkel AH et al.; We have cloned a human MDR3 complementary DNA, coding for a P-glycoprotein, into a mammalian expression vector and cotransfected it with a selectable marker into drug-sensitive human BRO melanoma cells . With low frequency we obtained stable, MDR3-expressing clones . Immunocytochemical and immunoblotting analysis of these clones using the monoclonal antibody C219 indicated that human MDR3 P-glycoprotein, like human MDR1 P-glycoprotein, was mainly localized in the plasma membrane and probably glycosylated . Although a significant fraction of the cells (5-10%) in one of the MDR3-expressing clones expressed as much P-glycoprotein as a clearly drug-resistant MDR1-transfected clone, we found no resistance against a range of drugs affected by multidrug resistance . The drugs tested included vincristine, colchicine, VP16-213, daunorubicin, doxorubicin, actinomycin D, and gramicidin D . We did not detect enhanced daunorubicin efflux either in any of the MDR3-expressing cells by fluorescence microscopy . Direct selection with vincristine, actinomycin D, gramicidin D, or daunorubicin of BRO cells transfected with expression constructs containing the regular MDR3 complementary DNA, or a complementary DNA representing a major MDR3 splice variant (C(-141)), likewise failed to yield resistant clones . Thus, although human MDR3 P-glycoprotein is highly similar to human MDR1 P-glycoprotein, we found no indications that it can transport drugs . We investigated the cross-reactivity of the monoclonal antibodies C219, C494, JSB-1, HYB-241, and MRK16, recognizing human MDR1 P-glycoprotein, with human MDR3 P-glycoprotein using immunocytochemistry and immunoblotting . Apart from monoclonal antibody C219, none of the monoclonal antibodies showed detectable cross-reactivity with human MDR3 P-glycoprotein . In our hands, monoclonal antibodies MRK16 and HYB-241 were most suitable for sensitive and specific cytochemical detection of human MDR1 P-glycoprotein.

Cancer Res, 1991 May 15, 51(10), 2582 - 6
Inhibition by erythroid differentiation factor (activin A) of P-glycoprotein expression in multidrug-resistant human K562 erythroleukemia cells; Okabe-Kado J et al.; The K562/VCR cell line, exhibiting acquired multidrug resistance (MDR) with increased expression of a cell surface glycoprotein (P-glycoprotein), was isolated from human erythroleukemia K562 cells . Various compounds that induced erythroid differentiation of K562 cells were tested for their effects on growth and differentiation of these K562/VCR cells . Sodium butyrate, hemin, 1-beta-D-arabinofuranosylcytosine, and erythroid differentiation factor (EDF) induced erythroid differentiation of K562/VCR cells as well as K562 cells . The MDR of K562/VCR cells was partly overcome by treatment with EDF but not with the other inducers . Expression of P-glycoprotein by K562/VCR cells was measured by radioimmunoassay using MRK16 monoclonal antibody . Results showed that EDF caused down-regulation of P-glycoprotein in K562/VCR cells, whereas the other inducers did not cause its down-regulation . Thus, in addition to inducing erythroid differentiation, EDF enhanced the sensitivity of K562/VCR cells to multidrugs and suppressed expression of P-glycoprotein . These results suggest that differentiation inducers may be useful in chemotherapy of leukemic MDR cells.

J Natl Cancer Inst, 1991 May 1, 83(9), 621 - 6
Phase I and pharmacokinetic trial of intraperitoneal etoposide in combination with the multidrug-resistance-modulating agent dipyridamole; Isonishi S et al.; Dipyridamole synergistically enhances the sensitivity of human ovarian carcinoma cells to etoposide in vitro . We conducted a phase I trial to investigate the feasibility of using dipyridamole to selectively increase the sensitivity to etoposide of tumors confined to the peritoneal cavity . Etoposide and dipyridamole were administered as a continuous 72-hour intraperitoneal infusion to 16 patients . The maximum tolerated dose of etoposide was 175 mg/m2 per day when administered with dipyridamole at a fixed dose of 24 mg/m2 per day . Dose-limiting toxic effects were leukopenia and thrombocytopenia . No other major toxic effects were observed . The free peritoneal etoposide and dipyridamole concentrations varied with the etoposide dose rate, reaching 218.9 and 25.3 microM, respectively, at an etoposide dose rate of 175 mg/m2 per day . The free etoposide and dipyridamole concentrations attained were well within the range needed for synergistic interaction.

Biochem Pharmacol, 1991 May 1, 41(9), 1283 - 92
Menadione: spectrum of anticancer activity and effects on nucleotide metabolism in human neoplastic cell lines; Nutter LM et al.; The spectrum of cytotoxicity of menadione (MD) was examined in a panel of human cancer cell lines . MD was equipotent against multidrug-resistant and parental leukemia cell lines with IC50 values of 13.5 +/- 3.6 and 18 +/- 2.4 microM respectively . A cervical carcinoma cell line resistant to the antimetabolite, methotrexate (MTX), was as sensitive to MD as its parental cell line . The interactions of fifteen clinically utilized anticancer drugs with MD were examined in vitro and the majority were found to be additive, with four agents exhibiting synergism and one agent exhibiting antagonism . MD inhibited the incorporation of radioactive thymidine, uridine and amino acids into DNA, RNA and protein, respectively, in three human cancer cell lines . Some possible reasons for the inhibition of DNA synthesis including effects of MD on intracellular deoxyribonucleoside triphosphate pools were examined and ruled out . Although results from previous studies using rat hepatocytes suggested that mitochondria may be a target of MD, no significant effect of this compound on total intracellular adenosine triphosphate (ATP) pools in human cancer cell lines was observed . Collectively, these in vitro results demonstrate that MD possesses a broad spectrum of anticancer activity and suggest the potential utility of this agent in cancer therapy . Future studies directed at elucidation of the mechanism of MD action in human cancer cells are warranted and are under study.

Anticancer Res, 1991 May-Jun, 11(3), 1301 - 4
Cross-resistance of human multidrug-resistant cells to mitomycin C; Yusa K et al.; Human multidrug-resistant cells, K562/ADM, KB-C-4, AdrRMCF-7 and CEM/VLB100 showed 21-, 7.5-, 105- and 3.4-fold cross-resistance to mitomycin C (MMC) . The resistance to MMC in K562/ADM, KB-C-4, AdrRMCF-7, CEM/VLB100 cells was reversed by 6.6 microM verapamil . Accumulation of {3H}MMC in K562/ADM, AdrRMCF-7 and CEM/VLB100 cells also decreased by 37, 26 and 33%, as compared with their drug-sensitive counterparts . In KB-C-4 cells, accumulation of {3H}MMC decreased by 60%, and efflux rate of {3H}MMC was slightly increased as compared to their parental KB-3-1 cells . Verapamil at 6.6 microM increased accumulation of {3H}MMC in these multidrug-resistant sublines . K562/ADM10, K562/ADM50, K562/ADM100 and K562/ADM250 cells, which showed 17- to 230-fold resistance to Adriamycin, also showed 0.8- to 7.3-fold cross-resistance to MMC . In these cell lines, the extent of resistance to Adriamycin (ADM) that was consistent with expression levels of P-glycoprotein shown by immunoblotting was directly proportional to the extent of their resistance to MMC . Regression analysis indicated that relative resistance to Adriamycin was correlated with relative resistance to MMC (r = 0.98) . These results indicate that MMC can be transported by P-glycoprotein overexpressed in multidrug-resistant cells.

Jpn J Cancer Res, 1991 May, 82(5), 593 - 8
Establishment of drug resistance in human gastric and colon carcinoma xenograft lines; Satta T et al.; We established multidrug-resistant human gastric and colon xenograft lines by means of intratumoral injections of four agents, doxorubicin (DXR), cisplatin (CDDP), 5-fluorouracil (5-FU) and mitomycin C (MMC), into subcutaneous SC1NU and SW480 tumors once a week or less . Such intermittent drug exposure is commonly used in clinical chemotherapeutic protocols . All xenograft lines acquired resistance to the injected drugs as evaluated by in vivo drug-resistance tests . Many of the drug-resistant lines showed various patterns of cross resistance to other drugs . In order to analyze the mechanism of resistance in vivo, we investigated the expression of drug resistance gene, which has been extensively studied in vitro . We used four complementary DNAs (cDNAs) for multidrug resistance (MDR1), glutathione S-transferase-pi (GST-pi), thymidylate synthase (TS) and dehydrofolate reductase (DHFR), as probes . We observed GST-pi, DHFR and TS mRNA expression at various levels, but MDR1 mRNA expression was found only in SW480/DXR by the method of poly (A+) RNA selection . Four resistant SW480 lines had higher TS mRNA expressions . Six resistant lines had stronger GST-pi mRNA expression . Five resistant lines had higher DHFR mRNA expression . Drug resistance genes related to the treated drug were also expressed in this in vivo model; MDR1 in SW480/DXR, GST-pi in SW480/CDDP and in SC1NU/CDDP and TS in SW480/5-FU . In contrast to in vitro resistant lines which have been reported as models of drug resistance, the expression of drug resistance genes in vivo was not always correlated to the acquisition of cross resistance . These resistant xenograft lines and the methods developed to induce drug resistance in vivo should be useful for studies on the mechanism of drug resistance in the clinical setting.

Anticancer Res, 1991 May-Jun, 11(3), 1275 - 9
Reversing multidrug resistance in L1210 tumor cells by hycanthone or chlorophenoxamine in vitro and in vivo; Efferth T et al.; In the presence study we demonstrated that hycanthone and chlorophenoxamine can modulate the resistance of multidrug resistant (MDR) murine L1210 leukemia tumor lines in vitro and in vivo . The circumvention of MDR by hycanthone and chlorophenoxamine in vitro was demonstrated by a short-term test using tritiated nucleic acid precursors and by flow cytometrical measurement of accumulation of rhodamine 123 . Furthermore, we treated mice bearing resistant L1210 ascites cells with doxorubicin and hycanthone or chlorophenoxamine . Hycanthone in combination with doxorubicin significantly inhibited tumor growth . We also found an improved therapeutic effect of doxorubicin plus chlorophenoxamine . Our results in vitro and in vivo indicate that hycanthone and chlorophenoxamine might be appropriate tools for the circumvention of MDR in human tumors.

Anticancer Res, 1991 May-Jun, 11(3), 1065 - 8
Intrinsic drug resistance in a human medullary thyroid carcinoma cell line: association with overexpression of mdrl gene and low proliferation fraction; Yang KP et al.; Medullary thyroid carcinoma (MTC) is a cancer that is relatively insensitive to clinical chemotherapy . Our previous studies have demonstrated that an established human MTC cell line, TT, seems to possess an intrinsic resistance phenotype when tested for its chemosensitivity to multiple antineoplastic agents . We now report our investigation on the potential mechanisms responsible for the chemoresistance of TT cells . Northern analysis showed an increased level of multidrug resistance gene (mdrl) mRNA in TT and in an inherently drug-resistant colon carcinoma cell line, LoVo, when compared with CEM, a drug-sensitive leukemic lymphoblastic cell line; the latter two cell lines were included here as a control . Verapamil (10 microM) partially reversed resistance to doxorubicin in TT and LoVo cells, but had no effect on doxorubicin cytotoxicity to CEM cells . Expression of glutathione-S-transferase-pi (GST pi) gene was undetectable in TT, whereas, under similar conditions, GST pi mRNA was detectable in LoVo . Growth kinetics studies revealed that doubling times of the 3 cell lines in exponential growth were 95, 37, and 24 h for TT, LoVo and CEM, respectively . Flow-cytometric analysis showed that the percentage of TT population is S phase was 49% and 33% of the LoVo and CEM cell populations, respectively, while the G1/G0 fraction of TT was about 63% and 61% higher than that of LoVo and CEM, respectively . Our data suggest that the intrinsic chemoresistance in TT cells may be attributed to the combined factors of overexpression of the mdrl gene, a slower growth rate and a smaller proliferation fraction, although other factors or mechanisms that are yet to be investigated may also act in concert to contribute to the resistance phenotype of TT.

Invest New Drugs, 1991 May, 9(2), 169 - 79
A novel N-myristylated synthetic octapeptide inhibits protein kinase C activity and partially reverses murine fibrosarcoma cell resistance to adriamycin; O'Brian CA et al.; This report shows that N-acylation of the protein kinase C (PKC) substrate Arg-Lys-Arg-Thr-Leu-Arg-Arg-Leu (RKRTLRRL) provides it with a potent inhibitory activity against PKC . N-myristoyl-RKRTLRRL inhibited Ca2(+)- and phosphatidylserine (PS)-dependent histone phosphorylation catalyzed by PKC with a 50% inhibitory concentration (IC50) of 5 microM, whereas neither RKRTLRRL nor myristic acid inhibited PKC-catalyzed histone phosphorylation at concentrations as high as 50 microM . A fully active, Ca2(+)- and PS-independent catalytic fragment of PKC can be generated by limited proteolysis . N-myristoyl-RKRTLRRL inhibited histone phosphorylation catalyzed by the catalytic fragment of PKC (IC50 = 80 microM), but neither myristic acid nor the nonmyristylated peptide inhibited the activity of the catalytic fragment at concentrations up to and including 200 microM . The Km app and Vmax app for N-myristoyl-RKRTLRRL were similar to those of RKRTLRRL . Thus, N-myristylation provided the octapeptide with an inhibitory activity against PKC but had only minor effects on its Km app and Vmax app . Kinetic analysis provided evidence that the peptide inhibited PKC noncompetitively with respect to ATP . Previously, we reported that the protein kinase inhibitor H7 partially reverses Adriamycin resistance in the multidrug resistant (MDR) murine fibrosarcoma line UV-2237M-ADRR . In this report, we show that N-myristoyl-RKRTLRRL also partially reverses Adriamycin resistance in UV-2237M-ADRR cells . These results suggest that potent and selective cell permeable PKC inhibitors may be designed by N-acylating small PKC peptide substrates.

Anticancer Res, 1991 May-Jun, 11(3), 1309 - 12
Establishment of multidrug resistant human colorectal carcinoma HCT-15 cell lines and their properties; Iwahashi T et al.; A series of MDR cell lines with various levels of P-glycoprotein have been established from a human colorectal carcinoma cell line, HCT-15, by stepwise exposure to adriamycin . The relative drug resistance of these cell lines correlated directly with both MDR1 mRNA levels and P-glycoprotein expression levels . Intracellular accumulation of adriamycin decreased inversely to their resistance . Drug sensitivities of these lines were reversed using verapamil . Since these cell lines are transplantable to nude mice, they may provide a useful animal model of MDR solid tumors for therapeutic experiments.

Br J Haematol, 1991 May, 78(1), 28 - 34
Expression of the multidrug resistance gene product (P-glycoprotein) in myelodysplasia is associated with a stem cell phenotype; List AF et al.; Previous studies have indicated relative resistance to chemotherapy in the myelodysplastic syndromes (MDS) and associated acute leukaemia . To determine if multidrug resistance may contribute to chemoresistance in these disorders, we studied bone marrow specimens for P-glycoprotein expression (P-GP) by immunocytochemical staining with monoclonal antibodies reactive with cytoplasmic (C219) or surface epitopes (MRK16) of P-GP . Forty-five case specimens from 43 patients were studied, including 32 cases of primary MDS, seven cases of acute myeloid leukaemia (AML) following MDS, and six therapy-related haematological disorders . Cytogenetic analysis was available on 36 specimens . Two staining patterns were detected: (1) cytoplasm and plasma membrane, and (2) staining restricted primarily to the nuclear-cytoplasmic junction . P-GP was detected in seven (22%) cases of primary MDS, four (57%) cases of AML evolving from MDS, and five (83%) cases of therapy-related haematological disorders . Expression of P-GP was restricted to blasts and leukaemic monocytes, and was otherwise absent from terminally differentiated blood cells . Analysis of the relation between P-GP expression and reactivity with the human progenitor cell antigen CD34, revealed a highly significant association (P = 0.001) . P-GP reactivity was distributed equally among normal and abnormal karyotypes and did not correlate with specific cytogenetic abnormalities . These findings indicate that multidrug resistance in MDS and karyotypically-related haematological disorders is closely linked to a stem cell phenotype and may contribute to chemoresistance in these disorders.

Cancer Commun, 1991 May, 3(5), 159 - 65
P-glycoprotein content and mediation of vincristine efflux: correlation with the level of differentiation in luminal epithelium of mouse small intestine; Meyers MB et al.; Luminal epithelium from mouse small intestine was fractionated into four distinct components ranging from the most differentiated (absorptive epithelial cells, fraction I) to the least differentiated (proliferative crypt cells, fraction IV) . Immunoblot analysis of these fractions with monoclonal antibodies to P-glycoprotein, C219, and HYB-241, indicated that the amount of P-glycoprotein increased in cells as they progressed in level of differentiation from crypt to the villar surface of the lumen of the small intestine . P-glycoprotein in these normal intestinal cells functioned as a transporter of vincristine, one of a group of cancer chemotherapeutic agents to which cells can develop multidrug resistance . Transport ability was shown by efflux studies with vincristine as substrate . Efflux increased with differentiation and correlated with the increase in the amount of P-glycoprotein . Fraction I cells effluxed vincristine about seven times more rapidly than fraction IV cells . Efflux of vincristine was almost entirely inhibited from fraction I cells after treatment with antibody HYB-241, which recognizes an external epitope of P-glycoprotein . This finding would appear to demonstrate that P-glycoprotein was directly responsible for at least the majority of efflux of vincristine in these cells . The observed Mr of P-glycoprotein in fraction I cells was 140,000 when cells were solubilized with sodium dodecyl sulfate at room temperature before electrophoresis and immunoblot analysis . When solubilized fraction I protein was heated with 2-mercaptoethanol before electrophoresis, P-glycoprotein was seen by immunochemical procedures as an Mr 50,000-55,000 protein . No Mr 170,000 or 180,000 forms of P-glycoprotein (forms that are found in multidrug-resistant cell lines and are stable to heat or sulhydryl agents) were detected in any fraction of these epithelial cells under the conditions used . Functional P-glycoprotein was, therefore, associated with differentiation of luminal epithelium in small intestine, and the P-glycoprotein produced by these cells may be a type not previously described.

Cancer Lett, 1991 May 1, 57(2), 165 - 71
Reversal of multidrug resistance in HL-60 cells by verapamil and liposome-encapsulated doxorubicin; Sadasivan R et al.; The means of circumventing multidrug resistance was investigated in HL-60 and HL-60R (a drug resistance variant) cell lines . The HL-60R cell line was developed from the parent line by serial exposure to increasing concentrations of doxorubicin over a 4-month period . This drug resistant cell line expressed P-glycoprotein in its cell surface and is 80-fold more resistant than the parent cell line . Multidrug resistance, as evaluated by a cell cytotoxicity assay using doxorubicin, can be overcome by use of verapamil . Multidrug resistance can also be circumvented when doxorubicin is encapsulated in liposomes . The combination of verapamil and doxorubicin-encapsulated liposomes does enhance circumvention of multidrug resistance beyond the effect of each agent alone, implying a synergistic effect . The lipid composition of the liposomes does affect the rate of drug uptake but not the overall cytotoxic effect of doxorubicin . The synergistic reversal of multidrug resistance by doxorubicin-encapsulated liposomes and verapamil suggests a multifactorial basis for drug resistance in this cell line.

Blood, 1991 May 1, 77(9), 2079 - 84
A combined approach for purging multidrug-resistant leukemic cell lines in bone marrow using a monoclonal antibody and chemotherapy; Aihara M et al.; Selective removal of malignant cells (purging) from bone marrow (BM) is a concern in autologous BM transplantation (ABMT) . Use of vincristine, etoposide, or doxorubicin for purging could be rendered ineffective by the presence of multidrug-resistant (MDR) tumor cells . To circumvent this particular problem, we investigated whether 17F9, a monoclonal IgG2b antibody directed against the cell surface product of the MDR gene, P-glycoprotein, is effective in selective removal of MDR cells from BM when used with rabbit complement (C') . Using two different cell lines we have demonstrated that 17F9 + C' selectively lyses MDR-positive cells . Three rounds of antibody + C' resulted in 96.4% +/- 3.6% kill of K562/DOX and 100% +/- 0% of CEM/VLB cells . Mixtures of malignant cells and normal BM resulted in 99.85% removal of K562/DOX and 99.91% removal of CEM/VLB clonogenic cells . This treatment did not affect normal committed precursors compared with C' alone . The addition of the cytotoxic agent etoposide (VP-16) following antibody purging results in a 4.6 log reduction of malignant cells . Furthermore, this antibody was effective when used against patients' leukemic blasts . These results suggest the use of 17F9 + C' is effective and selective for removal of MDR cells from BM before ABMT and the addition of VP-16 enhances the purging efficacy.

Cancer Res, 1991 May 1, 51(9), 2420 - 4
Circumvention of multidrug resistance by a newly synthesized quinoline derivative, MS-073; Sato W et al.; Newly synthesized quinoline derivatives were investigated for their efficacy to reverse multidrug resistance (MDR) . In this study, one of the most effective quinoline derivatives, MS-073, was compared with verapamil with regard to its ability to overcome MDR in vitro and in vivo . MS-073 at 0.1 microM almost completely reversed in vitro resistance to vincristine (VCR) in VCR-resistant P388 cells . The compound also reversed in vitro VCR, adriamycin (ADM), etoposide, and actinomycin D resistance in ADM-resistant human myelogenous leukemia K562 (K562/ADM) cells, ADM-resistant human ovarian carcinoma A2780 cells, and colchicine-resistant human KB cells . MS-073 administered i.p . daily for 5 days with VCR enhanced the chemotherapeutic effect of VCR in VCR-resistant P388-bearing mice . Increases in life span of 19-50% were obtained by the combination of 100 micrograms/kg of VCR with 3-100 mg/kg of MS-073, as compared to the control . The ability of MS-073 to reverse MDR was remarkably higher, especially at low MS-073 doses, than that of verapamil, both in vitro and in vivo . MS-073 enhanced accumulation of {3H}VCR in K562/ADM cells . Photolabeling of P-glycoprotein with 200 nM {3H}azidopine in K562/ADM plasma membranes was completely inhibited by 10 microM MS-073, indicating that MS-073 reverses MDR by competitively inhibiting drug binding to P-glycoprotein.

J Natl Cancer Inst, 1991 Apr 17, 83(8), 565 - 9
Magnetic-affinity cell sorting of human multidrug-resistant cells; Padmanabhan R et al.; We have developed a method for isolating multidrug-resistant cells from a heterogeneous cell population using the magnetic-affinity cell-sorting system . Human KB carcinoma cell lines expressing different amounts of P-glycoprotein have been selected by use of the monoclonal antibody MRK-16 coupled to magnetic particles . This specific, rapid, and sensitive method allows the selection of viable and clonable drug-resistant cells from various drug-resistant human and murine cell populations . This method may prove useful in isolating drug-resistant cells from tumors with heterogeneous P-glycoprotein expression for further analysis.

Biochem Pharmacol, 1991 Apr 15, 41(8), 1217 - 25
Modulation by verapamil of vincristine pharmacokinetics and sensitivity to metaphase arrest of the normal rat colon in organ culture; Ince P et al.; The in-vitro pharmacokinetics of vincristine (VCR) in normal rat colonic mucosa were studied . Two complementary approaches were adopted using an explant organ-culture system . Firstly {G-3H}vincristine (3HVCR) accumulation, retention and efflux were characterized under basal conditions and compared with measurements made either under energy-depleted conditions, or in the presence of VRP . Secondly, a histological method--the postmetaphase index (PMI)--was used to compare the sensitivity of explants to VCR in the presence or absence of verapamil (VRP) . This latter technique involves the measurement, by counting, of the proportion of mitotic figures escaping from metaphase arrest . The studies yielded the following results: 3HVCR accumulation in colonic mucosa showed no evidence of saturability up to the maximum dose studied (130 nM), at a dose of 52 nM accumulation was enhanced in energy-depleted conditions by a factor of 1.8, and in the presence of VRP (6.6 microM) by a factor of 1.4 . In the presence of VRP (6.6 microM) retention of 3HVCR was increased by a factor of 1.3 and efflux was reduced by a factor of 0.8 after 2 hr . VRP (6.6 microM) reduced the PMI of colonic mucosal epithelial cells exposed to 11 nM VCR from 18.8% to 11.4% (i.e . 40% reduction) indicating sensitization of the cells to this property of VCR . These results provide evidence that the sensitivity of normal colonic mucosa to vincristine is, at least in part, regulated by drug transport . Qualitatively our observations resemble those described in multidrug resistance . Given that P-glycoprotein has been demonstrated by several groups in colonic mucosal cells, the results support a normal role for this membrane transport molecule in the protection of intestinal cells from plant alkaloids and other xenobiotic agents ingested in the diet.

Cancer Res, 1991 Apr 15, 51(8), 2092 - 7
Assessment of P-glycoprotein, glutathione-based detoxifying enzymes and O6-alkylguanine-DNA alkyltransferase as potential indicators of constitutive drug resistance in human colorectal tumors; Redmond SM et al.; Drug resistance is a major problem in cancer chemotherapy . Treatment protocols generally include a number of different cytotoxic drugs given in combination . Therefore, drug resistance in the tumor is likely to result from the coexpression of several cellular activities able to prevent cell killing by any of the drugs used . In this study we have measured several potential drug resistance mechanisms consisting of the multidrug resistance gene product P-glycoprotein, glutathione, glutathione-transferase and -peroxidase, and the DNA repair enzyme O6-alkylguanine-DNA-alkyltransferase in samples of colon carcinoma and normal adjacent mucosa from 23 untreated patients . All of these, with the exception of P-glycoprotein, showed significant increases in tumor tissue levels when compared with normal tissue from the same patient . The significance was highest for glutathione peroxidase (P less than or equal to 0.0005) . Individual patients, however, showed very different patterns, with none, several, or all monitored resistance mechanisms elevated in the tumor . The implications both in the choice of drugs and in the use of resistance modifying agents to improve therapy for the individual patient are discussed.

