Int J Cancer, 1990 Oct 15, 46(4), 727 - 32 Modulation of multidrug resistance by verapamil or mdr1 anti-sense oligodeoxynucleotide does not change the high susceptibility to lymphokine-activated killers in mdr-resistant human carcinoma (LoVo) line; Rivoltini L et al.; Two sublines were derived from the colon adenocarcinoma line LoVo, the first one was sensitive (LoVo/H) and the second one was made resistant to doxorubicin (LoVo/Dx) . When tested for susceptibility to lysis by different types of immune effectors, LoVo/Dx appeared more sensitive than LoVo/H to the killing of CD3+CD5+CD16-, CD3- CD16+)-enriched lymphokine activated killers