Hepatology, 1991 Apr, 13(4), 722 - 7
Ethanol interferes with regeneration-associated changes in biotransforming enzymes: a potential mechanism underlying ethanol's carcinogenicity?
Diehl AM, Bisgaard HC, Kren BT, Steer CJ.
The effects of chronic ethanol consumption on enzyme systems involved in carcinogen activation and detoxification were studied in a rat model of liver regeneration . In control rats, steady-state messenger RNAs of cytochrome P450j decreased 12 to 24 hr after partial hepatectomy but were fully recovered by 48 to 72 hr . In contrast, messenger RNA levels of cytochrome P450b and P450d did not vary significantly during that period . Steady-state messenger RNA levels for the placental form of glutathione S-transferase decreased within 30 min after partial hepatectomy but fluctuated until levels returned to normal by 48 hr . Preliminary nuclear run-on analyses suggest that the regulation of cytochrome P450j and the placental form of glutathione S-transferase messenger RNA levels involves posttranscriptional control in these animals . In ethanol-fed rats, as in controls, expression of cytochrome P450j and the placental form of glutathione S-transferase decreased transiently after partial hepatectomy . However, compared with control values, messenger RNA levels for cytochrome P450j were greater in ethanol-fed rats at each time point . Similar results were noted for placental glutathione S-transferase levels from 12 to 48 hr after partial hepatectomy . Ethanol feeding had no apparent effect on steady-state messenger RNA levels of cytochrome P450d, P450b or the multidrug-resistant gene . In both ethanol and control rats, only prehepatectomy levels of cytochrome P450 transcripts correlated with levels of the respective P450 isoenzymes . These data indicate that liver regeneration selectively decreases the steady-state messenger RNA expression of certain isoenzymes of cytochrome P450 and glutathione S-transferase.(ABSTRACT TRUNCATED AT 250 WORDS)

Pediatr Clin North Am, 1991 Apr, 38(2), 299 - 315
The genetics of retinoblastoma . Relevance to the patient; Gallie BL et al.; The understanding of the molecular biology of human cancer has advanced rapidly in the last decade, in part due to discoveries in the rare, pediatric ocular tumor, retinoblastoma . RB studies have led to recognition of a class of human genes, the tumor suppressor genes, that are critical in the initiation and progression of the malignant process . Mutations in the RB1 gene initiate RB and other specific tumors . They may also contribute to progressive stages of many other malignancies . The protein product of RB1 (p110RB1) is a basic regulator of the cell cycle . In the absence of normal protein, the cell proceeds to the next cell division without the potential to become quiescent . Understanding the genetics of RB has benefited the patients, as the precise identification of the RB1 mutations in families has led to accurate prediction of individuals at risk for RB tumors . It seems unlikely, in the foreseeable future, that direct genetic manipulation of mutant RB1 genes will play a role in therapy, but complete understanding of the function of p110RB1 may eventually allow exploitation of its powerful antiproliferative effect . Other molecular genetic events in addition to RB1 mutations are documented in RB tumors, and may play a critical role in the full malignant phenotype . The oncogene, N-myc, is amplified in some RB tumors and is expressed in normal fetal retina . The cytogenetic abnormality, i(6p), is almost unique to RB tumors . The molecular and tissue-specific roles of these abnormalities are not yet known . Many RB tumors also acquire excessive expression of the cell surface membrane glycoprotein, p170, linked to multidrug resistance, whether or not the RB tumor has been exposed to chemotherapy . We anticipate that ways to avoid or counteract the drug resistance of excessive p170 expression will be developed for other pediatric tumors and eventually will be applied to chemotherapy for RB patients.

Chest, 1991 Apr, 99(4), 1025 - 6
Amoxicillin-clavulanic acid for treating drug-resistant Mycobacterium tuberculosis; Nadler JP et al.; This report describes two patients with multidrug resistant tuberculosis who were successfully treated with the addition of amoxicillin-clavulanic acid to second-line drugs . Mycobacterium tuberculosis possesses a beta-lactamase contributing to its resistance to beta-lactam antibiotics . The combination of clavulanic acid, a beta-lactamase inhibitor, and amoxicillin has been shown bactericidal for M tuberculosis in vitro . These data suggest that resistant tuberculosis may warrant a trial of treatment including amoxicillin-clavulanic acid.

J Clin Invest, 1991 Apr, 87(4), 1467 - 9
Reversal of daunorubicin resistance in P388/ADR cells by itraconazole; Gupta S et al.; Itraconazole is a recently developed triazole antifungal agent that inhibits cell membrane sterol biosynthesis . Itraconazole, in a dose-dependent manner, enhanced intracellular accumulation of daunorubicin and reversed the drug resistance in murine leukemia P388/ADR cells . In addition, itraconazole corrected the altered plasma membrane potentials of P388/ADR cells . The concentrations of itraconazole that reversed drug resistance are comparable to the plasma levels achieved by therapeutic dosage used in the treatment of fungal infections . Therefore, itraconazole is a potential candidate for in vivo use to reverse multidrug resistance in cancer with added benefit of its antifungal property.

Am J Pathol, 1991 Apr, 138(4), 799 - 806
Immunohistochemical detection of P-glycoprotein in endometrial adenocarcinoma; Axiotis CA et al.; P-glycoprotein (Pgp) has emerged as the central mediator in classic multidrug resistance in model systems in vitro . High levels of Pgp also have been detected in many normal human tissues and tumors; and its role in clinical drug resistance is currently under investigation . Recently significant levels of Pgp were localized to gravid and secretory endometrium; and it was demonstrated that the combination of estrogen and progesterone is sufficient to induce high levels of both Pgp mRNA and Pgp in uterine secretory epithelium . These findings suggest that increased Pgp expression also may be present in hormone-responsive malignancies such as endometrial adenocarcinoma . To determine whether Pgp is expressed in endometrial adenocarcinoma, 36 endometrial adenocarcinomas (grade I {n = 17}; grade II {n = 6}; grade III {n = 13}) were investigated retrospectively by the avidin-biotin-complex immunohistochemical procedure using three murine monoclonal antibodies (MAb) MAb C219, MAb C494, and MAb JSB-1, which recognize spatially distinct cytoplasmic epitopes of Pgp . Seventy-two percent of the tumors showed positive immunostaining with at least one MAb; 67% showed immunostaining with MAb C219, 50% with MAb C494, and 62% with MAb JSB-1 . Forty-six percent of tumors were immunoreactive to two and 29% to all three antibodies . Membranous and Golgi/paranuclear type staining patterns were observed . Overall the intensity of immunostaining varied from one sample to another for a given tumor type, and considerable heterogeneity of expression was commonly seen within a given tumor . Strong to moderate immunoreactivity was seen in diffusely infiltrating, adenosquamous, and serous papillary carcinomas . In general, immunoreactivity to MAb C494 was weaker than MAb C219 or MAb JSB-1 . Adenomatous and non-neoplastic endometrium adjacent to the tumors displayed strong membranous immunostaining with MAb JSB-1 . Endometrial capillaries showed weak-to-moderate immunostaining to all three antibodies . It is concluded that Pgp is commonly expressed in endometrial adenocarcinoma and may be a significant factor responsible for their drug-resistant nature subject to modulation by progesterone.

Anticancer Drugs, 1991 Apr, 2(2), 159 - 68
Modulation of thiol pools by vitamin K3 and its effect on survival of sensitive and resistant murine tumor cells; Parekh H et al.; Cytotoxic effects of vitamin K3 were evaluated utilizing the P388/S, L1210, EAT, S-180 and a multidrug-resistant variant of the P388 leukemia cells (P388/ADR) . Antitumorigenic potential of vitamin K3 was assessed by MTT and DNA and RNA biosynthesis inhibition assay . A dose-dependent inhibition of P388/S and P388/ADR cell survival and {3H}thymidine and {3H}uridine incorporation (as a function of DNA and RNA biosynthesis) was observed in tumor cell types exposed to vitamin K3 concentrations ranging from 1 to 100 microM . One hundred mg/kg vitamin K3 caused a 32 and 52% increase in life span of the sensitive and resistant P388 leukemia tumor-bearing mice . Induction of DNA strand breaks at 100 microM vitamin K3 was greater in P388/S than in P388/ADR cells . In vitro treatment with vitamin K3 (100 microM) reduced the intracellular levels of GSH by 40, 47, 6, 15 and 14% in P388/S, P388/ADR, EAT, S-180 and L1210 tumor cells, respectively . In vivo treatment with 100 mg/kg vitamin K3 reduced the GSH content by 18 and 38% and increased the activity of the enzyme GSH-S-transferase and gamma-glutamyl transpeptidase . Effects of free radical scavengers and of compounds that modulate the GSH metabolism on the cytotoxicity of vitamin K3 were also investigated . Results indicate that vitamin K3 interacts with the tumor cell thiol pools while eliciting its antitumor effects and suggest the utility of vitamin K3 in dealing with the growing problem of multidrug resistance.

Eur J Pediatr, 1991 Apr, 150(6), 416 - 8
Detection of multidrug-resistant protein, P-glycoprotein in childhood leukaemia and lymphoma; Mizuno Y et al.; Flow cytometric detection of surface P-glycoprotein, a multidrug-resistant gene product, with a monoclonal antibody, MRK 16, was performed on cells obtained from 18 children with leukaemia and lymphoma . Of 18 patients examined, 1 with malignant lymphoma at relapse showed a significant increase in P-glycoprotein-positive cells and a strong resistance to chemotherapy . Overexpression of P-glycoprotein in a case with B-cell type malignant lymphoma was confirmed by immuno-precipitation and Northern hybridization analysis . The present study suggests that an increased expression of surface P-glycoprotein might be involved in multidrug resistance at least in a certain case of childhood leukaemia and lymphoma.

Br J Haematol, 1991 Apr, 77(4), 460 - 5
The expression of the multidrug resistance-associated glycoprotein in B-cell chronic lymphocytic leukaemia; Michieli M et al.; The expression of the multidrug resistance (MDR)-associated 170 kDa glycoprotein (p170) was investigated in 63 cases of B-cell chronic lymphocytic leukaemia (CLL), with two monoclonal antibodies (MRK-16 and C-219) . By immunocytochemistry with MRK-16 (63 cases), the great majority of the cells was positive, with a weak reaction in 61% of cases and a strong reaction in 39% of cases . By flow cytometry, the proportion of positive cells was 39 +/- 25% with MRK-16 (63 cases), and 23 +/- 22% with C-219 (36 cases) . The expression of p170 in leukaemic B-lymphocytes suggests that also in B-CLL the development of MDR can have some therapeutic relevance . By either method the proportion of positive cells was not related to prior treatment, time from diagnosis, absolute lymphocyte count, and clinical stage (Rai's and workshop classifications), but 12 patients who were under treatment with alpha-interferon had more positive cells than the other ones.

Curr Opin Biotechnol, 1991 Apr, 2(2), 278 - 81
Molecular basis of multidrug resistance mediated by P-glycoprotein; Higgins CF; In the past year, our understanding of the biology and molecular basis of multidrug resistance of tumours has advanced on several fronts . Intriguing clues to some of the key questions in the area provide optimism for future understanding and, with luck, eventual prevention and/or treatment.

Biochim Biophys Acta, 1991 Mar 19, 1092(1), 7 - 14
Amplification and expression of mdr genes and flanking sequences in verapamil hypersensitive hamster cell lines; Stow MW et al.; The properties of several multidrug resistant (MDR) Chinese hamster ovary (CHO) cell lines which are verapamil hypersensitive have been investigated, extending our earlier study of two such cell lines . It was observed that increasing levels of multidrug resistance are associated with increasing verapamil and nicardipine sensitivity, although the cell lines are not hypersensitive to cyclosporin A . Although there is appreciable amplification of the P-glycoprotein gene at higher levels of multidrug resistance/verapamil hypersensitivity, there is only very low or no amplification of five flanking genes, including the sorcin gene . Low levels of resistance (3-10 fold) appear to involve increased P-glycoprotein gene expression at the level of transcription . P-glycoprotein levels of the cell lines have been measured by flow cytometry using the monoclonal antibody C219, and there is a general correlation between P-glycoprotein overexpression, increased levels of the corresponding mRNA and the level of verapamil hypersensitivity.

J Biol Chem, 1991 Mar 5, 266(7), 4309 - 14
Uptake and concentration of bioactive macromolecules by K562 cells via the transferrin cycle utilizing an acid-labile transferrin conjugate; Wellhoner HH et al.; The transferrin cycle was used to attempt the import of bioactive macromolecules into cells with the aid of an acid-labile cross-linking agent . Anti-tetanus F(ab')2 fragments were iodinated and then conjugated to transferrin with a newly developed acid-labile cleavable cross-linking reagent, bismaleimidoethoxy propane, following thiolation of both proteins . Noncleavable conjugates were also prepared . At saturating conjugate concentrations, the uptake rate for both conjugates averaged over the first 2 h is about 6.5 fmol/million cells/min . Incubation of loaded cells in fresh medium for 30 min and analysis of cell pellets and supernatants reveal that 1) of the previously cell-associated label, only intact conjugate (about 50% of the label) is returned to the medium; 2) most of the remaining cell-associated material for the cleavable conjugate is chromatographically coincident with free Fab with some contribution from free F(ab')2 fragments . In contrast, the cell pellets loaded with noncleavable conjugates contained intact transferrin-F(ab'), conjugates . These results are consistent with transferrin receptor-mediated uptake of acid-labile conjugate followed by hydrolysis in acidified endosomes and resulting in concentration of free F(ab')2 and Fab within a prelysosomal intracellular compartment . A protein shuttle such as transferrin may therefore be used with ketal based acid-labile cross-linkers to load foreign molecules into an intracellular compartment . In addition, these data provide independent confirmation of the low pH compartment within the transferrin cycle . This new methodology is applicable to other cases of receptor/ligand trafficking to report low pH compartments independent of morphological analysis . Since transferrin receptors are overexpressed in tumors, antineoplastic agents could be targeted to tumors as transferrin acid-labile conjugates . This import system might be particularly useful in combatting the tumor cell export of antitumor agents occurring in multidrug resistance.

Toxicol Appl Pharmacol, 1991 Mar 15, 108(1), 140 - 9
Increased toxicity of anthracycline antibiotics induced by calcium entry blockers in cultured cardiomyocytes; Santostasi G et al.; Calcium channel blocking drugs have been reported to reduce survival rate of laboratory animals treated with cardiotoxic antitumor anthracyclines . In order to elucidate the mechanisms of this drug interaction, cell toxicity of the anthracyclines, doxorubicin and daunorubicin, was evaluated in primary cultures of cardiac myocytes isolated from neonatal rats . Low concentrations of extracellular calcium ({Ca2+}0) and addition of calcium entry blockers (nifedipine or flunarizine) potentiated myocardial toxicity of anthracyclines as assessed by the release of lactate dehydrogenase from the cells . Accumulation of anthracyclines in the cardiomyocytes was increased by calcium entry blockers (nifedipine, flunarizine, and verapamil) and by low {Ca2+}0; efflux of {3H}daunorubicin from myocardial cells was inhibited by nifedipine . At a dose that exerts only modest calcium channel activity, R-verapamil failed to affect doxorubicin accumulation in cardiomyocytes, whereas the calcium channel activator, (+/-)-Bay K-8644, reduced the retention of anthracyclines; the calcium channel activity is thus required in order to increase the accumulation of anthracyclines in myocardial cells . Calcium channel blockers are also known to increase intracellular retention and toxicity of chemotherapeutic drugs in multidrug resistant tumor cells by inhibiting the efflux of cytotoxic agents from cells; however, the ability of the interacting drugs to inhibit the efflux of chemotherapeutic agents from tumor cells is not dependent on the calcium channel blocking activity . Therefore, the mechanism(s) by which calcium channel blocking drugs increase the accumulation of anthracyclines in resistant tumor cells and myocardial cells may be different . In accordance with previous investigations, the present in vitro study confirmed that anthracycline-induced cardiotoxicity may be potentiated by calcium channel blocking drugs . This indicates that, in the association of antineoplastic drugs with agents that reverse multidrug resistance, the potential exists for enhanced damage of normal cells and tissues; further studies are needed to evaluate the relevance of this adverse interaction.

Cancer Res, 1991 Mar 15, 51(6), 1638 - 44
The multidrug resistance phenotype: 31P nuclear magnetic resonance characterization and 2-deoxyglucose toxicity; Kaplan O et al.; In order to identify changes in 31P nuclear magnetic resonance (NMR) spectra associated with multiple drug resistance (MDR), a number of wild type and drug-resistant cancer cell lines were studied . The resistant cells included cells selected with various drugs, mainly Adriamycin, as well as cells transfected with the human multidrug resistance gene (MDR1 gene), which encodes P-glycoprotein . In most cases, 31P NMR spectra were significantly different from those of parental, drug-sensitive lines . The spectra of resistant cells generally indicated increased levels of ATP and phosphocreatine in the cytoplasm . These changes are compatible with the increased glucose utilization rate previously described for resistant cells . Major changes were also observed in the levels of glycerophosphocholine and glycerophosphoethanolamine . Changes in cellular metabolism reflected by 31P NMR spectra depend on the drug used to select the cells for MDR . The direction of these changes was not consistent for all cell lines studied and could not be directly attributed to expression of P-glycoprotein, suggesting that the changes may be related to alterations in metabolism and membrane function associated with other mechanisms of MDR . The results demonstrate the suitability of 31P NMR for studies of biochemical changes associated with MDR . The toxicity of 2-deoxyglucose, a glucose antimetabolite, was investigated in addition to the NMR studies and was found to be consistently higher in multidrug-resistant cells than in the parental drug-sensitive lines . For MCF-7 breast cancer cells, where several sublines with different levels of resistance were available, the toxicity was highest for the most resistant lines.

Eur J Biochem, 1991 Mar 14, 196(2), 483 - 91
Comparison of the membrane transport of anthracycline derivatives in drug-resistant and drug-sensitive K562 cells; Frezard F et al.; One of the phenotypes of multidrug resistance is characterized by a decrease in the intracellular concentration of drug in resistant cells as compared to sensitive cells . This is correlated with the presence in the membrane of resistant cells of a 150-180-kDa glycoprotein, P-glycoprotein, responsible for an active efflux of the drug . The fluorescence emission spectra from anthracycline-treated cells suspended in buffer have been used to compare the membrane transport of five anthracycline derivatives: adriamycin, daunorubucin, 4'-o-tetrahydropyranyladriamycin, carminomycin and aclacinomycin in drug-sensitive and drug-resistant K562 cells . The initial rate of uptake of these five drugs has been measured as a function of the extracellular pH, pHe . The data show that the uptake occurs through free permeation of the neutral form of the drug . For each drug an influx coefficient kpHe, characteristic of the drug and of the cell type has been defined and calculated: k+(7.2) = V+/{D}e.n where V+ and {D}e are the initial rate of uptake and the concentration of drug in the medium at pHe = 7.2 respectively and n is the number of cells . This coefficient is characteristic of a passive diffusion of the neutral form of the drug through the lipid bilayer . Efflux coefficients k-(7.2)- at pHi = 7.2 (the intracellular pH value) have also been calculated . In the case of sensitive cells, k+(7.2) and k-(7.2)- are equal . For resistant cells, the efflux coefficient is composed of two terms: (a) (k-)p corresponding to the passive diffusion of the neutral form of the drug and (k-)p = k+; (b) (k-)a corresponding to an active efflux mediated by the P-glycoprotein . Our data suggest that the anthracycline derivatives efflux actively in the neutral form.

J Biol Chem, 1991 Mar 5, 266(7), 4545 - 55
Full length and alternatively spliced pgp1 transcripts in multidrug-resistant Chinese hamster lung cells; Devine SE et al.; In an effort to better understand the preferential resistance to actinomycin D displayed by the multidrug-resistant Chinese hamster lung cell line DC-3F/ADX, we have cloned from those cells a number of cDNAs representing p-glycoprotein gene transcripts . Of the 12 clones isolated, all represent pgp1 transcripts and one, pADX165, contains a 4304-base pair insert with an open reading frame encoding a 1276-amino acid protein that is the homolog of the mouse mdr3/mdr1a gene product . A domain by domain comparison of this protein with p-glycoproteins capable of supporting multidrug resistance, i.e . human mdr1, mouse mdr1/mdr1b, and mouse mdr3/mdr1a, shows that, in addition to the ATP binding sites, the second, fourth, and eleventh transmembrane domains and the four small intracellular loops, IC-1, IC-2, IC-4, and IC-5, are highly conserved and are therefore likely to be important for the maintenance of p-glycoprotein function . Of the remaining 11 cDNA clones, 9 were found to be truncated versions of pADX165 . Two others, however, pADX185 and pADX124, contained internal deletions resulting in open reading frames capable of encoding lnovel forms of p-glycoprotein . S1 nuclease and RNase protection analysis demonstrated that these cDNAs represent transcripts present in a number of different multidrug-resistant Chinese hamster lung cell lines . Hence, both are considered to be splicing variants of the hamster pgp1 gene primary transcript.

Biochim Biophys Acta, 1991 Mar 4, 1073(2), 309 - 15
Glycosylation of P-glycoprotein in a multidrug-resistant KB cell line, and in the human tissues; Ichikawa M et al.; P-glycoprotein (P-gp) is thought to transport anti-cancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype . Immunohistochemistry reveals that P-gp is also expressed in normal human tissues, such as the adrenal gland, kidney, liver, and the capillary endothelium of the brain and testis . However, little is known about the structural and functional variations of P-gp in these tissues . With immunoblotting and photoaffinity labeling, we found that the molecular mass of P-gp in these tissues varied between 130-140 kDa . To clarify the post-translational modification of P-gp, we studied the biosynthesis of P-gp in a human multidrug-resistant cell line (KB-C2) . We found that P-gp was produced in KB-C2 cells as a 125 kDa precursor and was slowly processed (t1/2 = 45-60 min) to the mature form of 140 kDa . In the presence of tunicamycin, a 120 kDa form of P-gp was synthesized and this form was no longer processed . Treating the 125 kDa precursor form with endo-beta-N-acetylglucosaminidase H (Endo H) and the 140 kDa mature form with N-glycanase diminished the molecular size of P-gp to that of the tunicamycin-treated form . N-Glycanase almost completely removed {3H}glucosamine labeling from P-gp . These data indicate that the major modification of P-gp is N-linked glycosylation . P-gps from KB-C2 cells, kidney and adrenal gland had a different lectin-binding capacity . There seems to be a variety of N-linked glycosylations in tissue and tumor P-gps.

Res Commun Chem Pathol Pharmacol, 1991 Mar, 71(3), 321 - 36
Potentiation of pirarubicin cytotoxicity by dipyridamole in doxorubicin-resistant mouse P388 leukemia cells; Furusawa S et al.; The activity of dipyridamole and its possible mechanisms which reverse the resistance of pirarubicin were studied in a P388 mouse leukemia cell lines . Dipyridamole alone was minimally cytotoxic in both of the doxorubicin-resistant cell line (P388/DOX) and the sensitive parent cell line (P388/S), but reversed pirarubicin-resistance in a dose-related manner in P388/DOX cells . A similar dose-response relationship was observed for dipyridamole by increasing net intracellular pirarubicin accumulation . The increase was a result of secondary blocking of enhanced pirarubicin efflux from P388/DOX cells . In contrast, dipyridamole did not affect cytotoxicity and transport in the drug-sensitive cell line . It is suggested that dipyridamole is a useful drug for modulation of the multidrug-resistance of cells.

Biochem Pharmacol, 1991 Mar 1, 41(5), 797 - 806
Transient enhancement of multidrug resistance by the bile acid deoxycholate in murine fibrosarcoma cells in vitro; O'Brian CA et al.; Recent studies have implicated protein kinase C (PKC) activation in drug resistance in vitro . PKC can be activated directly by phorbol-ester tumor promoters as well as by the bile acid deoxycholate . In this report, we demonstrate that deoxycholate, at concentrations that are chronically present in the lumen of the colon in vivo, mimicked phorbol-ester tumor promoters by protecting Adriamycin (ADR)-sensitive and multidrug-resistant (MDR) murine fibrosarcoma UV-2237M cells from ADR cytotoxicity . Deoxycholate also enhanced the resistance of the MDR cell line UV-2237M-ADRR to the cytotoxic effects of vincristine and vinblastine . In contrast to cytotoxic drug-selected MDR phenotypes, deoxycholate-induced drug resistance was transient and required continuous exposure to the bile acid . The protein kinase inhibitor H7 completely reversed the protection against ADR cytotoxicity conferred on UV-2237M-ADRR cells by deoxycholate, providing evidence that deoxycholate exerts its protective effects by a mechanism that involves stimulation of protein phosphorylation and not merely by detergent effects on membrane permeability . PKC consists of a family of at least seven isozymes with distinct modes of activation and substrate specificities . We previously reported that MDR UV-2237M cell lines contain higher levels of PKC activity than the parental ADR-sensitive UV-2237M cell line (O'Brian et al., FEBS Lett 246: 78-82, 1989) . The present report shows that PKC-III is a major PKC isozyme in ADR-sensitive and MDR UV-2237M cell lines . Thus, the resistance to ADR induced by the phorbol esters in UV-2237M cell lines provides strong evidence that PKC-III activation confers protection against ADR on ADR-sensitive and MDR UV-2237M cell lines . Furthermore, since deoxycholate is an endogenous molecule in the colonic epithelium, our finding that physiological concentrations of deoxycholate can render cells more resistant to chemotherapeutic drugs in vitro may have implications for the biology and therapy of intestinal cancers.

Anticancer Res, 1991 Mar-Apr, 11(2), 917 - 9
Drug resistance, the last frontier?
Kellen JA.
A brief review of the (1990) "state of the art" knowledge concerning multidrug resistance and P-glycoprotein function is presented . Modification and manipulation of the efflux pump by calcium channel blockers and antiestrogens are discussed.

Anticancer Res, 1991 Mar-Apr, 11(2), 817 - 23
Induction of in vitro resistance to 4'-epidoxorubicin and cis-dichlorodiammineplatinum in hepatoma cells; Hall KS et al.; We have developed in vitro resistance to 4'-epidoxorubicin (Epi-A) and cis-dichlorodiammineplatinum (cis-DDP) in one rat (MH1C1) and one human hepatoma cell line (HepG2) . When compared to their parental cells, the Epi-A resistant rat cells were 17 times and the resistant human cells 27 times more resistant to Epi-A in terms of GI50 in the cell growth inhibition assay . The cis-DDP resistant rat cells were 20 times and the resistant human cells 12 times more resistant to cis-DDP . Cross-resistance to cis-DDP was observed in the Epi-A resistant rat cells but not in the human cells . The multidrug resistant gene product, GP 170, was markedly expressed in both Epi-A resistant substrains compared with their parent lines, suggesting a role of this protein in the development of resistance to Epi-A . Cadmium-binding proteins of metallothionein (MT) size bound 52% of cytosolic 109cadmium in the cis-DDP resistant human cells compared with 8% in the parental cells . This may indicate that these proteins contribute to the observed cis-DDP resistance.

Anticancer Res, 1991 Mar-Apr, 11(2), 579 - 85
Time course of MDR gene amplification during in vivo selection for doxorubicin-resistance and during reversal in murine leukemia L 1210; Volm M et al.; MDR gene expression in murine leukemia L 1210 cells was investigated during treatment in vivo with 0.5 mg doxorubicin/kg body weight (BW) . Drug resistance (measured by an in vitro short-term test and immunohistochemistry) increased with the number of treatments and the maximum resistance reached after 8 treatments was similar with that of an established multidrug- resistant cell line (20 treatments, 2 mg/kg BW) . Southern-blot and DNA dot-blot analyses show that development of MDR is associated with MDR-gene amplification and correlates with the degree of drug resistance and P-glycoprotein-expression . After cessation of doxorubicin treatment, resistance decreased continuously and disappeared after 20 passages . This decrease in resistance is accompanied by a loss of MDR gene amplification and P-glycoprotein expression . Furthermore, P-glycoprotein expression was analyzed in the first hours after treatment with doxorubicin in vivo (0.5 mg/kg BW) . Expression was markedly increased and peaked at about 24 hours after treatment . In contrast, only slightly increased resistance and no MDR gene amplification could be detected.

J Cell Sci, 1991 Mar, 98 ( Pt 3), 317 - 22
Human hepatocellular carcinoma cell lines exhibit multidrug resistance unrelated to MRD1 gene expression; Shen DW et al.; Multidrug resistance of human cancer cells may result from expression of P-glycoprotein, the product of the MRD1 gene, acting as an energy-dependent drug efflux pump . However, direct evidence that expression of the MDR1 gene contributes to the multidrug resistance of human liver carcinomas has not been established . In this study, we tested five cell lines derived from human hepatocellular carcinomas for sensitivity to a variety of drugs used widely as anticancer agents; these included vinblastine, doxorubicin, actinomycin D, mitomycin C, 5-fluorouracil, 6-mercaptopurine, melphalan, methotrexate, cis-platinum and etoposide (VP-16) . All five hepatoma cell lines were resistant at different levels to these chemicals compared to human KB cells . Although it has been demonstrated that resistance to vinblastine, colchicine, doxorubicin and actinomycin D in human multidrug-resistant cells is associated with overexpression of P-glycoprotein, very little expression of P-glycoprotein was found in these human hepatoma cells . Neither verapamil nor quinidine, inhibitors of the drug efflux pump, were able to overcome multidrug resistance in hepatoma cells . These results indicate that the multidrug resistance phenotype in human hepatocellular carcinoma cells cannot be attributed to expression of the MDR1 gene, but that novel mechanisms may account for the resistance of these cancer cells.

Blood Rev, 1991 Mar, 5(1), 38 - 41
Multidrug resistance: clinical relevance in haematological malignancies; Kaye SB et al.; Multidrug resistance describes an experimental observation which appears to explain cross-resistance to certain structurally unrelated cytotoxic agents, including anthracyclines, vinca alkaloids and podophyllotoxins . It is now clear that a major factor responsible for its development is increased expression of a membrane glycoprotein--P-glycoprotein, which functions as an energy-dependent efflux pump . Recent data, particularly in haematological malignancies such as acute non-lymphocytic leukaemia, myeloma and non-Hodgkin's lymphoma, indicate that P-glycoprotein may be involved in the development of clinical drug resistance . The potential therefore exists for new therapeutic studies aimed at circumventing resistance which develops through this mechanism, by using modulators, such as verapamil, quinidine and several others, which prevent cellular drug efflux by competitive binding to P-glycoprotein.

J Heart Lung Transplant, 1991 Mar-Apr, 10(2), 201 - 10
Multidrug resistance in heart transplant patients: a preliminary communication on a possible mechanism of therapy-resistant rejection; Kemnitz J et al.; Multidrug resistance refers to a complex cellular phenotype, the hallmark of which is cross-resistance to multiple drugs, for example, chemotherapeutic agents, that are unrelated to the selecting agent in structure, cellular target, and mode of action . The expression of this multidrug resistance is connected with the overexpression of P-glycoprotein . By applying the method of immunocytochemical assay, we have demonstrated the appearance of the multidrug-resistant phenotype (P-glycoprotein+ cells, multidrug-resistant cells) in mononuclear cells of the peripheral blood from 32/49 patients receiving triple-drug (azathioprine, steroids, cyclosporine) immunosuppressive therapy after heart transplantation . In the group of patients showing not only the presence of cells with multidrug-resistant phenotype in the peripheral blood, but also a significant increase in the number of these cells during the interval of observation (0 to 767 days)-16/32/49 cases--a significantly increased incidence of acute rejection episodes could be demonstrated . This supports the hypothesis of a possible existence of a therapy-resistant form of acute rejection, with an involvement of mechanisms of multidrug-resistance playing a role in its causal development.

Jpn J Cancer Res, 1991 Mar, 82(3), 351 - 6
Enhancement by verapamil of neocarzinostatin action on multidrug-resistant Chinese hamster ovary cells: possible release of nonprotein chromophore in cells; Miyamoto Y et al.; Multidrug-resistant CHRC5 cells were about 10-fold more resistant to the proteinaceous anticancer drug neocarzinostatin (NCS) and its nonprotein chromophore (NPC) than the parental AUXB1 cells . There was little difference in cell growth, glutathione content, or activities of several antioxidant enzymes between the two cell lines . The degree of intracellular incorporation and extracellular excretion of fluorescein isothiocyanate-labeled NCS by CHRC5 cells was similar to that of AUXB1 cells . On the other hand, 20 microM verapamil or 27 microM cepharanthine restored the susceptibility of CHRC5 cells to NCS and NPC to the level of AUXB1 cells . In addition, NPC was found to suppress the photolabeling of {3H}azidopine (a known P-glycoprotein-binding ligand) to plasma membranes of CHRC5 cells . All these findings favor the possibility that NPC was excreted via P-glycoprotein, which may contribute to the resistance of CHRC5 cells to NCS.

Br J Cancer, 1991 Mar, 63(3), 351 - 7
Retention of activity by selected anthracyclines in a multidrug resistant human large cell lung carcinoma line without P-glycoprotein hyperexpression; Coley HM et al.; A subline (COR-L23/R) of the human large cell lung line {corrected} COR-L23, derived by in vivo exposure to doxorubicin, exhibits an unusual multidrug resistant (MDR) phenotype . This subline shows cross-resistance to daunorubicin, vincristine, colchicine and etoposide but does not express P-glycoprotein . Interestingly, COR-L23/R {corrected} shows little or no resistance to a range of structurally-modified analogues of doxorubicin comprising 9-alkyl and/or sugar modified anthracyclines . We have previously identified these same compounds as effective agents against P-glycoprotein-positive MDR cell lines . In contrast to typical MDR cell lines, COR-L23/R {corrected} shows only minimal chemosensitisation by verapamil and no collateral sensitivity to verapamil . Compared to the parental cell line, COR-L23/R {corrected} displays reduced accumulation of doxorubicin and daunorubicin . Accumulation defects were apparent only after 0.5-1 h of incubation of cells with these agents . The rate of daunorubicin efflux was shown to be enhanced by COR-L23/R {corrected} and this efflux was demonstrated to be energy-dependent . The use of anthracyclines which retain activity in MDR cells thus appears to be a valid approach for the circumvention of MDR, not only in cells which express P-glycoprotein, but also where defective drug accumulation is due to other mechanisms possibly involving an alternative multidrug transporter.

Cancer Res, 1991 Feb 15, 51(4), 1190 - 5
Intercellular transfer of drug resistance; Frankfurt OS et al.; The effect of L-phenylalanine mustard (L-PAM) on heterogeneous cell populations containing sensitive and resistant cells was evaluated by flow cytometric analysis of DNA damage . Cell cultures were treated with L-PAM for 1 h, fixed, and stained with anti-DNA monoclonal antibody which detects DNA damage induced by alkylating agents . DNA damage was significantly lower in sensitive A2780 cells cocultured with resistant A549 or A2780/PAM cells than in A2780 cells grown separately . Decrease of DNA damage in sensitive cells did not occur when sensitive and resistant cells were grown in common medium without direct contact . Transfer of drug resistance in cocultures was prevented by phorbol ester which is known to inhibit metabolic cooperation via cell junctions . Treatment of cocultures with buthionine sulfoximine increased DNA damage in resistant cells and prevented decrease of DNA damage in sensitive cells . Glutathione (GSH) content in A2780 cells cocultured with A549 cells was significantly higher than GSH content in A2780 cells grown separately . We conclude that decreased response of sensitive cells in cocultures was induced by contact transfer of GSH from GSH-rich resistant cells to sensitive cells . Intercellular transfer of drug resistance demonstrated by analysis of DNA damage was confirmed by colony formation assay . Treatment with L-PAM and Adriamycin killed all cells in A2780/MDR and A549 cultures . Coculture of these lines survived combination treatment because transfer of GSH to multidrug-resistant cells from GSH-rich A549 cells induced resistance to L-PAM and Adriamycin in a single cell . The presence of 2% A549 cells increased resistance of A2780/MDR cells to L-PAM . Phorbol ester eliminated resistance of coculture to combination treatment . Metabolic cooperation between cell subsets with different mechanisms of drug resistance induced resistance to treatment with drugs of different classes (multiclass drug resistance) . Inhibition of cell cooperation may improve the response of tumors to combination chemotherapy.

Blood, 1991 Feb 15, 77(4), 818 - 25
Effect of tamoxifen on cell lines displaying the multidrug-resistant phenotype; Berman E et al.; We examined the effect of tamoxifen (Tmx), verapamil, and daunorubicin (DNR) in two cell lines that displayed the multidrug-resistant (MDR) phenotype and used laser flow cytometry to quantitate intracellular DNR content . In the vinblastine-resistant human lymphoblastic lymphoma cell line CEM-VBL, simultaneous incubation of DNR with Tmx 10 mumol/L or Tmx 50 mumol/L increased intracellular DNR fluorescence in a dose-dependent manner and demonstrated an uptake pattern similar to that seen with DNR and verapamil . Similar results were obtained in the vincristine-resistant human myeloid leukemia cell line HL-60/RV+ . Cellular retention of DNR was also measured in both cell lines and results suggested that continuous exposure of the cells to Tmx resulted in higher intracellular DNR content compared with cells resuspended in fresh medium . No effect of Tmx or verapamil was observed in the drug-sensitive parent cell lines CEM or HL-60 . Clonogenic experiments were then performed to determine whether Tmx was itself inhibitory to cell growth or whether Tmx potentiated DNR cytotoxicity . Tmx 10 mumol/L did not significantly inhibit either CEM-VBL or HL-60/RV+ cells after a 3-hour exposure followed by culture in methylcellulose . Tmx 50 mumol/L was significantly more inhibitory in both cell lines . However, cells that had been incubated with DNR and Tmx 10 mumol/L demonstrated a marked increment in growth inhibition compared with cells that had been incubated with DNR alone or Tmx 10 mumol/L alone . Based on the data presented here, we suggest that clinical testing of Tmx and DNR be pursued in the setting where MDR may play a role.

Br J Cancer, 1991 Feb, 63(2), 247 - 51
Reversal of intrinsic multidrug resistance in Chinese hamster ovary cells by amiloride analogs; Epand RF et al.; A number of amiloride analogs can sensitise wild type Chinese Hamster ovary (CHO) cells to the cytotoxic action of vinblastine, daunomycin, puromycin or colchicine . Some of these analogs also have weak sensitising effects on the multidrug resistant CHO cell line, CHRC5 . The unusual feature of most of the active amiloride analogs is that they are more potent in reversing the intrinsic multidrug resistance (MDR) phenotype of CHO cells than their acquired MDR characteristic . Human HeLa cells that do not exhibit intrinsic MDR are not affected by these agents . Several of the amiloride analogs have a greater effect in increasing adriamycin uptake in wild type CHO cells than they do with CHRC5 cells . The differential effect of amiloride analogs on intrinsic versus acquired MDR characteristics of Chinese hamster cells suggests some differences in the underlying resistance mechanisms.

J Biol Chem, 1991 Feb 5, 266(4), 2239 - 44
Structural and functional analysis of the mouse mdr1b gene promoter; Cohen D et al.; The overproduction of P-glycoprotein, an integral membrane protein thought to function as a drug efflux pump, is the hallmark of the multidrug resistance phenotype . In murine multidrug resistant J774.2 cell lines, distinct mdr genes, mdr1a and mdr1b, encode unique P-glycoprotein isoforms . To examine the transcriptional regulation of the mdr1b gene, its promoter was isolated and characterized . The transcription initiation site was mapped by primer extension, and the 5'-flanking region was sequenced . Several potential regulatory elements were identified in this region . A transient expression vector was constructed by fusion of 540 base pairs of 5'-flanking sequence and part of the first untranslated exon to the chloramphenicol acetyltransferase (CAT) gene . When transfected into monkey kidney COS-1, rat pituitary GH3 or T47D human breast cells, the mdr1b 5'-flanking sequences were capable of driving CAT expression . Transient transfection studies using deletion subclones of the mdr1b-CAT construct were done to locate potential cis-acting sequences . The studies indicate the presence of cis-acting elements in the 5'-flanking region of the mdr1b gene . The implications of these findings for expression and regulation of the mdr1b gene are discussed.

J Neurosci Res, 1991 Feb, 28(2), 310 - 4
Multidrug transport in human glioblastoma cells is inhibited by transforming growth factors type beta 1, beta 2, and beta 1.2; Schluesener HJ; The transforming growth factors type beta 1, beta 2, and beta 1.2 suppress multidrug transport in human pat-1 glioblastoma cells and even in cells that strongly over-express mdr genes and are resistant to inhibition of multidrug transport by chemosensitizers . Thus, inhibition of multidrug transport by cytokines might be a new approach to increase cellular accumulation of chemotherapeutic agents in multidrug resistant glial tumor cells . Interestingly, a member of the more distantly related decapentaplegic subgroup of transforming growth factors, the bone morphogenetic protein BMP 2, did not inhibit multidrug transport.

Anticancer Drug Des, 1991 Feb, 6(1), 47 - 57
Potentiation of etoposide and vincristine by two synthetic 1,4-dihydropyridine derivatives in multidrug-resistant and atypical multidrug-resistant human cancer cells; Watanabe Y et al.; Newly synthesized 1,4-dihydropyridine derivatives had been screened to determine whether they could overcome vincristine (VCR)-resistance in VCR-resistant (P388/VCR) leukemia-bearing mice, and six compounds had strong reversing ability among the screened compounds . We further determined whether NK-250 and NK-252 among the six compounds could potentiate cytocidal activities of etoposide (VP16) as well as VCR against both multidrug-resistant (MDR) cell line (VJ-300) and atypical MDR cell line (KB/VM-4) . Both VJ-300 and KB/VM-4 were derived from the same parental human cancer KB cell line: VJ-300 cells showed enhanced expression of a MDR-specific glycoprotein of molecular weight of 170,000 Da (gp170) while KB/VM-4 cells were selected as teniposide (VM26)-resistant cell line with no expression of gp170 . NK-250 and NK-252 potentiated the cytotoxic action of VCR about 2- to 10-fold against KB and KB/VM-4 cells, and they almost completely reversed VCR-resistance in VJ-300 cells . By contrast, NK-250 and NK-252 potentiated the cytotoxic action of VP16 about 2-fold against KB cells while they reversed 5- to 10-fold VP16-resistance in both VJ-300 and KB/VM-4 cells . The reversal effect by NK-250 and NK-252 of VCR-resistance in VJ-300 cells appeared to be due to enhanced cellular accumulation of radioactive VCR through interaction to 170-kDa P-glycoprotein . The potentiation effects by these dihydropyridines of VCR and VP16 on KB or KB/VM-4 cells also appeared to be due to enhanced accumulation of radioactive VP16 or VCR, but the effects might be mediated through other mechanisms, plausibly enhanced cellular uptake of the drugs.

Cancer Commun, 1991 Feb, 3(2), 37 - 44
Trifluoperazine modulation of resistance to the topoisomerase II inhibitor etoposide in doxorubicin resistant L1210 murine leukemia cells; Kamath N et al.; Murine leukemia L1210 cells selected for progressive resistance to doxorubicin (DOX) display both the multidrug resistant (MDR) phenotype and reductions in drug induced topoisomerase II-mediated DNA cleavage in nuclear extracts (Ganapathi, R.; Grabowski, D.; Ford, J.; Heiss, C.; Kerrigan, D.; Pommier, Y., Cancer Commun . 1:217-224; 1989) . The present study was performed to characterize the results of exposure of the sensitive (S) and progressively DOX-resistant (10-fold, R1, and 40-fold, R2) L1210 cells to the topoisomerase II inhibitor, etoposide, and to investigate the modulating effects of the calmodulin inhibitor, trifluoperazine (TFP) . Immunoblotting experiments indicated no apparent decrease in the p170 or p180 isoforms of topoisomerase II in the resistant sublines versus parental sensitive cells . Cross-resistance to etoposide (VP-16) was similar to that of DOX (10- and 40-fold) . A non-cytotoxic concentration of 5 microM TFP enhanced cell kill 1.5- fold in the sensitive and 3- to 5-fold in the progressively DOX-resistant cells . Accumulation of VP-16 was 30% to 50% lower in the resistant sublines versus similarly treated sensitive cells, and a marked enhancement of drug uptake in the presence of TFP was observed in the sensitive but not in the resistant cells exposed to equivalent extracellular levels of VP-16 . Although equimolar concentrations of VP-16 produced fewer DNA single strand breaks (SSB) and DNA protein crosslinks (DPC) in the resistant versus sensitive cells, similar DNA damage was apparent when S and R1, but not R2, cells were treated at VP-16 concentrations that produced equivalent cell death.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Biol, 1991 Feb, 11(2), 595 - 603
Functional analysis of chimeric genes obtained by exchanging homologous domains of the mouse mdr1 and mdr2 genes; Buschman E et al.; A full-length cDNA clone for the mouse mdr1 gene can confer multidrug resistance when introduced by transfection into otherwise drug-sensitive cells . In the same assay, a full-length cDNA clone for a closely related member of the mouse mdr gene family, mdr2, fails to confer multidrug resistance . To identify the domains of mdr1 which are essential for multidrug resistance and which may be functionally distinct in mdr2, we have constructed chimeric cDNA molecules in which discrete domains of mdr2 have been introduced into the homologous region of mdr1 and analyzed these chimeras for their capacity to transfer drug resistance . The two predicted ATP-binding domains of mdr2 were found to be functional, as either could complement the biological activity of mdr1 . Likewise, a chimeric molecule in which the highly sequence divergent linker domain of mdr2 had been introduced in mdr1 could also confer drug resistance . However, the replacement of either the amino- or carboxy-terminus transmembrane (TM) domain regions of mdr1 by the homologous segments of mdr2 resulted in inactive chimeras . The replacement of as few as two TM domains from either the amino (TM5-6) or the carboxy (TM7-8) half of mdr1 by the homologous mdr2 regions was sufficient to destroy the activity of mdr1 . These results suggest that the functional differences detected between mdr1 and mdr2 in our transfection assay reside within the predicted TM domains.

Cancer Res, 1991 Feb 1, 51(3), 897 - 902
Solutol HS 15, nontoxic polyoxyethylene esters of 12-hydroxystearic acid, reverses multidrug resistance; Coon JS et al.; A recently developed non-ionic surfactant called Solutol HS 15 (poly-oxyethylene esters of 12-hydroxystearic acid), with low toxicity in vivo, was shown to reverse completely the multidrug resistance of KB 8-5 and KB 8-5-11 human epidermoid carcinoma cells in vitro but did not potentiate drug toxicity in drug-sensitive KB 3-1 cells . At a concentration of 10% of its own IC50 (mean concentration of drug that causes 50% inhibition of cell growth compared to controls), Solutol HS 15 produced a 35-, 28-, and 42-fold reduction in the resistance of KB 8-5-11 cells to colchicine, vinblastine, and doxorubicin, respectively . Solutol HS 15 was relatively much more potent than the prototypic reversing agent, verapamil, for reversing colchicine resistance, compared to the ability of each agent to reverse colchicine resistance, compared to the ability of each agent to reverse vinblastine resistance . Like verapamil, Solutol HS 15 promoted a 50-fold accumulation of rhodamine 123 in KB 8-5-11 cells, as measured by flow cytometry . Also, Solutol HS 15 and verapamil reduced the efflux of rhodamine 123 from KB 8-5-11 cells previously loaded with rhodamine 123 to a similar low rate . Solutol HS 15 did not affect the transport of alanine or glucose into KB 8-5-11 cells, indicating that its effect upon membrane active transport is not entirely nonspecific . Considering their different structure and different relative potency for reversing colchicine resistance, Solutol HS 15 and verapamil probably reverse multidrug resistance by different mechanisms . Solutol HS 15 merits consideration as a potential therapeutic agent because of its effectiveness for reversing multidrug resistance in vitro and its low toxicity in vivo.

Clin Biochem, 1991 Feb, 24(1), 3 - 7
The mechanism of action of cyclosporine: a perspective for the 90's; Halloran PF et al.; The introduction of cyclosporine (CyA) as a pharmacological agent has resulted not only in a dramatic improvement in the clinical management of transplant recipients but also in a better understanding of the molecular basis of the immune response, especially T cell function . Knowledge of the mechanism of action of CyA has led to exciting areas of study . Among these are the sequence of regulatory events leading to T cell activation, the potential relevance of isomerases in signal transduction pathways (as the receptor for CyA, cyclophilin has been shown to be an isomerase), the blocking effect of CyA on the development of multidrug resistance, and the striking parallelism between CyA and the newer immunosuppressive agent FK-506 . These fields promise to be relevant in solving some of the crucial questions in transplantation immunology, and developing better strategies for immunosuppression.

Mol Pharmacol, 1991 Feb, 39(2), 136 - 43
Induction of thymidylate synthase associated with multidrug resistance in human breast and colon cancer cell lines; Chu E et al.; A series of Adriamycin-resistant human breast MCF-7 and human colon DLD-1 cancer cell lines were established by stepwise selection . The concentration of Adriamycin required to inhibit cell proliferation by 50% (IC50) in the parent breast line (MCF-7), Adriamycin-resistant lines (MCF-Ad5 and MCF-Ad10), and a 5-fluorouracil (5-FU)-revertant line (MCF-R) was 0.005, 3.3, 6, and 4.9 microM, respectively . The Adriamycin IC50 value for the resistant colon line (DLD-Ad) was 8.2 microM, 68-fold higher than that for its parent line (DLD-1) (IC50 = 0.12 microM) . The MCF-Ad5 and MCF-Ad10 cells were cross-resistant to 5-FU, with respective 5-FU IC50 values of 11.7 and 22.5 microM, or 7.3- and 14-fold less sensitive than their parent MCF-7 (IC50 = 1.6 microM) line . The MCF-R line completely reverted in sensitivity to 5-FU, with an IC50 of 1.7 microM . The resistant DLD-Ad line was 3.5-fold more resistant to 5-FU than was the parent DLD-1 line . Using both the 5-fluoro-2'-deoxyuridine-5'-monophosphate binding and catalytic assays for measurement of thymidylate synthase (TS) activity, there was significantly increased TS activity in the resistant MCF-Ad5 (2.4- and 2.5-fold), MCF-Ad10 (11.5- and 6.8-fold), and DLD-Ad (4.8- and 10.7-fold) lines, for binding and catalytic assays, respectively, compared with their parent MCF-7 and DLD-1 lines . The level of TS in cytosolic extracts, as determined by Western immunoblot analysis, was markedly increased for the resistant MCF-Ad5 (31-fold), MCF-Ad10 (46-fold), and DLD-Ad (52-fold) cells . Measurement of TS mRNA levels by Northern analysis revealed elevation of TS mRNA in the resistant MCF-AD5 (16.7-fold), MCF-Ad10 (31-fold), and DLD-Ad (55-fold) cells . Southern analysis showed that this increase in TS mRNA was not accompanied by any major rearrangements or amplification of the TS gene . Incorporation of 5-FU into the RNA and DNA of the resistant MCF-Ad10 cells was not significantly different, compared with that for parent MCF-7 cells . These studies suggest that exposure of human breast and human colon cancer cells to Adriamycin leads to overexpression of TS, with concomitant development of resistance to 5-FU.

Ann Oncol, 1991 Feb, 2 Suppl 2, 151 - 5
Expression of growth-related genes and drug-resistance genes in HTLV-I-positive and HTLV-I-negative post-thymic T-cell malignancies; Su IJ et al.; This study was designed to investigate the biologic and molecular basis of the aggressive behavior of high-grade post-thymic T-cell malignancies . Freshly frozen tumor tissues from (1) human T-cell leukemia/lymphoma virus type I (HTLV-I)-positive adult T-cell lymphoma (ATL) (7 cases), (2) HTLV-I-negative aggressive T-cell lymphoma (12 cases), and (3) HTLV-I-negative nonaggressive T-cell lymphoma (11 cases) were studied for the expression of several growth-related genes or proliferation antigens including interleukin-2 receptor (IL-2R), Ki-67, transforming growth factor-beta (TGF-beta), topoisomerase, and the multidrug resistance (MDR) gene by immunohistochemistry and Northern blot hybridization . Our results showed that tumor cells associated with HTLV-I and anaplastic morphology had an enhanced expression of Ki-67, TGF-beta, and topoisomerase, as compared to nonaggressive T-cell lymphoma . The expression of IL-2R was limited to ATL and one Ki-1 lymphoma . The MDR gene was frequently expressed in ATL, but only infrequently in other, HTLV-I-negative, malignancies . Clinical progression or relapse was associated with the expression of MDR, in addition to an increased expression of Ki-67 . We therefore conclude that the aggressive clinical behavior of high-grade T-cell lymphoma may result mainly from the high proliferative activity of tumor cells, but the association with HTLV-I and clinical relapse is further complicated by the development of drug resistance.

Curr Opin Oncol, 1991 Feb, 3(1), 21 - 9
Cellular and pharmacologic aspects of drug resistance in acute myeloid leukemia; Ross DD; Modern treatments and supportive care regimens for acute myeloid leukemia have produced some cures of what was once a uniformly fatal disease . To enhance the cure rate, intense efforts are now being put forth to understand the mechanisms of resistance to chemotherapeutic regimens that actually arise in clinical cases of acute myeloid leukemia, and to develop ways and means to circumvent such resistance . This review focuses on such efforts involving daunorubicin and cytarabine, the two most effective agents currently available for treatment of the disease . Most current published studies of daunorubicin have focused on detecting the classic form of multidrug resistance . For cytarabine, pharmacologically directed treatment approaches and enhancement of cytarabine leukemic cell kill by increasing the distribution of cells in S-phase and manipulating cellular cytarabine transport are currently under investigation.

Biochem Pharmacol, 1991 Feb 1, 41(3), 349 - 59
P-glycoprotein drug efflux pump involved in the mechanisms of intrinsic drug resistance in various colon cancer cell lines . Evidence for a saturation of active daunorubicin transport; Spoelstra EC et al.; We studied the resistance of colon tumors to anticancer agents in vitro . Using daunorubicin (DN), a number of cellular parameters which normally indicate acquired or multidrug resistance (MDR), were compared for several human wild-type colon cell lines, i.e . HT29, SW1116 and COLO 320, and the murine colon cell line C-26 . The sensitive/MDR human ovarian cancer cell line couple A2780/2780AD was used as a reference . The amount of P-glycoprotein (P-gp) was in the order HT29, A2780 less than or equal to SW1116 less than C26 less than or equal to COLO 320 less than 2780AD . The MDR modifiers verapamil, Cremophor EL, cyclosporin A and Ro 11-2933/001 had significant effects on DN cytotoxicity, total DN accumulation and efflux, only if P-gp was present . A flow-through system was used to study the mechanism of DN transport . For the first time, evidence for saturation of an active transport of DN from the cells is reported . We discussed the possible presence of cooperative activity between at least two binding sites on the protein responsible for DN efflux, likely to be P-gp.

Int J Radiat Oncol Biol Phys, 1991 Feb, 20(2), 361 - 7
Keynote address: multidrug resistance: a pleiotropic response to cytotoxic drugs; Fairchild CR et al.; Tumor cells exposed in tissue culture to one of several different classes of antineoplastic agents, including anthracyclines, vinca alkaloids, epipodophyllotoxins, and certain antitumor antibiotics, can develop resistance to the selecting agent and cross resistance to the other classes of agents . This phenomena of multidrug resistance is generally associated with decreased drug accumulation and overexpression of a membrane glycoprotein . This membrane protein, referred to as P-glycoprotein, apparently acts as an energy-dependent drug efflux pump . Multidrug resistance in human MCF-7 breast cancer cells selected for resistance to adriamycin (AdrR MCF-7) is associated with amplification and overexpression of the mdr1 gene which encodes P-glycoprotein . A number of other changes are also seen in this resistant cell line including alterations in Phase I and Phase II drug metabolizing enzymes . Similar biochemical changes occur in a rat model for hepatocellular carcinogenesis and are associated in that system with broad spectrum resistance to hepatotoxins . The similar changes in these two models of resistance suggests that these changes might be part of a battery of genes whose expression can be altered in response to cytotoxic stress, thus rendering the cell resistant to a wide variety of cytotoxic agents.

J Biol Chem, 1991 Jan 25, 266(3), 1641 - 5
Evidence for cAMP and cholate extrusion in C6 rat glioma cells by a common anion efflux pump; Henderson GB et al.; C6 rat glioma cells were investigated for a shared unidirectional efflux system for cAMP and cholate . {3H}Cholate was accumulated (at pH 7.3) by scraped C6 cell monolayers via a process which was rapid initially and then slowed to a steady state after 10 min at 37 degrees C . Release of the accumulated label was also rapid (t1/2 = 2 min), was essentially complete within 15 min, and exhibited energy dependence since it could be blocked by antimycin A . Half-maximal inhibition by antimycin A occurred at 0.87 microM, and maximal inhibition exceeded 90% . Various other compounds also inhibited {3H}cholate efflux . The most effective was prostaglandin A1, which reduced efflux half-maximally at a concentration of 0.14 microM . Other inhibitors, prostaglandin B1, verapamil, probenecid, and bromosulfophathalein, produced half-maximal inhibition at 5.3, 42, 78, and 110 microM, respectively . Cholate efflux was also blocked by 40 microM vincristine . Initial influx of {3H}cholate was not affected by antimycin A, prostaglandin A1, or vincristine and hence was attributed to a process separate from efflux . C6 rat glioma cells also have the ability to produce high intracellular levels of cAMP in response to isoproterenol and to release cAMP into the medium via a carrier-mediated efflux system . When measured under the same conditions employed for cholate efflux, the efflux of cAMP was found to be sensitive to each of the inhibitors of cholate efflux . Moreover, plots of cAMP efflux versus varying concentrations of prostaglandin A1, antimycin A, prostaglandin B1, verapamil, and probenecid showed similar response curves and comparable values for half-maximal These results indicate that C6 rat glioma cells contain a unidirectional efflux pump for cholate and that this same system also appears to mediate the unidirectional efflux of cAMP . These findings support the hypothesis that various cells contain efflux pumps which exhibit a broad specificity for large organic anions of diverse structure and that the function of these efflux pumps resides primarily in cellular anion detoxification . Analogous efflux pumps for hydrophobic drugs are overproduced in tumor cells exhibiting multidrug resistance.

J Natl Cancer Inst, 1991 Jan 16, 83(2), 105 - 10
Systemic toxic effects associated with high-dose verapamil infusion and chemotherapy administration; Pennock GD et al.; Aside from its more conventional uses as a cardiovascular drug, the calcium channel blocker verapamil has recently been added to chemotherapeutic regimens to reduce drug resistance in B-cell and other neoplasms that express the P-glycoprotein . We recently treated patients with continuous-infusion verapamil (0.15 mg/kg per hour to 0.60 mg/kg per hour) over a 5-day period in combination with continuous-infusion vincristine and doxorubicin plus oral dexamethasone . Seventy-one courses involving 35 hospitalized patients were prospectively studied for cardiovascular and other side effects . Cardiovascular side effects were observed most frequently and consisted of first-degree heart block, hypotension, sinus bradycardia, and junctional rhythms . We observed higher degree heart block, but the QRS interval remained narrow and the ventricular escape rate remained relatively normal . Effects on mean arterial pressure, heart rate, and PR interval were both time and dose related . Severe, symptomatic congestive heart failure was rarely observed . The most common noncardiovascular side effects were constipation, peripheral edema, and weight gain . All systemic toxic effects observed were easily treated or disappeared with either temporary or permanent discontinuation of the verapamil infusion or by a decrease in the dose of verapamil . We conclude that the cardiovascular side effects associated with continuous, high-dose intravenous verapamil therapy are significant and dose limiting but are rapidly reversible . Less cardiotoxic chemosensitizers are needed to reverse multidrug resistance in cancer.

J Natl Cancer Inst, 1991 Jan 16, 83(2), 111 - 6
Clinical relevance of immunohistochemical detection of multidrug resistance P-glycoprotein in breast carcinoma; Verrelle P et al.; In 20 women with breast carcinoma, 17 of whom had locally advanced cancer and 3 of whom had confirmed metastases, the expression of P-glycoprotein was evaluated before the start of a chemotherapy regimen that included multidrug resistance-related drugs . With the use of the C494 monoclonal antibody in an avidin-biotin-immunoperoxidase technique, P-glycoprotein was detected in 17 of 20 tumor samples . Results were expressed in a semiquantitative manner, taking into account the number of positive tumor cells (N index) and the specific staining intensity (I index) . The 17 patients with nonmetastatic cancer were followed from the first cycle of chemotherapy to cancer recurrence; subsequent to six cycles of chemotherapy, all of these patients except one were rendered clinically disease-free through surgery and/or radiation . The end point was defined as either local/regional recurrence or metastasis . Strong P-glycoprotein-positive staining in a majority of tumor cells (the N+/I+ phenotype) was significantly correlated with no initial response to chemotherapy (P less than .02) and with a shorter progression-free survival (P less than .02) . Thus, the pretreatment evaluation of P-glycoprotein expression may be of prognostic value in patients with locally advanced breast cancer.

Cancer Res, 1991 Jan 15, 51(2), 521 - 7
Increased glutathione peroxidase activity in a human sarcoma cell line with inherent doxorubicin resistance; Samuels BL et al.; Several mechanisms of drug resistance have been defined using cell lines selected for resistance in vitro . However, the relevance of these to tumor cell resistance in vivo remains unclear . We established tumor cell lines from biopsies of human sarcomas before and after doxorubicin therapy . One pretreatment sarcoma line, STSAR90, was 6-fold less sensitive to doxorubicin than was a normal fibroblast line, AG1522 . The sensitivities of six other sarcoma lines were similar to that of AG1522 . STSAR90 cells did not overexpress P-glycoprotein mRNA, by Northern analysis with the pCHP1 complementary DNA fragment . Photoaffinity labeling with the vinblastine analogue N-(p-azido-3-125I-salicyl)-N'-beta-aminoethylvindesine did not show increased P-glycoprotein concentrations . Accumulation of {3H}daunomycin was not decreased in STSAR90 compared with a less resistant sarcoma line, STSAR11, nor was the doxorubicin sensitivity of STSAR90 increased by coincubation with verapamil . Glutathione levels were twice as high in STSAR90 as in STSAR11, and glutathione peroxidase activity was 3.5- to 6-fold higher . This was due mostly to an increase in selenium-dependent peroxidase activity . After exposure to doxorubicin, STSAR90 cells formed only half as much measurable hydroxyl radical as STSAR11, as detected by electron spin resonance spectrometry . Doxorubicin sensitivity was increased in STSAR90 cells when intracellular glutathione levels were reduced by buthionine sulfoximine . These results indicate that multidrug resistance due to P-glycoprotein-mediated drug efflux is not the only mechanism of doxorubicin resistance that occurs in sarcomas and that glutathione peroxidase-dependent detoxification of doxorubicin-induced oxygen radicals may contribute to clinical doxorubicin resistance.

Proc Natl Acad Sci U S A, 1991 Jan 15, 88(2), 547 - 51
Transgenic mice that express the human multidrug-resistance gene in bone marrow enable a rapid identification of agents that reverse drug resistance; Mickisch GH et al.; The development of preclinical models for the rapid testing of agents that circumvent multidrug resistance in cancer is a high priority of research on drug resistance . A common form of multidrug resistance in human cancer results from expression of the MDR1 gene, which encodes a Mr 170,000 glycoprotein that functions as a plasma membrane energy-dependent multidrug efflux pump . We have engineered transgenic mice that express this multidrug transporter in their bone marrow and demonstrated that these animals are resistant to leukopenia by a panel of anticancer drugs including anthracyclines, vinca alkaloids, etoposide, taxol, and actinomycin D . Differential leukocyte counts indicate that both neutrophils and lymphocytes are protected . Drugs such as cisplatin, methotrexate, and 5-fluorouracil, which are not handled by the multidrug transporter, produce bone marrow suppression in both normal and transgenic mice . The resistance conferred by the MDR1 gene can be circumvented in a dose-dependent manner by simultaneous administration of agents previously shown to be inhibitors of the multidrug transporter in vitro, including verapamil isomers, quinidine, and quinine . Verapamil and quinine, both at levels suitable for human trials that produced only partial sensitization of the MDR1-transgenic mice, were fully sensitizing when used in combination . We conclude that MDR1-transgenic mice provide a rapid and reliable system to determine the bioactivity of agents that reverse multidrug resistance in animals.

J Biol Chem, 1991 Jan 15, 266(2), 903 - 8
Interaction of organic chemicals with P-glycoprotein in the adrenal gland, kidney, and a multidrug-resistant KB cell; Ichikawa M et al.; P-glycoprotein (P-gp) is thought to mediate the transport of anti-cancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype in tumor cells . However, the function of P-gp in normal tissues is still not well understood . We present evidence indicating that the active efflux of several structurally unrelated organic compounds is mediated by P-gp in multidrug-resistant KB (KB-C2) cells and that these compounds interact with P-gp in the kidney and adrenal gland . The photoactive radioactive calcium channel blocker {3H}azidopine labels a protein of approximately 140 kDa in crude membrane fractions from human kidney and adrenal gland and a 130-kDa protein from bovine adrenal gland . These photolabeled proteins are immunoprecipitated with an anti-P-gp antibody . Photolabeling is inhibited by vinblastine, reserpine, and several other organic chemicals . These data indicate that the kidney and adrenal gland express P-gp (or a protein closely related to P-gp) that can interact with several organic compounds and that the P-gp expressed in these tissues has a drug-binding site similar to that of P-gp in KB-C2 cells . Our findings thus strongly support the hypothesis that P-gp can transport a wide variety of organic chemicals as well as anti-cancer drugs and that one of the physiological functions of P-gp is the excretion of certain classes of organic compounds.

Blood, 1991 Jan 15, 77(2), 348 - 54
Synergistic inhibition by verapamil and quinine of P-glycoprotein-mediated multidrug resistance in a human myeloma cell line model; Lehnert M et al.; In an effort to develop a clinically useful approach to overcoming P-glycoprotein-mediated multidrug resistance (MDR1), we evaluated combined chemosensitization with verapamil and quinine in a multidrug-resistant (MDR) human myeloma cell line model . In clonogenic assay, verapamil was used at concentrations from 0.1 to 1.0 micrograms/mL, bracketing the plasma levels achieved by oral administration and high-dose intravenous (IV) infusion, respectively . The dose of quinine was held constant at 1.0 micrograms/mL, a plasma concentration readily achieved by oral administration . At each dose level of verapamil tested, the combination with quinine proved more effective than either drug individually in reversing resistance to doxorubicin and vinblastine and synergistic chemosensitizing interaction was observed . Verapamil at 0.1 microgram/mL combined with quinine was capable of restoring sensitivity to doxorubicin fully and reduced resistance to vinblastine as effectively as verapamil alone at 1.0 micrograms/mL . Furthermore, the combination of 1.0 mumol verapamil with 10 mumols quinine increased accumulation and retention of anthracycline in the resistant cells to a greater extent than did either drug individually (P less than .001) and inhibited drug efflux as effectively as verapamil alone at 10 mumols . Our findings suggest that combined chemosensitization with verapamil and quinine may prove useful for overcoming MDR1 in patients with drug-refractory B-cell neoplasms such as multiple myeloma or non-Hodgkin's lymphomas.

Cancer Chemother Pharmacol, 1991, 28(2), 93 - 101
Membrane glycoprotein changes associated with anthracycline resistance in HL-60 cells; Gervasoni JE Jr et al.; The glycoproteins on the surface of HL-60/S wild-type, drug-sensitive human leukemia cells and HL-60/AR anthracycline-resistant cells which do not overexpress the P-glycoprotein, were characterized by labeling with {35S}-methionine, NaB{3H4}, phosphorus 32, or sodium iodide I 125 . HL-60/S and HL-60/AR cell lysates and membrane fractions tagged with {35S}-methionine or phosphorus 32 showed no significant differences in their protein patterns as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by autoradiography . HL-60/S cells labeled with NaB{3H4} yielded glycoproteins that were smeared predominantly in the molecular-weight range of 210,000 and 160,000 Da, with pI values ranging between pH 4 and pH 4.4 . In contrast, NaB{3H4}-labeled HL-60/AR cells showed 7-8 discrete glycoproteins within a molecular-weight range of 170,000 and 140,000 Da, with pI values also ranging between pH 4 and pH 4.4 . In addition, {3H}-glucosamine incorporation into HL-60/S and HL-60/AR cells revealed that the latter showed lower uptake of {3H}-glucosamine than did the former . Following treatment with tunicamycin, {3H}-glucosamine uptake in HL-60/S cells decreased, whereas that in HL-60/AR cells remained unchanged . Surface-membrane radioiodination of HL-60/S and HL-60/AR cells showed two distinct protein electrophoretic patterns, with differences being observed in both the high-(220-95 kDa) and low-molecular-weight ranges (21 kDa) . Flow cytometric analysis of HL-60/S and HL-60/AR cells using myeloid and lymphoid antigen-specific antibodies demonstrated no antigenic differences between HL-60/S and HL-60/AR cells . HL-60/S cells incubated in the presence of tunicamycin, an inhibitor of N-linked glycosylation, or the protein kinase C agonist phorbol 12-myristate 13-acetate (PMA) developed a glycoprotein pattern similar to that observed in HL-60/AR cells . In addition, tunicamycin treatment of HL-60/S cells decreased daunorubicin (DNR) retention and altered its intracellular distribution as compared with that in HL-60/AR cells . These data indicate that HL-60/AR cells do not possess either de novo or amplified high-molecular-weight surface-membrane proteins; instead, existing proteins are hypoglycosylated . These results also show that HL-60/AR cells exhibit the multidrug-resistant phenotype in association with altered membrane glycoproteins of both high (220-95 kDa) and low molecular weight (21 kDa), but without overexpression of the P-glycoprotein . Furthermore, in HL-60/S cells, the multidrug-resistant phenotype is partially inducible by inhibition of N-linked glycosylation of cell-surface proteins.

Biochim Biophys Acta, 1991 Jan 9, 1061(1), 106 - 10
Agents which reverse multidrug-resistance are inhibitors of {3H}vinblastine transport by isolated vesicles; Horio M et al.; Resistance of human cancer cells to multiple cytotoxic hydrophobic agents (multidrug resistance) is due to overexpression of the MDR1 gene whose product is the ATP-dependent multidrug transporter, P-glycoprotein . We have previously reported that plasma membrane vesicles partially purified from multidrug-resistant human KB carcinoma cells, but not from drug-sensitive cells, accumulated {3H}vinblastine in an ATP-dependent manner (Horio, M., Gottesman, M.M . and Pastan, I . (1988) Proc . Natl . Acad . Sci . USA 85, 3580-3584) . Certain calcium-channel blockers, quinidine, and phenothiazines are able to overcome multidrug resistance in cultured cells . In this work, the effect of these reversing agents on ATP-dependent vinblastine (VBL) transport by vesicles from drug-resistant KB cells has been characterized . Azidopine was the most potent inhibitor of ATP-dependent VBL uptake tested (ID50: concentration of inhibitor such that the transport of vinblastine is inhibited by 50%, less than 1 microM) . Verapamil, quinidine, and the tiapamil analogue RO-11-2933 were potent but less effective inhibitors (ID50 less than 5 microM) . Diltiazem, nifedipine and trifluoperazine were even less effective . These agents had no effect on Na(+)-dependent and Na(+)-independent L-leucine uptake by the vesicles, indicating that the inhibition of ATP dependent VBL transport by these agents is not a non-specific effect, as might result from leaks in the vesicle membrane . Verapamil, quinidine, azidopine and trifluoperazine increased the apparent Km value of vinblastine transport, suggesting that these agents may be competitive inhibitors of vinblastine transport.

Annu Rev Med, 1991, 42, 277 - 86
Multidrug resistance; Pastan I et al.; Laboratory investigations indicate that cancer cells can become simultaneously resistant to many different chemotherapeutic drugs that are natural products via the expression of an energy-dependent drug efflux pump . This multidrug transporter is a plasma membrane glycoprotein encoded in the human by the MDR1 gene . Recent clinical studies indicate that expression of the multidrug transporter plays a major role in the intrinsic and acquired resistance to chemotherapy of many human cancers . Strategies aimed at inactivating this drug efflux pump may have significant impact on the treatment of human cancer.

Bull Cancer, 1991 Jan, 78(1), 91 - 7
{Activation of N-myc oncogene and associated genes to chemoresistance, prognostic value for neuroblastoma}; Benard J et al.; N-myc amplification and ploidic index are important prognosis factors in neuroblastoma . Combined overexpression of the multidrug resistance gene, MDR1 and of Glutathion-S-Transferase, GST-pi may be related to the response of tumors to chemotherapy . Measurements of these parameters may contribute to define responders and non-responders to chemotherapy and provide the clinician with additional criteria to manage treatment.

Anticancer Res, 1991 Jan-Feb, 11(1), 473 - 80
Oncogene and MDR1 gene expression in rat rhabdomyosarcoma sublines of different metastatic potential; Hanania N et al.; The expression of various protooncogenes (myc, Ki-ras, Ha-ras, erbB, fms, sis, jun and fos) and a gene implicated in multidrug resistance (mdr1) was investigated in cell sublines, isolated from a rat rhabdomyosarcoma cell line, and in the corresponding tumors induced by injection of these cells into syngeneic rats . These cell lines and tumors, selected or not by treatment with chlorozotocin (Czt) or adriamycin (Adr), differed in their tumorigenicities and metastatic potentials . Our results showed that 1) an increased expression of some protooncogenes could be correlated with the metastatic potential of tumors; 2) such a correlation was not observed in the cultured cells from which these tumors were derived; 3) mdr1 expression, similarly to that of protooncogenes, was correlated with metastatic potential in all tumors except the Adr-selected tumors.

Anticancer Res, 1991 Jan-Feb, 11(1), 241 - 8
Simultaneous amplification of epidermal growth factor-receptor and multidrug-resistance genes in a newly . Established human lung cancer cell line; Shin HJ et al.; We have investigated amplification and expression of the epidermal growth factor (EGF)-receptor gene and the multidrug-resistance (MDRI) gene in a newly established lung adenocarcinoma cell line and its subcell lines . Of these cell lines, the EGF-receptor was amplified (64-fold) and rearranged (6.5 and 5.4 Kb with EcoRI restriction) in an early passage cell line, DMS-4CS . MDRI gene amplification (10.5-fold), accompanied by rearrangement (4.6 and 3.7 Kb with Hind III restriction), was found in the same cell line . All other subcell lines derived from DMS-4C5 failed to demonstrate any amplification of these genes . Moreover, mRNA expression of these two genes was not overproduced . Our findings seem to indicate that highly selected cells at an early stage have undergone spontaneous amplification of these genes . However, cells containing such amplified DNA sequences may exhibit growth disadvantage, and, therefore, the amplified DNA sequences were not present in the sublines . The cytogenetic study demonstrated that early passage cells, DMS-4C6, only showed one unique marker chromosome, M10, which may represent amplification of these genes in question.

Acta Oncol, 1991, 30(1), 87 - 105
Genetic mechanisms of drug resistance . A review; Borst P; An overview of our present understanding of mechanisms of resistance against cytotoxic drugs is presented . Most of this understanding has come from studies on tumor cells made resistant in vitro, but there is reason to think that similar mechanisms are responsible for resistance in patients . After a brief overview of biochemical mechanisms of drug resistance, the types of mutations in tumor cells that can alter drug handling are discussed . Three examples of resistance are analysed in more detail: resistance to the folate analogue methotrexate; the multidrug resistance caused by increased levels of P-glycoprotein, which extrudes drugs from the cell; and resistance to alkylating agents.

Cancer Chemother Pharmacol, 1991, 27(5), 413 - 5
Effect of verapamil on daunorubicin accumulation in lymphocytes isolated from patients undergoing chemotherapy; McGown AT et al.; Verapamil was shown to be capable of increasing intracellular daunorubicin levels in normal lymphocytes isolated from patients undergoing chemotherapy for epithelial ovarian cancer . The extent of the increase in daunorubicin accumulation was variable, occurring in the range of 0-123% as compared with intracellular daunorubicin levels attained in the absence of verapamil . No similar effect was seen in lymphocytes isolated from healthy volunteers . A tentative explanation of these data may be the induction of multidrug resistance (mdr)-like characteristics in normal lymphocytes following cytotoxic chemotherapy.

Cancer Res, 1991 Jan 1, 51(1), 67 - 72
Effect of buthionine sulfoximine on toxicity of verapamil and doxorubicin to multidrug resistant cells and to mice; Ford JM et al.; Resistance of tumor cells to chemotherapeutic drugs may be due to several mechanisms within a single cell line . Resistance to doxorubicin in the human multidrug resistant breast cancer cell line, MCF-7 AdrR, has been attributed to increased glutathione (GSH) S-transferase and GSH peroxidase activity, as well as to increased expression of the mdr1 gene product, P-glycoprotein . We studied the potentiation of doxorubicin activity in these cells by buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, and by verapamil and trans-flupenthixol, agents which interact with P-glycoprotein . Treatment with BSO enhanced the effect of doxorubicin by 1.5-fold, while verapamil or transflupenthixol caused a greater reversal of drug resistance . The combination of BSO with trans-flupenthixol produced no further potentiation of doxorubicin activity . However, the combination of BSO with verapamil and doxorubicin caused up to a 10-fold increment in antiproliferative effect . To explore the mechanism by which BSO interacted with this drug combination, we determined whether or not BSO might potentiate the effects of verapamil . These studies demonstrated that the effects of BSO were predominantly due to an increase in verapamil toxicity rather than to doxorubicin toxicity . In addition, when mice received concentrations of BSO in their drinking water sufficient to deplete GSH and were treated with verapamil, the calcium channel blocker was lethal to 9 of 12 mice receiving BSO compared to 1 of 10 control animals receiving verapamil alone . These studies demonstrate that BSO does not markedly increase the pharmacological effect of doxorubicin against MCF-7 AdrR cells and suggest that alterations in GSH and related enzymes are not a major factor in drug resistance in this cell line . Furthermore, BSO can increase the toxicity of verapamil, a finding which may have important implications for clinical trials.

Cancer Res, 1991 Jan 1, 51(1), 157 - 61
Cellular pharmacology of MX2, a new morpholino anthracycline, in human pleiotropic drug-resistant cells; Watanabe M et al.; We previously reported that MX2, a new morpholino anthracycline, showed marked effects on pleiotropic drug-resistant sublines of murine P388 leukemia in vivo as well as in vitro . In this study we examine the in vitro cytotoxicity against pleiotropic drug-resistant sublines of human tumor cell lines . MX2 was effective against multidrug-resistant sublines of four human tumor cell lines; these cells, having a 4.8- to 200-fold cross-resistance to Adriamycin (ADM) showed only a 0.7- to 2.3-fold resistance to MX2 compared with the sensitive cells . To elucidate the mechanism by which MX2 overcomes multidrug resistance, the intracellular pharmacology of MX2 in human myelogenous leukemia K562 and its ADM-resistant subline (K562/ADM) was examined . Both K562 and K562/ADM cells accumulated MX2 more easily than ADM, and the intracellular accumulation of MX2 attained a steady state in both cell lines within 30 min of incubation at 37 degrees C . The amount of MX2 that accumulated in K562/ADM at a steady state was only 1.3 times lower than that in K562 . However, ADM was accumulated slowly in both cell lines compared with MX2, and the intercellular concentration reached a steady state in K562/ADM after 90 min of incubation and in K562 after more than 120 min . K562/ADM cells accumulated a 3.3-fold lower concentration of ADM than K562 after 120 min of exposure . The steady-state concentration of ADM in K562/ADM was 8.3 times lower than that of MX2 . In addition, greater than 70% of MX2 was retained in both cell lines after 150 min of incubation in the absence of this drug . Verapamil, a calcium antagonist, hardly augmented the cytotoxicity of MX2 against K562/ADM, and no distinct effect of this drug on both the time course and the maximal level of accumulation of MX2 was observed . Interestingly, MX2 effectively inhibited ATP/Mg2(+)-dependent {3H}vincristine binding to K562/ADM membrane preparations, indicating that MX2 could be transported outside the cell by an active efflux pump . The high intracellular accumulation and retention of MX2 in K562/ADM through the rapid influx of the drug into the cells may be one of the reasons why MX2 circumvents pleiotropic drug resistance.

Proc Natl Acad Sci U S A, 1991 Jan 1, 88(1), 84 - 8
Structural model of the nucleotide-binding conserved component of periplasmic permeases; Mimura CS et al.; The amino acid sequences of 17 bacterial membrane proteins that are components of periplasmic permeases and function in the uptake of a variety of small molecules and ions are highly homologous to each other and contain sequence motifs characteristic of nucleotide-binding proteins . These proteins are known to bind ATP and are postulated to be the energy-coupling components of the permeases . Several medically important eukaryotic proteins, including the multidrug-resistance transporters and the protein encoded by the cystic fibrosis gene, are also homologous to this family . By multiple sequence alignment of these 17 proteins, the consensus sequence, secondary structure, and surface exposure were predicted . The secondary structural motifs that are conserved among nucleotide-binding proteins were identified in adenylate kinase, p21ras, and elongation factor Tu by superposition of their known tertiary structures . The equivalent secondary structural elements in the predicted conserved component were located . These, together with sequence information, served as guides for alignment with adenylate kinase . A model for the structure of the ATP-binding domain of the permease proteins is proposed by analogy to the adenylate kinase structure . The characteristics of several permease mutations and biochemical data lend support to the model.

C R Acad Sci III, 1991, 313(3), 171 - 4
{Vectorisation of doxorubicin in nanospheres and reversion of pleiotropic resistance of tumor cells}; Treupel L et al.; A multidrug resistance human cell line (Dox-R-MCF7), originating from mammary adenocarcinoma was compared to the drug sensitive parent line (MCF7) to determine whether or not the use of doxorubicin-loaded biodegradable nanospheres (NS-Dox) could circumvent drug resistance . The Dox-R-MCF7 line was shown to resist free doxorubicin (Dox) as the 50% lethal dose for them was 150 times higher than for the sensitive cell-line . Isohexylcyanoacrylate nanospheres, porous matrices of biodegradable innocuous polymer, diameter 300 nm, were loaded with doxorubicin . Free Dox, NS-Dox and Dox-free polymer (NS) were added to the culture medium for 6 hrs . In terms of 50% lethal dose, NS-Dox were cytotoxic for the resistant cell line to an identical extent as for the sensitive parent line . Dox-free nanospheres alone or mixed with free-Dox were noncytotoxic for the resistant line . Drug targetting could be of main importance to overcome multidrug resistance.

Cancer Chemother Pharmacol, 1991, 28(4), 255 - 8
Quercetin inhibits the growth of a multidrug-resistant estrogen-receptor-negative MCF-7 human breast-cancer cell line expressing type II estrogen-binding sites; Scambia G et al.; It has been demonstrated that the flavonoid quercetin (3,3',4',5,7-pentahydroxyflavone; Q) inhibits the growth of several cancer cell lines . There is evidence suggesting that the antiproliferative activity of this substance is mediated by the so-called type II estrogen-binding site (type II EBS) . We looked for the presence of type II EBS and the effect of Q on the proliferation of an Adriamycin-resistant estrogen-receptor-negative human breast-cancer cell line (MCF-7 ADRr) . By whole-cell assay using estradiol labelled with 6,7-tritium {( 3H}-E2) as a tracer, we demonstrated that MCF-7 ADRr cells contain type II EBSs . Competition analysis revealed that diethylstilbestrol (DES) and Q competed with similar potency for {3H}-Es binding to type II EBSs . The antiestrogen tamoxifen (TAM) competed for type II EBSs, albeit to a lesser extent than either DES or Q . Growth experiments demonstrated that Q and DES exerted a dose-dependent inhibition of cell proliferation in the range of concentrations between 10 nM and 10 microM, whereas TAM was less effective . Q could also inhibit colony formation in a clonogenic assay . Our results indicate that multidrug-resistant estrogen-receptor-negative MCF-7 cells express type II EBSs and are sensitive to the inhibitory effect of Q . This substance could be the parent compound of a novel class of anticancer agents.

Acta Paediatr Hung, 1991, 31(2), 223 - 31
Decreased sensitivity of cytostatic drugs in glucocorticoid receptor-free acute myeloid leukaemia cells . Clinical and experimental observations; Kiss C et al.; Preliminary clinical observations have suggested that low cellular glucocorticoid receptor (GR) levels might have been connected with multidrug resistance in children with acute myeloblastic leukaemia (AML) . To test this possibility, we have developed glucocorticoid resistant subclones of two recently established human myeloid leukaemic cell lines . The cause of glucocorticoid resistance was GR negativity in these subclones . GR positive parent cell lines or GR negative subclones were incubated for 1 h in the presence of Adriamycin, Cytosine-arabinosid, Etoposide or Vincristine, respectively . After short-term (1 h) incubation in suspension cultures cells were washed and plated in clonogenic agar cultures . Each anticancer drug was more potent against both GR positive parent cell lines than against the GR negative subclones . The results of this study suggest that the absence of GRs is a useful marker of multidrug resistance in childhood AML.

Anticancer Res, 1991 Jan-Feb, 11(1), 455 - 9
Combination of cyclosporin A and buthionine sulfoximine (BSO) as a pharmacological strategy for circumvention of multidrug resistance in small cell lung cancer cell lines selected for resistance to doxorubicin; Larsson R et al.; The small cell lung cancer (SCLC) cell lines U-1285 and U-1690 were adapted to growth in continuous presence of doxorubicin (Dox) . The resulting cell lines U-1285R and U-1690R were investigated with respect to sensitivity to the glutathione (GSH) depleting agent buthionine sulfoximine (BSO) and the immunosuppressant cyclosporin A (CsA) as well as the Dox resistance modifying ability of these agents . The parental U-1285 cells were more sensitive to BSO compared to parental U-1690 and the multidrug resistant (MDR) sublines, whereas no difference in sensitivity to CsA was observed between parental and MDR lines . BSO (10 microM) or CsA (1 microgram/ml) alone were able to partially reverse Dox resistance in the MDR cell lines, CsA being only marginally active in U-1285R cells . However, the combination of these two drugs at the same concentrations completely reversed Dox resistance in the MDR U-1690R cells whereas the combination was less effective in the U-1285R cells . The results demonstrate that a combination of low concentrations of BSO and CsA, only partially active by themselves in modifying Dox resistance, may be used as a pharmacological strategy to increase Dox sensitivity in some MDR SCLC cells.

Cancer Chemother Pharmacol, 1991, 27(6), 445 - 50
Detection of multidrug resistance and quantification of responses of human tumour cells to cytotoxic agents using flow cytometric spectral shift analysis of Hoechst 33,342-DNA fluorescence; Smith PJ et al.; We describe the application of a flow cytometric technique for assessing the radiation or drug sensitivity characteristics of human tumour cells . The technique makes use of the phenomenon that a red shift occurs in the fluorescence emission spectrum of a DNA-specific dye (Hoechst 33,342) as an increasing number of dye molecules bind to nuclear DNA . Intact, viable cells undergo a time-dependent spectral shift that can be distinguished from the rapid shift observed in cells with damaged membranes by the use of multiparametric flow cytometry . The responses of various human cell lines were compared, namely, those of normal and ataxia-telangiectasia (A-T) lymphoblastoid lines, a small-cell lung carcinoma line and its (in vitro) derived multidrug-resistant variants . A close correlation was found between dye toxicity and the degree of DNA binding of Hoechst 33,342 independent of cellular DNA content, with lymphoblastoid and multidrug-resistant small-cell lung cancer cells showing enhanced and restricted dye-binding rates, respectively . VP16- and radiation-induced cell kill was found to result in a quantifiable increase in the fraction of cells undergoing a rapid spectral shift and was capable of detecting the increased radiation sensitivity of A-T-derived cells . Spectral shift analysis provides a rapid method for assessing the responses of tumour cells to cytotoxic agents and for determining the general ability of cells to protect cellular DNA from a model DNA-binding agent (Hoechst 33,342) that participates in the multidrug resistance phenotype.

Eur J Cancer, 1991, 27(1), 6 - 8
Failure of GM-CSF to influence the growth of small cell and non-small cell lung cancer cell lines in vitro; Twentyman PR et al.; The effects of human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) 1-1000 U/ml on the growth of human lung cancer cell lines have been studied in vitro . A panel of 10 small cell, 1 adenocarcinoma and 1 large cell lines was used with multidrug resistant sublines of 3 of the panel . The MTT assay was used to quantify cell numbers after 6-8 days' growth in the presence of GM-CSF . Neither growth inhibition nor stimulation of any of the cell lines in the presence of GM-CSF was observed . Any effects of this agent on residual tumour cells may not therefore present a problem during its clinical use to stimulate marrow regeneration after high-dose chemotherapy for lung cancer.

Br J Cancer, 1991 Jan, 63(1), 75 - 83
Chemosensitivity testing of small cell lung cancer using the MTT assay; Campling BG et al.; A simple colorimetric test, the MTT assay, has been adapted for chemosensitivity testing of human small cell lung cancer cell lines, and fresh tumour samples . Optimal conditions for clinical chemosensitivity testing were determined using established SCLC lines . Nineteen different chemotherapeutic agents were tested, and sixteen of them were found to be cytotoxic in this assay system . The drug sensitivity of a panel of 16 SCLC cell lines was measured and compared . There was very little intraexperiment variation, but the interexperiment variation was significant . Cell lines which were derived from patients who had not received chemotherapy at the time the cell line was established were more sensitive (to all but one of the drugs) than lines derived from treated patients, and the differences were statistically significant for two of the drugs . One cell line, NCI-H209, which was derived from an untreated patient, stood out as being the most sensitive or among the most sensitive to all of the drugs tested . Another cell line, H69AR, which is a multidrug resistant subline of the cell line NCI-H69, was the most resistant to many of the natural product drugs tested . Multiple drug chemosensitivity testing was performed on eight fresh tumour samples from SCLC patients (five pleural effusions, one lymph node, and two primary tumours) . It was possible to perform chemosensitivity testing on all of the clinical samples in which sufficient tumour cells were available . The drug sensitivity of the clinical samples was, in most cases, within the same range as for the cell lines . Since this assay is very rapid and simple to perform, it may have practical applications in clinical drug sensitivity testing of human tumours.

Crit Rev Oncol Hematol, 1991, 11(1), 43 - 64
New anthracycline antitumor antibiotics; Muggia FM et al.; Doxorubicin is an essential component of the treatment of aggressive lymphoma, childhood solid tumors, bone and soft tissue sarcomas, and breast cancer and additional indications are emerging . On the other hand, daunorubicin has occupied the central position of interest in the treatment of acute leukemia . Epirubicin has a spectrum very similar to doxorubicin but lesser toxicity . The ability to protect against cardiotoxicity with ICRF-187 further enhances clinical interest in exploiting modifications in doze intensity to therapeutic advantage . Idarubicin has at least equivalent activity to daunorubicin and doxorubicin in leukemia . New areas of research in relation to anthracycline antibiotics include introduction of new the analogs, insight into mechanisms of resistance, the reversal of multidrug resistance in vitro, the protection of cardiac toxicity, and the study of other important biochemical reactions relevant to cytotoxicity . Orally active anthracyclines such as idarubicin and compounds which lack cross-resistance with the parent drugs or have other mechanisms for cytotoxicity are being developed . It is likely that these modifications will lead to an expanding therapeutic spectrum for these already widely useful drugs.

Eur J Cancer, 1991, 27(6), 739 - 44
Bepridil in combination with anthracyclines to reverse anthracycline resistance in cancer patients; van Kalken CK et al.; The use of calcium antagonists as multidrug resistance reversing agents is limited by acute cardiac toxicity which, for verapamil, becomes prohibitive when concentrations in plasma approach those required in vitro for its action . A new calcium antagonist, bepridil, is as active as verapamil in reversing drug resistance in vitro . In addition, bepridil has some more favourable pharmacological properties compared with verapamil and other calcium antagonists . 14 patients with progressive advanced cancer, resistant to doxorubicin or epirubicin, were treated with the same anthracycline in combination with bepridil . Bepridil was administered in a continuous 36 h infusion at 22 mg/kg/36 h, with a dose scheme which should result in a steady state plasma concentration of approximately 5 mumol/l, able to reverse anthracycline resistance in vitro . Pharmacokinetic studies demonstrated a median bepridil plasma concentration of 5.3 mumol/l (range 2.6-19.3 mumol/l), at the time of administration of the anthracycline . No acute cardiac toxicity was observed and apparently bepiridil did not induce an increase or change in anthracycline toxicity . However, 2 patients developed overt chronic heart failure after treatment discontinuation, which caused 1 patient's death, and a significant reduction in left ventricular ejection fraction was seen in 4 patients . This chronic cardiac toxicity could be related to the total anthracycline dose received . 5 patients attained short lasting minor responses, 3 had stable disease and 6 progressed . Immunohistochemical studies in 7 tumours failed to reveal P-glycoprotein expression . Further trials with escalating doses of bepridil in combination with multiple drug resistance related anticancer agents are warranted.

Eur J Cancer, 1991, 27(4), 482 - 7
Inhibition of growth of colon 38 adenocarcinoma by vinblastine and colchicine: evidence for a vascular mechanism; Baguley BC et al.; Vinblastine or colchicine, administered intraperitoneally to B6D2F1 mice with advanced subcutaneous colon 38 tumours, induced substantial tumour growth delays with progressive development of haemorrhagic necrosis beginning within 8 hours of treatment . Two multidrug-resistant P388 leukaemia sublines, refractory to vinblastine and vincristine when grown as intraperitoneal ascites, were sensitive to necrosis induction when grown as subcutaneous tumours . Vascular labelling with two fluorescent markers indicated that vincristine substantially reduced tumour blood flow within 4 hours after treatment . The effects of vinblastine, vincristine and colchicine were similar to those of tumour necrosis factor alpha in that: (a) similar tumour necrosis and blood flow changes were induced, (b) coadministration of the serotonin antagonist cyproheptidine prevented tumour necrosis and (c) plasma nitrate levels were elevated, indicative of the stimulation of oxidation of L-arginine to nitric oxide . The results suggest that vinca alkaloids and colchicine act on solid tumours by host cell-mediated vascular effects as well as by direct tubulin-mediated cytotoxicity.

Cancer Res, 1991 Jan 1, 51(1), 55 - 61
Protein kinase C activity and multidrug resistance in MOLT-3 human lymphoblastic leukemia cells resistant to trimetrexate; Schwartz GK et al.; It has been suggested that protein kinase C (PKC) plays a role in multidrug resistance (MDR) . In this study we assayed PKC activity in MOLT-3 human acute lymphoblastic leukemia cells and found an approximately 50% decrease in activity in MDR sublines made resistant to the lipophilic antifolate trimetrexate, when compared with trimetrexate-sensitive parent cells . The PKC activity of a methotrexate-resistant subline without MDR (MOLT-3/MTX10,000) was identical to that of parent cells . Although a downward trend was noted in PKC activity in the membrane fraction of cells with increasing trimetrexate resistance, there was no absolute correlation between the degree of MDR and the relative decrease in PKC activity . Using the same method, we also confirmed an over 6-fold increase in PKC activity in the MDR human breast cancer subline MCF-7/DOXR when compared with the sensitive parent cell line, MCF-7/WT . Because of this divergent relationship between relative PKC activity and MDR, we tested the effect of PKC inhibition and activation on drug resistance . The PKC inhibitor staurosporine, at both subtoxic and toxic concentrations as well as at concentrations shown to be inhibitory to PKC, failed to increase drug resistance of parent and resistant MOLT-3 cells and decrease drug resistance of MCF-7/WT and MCF-7/DOXR cells . Short-term exposure to 3-phorbol-12-myristate-13-acetate, which activated PKC 7.0-fold and 4.7-fold, respectively, in the membrane of MOLT-3 and resistant cells, resulted in small (1.3- to 1.8-fold), approximately equivalent, increases (rather than decreases) in resistance to doxorubicin, whereas for vincristine no consistent trend was observed . Identical results were also obtained with phorbol-12,13-dibutyrate . These results indicate that PKC activity can be decreased and increased in MDR cells . Both staurosporine inhibition and phorbol ester activation failed to produce changes in drug resistance that would be considered consistent with the resulting degree of PKC activity . Short-term phorbol ester exposure can change the sensitivity of the cells to doxorubicin without changing the relative drug resistance . PKC activity in these cells may then be unrelated to MDR.

Eur J Cancer, 1991, 27(12), 1639 - 42
Resistance modification by PSC-833, a novel non-immunosuppressive cyclosporin {corrected}; Twentyman PR et al.; A novel non-immunosuppressive cyclosporin {corrected}, PSC-833, has been tested for its ability to circumvent resistance to doxorubicin, vincristine and colchicine in human and murine multidrug resistant (MDR) cell lines . This compound is shown to be a highly potent resistance modifier, being 7-10-fold more potent than the parent compound, cyclosporin A, whilst approximately equal to cyclosporin A in the growth inhibitory effects of compound alone . Reversal of the P-glycoprotein-associated MDR drug accumulation defect is a major component of resistance reversal for PSC-833, as it is for cyclosporin A.

Sel Cancer Ther, 1991 Fall, 7(3), 103 - 12
Overcoming murine tumor cell resistance to vinblastine by presentation of the drug in multilamellar liposomes consisting of phosphatidylcholine and phosphatidylserine; Seid CA et al.; We determined whether vinblastine (VLB) encapsulated within multilamellar vesicle-liposomes (MLV) would reverse target cell resistance to the drug exhibited by the UV-2237M murine fibrosarcoma and its Adriamycin (ADR)-selected multidrug resistant (MDR) variants . Resistant fibrosarcoma cells were grown in medium containing 1 and 10 micrograms/ml ADR to yield the MDR lines UV-2237M/ADRR (ADR-1) and UV-2237M/ADRRR (ADR-10), respectively . VLB encapsulated in MLV composed of phosphatidylcholine (PC) and phosphatidylserine (PS) (7:3 molar ratio) was hydrophobic, occupied an internal space equivalent of 6.13 microliters/mumol, and was stable in medium at 37 degrees C for up to 6 days . The 50% inhibitory concentrations (IC50) of VLB were 2, 25, and 70 ng/ml for the parent, ADR-1, and ADR-10 cell lines, respectively . VLB in MLV significantly enhanced sensitivity of tumor cells to VLB . The respective IC50 of liposomal VLB were 0.5, 5.7, and 12 ng/ml for the parent, ADR-1, and ADR-10 lines . MLV containing saline were not toxic to the cells . These data indicate that presentation of VLB entrapped in PC:PS MLV provides a method to overcome tumor cell resistance to this drug.

Leuk Res, 1991, 15(12), 1139 - 43
Expression of a multidrug-resistance gene in human malignant lymphoma and related disorders; Dan S et al.; The expression of mdr1 gene was measured to determine whether it plays a role in clinical resistance to chemotherapy of human malignant lymphomas . mdr1 expression was found in 4 of 9 cases resistant to chemotherapy . Expression of mdr1 was not detectable in any of 7 chemotherapy-sensitive tumors . The 2 cases of reactive lymphadenitis and the 3 samples of normal mononuclear cells did not show any expression of mdr1 gene, either . These results indicate that expression of the mdr1 gene is not always detectable in cases of malignant lymphoma resistant to chemotherapy, but the detectable expression of mdr1 gene may predict clinical resistance to chemotherapy.

Cancer Chemother Pharmacol, 1991, 29(2), 127 - 32
Multidrug resistance in MCF-7 human breast cancer cells is associated with increased expression of nucleoside transporters and altered uptake of adenosine; Morgan PF et al.; The rate of adenosine uptake and the corresponding expression of nucleoside transporters were studied in several MCF-7 human breast-cancer cell lines that express different levels of multidrug resistance (MDR) . Kinetic studies of adenosine transport in these cell lines revealed that the mean apparent Km and Vmax values for the nucleoside transporters increased with increasing MDR . The apparent Km and the apparent Vmax of Adriamycin-resistant (ADR10) cell lines were respectively 3.2- and 1.8- fold those of Adriamycin-sensitive wild-type (WT) cells (P less than 0.001) . A partially revertant cell line (ADR10rev) that was derived from the ADR10 line and was partially sensitive to Adriamycin exhibited apparent Km and Vmax parameters that lay between those of the ADR10 and WT cells (P less than 0.001 vs ADR10 cells; P less than 0.05 vs WT cells) . ADR10 cell membranes bound greater than 4 times more of the nucleoside transporter blockers {3H}-nitrobenzylthioinosine {( 3H}-NBI) and {3H}-dipyridamole {( 3H}-DPR) than did WT cell membranes per unit protein (P less than 0.0001) . Scatchard analysis revealed a 2-3 times greater density for nucleoside transporters in ADR10 membranes as compared with those in WT membranes . ADR10rev membranes bound less {3H}-NBI and {3H}-DPR than did ADR10 membranes (P less than 0.001), but they bound more of the blockers than did WT membranes (P less than 0.05) . A 2.5-h exposure to 200 nM phorbol-12,13-dibutyrate (PDBu), which activates protein kinase C (PKC) and induces WT cells to exhibit a 4-fold increased transient MDR phenotype, increased the apparent Km of WT cells for adenosine transport by greater than 2 times (P less than 0.001) to a value close to that found for the ADR10 cells . An identical exposure of ADR10 cells to PDBu produced no significant effect . The apparent Km of ADR10rev cells was increased 1.4 times by a 2.5-h PDBu exposure . None of the cell lines were affected by a 2.5-h exposure to 200 nM phorbol-13,10-diacetate (PDA), a much less active phorbol, or vehicle . These results suggest that MDR in MCF-7 cells is associated with changes in nucleoside transport, including both the number of transporters and their rate of transport, and that such changes can be partially mimicked by stimulation of PKC.

Eur J Clin Pharmacol, 1991, 41(2), 161 - 4
Pharmacokinetics of halofantrine and n-desbutylhalofantrine in patients with falciparum malaria following a multiple dose regimen of halofantrine; Veenendaal JR et al.; Halofantrine is a new blood schizontocidal drug used for the treatment of multidrug-resistant falciparum malaria . The pharmacokinetics of halofantrine (HAL) and its principal metabolite, N-desbutylhalofantrine (BHAL), was investigated in 6 adult male patients of Melanesian origin with uncomplicated falciparum malaria . The patients received 500 mg of halofantrine hydrochloride at times 0, 6 and 12 h (total 1.5 g) . All patients responded to treatment with a mean parasite clearance time of 52.7 h and a mean fever clearance time of 33.8 h . The following kinetic parameters (mean values) were determined for HAL and BHAL, respectively: maximum plasma concentration (Cmax) = 896 and 491 ng.ml-1; time to reach the Cmax (tmax) = 15 and 56 h; elimination half-life (t1/2) = 91 and 79 h and the mean residence time (MRT) = 71 and 102 h . Based on the clinical response the plasma concentrations of HAL and BHAL were adequate for the treatment of uncomplicated falciparum malaria in the 6 patients.

Acta Neuropathol (Berl), 1991, 82(6), 516 - 9
The multidrug-resistance gene MDR1 is expressed in human glial tumors; Becker I et al.; The most consistantly reported alteration of multidrug-resistant carcinoma cells is the overexpression of a membrane glycoprotein, termed P-glycoprotein . In this study we examined whether the strong intrinsic chemotherapy resistance of glial tumors might be related to the expression of the MDR1 gene which codes for P-glycoprotein . Fourteen glial tumors were examined immunohistochemically using the monoclonal antibody C219 . In addition, RNA samples of 11 of these tumors were analysed using a sensitive Northern blot assay . P-glycoprotein is expressed in all 14 glial tumors; the number of stained tumor cells, however, varied considerably ranging from 0.3% to 15% . There was no correlation between the number of MDR1-positive cells and the histological malignancy . Varying amounts of MDR1 mRNA were detectable in 7 from 11 examined tumors . The results of our study show that the MDR1 gene is expressed in human glial tumors and suggest that the multidrug transporter may contribute to the clinical non-responsiveness of these tumors to chemotherapy.

Cancer Immunol Immunother, 1991, 33(4), 274 - 7
Differential growth inhibition and enhancement of major histocompatibility complex class I antigen expression by interferons in a small-cell lung cancer cell line and its doxorubicin-selected multidrug-resistant variant; Cole SP et al.; Expression of class I and class II major histocompatibility complex antigens on a human small-cell lung cancer cell line and its multidrug-resistant variant was examined before and after exposure to interferon alpha (IFN alpha) and IFN gamma by flow cytometry . Neither IFN alpha nor IFN gamma induced class II antigen expression on the drug-sensitive or resistant cell line . Induction of class I antigen expression along with an inhibition of proliferation was observed in both cell lines after IFN alpha treatment . On the other hand, IFN gamma treatment resulted in growth inhibition and enhancement of class I antigen expression in the sensitive cell line but not the resistant cell line . The differential response of the two cell lines to IFN gamma cannot be directly attributed to the acquisition of drug resistance but it suggests that further investigation of the possibility that drug-sensitive and resistant small-cell lung tumors may respond differently to immunotherapies that include IFN gamma is warranted.

Patol Pol, 1991, 42(4), 119 - 25
{P glycoprotein and multidrug resistance}; Kaczorowski S et al.; Drug resistance has been shown to be associated with the expression of P-glycoprotein (P-gp), the product of mdr-1 gene . One of the suggested mechanisms for the phenomenon called Multidrug Resistance (MDR) is related to the overexpression and amplification of the mdr-1 gene . The product of this gene is called P-gp and it has been considered as a potential marker for drug resistance . Transfection of the mdr-1 gene into drug-sensitive cells confers the role of mdr-1 gene in developing MDR phenotype . Structural analysis of homologous cDNA, responsible for production of transport proteins, from: MDR cell lines, bacteria and yeast, revealed high similarity . Molecular structure analysis indicate that P-gp has nucleotide binding sites . It has been established that P-gh has an internal ATP-ase activity, thus it can act as energy dependent transport protein . The expression of P-gp has been found in neoplastic and normal tissues (as adrenal glands, kidney, liver, pancreas, jejunum and large intestine), as well as in several cell lines which have been induced to become resistant to cytostatics . The aim of present study was to review the role of P-gp expression in laboratory and clinic.

Cancer Treat Res, 1991, 57, 57 - 99
Resistance to inhibitors of DNA topoisomerases; Sullivan DM et al.; Studies examining the mechanisms of resistance to camptothecin and its water-soluble analogs have been reported only recently . None of these studies have involved resistance derived in vivo in humans . Some of the mechanisms already describe could be predicted from the mechanism of action of the drug and from prior studies in yeast . It is interesting that, to date, the only mechanisms of resistance relate directly to the target of the drug, DNA topoisomerase I, and that the drugs are active in cell lines exhibiting the multidrug-resistant phenotype . Should camptothecin analogs prove as active in human clinical trials as animal tests predict, it will be interesting to see if additional mechanisms of resistance emerge from studies in treated patients . On the other hand, if clinical activity is similar to that demonstrated by camptothecin 15 years ago, the issue will be of academic interest only.

Cancer Treat Res, 1991, 57, 37 - 56
Molecular biology of P-glycoprotein; Cornwell MM; The molecular genetic characterization of MDR human, mouse, and hamster P-glycoprotein genes has identified several elements that may contribute to the diversity in multidrug-resistance phenotype associated with P-glycoprotein expression . First, spontaneous mutations within the MDR genes may alter the relative affinity of P-glycoprotein for certain drugs or alter the substrate specificity of the protein . Secondly, alternative splicing of MDR mRNA may result in isoforms with different substrate recognition or transport properties . Differential splicing has not thus far been demonstrated for human MDR1 or mouse mdr1a and mdr1b genes . Finally, differential expression of mdr genes encoding P-glycoprotein isoforms with distinct properties appears to be a possible mechanism for generating diversity in MDR rodent cells.

Mol Carcinog, 1991, 4(6), 499 - 509
Regulation of 2-acetylaminofluorene-and 3-methylcholanthrene--mediated induction of multidrug resistance and cytochrome P450IA gene family expression in primary hepatocyte cultures and rat liver; Gant TW et al.; Previous studies by this laboratory have indicated that expression of the multidrug resistance (mdr) gene can be increased in vivo by exposure to a variety of xenobiotics . Because of the nature of these compounds, it was proposed that mdr gene expression might, at least in part, be regulated by the arylhydrocarbon (Ah) receptor . In the present study, we used a primary hepatocyte culture model to examine the relationship between induction of cytochrome P450IA and mdr expression in vitro . Both 3-methylcholanthrene (MC) and 2-acetylaminofluorene (AAF) were efficient inducers of mdr expression in this model . Induction of mdr gene expression by both MC and AAF obeyed a log10 concentration/response relationship . In contrast, 2,3,7,8-tetrachlorodibenzo-P-dioxin did not induce mdr expression at concentrations that yielded maximum induction of cytochrome P450IA expression . These data suggest that mdr induction was not mediated via the Ah receptor . Nuclear run-off analysis indicated that both AAF and MC induced mdr expression by increasing transcription . Primer extension analysis indicated that mdr gene transcription was initiated at one major site 151 bp upstream of the ATG site in both the uninduced and induced state in vivo and in vitro . The sequence of the primer and the site of initiation of gene transcription indicate that the main gene induced was the mdr 1b gene.

Cytometry, 1991, 12(7), 636 - 44
Flow cytometric double labeling technique for screening of multidrug resistance; Gheuens EE et al.; We investigated the capabilities of flow cytometry in the analysis of a multidrug resistant (MDR) human ovarian cancer cell line 2780AD and its drug sensitive parental A2780 . A functional assay using daunorubicin (DNR) as a fluorescent probe was combined with an immunofluorescence assay of P-glycoprotein (P-gp) using the monoclonal antibody MRK-16 . Functionally MDR could be demonstrated by the lower DNR-content of MDR cells compared to DNR-content of drug sensitive cells . When incubation was performed with DNR in the presence of verapamil, DNR-content increased in the MDR cells . However the content of the A2780 cells was never attained . Differences in DNR-content were not related to differences in DNA-content . In experimental cell lines immunofluorescence data were inversely related with those of DNR-content: MDR cells had high levels of P-gp expression and low levels of DNR-content (and vice versa in drug sensitive cells) . Both assays can be easily combined in a multiparametric flow cytometric procedure to evaluate both parameters simultaneously in the same cells . Analysis of clinical samples demonstrates the existence of aberrant subpopulations which would not be detected by using a single parameter assay.

DNA Seq, 1991, 2(2), 89 - 101
Complete cDNA sequences encoding the Chinese hamster P-glycoprotein gene family; Endicott JA et al.; The only function of the transport protein P-glycoprotein (Pgp) that has been identified to date in mammals is its ability to mediate multidrug resistance (MDR) in tumour cell lines . Rodents have three P-glycoprotein (pgp) genes (termed pgp or mdr 1, 2 and 3), and humans have two (mdr1 and mdr3/mdr2) . Pgp isoforms differ in their drug transport capabilities: Pgp1 and Pgp2 can mediate MDR, while Pgp3 apparently cannot . The expression of the gene family members is tissue-specific, suggesting that they have unique physiological roles . We report in this paper the complete cDNA sequences for each of the three pgp genes in Chinese hamster . A comparison of the Chinese hamster cDNA sequences with those isolated from human and mouse confirms the identification of the gene family member homologues across these species . An analysis of mammalian Pgp sequences identifies conserved sequences which, it may be speculated, are important for Pgp activity . Previously, three different mdr3 (pgp3 homologous) transcripts, products of alternative splicing, have been reported in humans . Unexpectedly, we find no evidence for a similar alternative splicing event in Chinese hamster: it appears that the expression of pgp3 (mdr3) is different between rodents and humans.

Urol Int, 1991, 47(3), 118 - 25
Circumvention of multidrug resistance mediated by P-170 glycoprotein using calcium antagonists in primary human renal cell carcinoma; Mickisch GH et al.; In experimental cell lines and in some human tumors, calcium antagonists reversed multidrug resistance mediated by P-170 glycoprotein in vitro . So far, clinical trials have not been very rewarding as intrinsic cardiovascular activities of these compounds impeded sufficient dosage . Renal cell carcinomas are considered to be good models for the evaluation of this new therapeutic concept . In 35 primary human renal cell carcinomas, the potency of 7 different calcium antagonists in combination with vinblastine monotherapy was examined in a tetrazolium-based microculture assay (MTT test) in order to circumvent chemoresistance . Concomitantly, P-170 glycoprotein expression was traced immunohistochemically using moab C 219 . Substances derived from piperazine (flunarizine) showed only minor effects in this respect . The calcium antagonists of the papaverine type such as verapamil etc . revealed the strongest reversal of chemoresistance . Derivatives of benzothiazepine (diltiazem) or of dihydropyridine (nifedipine etc.) acted similarly and reached about 70% of the verapamil activity . All calcium antagonists lead to a significant enhancement of vinblastine cytotoxicity . An obvious link of P-170 glycoprotein to vinblastine chemoresistance was demonstrated . This particular resistance characteristic was detected in 19 of 27 resistant cases, but in none of the tumors displaying a chemoresponse . In particular, the new stereoisomer R-verapamil, which showed strong reversal of chemoresistance but which exerts 10 times lower cardiovascular side effects than racemic verapamil, seems to be suitable for further evaluation with regard to the clinical application.

Ann Med, 1991, 23(5), 509 - 20
Multidrug resistance; Tiirikainen MI et al.; Resistance of malignant cells to cytotoxic agents is often a limiting factor to successful chemotherapy . The classical multidrug resistance is characterised by overexpression of a membrane protein, P-glycoprotein, which acts like a drug extruding pump reducing accumulation of cytotoxic agents inside malignant cells, thereby preventing their function . Resistance is expressed simultaneously towards several structurally unrelated drugs . P-glycoprotein is also expressed in many normal human tissues, e.g., in the gastrointestinal tract, and this may be the reason for intrinsic resistance observed clinically in cancers derived from certain tissues . More often multidrug resistance is acquired during chemotherapy . The physiological function of P-glycoprotein is still unknown but it may have a role in cellular detoxification and secreting mechanisms . Interest in the phenomenon of multidrug resistance centres on the correlation of P-glycoprotein expression to clinical drug resistance . Another goal is to find mechanisms by which the function of P-glycoprotein as a multidrug transporter is prevented and drug resistance reversed.

J Cancer Res Clin Oncol, 1991, 117(6), 519 - 25
A human leukemia cell line made resistant to two folate analogues, trimetrexate and N10-propargyl-5,8-dideazafolic acid (CB3717); Takemura Y et al.; We established a novel human acute lymphoblastic leukemia cell line made resistant to two folate analogues, trimetrexate (TMQ) and N10-propargyl-5,8-dideazafolic acid (CB3717), by sequential exposure of the 200-fold TMQ-resistant cells (MOLT-3/TMQ200) to CB3717 . A 30-fold-resistant subline to CB3717 was selected from the TMQ-resistant cells and designated as MOLT-3/TMQ200-CB371730 . This double-folate-resistant cell line was 15-fold more resistant to methotrexate (MTX) than MOLT-3/TMQ200; however, TMQ resistance was decreased to 10-fold as compared to MOLT-3/TMQ200 . The doubly resistant cells also showed 2-fold cross-resistance to 5-fluorouracil (5-FU) . Equimolar concentrations of leucovorin almost completely reversed the inhibitory effect of MTX on the doubly resistant cells and partially that of CB3717 and TMQ; on the other hand, leucovorin enhanced the inhibitory effect of 5-FU . Thymidylate synthase activities demonstrated little or no difference among these three cell lines, being consistent with no overexpression of mRNA for this enzyme in the doubly resistant cells . MOLT-3/TMQ200 cells displayed classical multidrug resistance; sequential development of CB3717 resistance in the TMQ-resistant cells resulted in an enhancement of the multidrug-resistance phenotype and a concomitant increase of MDR1 mRNA . The development of a complex resistance pattern seen in this double-folate-resistant subline indicates intricacy in the study of drug resistance after multidrug chemotherapy.

Eur J Gynaecol Oncol, 1991, 12(5), 359 - 73
Chemoresistance in breast tumors; Giai M et al.; Resistance to multiple chemotherapeutic agents is a common clinical problem in the treatment of cancer: such resistance may occur in primary therapy or be acquired during treatment . The most commonly used antineoplastic agents in the treatment of disseminated breast cancer are adriamycin, methotrexate and cyclophosphamide . Cell lines selected for resistance to adriamycin often develop cross-resistance to structurally dissimilar antineoplastic drugs with different mechanisms of cytotoxic action; this phenomenon has been called pleiotropic or multidrug resistance (MDR) . In vitro models of MDR have shown that this type of resistance is accompanied by a decrease in cellular drug accumulation, mediated by the over-expression of a 170 kD plasma membrane glycoprotein referred to as P170 . Glycoprotein P170 is an energy-dependent multidrug efflux pump, whose activity can be inhibited in vitro by a variety of agents including verapamil, quinidine and reserpine . P170 is over-expressed also in some human malignancies, and evidence exists about its role in examples of clinical resistance in vitro . Clinical trials using verapamil, a calcium channel blocker which selectively enhances drug cytotoxicity in MDR cell lines, have been prompted for leukemia and ovarian cancer . In addition other approaches are the subject of current preclinical investigations . Several observations as well the phenomenon of "atypical" MDR in cell lines which do not overexpress P170, suggest that also other factors are involved in multidrug resistance . Qualitative or quantitative changes in the activity of topoisomerases, protein kinase-related systems and glutathione S-transferase, may confer pleiotropic resistance . As the role of these genes and their regulation is clarified, they may also serve as useful targets for pharmacologic intervention in the treatment of drug-resistant human tumors . The mechanisms involved in resistance to methotrexate and cyclophosphamide are less studied, particularly in vivo samples . Methotrexate resistance is probably a complex multifactorial phenomenon; in some cases it is due to an increase in the expression of the drug target dihydrofolate reductase, often as a result of gene amplification, but in other cases a transport defect of the methotrexate or alterations of the activity of different enzymes have been reported . Cyclophosphamide (CP) resistance has been attributed to an increased activity of two different enzymes, glutathione S-transferase, also involved in MDR phenotype, and aldehyde dehydrogenase, which catalyzes inactivation of CP in non cytotoxic metabolites . This paper reviews the current state of our knowledge of chemo-resistance and the utility of available markers to identify potentially resistant tumors in vivo; the strategies that might be used to overcome this phenomenon are also described.

Mol Carcinog, 1991, 4(5), 358 - 61
Coordinate activation of multidrug-resistance (P-glycoprotein) genes mdr2 and mdr3 during mouse liver regeneration; Teeter LD et al.; We previously reported that only one of the three mouse multidrug-resistance (mdr) genes, mdr3, is activated in hepatocellular carcinomas (HCCs) . The present study examined the expression of mdr family members during mouse liver regeneration after partial hepatectomy to determine whether the regeneration that occurs during hepatic tumorigenesis is responsible for mdr3 elevation in HCC . We demonstrated that in both C3H/HeN and B6C3/F1 mice strains, the levels of both mdr2 and mdr3 mRNAs coordinately increased five- to sevenfold 24 h after partial hepatectomy, whereas the levels of mdr1 mRNA were not statistically different from those in the controls . Forty-eight hours after partial hepatectomy, mdr mRNA levels decreased and in most cases returned to normal levels after 72 h . These results indicate that mdr3 induction during hepatocarcinogenesis is not due to liver regeneration alone.

J Recept Res, 1991, 11(1-4), 675 - 86
Identification of the multidrug resistance-related membrane glycoprotein as an acceptor for cyclosporine; Ryffel B et al.; The immunosuppressive agent cyclosporine A (CSA) has been shown to reverse multidrug resistance (MDR) in malignant cells . In the present study, a 3H-cyclosporine diazirine analogue (3H-PL-CS) was used to photolabel viable MDR cells . The 170 kDa membrane P-glycoprotein, which functions as a drug efflux pump, was strongly labeled . The binding of 3H-cyclosporine diazirine analogue to P-glycoprotein was competable by excess cyclosporine A and by the nonimmunosuppressive cyclosporine H . These results suggest that cyclosporine reverses the MDR phenotype by binding directly to P-glycoprotein and that this binding is not dependent on the immunosuppressive potential of the cyclosporine derivative . The identification of P-glycoprotein as a cyclosporine binding protein has obvious implications for cancer chemotherapy.

Cancer Chemother Pharmacol, 1991, 28(4), 259 - 65
Sensitization of multidrug-resistant colon cancer cells to doxorubicin encapsulated in liposomes; Oudard S et al.; The effectiveness of liposome-encapsulated doxorubicin in overcoming multidrug resistance was studied in various human colon cancer cells . Colon-cancer cell lines SW403, HT29, SW620, and SW620/R overexpressed P-glycoprotein as determined by immunoflow cytometry, thereby confirming the presence of the multidrug-resistant phenotype . Important differences were observed in the cytotoxicity of free doxorubicin as represented by IC50 values of 0.168, 0.058, 0.023, and 9.83 microM for SW403, HT29, SW620, and SW620/R, respectively . Liposomally encapsulated doxorubicin provided an IC50 that was 1.4 times lower than that of the free drug in the doxorubicin-resistant SW 620/R cell line, whereas no difference was evident in the sensitive parental SW620 cells . In addition, liposome-encapsulated doxorubicin exhibited 1.31- and 2.33-fold cytotoxicity to HT-29 and SW403 cells, respectively . The intracellular drug accumulation in SW620/R cells was enhanced by liposomally encapsulated doxorubicin, whereas it was reduced in all other cell lines as compared with that of free drug . The colon-cancer cell lines demonstrated different degrees of doxorubicin-induced DNA strand breakage that correlated with their sensitivities to drug-induced cytotoxicity . However, no difference was observed between DNA breakage caused by the free drug and that induced by liposome-encapsulated doxorubicin in any of the cell lines . The results suggest that the enhanced cytotoxicity of liposomal doxorubicin to colon cancer cells was due to some secondary non-DNA target . However, liposomally encapsulated doxorubicin appears to be effective in diminishing the multidrug-resistant phenotype and may have clinical applications.

Urol Res, 1991, 19(2), 99 - 103
Cross-resistance patterns related to glutathione metabolism in primary human renal cell carcinoma; Mickisch G et al.; In 59 cases of primary human renal cell carcinoma (RCC), cross-resistance and collateral susceptibility patterns were determined in an MTT microculture assay . Concomitantly, the glutathione (GSH) content and the enzymatic activity of gamma-glutamyl transpeptidase (GGT) were measured as distinct resistance characteristics . Resistance or chemoresponse towards Vinca alkaloids and anthracyclines were found to be highly coincident, suggesting that the classical multidrug resistance mechanism is active in human RCC . Strong resistance to platinum complexes combined with relative sensitivity to bleomycin was significantly associated with elevated glutathione levels, providing evidence for another pathway instigating chemoresistance . In contrast, despite substantial enzymatic activity, GGT effects revealed no correlation to the chemoresistance pattern . This result implies that it is the GSH-linked binding and reduction potential rather than the GGT-associated transportation capacity that has an impact on the expression of chemoresistance in human RCC.

Pharmacol Ther, 1991, 49(3), 283 - 92
Multidrug resistance gene family and chemical carcinogens; Thorgeirsson SS et al.; The data discussed in this review indicate that the coordinated induction of both the mdr gene family and a subfamily of the cytochrome P-450 supergene family provide a unified response of the organism to prevent lethal accumulation of xenobiotics . Consequently, a distinct physiological role for the mdr multigene family now exists . Furthermore, recent evidence suggests the existence of multiple receptors with overlapping substrate specificity that are involved in the induction of both mdr and P-4501A gene families . The increased expression of mdr gene(s) in the early stages of liver carcinogenesis and presumably in other tissues is associated with the development of xenobiotic resistance that is observed in the preneoplastic cell populations . These observations may have important clinical implications and may provide an explanation for resistance to chemotherapy of tumors in organs such as liver and colon that are frequently exposed to both environmental and dietary xenobiotics.

Virchows Arch B Cell Pathol Incl Mol Pathol, 1991, 60(2), 133 - 8
Multidrug resistance gene and P-glycoprotein expression in gastric adenocarcinoma and precursor lesions; Vollrath V et al.; Overexpression of the Multiple Drug Resistance gene (MDR1) has been proposed as a major mechanism related to both intrinsic and acquired resistance to chemotherapeutic agents . The gene product is a membrane protein (P-glycoprotein), that acts as an energy-dependent drug efflux pump decreasing drug accumulation in resistant tumor cells . We have characterized MDR1 and P-Glycoprotein expression in human gastric adenocarcinoma and in precursor lesions . MDR1 mRNAs, analyzed by dot-blot technique, were detected in 9 of 10 non-tumoral gastric mucosae and in 8 of 10 gastric adenocarcinomas . Immunohistochemical analysis, using the MRK16 monoclonal antibody, revealed heterogeneous expression of P-Glycoprotein in individual cells . The P-Glycoprotein was found on the surface of cells of gastric areas with intestinal metaplasia subtype III . This type of intestinal metaplasia, also called "colonic metaplasia", has been strongly associated with a high risk for the development of gastric cancer . The fact that the P-Glycoprotein was detected in this precursor lesion is consistent with the intestinal metaplasia-dysplasia and carcinoma sequence proposed in the histogenesis of this tumour . The finding that P-Glycoprotein was heterogeneously expressed in malignant cells of some gastric adenocarcinomas also suggests that this transporter system probably contributes to primary and secondary multidrug resistance in this neoplasm.

Eur J Cancer, 1991, 27(2), 155 - 8
Expression of P-glycoprotein in breast cancer tissue and in vitro resistance to doxorubicin and vincristine; Sanfilippo O et al.; Expression of P-glycoprotein was evaluated by C219 monoclonal antibody immunoblots in 34 previously untreated and 14 pretreated breast cancers and in benign breast lesions or histologically normal breast glands . P-glycoprotein was not detectable in the few cases of normal or benign tissue . P-glycoprotein was expressed in the 170 kD areas of 29% (10/34) of untreated and 64% (9/14) of previously treated tumours (P = 0.02) . In treated tumours, high intensity expression was observed more frequently than in untreated breast cancer (40% vs . 9%) . Moreover, there was a significant association between P-glycoprotein expression and in vitro resistance to doxorubicin and vincristine . Simultaneous resistance was observed in all of the P-glycoprotein positive and in only 56% of the P-glycoprotein negative tissues (P less than 0.01) . Some aspects of the typical multidrug resistant phenotype, such as P-glycoprotein expression and simultaneous resistance to doxorubicin and vincristine, could be detected in small subsets of breast cancer patients . No relation between P-glycoprotein expression and the type of previous clinical treatment was observed.

Anticancer Res, 1991 Jan-Feb, 11(1), 423 - 8
Molecular analysis of two human doxorubicin-resistant cell lines: evidence for differing multidrug resistance mechanisms; Slovak ML et al.; The molecular characteristics of two human doxorubicin-resistant cell lines were examined specifically for MDR1 gene amplification by Southern analysis and for overexpression of its messenger RNA . The 285-fold doxorubicin-resistant colon adenocarcinoma subline, LoVo/DR5, was found to overexpress the mRNA for P-glycoprotein without the concomitant requirement of MDR1 gene amplification, suggesting that relatively high levels of P-glycoprotein mediated multiple drug resistance may occur by transcriptional activation of the gene . Despite a similar in vitro selection strategy and in contrast to LoVo/DR cells, the 220-fold doxorubicin-resistant fibrosarcoma subline, HT1080/DR4, did not overexpress P-glycoprotein mRNA nor was the MDR1 gene amplified . In-gel renaturation studies were performed to determine the nature of a putative HSR-bearing chromosome 7 found in HT1080/DR4 cells; however, at a level of sensitivity nearing 20 copies of an amplified DNA fragment per haploid genome, no amplified sequences could be detected . These results suggest that doxorubicin resistance is multifactorial and alternative mechanisms of multiple drug resistance remain to be determined . LoVo/DR5 cells should prove to be a useful model for investigating transcriptional activation of the MDR1 gene; HT1080/DR4 cells should be an excellent model for the study of non-P-glycoprotein mediated multiple drug resistance.

Chemotherapy, 1991, 37(1), 57 - 61
Resistance to doxorubicin in tumor cells in vitro and in vivo after pretreatment with verapamil; Donenko FV et al.; Sarcoma 180 (S180) cells pretreated with verapamil increase their resistance to doxorubicin in vitro . This result could be confirmed by in vivo experiments using P388 cells . The mechanism for this resistance is different from the P-glycoprotein-mediated mechanism characteristic for multidrug-resistant cells.

J Cancer Res Clin Oncol, 1991, 117(2), 168 - 71
p-glycoprotein expression in malignant melanoma; Fuchs B et al.; In some human malignancies resistance to chemotherapy is caused by an energy-dependent efflux system, responsible for the removal of chemotherapeutics out of the resistant tumor cells . A major component of this efflux system is the permeability glycoprotein (p-glycoprotein), which depends on the multidrug-resistance gene MDR1 . We have tested p-glycoprotein in primary and metastatic human melanoma by use of the monoclonal antibody C219; a substantial expression was only observed in 1/37 primary melanomas and in 1/27 melanoma metastases . None of the patients with negative metastases responded to chemotherapy . Moreover a complete remission of metastatic growth was observed in the patient with the metastasis significantly expressing the p-glycoprotein . Sequential studies revealed no significant increase of p-glycoprotein-positive cells during and after chemotherapy . We conclude that drug resistance in human melanoma does not usually depend on the p-glycoprotein-related efflux system . Other mechanisms are obviously responsible for drug resistance in this human malignancy.

Jpn J Cancer Res, 1991 Jan, 82(1), 127 - 33
Novel mechanism of N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine in potentiation of antitumor drug action on multidrug-resistant and sensitive Chinese hamster cells; Tomida A et al.; The mechanism of the synthetic isoprenoid N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine (SDB-ethylenediamine) in potentiating antitumor drug action against multidrug-resistant cells was comparatively studied with other potentiators such as verapamil and cepharanthine . SDB-ethylenediamine increased the accumulation of {3H}daunorubicin (DNR) in Chinese hamster V79 (V79/S) and its multidrug-resistant mutant (V79/ADM) cells . Even after SDB-ethylenediamine was removed from the medium, its effect continued . But when verapamil was removed from the medium, its effect disappeared immediately . Unlike verapamil and cepharanthine, SDB-ethylenediamine did not greatly inhibit the efflux of {3H}DNR from V79/ADM, the binding of {3H}vinblastine to membrane vesicles of V79/ADM, or the binding of {3H}azidopine to P-glycoprotein in the cytoplasmic membrane of V79/ADM . It did stimulate the influx of {3H}DNR into the ATP-depleted cells of V79/S and V79/ADM . Thus, SDB-ethylenediamine uniquely potentiates antitumor drugs . The increased intracellular accumulation of antitumor drugs in the presence of SDB-ethylenediamine is due not only to the inhibition of active efflux but also to the stimulation of the influx of antitumor drugs.

Cancer Chemother Pharmacol, 1991, 27(4), 290 - 4
Modulation of mitomycin C-induced multidrug resistance in vitro; Dorr RT et al.; A series of in vitro cytotoxicity studies were performed to achieve pharmacologic reversal of resistance to the alkylating agent mitomycin (MMC) in L-1210 leukemia cells . A multidrug-resistant (MDR), P-glycoprotein-positive cell line, RL-1210/.1 {11}, was exposed to potential MDR modulators in the absence or presence of MMC . The following compounds did not reverse MMC-induced MDR: quinine, quinidine, lidocaine, procaine, dimethylsulfoxide (DMSO), dexamethasone, hydrocortisone, prednisolone, estradiol, and testosterone . Three agents were capable of reversing MMC resistance: progesterone, cyclosporin A, and verapamil . The R- and S-isomers of verapamil were equipotent, although they showed a 10-fold difference in cardiovascular potency (S greater than R) . Some agents produced cytotoxic effects in MDR cells in the absence of MMC, including progesterone, quinine, and quinidine . The results suggest that R-verapamil and progesterone may have clinical utility in reversing MMC resistance in human tumors . Progesterone may be uniquely efficacious due to (a) its low toxicity in normal cells, (b) its selective cytotoxicity in MDR cells (in the absence of MMC), and (c) its ability to reverse MMC resistance in vitro . The findings also suggest that the P-glycoprotein induced by MMC differs from that induced by doxorubicin, which is highly sensitive to modulation by lysosomotropic amines such as quinine and quinidine.

Br J Haematol, 1991 Jan, 77(1), 50 - 3
High risk of early resistant relapse for leukaemic patients with presence of multidrug resistance associated P-glycoprotein positive cells in complete remission; Musto P et al.; The immunocytochemical detection of multidrug-resistance (MDR) associated P-glycoprotein (P-170) was longitudinally performed on bone marrow smears from 32 responsive patients with acute leukaemia in the different phases of the disease (at diagnosis, in complete remission, at relapse) by means of APAAP technique and monoclonal antibody C219 . The whole group of eight patients with presence of P-170 positive cells while in complete remission rapidly relapsed with a high proportion of blasts showing MDR phenotype; they were resistant to further treatments . Twelve out of 24 subjects without cells with MDR phenotype in complete remission remained in this condition, six had a responsive relapse (without significant expression of P-170 in 5/6 patients) and six a resistant relapse . Four patients of this last group significantly expressed P-170 . Our data indicate that the detection of scattered P-170 positive cells during complete remission might identify a subset of leukaemic patients with high risk of early and resistant relapse.

Br J Cancer, 1991 Jan, 63(1), 51 - 6
Pleiotropic-resistant phenotype is a multifactorial phenomenon in human colon carcinoma cell lines; Toffoli G et al.; The biochemical basis of multidrug-resistant (MDR) phenotype has been investigated in drug-resistant sublines independently obtained in our laboratories by single step doxorubicin (DOX) selection of LoVo, DLD1, and SW948 human colon carcinoma (HCC) cell lines . All the chemoresistant sublines have been found to be cross-resistant to DOX, actinomycin-D (ACT-D) and vincristine (VCR) but not to cis-diamminedichloroplatinum (CDDP), and have exhibited an increased expression level of mdr1 mRNA and gp170 glycoprotein . Comparative analyses in drug-resistant and sensitive cells of resistance index, extracellular and intracellular equitoxic DOX concentrations, and mdr1 gene products expression have indicated that MDR phenotype is a multifactorial phenomenon due to different and possibly independent biochemical mechanisms which cooperate, in varying degrees from cell line to cell line, in conferring cellular chemoresistance.

Mol Pharmacol, 1991 Jan, 39(1), 1 - 8
Relationship between expression of P-glycoprotein and efficacy of trifluoperazine in multidrug-resistant cells; Ganapathi R et al.; Tumor cell resistance due to enhanced efflux of drugs with diverse structures and/or mechanisms of action is termed multidrug resistance (MDR), and modulation of the MDR phenotype by calcium blockers or calmodulin inhibitors is suggested to involve P-glycoprotein . In drug-sensitive (S) and 5-fold doxorubicin (DOX)-resistant (R0) L1210 mouse leukemia cells, no obvious differences in mdr mRNA or P-glycoprotein expression or alterations in cellular uptake, retention, or cytotoxicity of vincristine (VCR) were observed . However, in the 10-fold (R1) and 40-fold (R2) DOX-resistant sublines, expression of P-glycoprotein was correlated with the level of resistance (R2 greater than R1) . An RNase protection assay revealed that elevated levels of mdr1 and mdr2 mRNA were detected in R1 and R2 cells, with an additional increase in mdr3 mRNA in the R2 subline . Further, in the R1 and R2 sublines, no VCR dose-dependent cytotoxicity was apparent, and cell kill of greater than 40% was not achievable following a 3-hr drug exposure . Cellular uptake and retention of VCR were 2- to 4-fold lower in the R1 and R2 sublines, compared with similarly treated S or R0 cells . Potentiation of VCR cytotoxicity by a noncytotoxic concentration of 5 microM trifluoperazine (TFP) was greater than 2-fold in S and R0 cells and less than 1.3-fold in the R1 and R2 sublines . Modulation of VCR uptake by 5 microM TFP in the S and R0 cells was 2-fold and it was 4- to 7-fold in the R1 and R2 sublines . The presence of 5 microM TFP, by competing for efflux, enhanced VCR retention 1.5-fold in S and R0 cells and 2- to 4-fold in the R1 and R2 sublines . In contrast to these results with VCR, dose-dependent cytotoxicity of DOX was apparent in all the resistant sublines, and modulation of DOX cytotoxicity by 5 microM TFP was dependent on the level of resistance . Cellular accumulation of DOX was 20 and 50% lower in the R1 and R2 sublines, respectively, compared with similarly treated S or R0 cells . Marked increases (greater than 1.5-fold) in cellular accumulation of DOX by TFP were apparent only in the R2 subline . Results suggest that a relationship between overexpression of P-glycoprotein isoforms and their role in affecting cellular drug levels and consequent cytotoxicity in MDR L1210 cells determines resistance to VCR but not DOX.(ABSTRACT TRUNCATED AT 400 WORDS)

J Clin Oncol, 1991 Jan, 9(1), 17 - 24
P-glycoprotein expression in malignant lymphoma and reversal of clinical drug resistance with chemotherapy plus high-dose verapamil; Miller TP et al.; P-glycoprotein is a transmembrane protein thought to function as an efflux pump to detoxify cells . It is associated with multidrug resistance in laboratory systems and has recently been found in human tumors associated with in vitro and clinical drug resistance . We used an immunohistochemical method employing two monoclonal antibodies, JSB-1 and C-219, to detect expression of P-glycoprotein in lymphoma patients . One of 42 newly diagnosed and untreated lymphoma patients (2%) and seven of 11 previously treated and drug-resistant patients (64%) had detectable levels of P-glycoprotein (P less than .001) . Based on prior reports suggesting that verapamil sensitizes drug-resistant cancer cells to chemotherapy by competitive inhibition of the P-glycoprotein, we tested the efficacy of verapamil as a chemosensitizer in 18 patients with drug-refractory disease . All patients had previously failed or relapsed within 3 months of a doxorubicin-vincristine-containing drug regimen . Patients received day-1 cyclophosphamide, and 4-day continuous infusion doxorubicin and vincristine and oral dexamethasone (CVAD) . CVAD was combined with 5-day continuous infusion verapamil given at maximally tolerated dose . Overall, 13 of 18 patients (72%) responded to treatment including five complete remissions (CRs; 28%) . The median duration of response was 200 days and median survival was 242 days . The dose-limiting toxicity of the verapamil infusion was temporary cardiac dysfunction including hypotension, congestive heart failure, and cardiac arrhythmia . We conclude that the P-glycoprotein is uncommonly expressed in untreated lymphomas and frequently expressed in clinically drug-resistant disease, and that chemotherapy using CVAD plus maximally tolerated doses of verapamil results in a high response rate in patients carefully selected for clinical drug resistance.

Cancer Chemother Pharmacol, 1991, 28(6), 461 - 4
Lack of cross-resistance to fostriecin in a human small-cell lung carcinoma cell line showing topoisomerase II-related drug resistance; de Jong S et al.; Cells exhibiting decreased topoisomerase II (Topo II) activity are resistant to several drugs that require Topo II as an intermediate . These drugs are cytotoxic due to the formation of a cleavable complex between the drug, Topo II and DNA . Fostriecin belongs to a new class of drugs that inhibit Topo II without inducing the formation of this cleavable complex . We tested fostriecin in three human small-cell lung carcinoma cell lines . GLC4 is the parent line . GLC4/ADR is the P-glycoprotein-negative multidrug-resistant subline, which is resistant to several Topo II inhibitors due to its decreased Topo II activity . GLC4/cDDP is the cisplatin-resistant subline, which displays increased Topo II activity . Topo II activity proved to be 100% in GLC4, 35% in GLC4/ADR and 130% in GLC4/cDDP . The fostriecin concentration causing inhibition of the growth of 50% of the cells (IC50) in the microculture tetrazolium assay following continuous incubation was 11.2, 4.1 and 14.9 microM, respectively . After 1-h incubations, the IC50 was 117.8, 101.3 and 219.8 microM, respectively . Our results indicate a relationship between Topo II activity and fostriecin sensitivity in these closely related cell lines . At least in vitro, fostriecin displayed the capacity to kill cells showing resistance to drugs due to decreased Topo II activity . There was no relationship between this capacity and an increase in the activity of the reduced-folate carrier system, the proposed mechanism for cellular entry of fostriecin, since we found no correlation between the cytotoxicity of fostriecin and that of methotrexate.

Int J Rad Appl Instrum B, 1991, 18(4), 437 - 43
Monoclonal antibodies for radioimmunoscintigraphy of breast cancer; Merino MJ et al.; Breast cancer is the leading cause of cancer deaths among females, and it is estimated that each year, one in ten American women will be newly diagnosed as having the disease . It is therefore not surprising, that a great deal of effort has been made to better understand the biology of breast cancer, and that investigators keep up the search for new tools to better characterize, diagnose and treat these tumours . In this regard, the introduction of the hybridoma technique in 1975 by Kohler and Milstein has lead to an extensive work in the characterization of monoclonal and polyclonal antibodies against breast cancers . A large number of antibodies has been raised to different epitopes present in normal and neoplastic breast tissue; but unfortunately we have yet to find a highly sensitive and specific monoclonal antibody for breast cancer that can successfully be used for scintigraphic detection of nodal metastases and for radioimmunotherapy treatment of this disease . As possible radioimmunodiagnostics, antibodies are known which react with the following antigens: (1) cytoskeletal proteins (2) breast cell products (3) steroid receptors (4) putative tumor-associated antigens (5) oncogene products (6) pregnancy-related products (7) basement membrane antigens (8) degradative enzymes (9) cell receptors for extracellular matrix molecules (10) multidrug resistance gene product (p-glycoprotein) (11) proliferative markers.

Br Med Bull, 1991 Jan, 47(1), 178 - 96
Drug resistance; Hochhauser D et al.; Chemotherapy cures a minority of adult tumours (e.g . Hodgkin's and non-Hodgkin's lymphoma, acute leukaemia, teratoma) and the majority of childhood tumours . Prolongation of survival by chemotherapy has been shown for small cell lung cancer, ovarian cancer and breast carcinoma (when used as an adjuvant) . However, in the majority of solid tumours there is a less than 20% response to chemotherapy and even curable tumours may relapse and become resistant . Resistance may be de novo, acquired as a stable change within the cell, or be rapidly inducible within the cell after drug administration . Several mechanisms have been described including multidrug resistance, glutathione transferases and DNA repair . Understanding these mechanisms may help to improve the therapeutic ratio and develop new approaches.

Life Sci, 1991, 48(23), 2195 - 205
The role of topoisomerase II in drug resistance; De Isabella P et al.; The conventional laboratory approach to study the mechanisms of drug resistance has been the selection of drug-resistant cell lines by continuous exposure to cytotoxic agents . Such lines, which are selected for resistance to a single agent, frequently display cross-resistance to a number of cytotoxic agents that are unrelated in both structure and proposed mechanism of action . Multidrug-resistant cells display reduced drug accumulation, which is the result of overexpression of a surface glycoprotein (P170) . Although resistance to multiple antitumor agents is a common clinical problem in the treatment of cancer, the precise role of the P-glycoprotein-mediated mechanism in human tumors remains to be established . Many alterations in multidrug-resistant cells selected in vitro have been identified . The concomitant expression of multiple phenotypic differences, which appear to be favored by continued and prolonged drug exposure, makes analysis of critical individual resistance pathways more difficult . However, multiple factors may also be involved in the development of clinical resistance . Recent studies have identified alterations in DNA topoisomerase II activity and function as an alternative mechanism that contributes to the multidrug-resistance phenomenon or is responsible for a different type of drug resistance . The precise nature of these changes remains unclear . Available evidence supports the view that expression of the enzyme is an important determinant of cell sensitivity to DNA topoisomerase poisons, but that other changes involved in regulation of enzyme function and/or in the cellular processing of drug-induced DNA damage may be critical in determining the differential pattern of cell response to antitumor agents.

SAAS Bull Biochem Biotechnol, 1991 Jan, 4, 13 - 6
New approaches to the study of tumor drug resistance; Mansouri A et al.; The development of tumor drug resistance is the major obstacle to successful systemic chemotherapy . Therefore, devising methods for reversing drug resistance is a high priority and could lead to significant improvements in cancer treatment . The mechanisms of tumor drug resistance are manifold and are not well understood . The phenomenon of multidrug resistance (MDR) represents the development of resistance to most drugs, regardless of their chemical structure . Several types of MDR are known, for example, the overexpression of a cell membrane glycoprotein (P-170), increased activity of glutathione S-transferase, elevated levels of glutathione (GSH), and alterations in topoisomerase action . A partial reversal of tumor drug resistance has been achieved by the use of competitive inhibitors for the function of glycoprotein P-170, or by the inhibition of GSH synthesis; however, this strategy has not been substantially successful for improving the response of human tumors to clinical therapy . We have recently used electroporation, in conjunction with the cytotoxic drug, cisplatin (cDDP), in an attempt to circumvent drug resistance in cDDP-resistant mouse tumor cells (RIF/Ptr1) . Electroporation is the application of a high-voltage electric shock which is known to create transient pores in plasma membranes of cultured cells . Electroporation plus cDDP treatment increased intracellular cDDP concentration and reversed cellular resistance to cDDP-induced cell killing.

Biochem Biophys Res Commun, 1990 Dec 31, 173(3), 1252 - 7
Staurosporine, a potent inhibitor of C-kinase, enhances drug accumulation in multidrug-resistant cells; Sato W et al.; Staurosporine, a potent inhibitor of C-kinase, enhances accumulation of vincristine (VCR) in multidrug-resistant cells . We investigated this enhancement by two methods: (I) ATP-dependent VCR binding system; (II) azidopine photolabeling system . The ATP-dependent VCR binding to the resistant cell membrane was inhibited more efficiently by staurosporine than by verapamil . Staurosporine also inhibited the azidopine photolabeling of P-glycoprotein . These results indicate that staurosporine, an inhibitor of C-kinase, might directly bind to P-glycoprotein as well as antitumor agents and Ca2+ channel blockers . These findings also indicate that C-kinase might be involved in the function of P-glycoprotein.

Nature, 1990 Dec 20-27, 348(6303), 741 - 4
Sequences encoded in the class II region of the MHC related to the 'ABC' superfamily of transporters; Trowsdale J et al.; Class I molecules of the major histocompatibility complex (MHC) bind and present peptides derived from the degradation of intracellular, often cytoplasmic, proteins, whereas class II molecules usually present proteins from the extracellular environment . It is not known how peptides derived from cytoplasmic proteins cross a membrane before presentation at the cell surface . But certain mutations in the MHC can prevent presentation of antigens with class I molecules . In addition, mutations possibly in the MHC can affect presentation by class II molecules . Here we report the finding of a new gene in the MHC that might have a role in antigen presentation and which is related to the ABC (ATP-binding cassette) superfamily of transporters . This superfamily includes the human multidrug-resistance protein, and a series of transporters from bacteria and eukaryotic cells capable of transporting a range of substrates, including peptides.

Nature, 1990 Dec 20-27, 348(6303), 738 - 41
MHC class II region encoding proteins related to the multidrug resistance family of transmembrane transporters; Deverson EV et al.; The T-cell immune response is directed against antigenic peptide fragments generated in intracellular compartments, the cytosol or the endocytic system . Peptides derived from cytosolic proteins, usually of biosynthetic origin, are presented efficiently to T-cell receptors by major histocompatibility complex (MHC) class I molecules, with which they assemble, probably in the endoplasmic reticulum (ER) . In the absence of recognizable N-terminal signal sequences, such cytosolic peptides must be translocated across the ER membrane by a novel mechanism . Genes apparently involved in the normal assembly and transport of class I molecules may themselves be encoded in the MHC . Here we show that one of these, the rat cim gene, maps to a highly polymorphic part of the MHC class II region encoding two novel members of the family of transmembrane transporters related to multidrug resistance . Other members of this family of transporter proteins are known to be capable of transporting proteins and peptides across membranes independently of the classical secretory pathway . Such molecules are credible candidates for peptide pumps that move fragments of antigenic proteins from the cytosol into the ER.

Cancer Res, 1990 Dec 15, 50(24), 7775 - 80
Site-dependent differences in response of the UV-2237 murine fibrosarcoma to systemic therapy with adriamycin; Staroselsky AN et al.; Murine fibrosarcoma UV-2237MM cells were implanted into different organs of syngeneic C3H/HeN mice . The resultant tumors were treated by i.v . administration of Adriamycin (ADR) . Despite the high sensitivity of the fibrosarcoma cells to ADR in vitro, the established tumors growing in vivo exhibited marked differences in their responses to ADR . Tumors growing in the subcutis and the spleen were ADR-sensitive, whereas lung metastases were not . The resistance of lung metastases to ADR was not due to selection of a drug-resistant population since tumor cells isolated from lung metastases were highly sensitive to ADR under in vitro conditions . The responsiveness of skin and spleen tumors to ADR was due neither to increased blood supply nor to preferential accumulation of ADR, since both parameters were higher in lung metastases . Protein kinase C activity levels correlated with ADR resistance in the closely related murine fibrosarcoma cell line UV-2237 and its ADR-selected multidrug-resistant variants . However, nearly identical levels of protein kinase C activity were found in UV-2237MM tumors growing in the lung, spleen, and subcutis, indicating that protein kinase C activity levels did not account for the different responses to ADR . The present studies suggest that the organ environment influences the response of UV-2237MM to ADR administered systemically . This finding may have implications for the design of animal models for therapy of disseminated cancer.

Biochem Pharmacol, 1990 Dec 15, 40(12), 2625 - 35
Evidence for impaired mitoxantrone and vinblastine binding in P388 murine leukemia cells with multidrug resistance; Pincus R et al.; Multidrug resistance is associated with a P170 glycoprotein efflux pump that limits net drug accumulation in resistant cell lines . Other evidence has suggested that diminished net drug uptake in multidrug resistant (MDR) cells is due to decreased drug binding as well . To assess the contribution of binding differences to net drug accumulation and retention in MDR cells, mitoxantrone and vinblastine, two agents commonly associated with the MDR phenotype but with different mechanisms of action and intracellular binding sites, were studied in P388 murine leukemia cells . For both drugs, resistance was associated with a marked reduction in tightly bound drug which can account for the diminished net drug accumulation in this cell line; even at 1 microM vinblastine when the exchangeable component was one-half that of the sensitive cells, the nonexchangeable component was only one-seventh . For mitoxantrone, the exchangeable drug component was greater in resistant cells at low drug levels (1 microM) and similar at high drug levels (10 microM) . For vinblastine, the exchangeable drug component was decreased in the resistant cells at 1 microM, but the difference compared to sensitive cells became neglible at 10 microM . The data indicate that diminished net drug uptake in the P388 MDR cell line was associated with a marked decrease in tightly bound, i.e . nonexchangeable, drug fractions for both mitoxantrone and vinblastine . Therefore, alterations in intracellular binding are in important factor in the decreased cellular uptake and retention of drugs in the multidrug resistance phenomenon . The relationship between these changes and the P170 efflux pump requires further clarification.

Biochim Biophys Acta, 1990 Dec 10, 1055(3), 217 - 22
Evidence for daunomycin efflux from multidrug-resistant 2780AD human ovarian carcinoma cells against a concentration gradient; Lankelma J et al.; Using a flow-through system human multidrug-resistant 2780AD ovarian carcinoma cells were exposed to flowing culture medium containing the anticancer agent daunomycin (5 microM) . A pulse of medium containing verapamil caused increased cellular daunomycin accumulation, resulting in a dip in the fluorescence signal from daunomycin in the effluent . After passage of this pulse we observed an efflux of more than 90% of this extra accumulated daunomycin within 10 min . This daunomycin efflux against a concentration gradient provides evidence for drug efflux from these cells being an active process . After the addition of methylamine to the medium to increase intravesicular pH, the dip in the fluorescence signal was not decreased, indicating that vesicular transport was not an important component of this efflux.

Cell Growth Differ, 1990 Dec, 1(12), 607 - 15
The human multidrug resistance gene: sequences upstream and downstream of the initiation site influence transcription; Cornwell MM; To identify the DNA sequences required for multidrug resistance (MDR1) gene transcription, we have optimized conditions for transcription of the MDR1 proximal promoter in vitro . Using HeLa cell nuclear extracts, the direction, initiation, and RNA polymerase II dependence of transcription in vitro accurately reflect events in cells . The DNA template concentration, reaction temperature, and MgCl2 concentration were critical parameters of the in vitro system . Using conditions optimized for these parameters, the effect of deletions in the 5' flanking region and deletions in sequences downstream of the initiation site were examined . We found that deletion of sequences 5' and 3' of the transcription initiation site modulated the level of transcription . Of particular interest was the deletion of exon 1 sequences +5 to +127, which completely inhibited accurately initiated MDR1 transcription . Reconstitution of the +5 site, used for initiation in vivo and in vitro, did not reverse the inhibition . MDR1 transcription was specifically inhibited by an oligonucleotide corresponding to sequences +46 to +58 . Our data indicate that sequences both upstream and downstream of the transcription initiation site modulate the efficiency of the MDR1 proximal promoter.

Radiother Oncol, 1990 Dec, 19(4), 297 - 305
Biochemical basis of resistance to chemotherapy; Masters JR; Recent progress in the understanding of drug resistance has led to the discovery of new targets for chemotherapy . By attacking the molecules that make cancer cells insensitive to chemotherapy, it is hoped that drug-resistant disease will respond to treatment . This review describes some of the latest advances in understanding of the biochemistry of drug resistance . Following a general introduction four areas of topical interest are discussed: (1) multidrug resistance and P-glycoprotein, (2) glutathione and its related enzymes, (3) topoisomerase II and (4) DNA repair.

Exp Hematol, 1990 Dec, 18(11), 1193 - 8
Modulation of etoposide (VP-16) cytotoxicity by verapamil or cyclosporine in multidrug-resistant human leukemic cell lines and normal bone marrow; Chao NJ et al.; We studied the effects of two modulators of multidrug resistance (MDR), cyclosporine and verapamil, on the cytotoxicity of etoposide (VP-16) in normal human bone marrow; two human leukemia cell lines, K562 and CEM; their MDR variants, K562/DOX and CEM/VLB; and mixtures of normal marrow and leukemic cells . VP-16 was selectivity toxic to the parental leukemic cells, with IC-50 values of 2 microM for CEM cells, 1.5 microM for K562 cells, and 12 microM for normal marrow CFU-GM . This selectivity was lost in the MDR variant leukemia cells, with IC-50s of 20 microM in K562/DOX and 8 microMs in CEM/VLB . Cyclosporine, 6 microMs, and verapamil, 20 microM, alone were nontoxic to bone marrow CFU-GM, and did not significantly increase the toxicity of VP-16 to normal marrow cells or to the two drug-sensitive leukemic cell lines . However, cyclosporine specifically enhanced the cytotoxicity of VP-16 in the MDR leukemia cells, reducing the IC-50 to the same level as the parental sensitive cells . Verapamil was considerably less effective . In a mixing experiment that included K562/DOX cells and normal bone marrow, cyclosporine increased the toxicity of VP-16 to the resistant leukemic cells by nearly 20-fold . Because the cytotoxic effect of cyclosporine is additive for resistant tumor cells, its combination with VP-16 may be useful in the purging of contaminating tumor cells prior to autologous bone marrow transplantation.

Cancer Treat Rev, 1990 Dec, 17 Suppl A, 11 - 20
Mechanisms of multidrug resistance in human tumor cells . The roles of P-glycoprotein, DNA topoisomerase II, and other factors; Beck WT; Multidrug resistance (MDR) associated with overexpression of P-glycoprotein (Pgp) is a well-described experimental phenomenon that appears to have clinical correlates . However, recent descriptions of non-P-glycoprotein forms of MDR have complicated efforts to detect and circumvent MDR in the tumors of patients . One major form of natural product MDR appears to be due to alterations in the amount of activity of DNA topoisomerase II . Compared to Pgp-MDR cells, cells expressing this form of MDR (at-MDR) do not overexpress the mdr1 gene or its product, Pgp, are unaltered in drug accumulation and retention, are unaffected by such 'modulators' of Pgp-MDR as verapamil, and express this phenotype recessively . Recently, other MDR cell lines have been described with some characteristics of Pgp-MDR (decreased drug accumulation and retention, increased drug cytotoxicity by modulators of MDR), but not others (no expression of the mdr1 gene or Pgp) . Whether any non-Pgp forms of MDR occur in patients' tumors remains to be determined.

Jpn J Cancer Res, 1990 Dec, 81(12), 1214 - 7
Increase in the level of P-glycoprotein mRNA expression in multidrug-resistant K562 cell lines treated with sodium butyrate is not accompanied with erythroid differentiation; Shibata H et al.; We examined the effect of hemin, sodium butyrate and mitomycin C on levels of P-glycoprotein mRNA in human myelogenous K562 cells by northern blot analysis . After treatment with sodium butyrate a dose-dependent increase of P-glycoprotein mRNA expression was observed in the adriamycin-resistant K562 and vincristine-resistant K562 lines . With 10 mM sodium butyrate, the level of P-glycoprotein mRNA reached 20 times that of control adriamycin-resistant K562 and with 30 mM sodium butyrate, it exceeded 5 times that of control vincristine-resistant K562 . In contrast, hemin and mitomycin C had almost no effect on P-glycoprotein mRNA . In this experiment, since expression of P-glycoprotein mRNA was not necessarily accompanied with induction of erythroid differentiation, the increased amount of P-glycoprotein mRNA is unlikely to be a result of differentiation.

Lab Invest, 1990 Dec, 63(6), 815 - 24
Optimization of immunocytochemical P-glycoprotein assessment in multidrug-resistant plasma cell myeloma using three antibodies; Grogan T et al.; Seeking to optimize the immunocytochemical assay of P-glycoprotein, a 170-kilodalton (P-170) molecule associated with multidrug resistance, we experimented with a variety of antibodies (JSB-1, C219, and MRK-16), fixation conditions, and titers using both human myeloma cell lines and clinical myeloma specimens . Under optimized conditions, using all three antibodies and the cell lines as standards and controls, the ICC method proved sensitive, specific, reliable, rapid, and within the realm of everyday hospital laboratory expertise . The 3 anti-P-glycoprotein antibodies revealed different reactivities with P-170 . Both C219 and JSB1 were optimized by fixation in cold acetone . With MRK-16 optimal results were obtained on unfixed or formalin fixed specimens . Under optimal fixation and titering conditions, low level (DOX 4) detection was possible . Given that the three antibodies differ in reactivity and recognize different P-170 epitopes, it follows that using the antibodies in a small panel is a useful strategy in increasing the likelihood of detecting true P-glycoprotein expression by the immunocytochemical method . In dilution experiments, the immunocytochemical method was as sensitive as RNase protection assay and more sensitive than Western blot detection . Immunocytochemistry coupled to computer-assisted single-cell densitometry, showed a strong correlation (R = 0.98) between cellular P-170 density and in vitro resistance to doxorubicin . Multidrug-resistant specific probes for RNA expression and Western blot assays confirmed the specificity of P-170 expression in both cell lines and clinical samples . Thus, a small panel of antibodies, under optimized immunocytochemical conditions, appears to have potential as a rapid, sensitive, clinically useful assay for multidrug resistance in myeloma.

Cancer Res, 1990 Dec 1, 50(23), 7634 - 40
In vivo selection of human tumor cells resistant to monoclonal antibody-Vinca alkaloid immunoconjugates; Starling JJ et al.; UCLA-P3 human lung adenocarcinoma cells were grown in nude mice and given repetitive treatments of a monoclonal antibody-Vinca alkaloid immunoconjugate . Although this therapy resulted in a greater than 4-fold reduction in mean tumor mass of the established tumors, some animals experienced a reinitiation of tumor growth after cessation of conjugate treatment . Two such animals were treated again with high doses of monoclonal antibody-Vinca but one of the tumors was no longer regressed by the drug conjugate . The tumor was excised, enzymatically dissociated, and grown in tissue culture . Cultured cells were reimplanted in nude mice and subjected to further therapy with a monoclonal antibody-Vinca conjugate . The resulting tumors were also refractory to the immunoconjugate therapy . This cycle was repeated for a total of three times and resulted in the serial in vivo selection of three conjugate resistant variants . The mechanism responsible for the in vivo resistance of human tumor cells to the monoclonal antibody-Vinca immunoconjugate is unknown but does not appear to involve antigen modulation, altered tumor cell growth rate, or an apparent decrease in tumor targeting in vivo . The resistance was also not accompanied by any detectable elevation in multidrug resistance 1 mRNA or P-glycoprotein expression . Significantly, the resistance pattern was observed only in vivo and was not maintained by cells grown in vitro.

Cancer Res, 1990 Dec 1, 50(23), 7537 - 43
Modulation of drug sensitivity by dipyridamole in multidrug resistant tumor cells in vitro; Shalinsky DR et al.; The concept of overcoming multidrug resistance using modulators is based on the hypothesis that there will be a synergistic interaction between the modulator and the cytotoxic agent . We examined the ability of dipyridamole (DPM) to synergistically enhance drug sensitivity in drug-sensitive KB-3-1 cells and their drug-resistant variants, KB-GRC1 and KBV1 cells, using median effect analysis to produce a quantitative measure of the extent of synergy . The drug-resistant variants were resistant to vinblastine (VBL), colchicine (COL), and etoposide (VP-16) in the order VBL greater than COL greater than VP-16 on the basis of 50% inhibitory concentration values obtained by clonogenic assay with continuous drug exposure . The extent of staining with the monoclonal antibody HYB-241, directed at a Mr 180,000 form of the mdrI gene product, correlated with drug resistance for all three drugs (r greater than or equal to 0.92) . DPM and verapamil elevated the steady state content (Css) of VBL, but there was no correlation between elevation of Css and the extent of synergy observed . DPM enhanced the cytotoxicity of VBL and COL in a synergistic manner in KB-GRC1 cells, and in KBV1 cells DPM interacted synergistically with VBL . VPL was synergistic with VBL only in KB-GRC1 cells . No synergy was observed in the parental KB-3-1 line . These data indicate that, although both DPM and verapamil can increase Css in cells not expressing P-glycoprotein, such an increase was not associated with synergy . In cells expressing mdrl, synergy was observed, and it was greatest for the cytotoxic agent for which expression of mdrl produced the greatest fold-resistance and enhancement of Css . However, neither the level of resistance, the level of expression of mdrl, nor the ability of the modulator to alter Css accurately predicted whether the interaction would be truly synergistic . We conclude that additional factors determine the nature of the drug interaction.

J Cell Physiol, 1990 Dec, 145(3), 398 - 408
Sex-dependent and independent expression of the P-glycoprotein isoforms in Chinese hamster; Bradley G et al.; P-glycoprotein (Pgp) is a small family of membrane proteins which belongs to a superfamily of energy-dependent membrane transport proteins identified in phylogenetically distant species, from bacteria to man . Among mammalian species, some of the Pgp isoforms can mediate multidrug resistance by acting as an energy-dependent drug efflux pump . However, the physiologic functions of the Pgp isoforms have not been defined . In this study we examined the expression of the three hamster Pgp isoforms in normal hamster tissues, by using isoform-specific monoclonal antibodies in a competitive immunohistochemical assay . We showed that each Pgp isoform is predominantly expressed in a small, distinct group of differentiated cells, where it is likely to function in specific secretory pathways . The expression of the Pgp isoforms appears to be tightly regulated and, at least in some cells, under complex hormonal control . Furthermore, there is a striking sex difference in Pgp content of the adrenal cortex . These findings are important for the ultimate understanding of the normal physiologic roles of the Pgp gene family members.

Cancer Lett, 1990 Nov 19, 55(1), 17 - 23
Activity of various amphiphilic agents in reversing multidrug resistance of L 1210 cells; Pommerenke EW et al.; Several compounds (bamipine, chlorphenoxamine, estracyt, hycanthone, quinidine, quinine, tamoxifen, trifluoperazine and verapamil) have a common basic structure with the following features: lipophilic aromatic ring system; linked chain hydrophilic N-alkyl group . They are used medically for varying diseases . Their activity in reversing multidrug-resistance (MDR) with other compounds (diethylstilbestrol, beta-estradiol, methylbiguanide, methylpiperazine, testosterone) lacking one of these chemical features is compared . The in vitro test system we used was the nucleoside incorporation assay using parental L 1210 ascites tumor cells and a doxorubicin resistant subline, which expresses the MDR phenotype . The substances lacking one of these features were not effective in reversing the MDR whereas all other tested substances demonstrated modulating potential in the MDR resistant L 1210 cells.

J Biol Chem, 1990 Nov 15, 265(32), 19690 - 6
Tissue-specific enhancer of the human multidrug-resistance (MDR1) gene; Kohno K et al.; Identification of cis-regulatory sequences is a first step in analyzing the regulation of the human multidrug-resistant 1 (MDR1) gene which encodes the 170-kilodalton membrane P-glycoprotein in normal tissues and tumor cells . We have studied several overlapping genomic clones containing the 5'-flanking region of the gene . These clones span about 30 kilobases (kb) of contiguous DNA containing 10 kb of the gene and 20 kb of the 5'-flanking sequence . The nucleotide sequence of the first exon and the 2 kb preceding the exon were determined . DNA sequences containing the 5'-flanking regions were linked to the chloramphenicol acetyltransferase (CAT) gene . For transient CAT assay, we have employed six cell lines, including human cancer KB, vincristine-resistant VJ-300 derived from KB, mouse adrenal tumor Y-1, African green monkey kidney CV-1, mouse fibroblast NIH3T3, and human adrenal carcinoma SW-13 cells . Promoter activity was very weak regardless of the length of the promoter region in mouse adrenal tumor Y-1 and monkey kidney CV-1 cells, in which endogenous P-glycoprotein was expressed . Introduction of a 700-base genomic DNA fragment from a site located at 10 kb far upstream of the initiation site increased the transcription of the CAT gene in Y-1, CV-1, and SW-13 cells . However, no significant increase in the CAT activity could be observed in NIH3T3, KB, and VJ-300 cells . This fragment markedly augmented the expression of the CAT gene regardless of orientation or position, and it acted in a cell type-specific manner even with heterogenous promoters . Our present study suggests that the 700-base pair fragment may carry a tissue-specific transcriptional enhancer that is active in at least some adrenal and kidney-derived cell lines.

Blood, 1990 Nov 15, 76(10), 2065 - 71
Expression of P-glycoprotein in adult T-cell leukemia cells; Kuwazuru Y et al.; We have examined the expression of P-glycoprotein (P-gp) in adult T-cell leukemia (ATL) samples from 25 patients . Based on immunoblotting with a monoclonal antibody against P-gp, C219, 8 of 20 ATL patients were P-gp positive at the initial presentation . All 6 patients at the relapsed stage were P-gp positive, and refractory to chemotherapy . The expression of MDR1 mRNA in P-gp-positive ATL cells was increased at the relapsed stage of one patient . P-gp of this patient was photolabeled with {3H}azidopine and the labeling was inhibited with nimodipine, vinblastine and progesterone . These results suggest that P-gp expressed in ATL cells from patients at relapsed stage has the same binding site(s) for the drugs as that in multidrug resistant cells, and is correlated with the refractory nature of the cells to chemotherapy.

Biochemistry, 1990 Nov 13, 29(45), 10351 - 6
Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression; Murphy LD et al.; The ability of the polymerase chain reaction (PCR) to quantitate expression of mRNA was examined in the present study . The model chosen was expression of the multidrug resistance gene mdr-1/Pgp in two colon carcinoma cell lines which express mdr-1/Pgp at levels comparable to those found in many clinical samples . PCR was utilized to evaluate differences in mdr-1/Pgp expression in the two cell lines after modulation by sodium butyrate . Thus, comparisons were made across a range of mdr-1/Pgp expression as well as comparisons of small differences . The PCR was found to be both sensitive and quantitative . Accurate quantitation requires demonstration of an exponential range which varies among samples . The exponential range can be determined by carrying out the PCR for a fixed number of cycles on serial dilutions of the RNA reverse transcription product, or by performing the reaction with a varying number of cycles on a fixed quantity of cDNA . By quantitation of the difference in PCR product derived from a given amount of RNA from the sodium butyrate treated and untreated cells, the difference in mRNA expression between samples can be determined . Normalization of the results can be achieved by independent amplification of a control gene, such as beta 2-microglobulin, when the latter is also evaluated in the exponential range . Simultaneous amplification of the control and target genes results in lower levels of PCR products due to competition, which varies from sample to sample . The PCR is thus a labor-intensive but sensitive method of quantitating gene expression in small samples of RNA.

Biochim Biophys Acta, 1990 Nov 12, 1055(2), 117 - 25
A model for computer simulation of P-glycoprotein and transmembrane delta pH-mediated anthracycline transport in multidrug-resistant tumor cells; Demant EJ et al.; Anthracycline resistance in multidrug-resistant (MDR) tumor cells is due in part to a reduced cellular drug accumulation . Using a simple kinetic model and numerical computer simulations, we have analyzed mathematically the following possible mechanisms controlling fluxes of the membrane permeable anthracyclines in MDR cells: (1) active outward transport via a specific drug transporter (P-glycoprotein), (2) exocytotic drug export via the endosomal vesicle system, and (3) pH-gradients across the plasma membrane . Model calculations were based on morphometric and kinetic data previously presented in the literature for daunorubicin transport in wild-type Ehrlich ascites tumor cells (EHR2) and the corresponding daunorubicin (DNR)-resistant cell line EHR2/DNR+ . The results confirm the possible importance of the cell-surface pH in controlling DNR accumulation in the cells . With P-glycoprotein as the main efflux pump, a catalytic constant of the protein greater than 40 mol DNR transported/mol protein per min is predicted by the model calculations . Changes in the drug binding affinity of P-glycoprotein (Km = 10(-9)-10(-6) M) is of little importance in influencing its effectiveness to reduce DNR accumulation, which could explain the broad substrate specificity of the MDR efflux pump system . The conditions to evaluate unidirectional fluxes of DNR across the plasma membrane in cells with active P-glycoprotein are defined . An efflux mechanism which relies solely on pH-dependent drug trapping in a pH 5 endosomal compartment by a simple diffusion process followed by exocytosis, appears inadequate to account for the high rate of DNR efflux in EHR2/DNR+ cells.

J Histochem Cytochem, 1990 Nov, 38(11), 1677 - 81
Immunohistochemical localization of P-glycoprotein in adult human ovary and female genital tract of patients with benign gynecological conditions; Finstad CL et al.; The multidrug-resistance gene, MDR1, encodes a plasma membrane glycoprotein termed P-glycoprotein, which mediates active cellular efflux of certain cytotoxic agents . Two mouse monoclonal antibodies (MAb), C219 and JSB-1, were used to identify P-glycoprotein in frozen tissue from the female genital tract of 14 women with benign gynecological conditions; multiple samples from several sites in the genital tract were available from seven patients . P-glycoprotein was detected in the ovarian surface epithelium in four of 14 cases, in the Fallopian tube in three of five cases, in occasional epithelial cells of the endometrial glands in two of five cases, in some endocervical glandular epithelium in three of five cases, in ectocervical squamous epithelium in one of the two cases, and in luteinized cells of the eight cases in which a corpus luteum was present in the specimen . Positive staining with these two MAb was also observed in some endothelial cells in the cortex of the ovary and in the stromal tissue of the myometrium, endometrium, and endocervix . These studies suggest that, if epithelial ovarian cancers are derived from the surface epithelial cells of the ovary, a small proportion of the cancers might be expected to retain the phenotype found in non-cancerous cells and to express P-glycoprotein.

J Biol Chem, 1990 Nov 5, 265(31), 18753 - 6
Progesterone photoaffinity labels P-glycoprotein in multidrug-resistant human leukemic lymphoblasts; Qian XD et al.; We show for the first time that {3H}progesterone ({3H}PRG) can directly photoaffinity label membrane proteins prepared from a multidrug-resistant human leukemic lymphoblastic cell line CEM/VLB5K . A 170-kDa protein in CEM/VLB5K cell membranes was specifically labeled by {3H}PRG, which we identified as P-glycoprotein (Pgp) by immunoprecipitation with monoclonal antibody C219 . The anticancer drug vinblastine and multidrug resistance reversing agent verapamil as well as several steroidal hormones were examined for their ability to interfere with {3H}PRG binding to Pgp . We found that 200-fold molar excess of vinblastine strongly inhibited (93%) the binding of {3H}PRG to Pgp compared with verapamil (80%), progesterone (78%), testosterone (46%), dexamethasone (25%), and aldosterone (56%) . The results of this study provide direct evidence that progesterone can bind to Pgp and support the hypothesis that under physiological conditions Pgp may play a role in the excretion of progesterone from certain cells . Importantly, our results show that under our conditions vinblastine and verapamil are better able to compete with {3H}PRG for binding to Pgp than are other steroids, including testosterone, corticosteroids, and mineralocorticoids.

Cancer Lett, 1990 Nov 5, 54(3), 125 - 31
Verapamil and cyclosporin A potentiate the effects of chemotherapeutic drugs in the human medullary thyroid carcinoma TT cell line not expressing the 170 kDa P-glycoprotein; Larsson R et al.; The TT-cell line, derived from a patient with metastatic medullary thyroid carcinoma (MTC), was found to exhibit intrinsic resistance to vincristine (VCR) despite the absence of immunohistochemically detectable 170 kDa P-glycoprotein (PGP 170) associated with multidrug resistance (MDR) . Verapamil and cyclosporin A, two well known resistance modifiers of MDR, were found to significantly potentiate the action of VCR (60-fold) and to a lesser degree also of VP-16 and daunorubicin (dnr) . The present results suggests that resistance of MTC to chemotherapy may be at least partly circumvented by the addition resistance modifiers to chemotherapeutic regimens.

Mol Cell Biol, 1990 Nov, 10(11), 6036 - 40
Cell-specific activity of cis-acting regulatory elements in the promoter of the mouse multidrug resistance gene mdr1; Raymond M et al.; To define cis-acting elements implicated in transcriptional regulation of the mouse multidrug resistance gene mdr1, we have cloned and characterized the 5' end of the gene . Nucleotide sequence analysis identified TATA, GGGCGG, and CCAAT consensus sequence elements at positions -27, -47, and -83, respectively . The transcriptional activities of 5' deletion fragments from the promoter linked to a reporter gene were tested in mouse cell lines of different tissue origins shown to express different levels of endogenous mdr1 RNA . Sequences located between nucleotides -93 and +84 were able to confer basal promoter activity and cell specificity to the reporter gene . The addition to the basal promoter of sequences upstream of position -141 was found to up or down regulate the basal level of expression of the reporter gene in a cell-specific manner.

Br J Cancer, 1990 Nov, 62(5), 712 - 7
Reversal of acquired resistance to adriamycin in CHO cells by tamoxifen and 4-hydroxy tamoxifen: role of drug interaction with alpha 1 acid glycoprotein; Chatterjee M et al.; Tamoxifen and 4-OH tamoxifen were used to reverse multidrug resistance (MDR) in CHO cells with acquired resistance to adriamycin (CHO-Adrr) . Because alpha 1 acid glycoprotein (AAG) can bind a range of calcium channel blockers that also reverse MDR and rises in malignancy, its interactions with tamoxifen and 4-OH tamoxifen were also studied . Tamoxifen decreased the IC50 of 10 microM adriamycin 4.8-fold in the parent CHO-K1 cell line and 16-fold in CHO-Adrr . Similarly 4-OH tamoxifen decreased the IC50 3-fold in the parent cells, but 13-fold in the resistant cells . Tamoxifen and 4-OH tamoxifen were similarly potent in reversing MDR, although their anti-oestrogen potency differs 100-fold . AAG was added in increasing concentrations to the combination of adriamycin and tamoxifen . As AAG concentrations increased from 0.5 to 2 mg ml-1 (the range found in vivo) the effect of tamoxifen on reversing MDR was gradually decreased . At the highest AAG concentrations, there was complete reversal of the effects of both tamoxifen and 4-OH tamoxifen . AAG was found to bind 3H-tamoxifen in a non-saturable non-specific manner, in contrast to the binding of tamoxifen to albumin . Thus the use of tamoxifen as a reversal agent for MDR in vivo may be impaired by high binding to AAG . However, at the lower range of normal values of AAG, there was still an effect of 10 microM tamoxifen . It may be desirable to select patients for modifier studies based on AAG plasma levels.

Cancer, 1990 Nov 1, 66(9), 1980 - 3
Increased expression of the multidrug-resistance gene in undifferentiated sarcoma; Tawa A et al.; We analyzed multidrug-resistance gene (mdr1 gene) expression in a patient with undifferentiated sarcoma of the liver using the cloned cDNA for the mdr1 gene . Tissue samples were available at the time of initial diagnosis and of two intracranial relapses after chemotherapy with a regimen including doxorubicin and teniposide . The level of mdr1 gene expression was increased sevenfold in the intracranial tumor at the time of first relapse and 11-fold at the second relapse . This case may be an example of acquired multidrug resistance associated with overexpression of the mdr1 gene.

Cancer Res, 1990 Nov 1, 50(21), 6936 - 43
Action of gossypol and rhodamine 123 on wild type and multidrug-resistant MCF-7 human breast cancer cells: 31P nuclear magnetic resonance and toxicity studies; Jaroszewski JW et al.; The action of gossypol, a polyphenolic bisnaphthalene aldehyde, on a number of drug-sensitive and multidrug-resistant cell lines, in particular MCF-7 WT and MCF-7 ADR cells, was studied and compared to the effects of rhodamine 123 . 31P nuclear magnetic resonance spectra of cells exposed to low concentrations of gossypol exhibited decreased levels of ATP, markedly increased levels of pyridine nucleotides, and decreased levels of glycerylphosphocholine . The latter effect may be related to the membrane viscosity-increasing effect of gossypol, whereas changes in the levels of pyridine nucleotides are probably due to an interference with NAD- and NADP-dependent enzymes . The effect of gossypol represents a rare example of selective and differentiated changes observed in 31P nuclear magnetic resonance spectra of cells following exposure to a drug; the effect was markedly different from that of rhodamine 123, which caused ATP depletion but no changes in the levels of glycerylphosphocholine or pyridine nucleotides . Also, the effects of gossypol and rhodamine 123 on glucose metabolism in the MCF-7 WT cells were different . Thus although both drugs caused a marked elevation of glucose uptake, an increase in lactate production exceeding that of glucose consumption, indicating an inhibition of oxidative phosphorylation, was observed only in the case of rhodamine 123 . Significantly, multidrug-resistant cells exhibited strong cross-resistance to rhodamine but practically no resistance to gossypol, which emphasizes the attractiveness of the latter as a potential anticancer drug . The resistance to rhodamine 123 and sensitivity to gossypol was also observed with cells transfected with the MDR1 gene, showing that the difference in toxicity is mainly due to the different response to the P-170 drug efflux pump.

Jpn J Cancer Res, 1990 Nov, 81(11), 1132 - 6
Inhibition of abl oncogene tyrosine kinase induces erythroid differentiation of human myelogenous leukemia K562 cells; Honma Y et al.; The human chronic myelogenous leukemia cell line K562 expresses a structurally altered c-abl protein with tyrosine kinase activity . Erythroid differentiation of K562 cells was induced by tyrosine kinase inhibitors, but not by other kinase inhibitors . Treatment of K562 cells with 5'd(TACTGGCCGCTG-AAGGGC)3', complementary to the second exon (codons 2 to 7) of c-abl mRNA, inhibited cell growth and induced benzidine-positive cells in a dose-dependent manner . However, exposure to the sense oligomer did not induce erythroid differentiation of the cells . These results suggest that inhibition of abl tyrosine kinase activity is closely related to induction of erythroid differentiation of K562 cells . A multidrug-resistant subline (K562R) was induced to undergo erythroid differentiation by tyrosine kinase inhibitors such as genistein or herbimycin A as effectively as the parent K562 cells were . Therefore, tyrosine kinase inhibitors might be useful as cancer chemotherapeutic agents against some multidrug-resistant leukemias having abnormally high activity of oncogene tyrosine kinase(s).

Mol Cell Biol, 1990 Nov, 10(11), 5728 - 35
Overexpression of the multidrug resistance gene mdr3 in spontaneous and chemically induced mouse hepatocellular carcinomas; Teeter LD et al.; Overexpression of a family of plasma membrane glycoproteins, known as P-glycoproteins, is commonly associated with multidrug resistance in animal cells . In rodents, three multidrug resistance (mdr or pgp) genes have been identified, but only two can confer the multidrug resistance phenotype upon transfection into animal cells . Using the RNase protection method, we demonstrated that the levels of three mdr gene transcripts differ among mouse tissues, confirming a previous report that the expression of these genes is tissue specific (J.M . Croop, M . Raymond, D . Huber, A . DeVault, R . J . Arceci, P . Gros, and D . E . Housman, Mol . Cell . Biol . 9:1346-1350, 1989) . The levels of mdr transcripts were determined for mouse liver tumors spontaneously arising in both C3H/HeN and transgenic animals containing the hepatitis B virus envelope gene and for tumors induced by two different carcinogenic regimens in C57BL/6N and B6C3-F1 mice . The mdr3 gene was overexpressed in all 22 tumors tested . Our results demonstrate that overexpression of the mdr3 gene in mouse liver tumors does not require exposure of the animals to carcinogenic agents and suggest that its overexpression is associated with a general pathway of hepatic tumor development . The overexpression of the mdr3 gene, which is the homolog of human mdr1 gene, in hepatocellular carcinomas may be responsible for the poor response of these tumors to cancer chemotherapeutic agents.

Anticancer Drug Des, 1990 Nov, 5(4), 319 - 35
Comparative cytotoxicities of a series of ellipticine and olivacine derivatives on multidrug resistant cells of human and murine origins; Chevallier-Multon MC et al.; The MDR P-glycoprotein has been described as a major factor of multidrug resistance . This transmembrane glycoprotein acts like an energy dependent efflux pump which possesses a broad specificity . It seems to be acting as a pump requiring drug fixation prior to extrusion . With the aim of investigating which parameters influence the recognition of drugs by the MDR system, we have determined the toxicities of different drugs on human and murine sensitive and resistant cell lines . For this purpose we have isolated and characterized a human adriamycin-resistant cell line, CEM/Adr, which presents an MDR phenotype . The tested drugs were ellipticine and olivacine derivatives which differ through discrete lateral chain substitutions . The influence of lateral chain lipophilicity and nitrogen quaternarization on drug recognition was studied . Small modifications in the chemical structure of the drugs have induced large changes in their toxicities and in the cross-resistance levels of the MDR cells to the tested compounds . The cross-resistances of the murine and human cells to the various compounds were strikingly different . The validity of murine screening models in the selection of anti-tumor drugs for human therapy must therefore be questioned.

Jpn J Cancer Res, 1990 Nov, 81(11), 1155 - 61
Monoclonal anti-P-glycoprotein antibody-dependent killing of multidrug-resistant tumor cells by human mononuclear cells; Heike Y et al.; Mouse monoclonal antibodies (MRK16 and MRK17) against human multidrug-resistant cancer cell lines were tested for antibody-dependent cytotoxicity mediated by human blood mononuclear cells, using a 4-h 51Cr release assay . MRK16 (IgG2a isotype) was shown to be more effective than MRK17 (IgG1 isotype) . Moreover, when four pairs of drug-resistant and their parent sensitive human cancer cells were tested for antibody-dependent cell-mediated cytolysis (ADCC) using MRK16, only the drug-resistant cell lines were susceptible to ADCC reaction . When highly purified lymphocytes (greater than 99%) and monocytes (greater than 97%) were isolated from blood mononuclear cells by centrifugal elutriation and adherence, MRK16 promoted both lymphocyte- and monocyte-mediated tumor cell killing, whereas MRK17 induced only a lymphocyte-mediated ADCC reaction . These results suggest that MRK16 of IgG2a subtype may be a useful therapeutic agent in eradication of drug-resistant cancer cells expressing P-glycoprotein through ADCC reaction.

Br J Cancer, 1990 Nov, 62(5), 758 - 61
P-glycoprotein expression in primary breast cancer detected by immunocytochemistry with two monoclonal antibodies; Wishart GC et al.; We have investigated P-glycoprotein (P-gp) expression in samples of primary breast cancer from 29 patients before therapy . We employed immunohistochemical techniques using two monoclonal antibodies (C219 and MRK16) and an indirect alkaline phosphatase method . Heterogeneous expression in epithelial cells was detected with both C219 (21 of 29) and MRK16 (16 of 29) . A surprising finding was P-glycoprotein expression in stromal cells with both C219 (26 of 29) and MRK16 (12 of 29) . Our results suggest that significant levels of P-glycoprotein expression may be present in breast cancer before exposure to drugs associated with multidrug resistance.

Cancer Res, 1990 Nov 1, 50(21), 6841 - 7
Response of mouse skin tumors to doxorubicin is dependent on carcinogen exposure; Keith WN et al.; To investigate the role of carcinogenesis in determining the response of tumors to anticancer drugs, we have used the in vivo model of multistage carcinogenesis of the mouse skin . Mice were initiated with Harvey murine sarcoma virus or single and repeated applications of dimethylbenzanthracene (DMBA) . The papillomas which developed as a result of these initiation protocols were monitored quantitatively for their response to the anticancer drug doxorubicin . A single dose of 10 mg/kg doxorubicin is relatively inefficient at reducing the frequency of papillomas arising as a result of either single or repeated applications of the chemical DMBA . However, virally initiated papillomas are sensitive to the single 10-mg/kg dose of doxorubicin and are reduced in frequency by greater than 80% . Repeat treatment with four doses of 5 mg/kg doxorubicin over a 4-week period also reveals differences in the responses of the papillomas to doxorubicin . As with the single dose of doxorubicin, papillomas initiated with multiple applications of DMBA showed only a limited response to four 5-mg/kg doses of doxorubicin . In comparison both the virally initiated and the single DMBA initiated papillomas responded to the four doses of doxorubicin and are reduced in frequency by about 80% . These data show that the response of papillomas to doxorubicin is related to the initiating event . Papillomas derived by viral initiation are most sensitive to doxorubicin while increasing the level of exposure to the chemical carcinogen DMBA increases the proportion of papillomas which do not respond to treatment with doxorubicin . There was no obvious relationship between the method of initiation or the treatment of the mice with doxorubicin and the levels of P-glycoprotein expression observed in the papillomas . All the papillomas expressed detectable levels of P-glycoprotein approaching that of the multidrug resistant cell line, CHRC5.

Anticancer Res, 1990 Nov-Dec, 10(6), 1571 - 7
Effect of lysosomotropic and membrane active substances on adriamycin uptake and histamine release; Klugmann FB et al.; It has recently been shown that adriamycin is accumulated in mast cells by an active transport system, which closely resembles the outward transport system of multidrug resistant (MDR) cells . The present study was undertaken in order to test the effect of substances which are known to limit or reverse resistance in MDR cells on adriamycin uptake and histamine release in rat peritoneal mast cells . The lysosomotropic amines chloroquine, nicotine, propranolol, atropine, methylamine, ammonium chloride and quinacrine were only slightly effective at very high concentrations; no effect could be observed with the lysosomotropic amine amantadine . The carboxylic ionophores monensin and nigericin were, on the contrary, extremely efficacious; in particular, the effect of monensin was more evident when mast cells were preincubated with the ionophore for 10 or 30 minutes while only a slight inhibition was evident when the two substances were added simultaneously . Removal of monensin from the incubation medium before challenge with adriamycin did not abolish the inhibitory action of the ionophore . Among the tested membrane active agents, Tween 80 and Triton WR-1339 were able to limit adriamycin uptake and histamine release . The microscopical examination showed that in mast cells treated with adriamycin, an intense fluorescence was present in cytoplasmic granules; on the contrary, mast cells preincubated with monensin showed hardly any fluorescence.

Jpn J Cancer Res, 1990 Nov, 81(11), 1184 - 90
Synergistic effect in culture of bleomycin-group antibiotics and N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine, a synthetic isoprenoid; Tomida A et al.; Like bleomycin and peplomycin, libromycin, a newly developed bleomycin-group antibiotic, was potentiated 130-fold against Chinese hamster V79 cells (V79/S) and 47-fold against its multidrug-resistant mutant (V79/ADM) by N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine (SDB-ethylenediamine) at 10 and 3 micrograms/ml, respectively . But neocarzinostatin, known to cause DNA strand scission as bleomycins do, was potentiated only twofold . This suggests that the high potentiation by SDB-ethylenediamine is unique to the bleomycin-group antibiotics . Isobologram analysis revealed that the combined effect of peplomycin and SDB-ethylenediamine was highly synergistic . SDB-ethylenediamine did not increase the intracellular accumulation of {3H}peplomycin in V79/S cells . Analyses by an alkaline elution method demonstrated that single strand scission in DNA of intact V79/S cells caused by 1-h incubation with peplomycin was greatly stimulated by pre- and co-existence of SDB-ethylenediamine, but DNA strand breaks in isolated nuclei were not affected . Apparently some cytoplasmic constituent(s) is involved in the potentiation mechanism . SDB-ethylenediamine did not block the DNA repair which occurred after the removal of peplomycin from the medium . Two fragments of SDB-ethylenediamine, solanesol (polyprenoid moiety) and a diamine component (verapamil-like moiety), were not synergistic with peplomycin, even when they were mixed together . This indicates that the steric conformation of the intact SDB-ethylenediamine molecule is important for the activity.

Cancer Res, 1990 Nov 1, 50(21), 6793 - 9
Susceptibility of multidrug-resistant human leukemia cell lines to human interleukin 2-activated killer cells; Kimmig A et al.; Considering the possibility to overcome drug resistance by other treatment strategies than chemotherapy we investigated the susceptibility of three independently selected multidrug-resistant sublines of the T-lymphoblastoid leukemic cell line CCRF-CEM to lymphokine-activated killer (LAK) cells . We found that two of the multidrug-resistant sublines were significantly less susceptible targets to LAK cells . A third one, however, was as susceptible as the parental CCRF-CEM cell line . Moreover, a multidrug-resistant subline that reverted to an almost drug-sensitive phenotype was observed to be also revertant for resistance against LAK cells . We found an inverse relationship between the expression of the mdr1 gene (P-glycoprotein) and the susceptibility to LAK cells . Verapamil, a calcium channel blocker, while increasing the drug sensitivity of a multidrug-resistant subline, did not induce a reversal of the suppression of LAK susceptibility . The possibility of enhanced resistance to LAK cells of multidrug-resistant cells should be taken into account when one is looking for therapy strategies to overcome multidrug resistance.

Biochem Biophys Res Commun, 1990 Oct 30, 172(2), 842 - 9
Increased mdr gene expression and decreased drug accumulation in a human colonic cancer cell line resistant to hydrophobic drug; Chao CC et al.; An adriamycin-resistant human colonic cancer cell line was characterized . This clone exhibits the classical multidrug resistance (MDR) phenotype, being cross-resistant to hydrophobic drugs such as colchicine, and vinblastine . In contrast, this clone shows a normal response to DNA-damaging agents . The appearance of MDR in these cells was linked to a decreased accumulation of the drug {3H}colchicine as compared to the drug-sensitive cells . This MDR line expressed 80-100 fold increased levels of the specific 4.5-kb mdr mRNA, and a gene amplification . Our results indicate that MDR in human colonic cancer cells can result from increased expression of at least one member of the mdr gene family.

Biochem Biophys Res Commun, 1990 Oct 15, 172(1), 262 - 7
Efflux of bis-carboxyethyl-carboxyfluorescein (BCECF) by a novel ATP-dependent transport mechanism in epithelial cells; Allen CN et al.; The efflux of the intracellular pH fluorochrome 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was quantified in four cultured epithelial cell lines; HCT-8, T84, HGT-1 and MDCK . BCECF efflux was time-dependent, and after 5 h 45-91% of the initial BCECF loaded was extracellular, efflux being greatest in MDCK cells . Depletion of cellular ATP approximately halved BCECF efflux . BCECF efflux was inhibited by indomethacin, vinblastine and verapamil, but not by nifedipine or reserpine . Certain features of BCECF efflux resemble drug efflux in multidrug resistant cells, but inhibition of efflux displays a distinct pharmacological profile suggesting BCECF is a substrate for a novel ATP-dependent transport system.

Cancer Res, 1990 Oct 15, 50(20), 6626 - 31
Mechanisms of resistance of confluent human and rat colon cancer cells to anthracyclines: alteration of drug passive diffusion; Pelletier H et al.; Two colon cancer cell lines, HT-29 (human) and DHD/K12/TRb (rat), were grown as monolayer cultures to various confluence degrees . The cytotoxic efficacies of doxorubicin and 4'-deoxydoxorubicin, evaluated by a survival assay, and the nuclear drug concentrations, measured by microspectrofluorometry, were shown to progressively decrease with the augmentation of confluence . This confluence dependent resistance (CDR) to anthracyclines was demonstrated independent of the multidrug resistance drug efflux mechanism . The cellular uptake of three compounds (sodium {51Cr}chromate, D-{14C}alanine, L-{14C}glucose) known to passively diffuse across the cell membrane as anthracyclines do was also reduced in confluent cells . After trypsin cell detachment, the kinetics of reversion of the sodium {51Cr}chromate uptake decrease and that of CDR were similar . Therefore, CDR may be attributed to a reduction of anthracycline cell intake due to a general alteration of passive diffusion across the cell membrane . However, CDR is only partly explained by this phenomenon since a reduced sensitivity of confluent cells was observed compared with nonconfluent cells for a similar amount of drug in their nuclei . CDR could explain the high resistance to anthracyclines of some solid tumors, such as colon tumors, in which cancer cells are tightly aggregated.

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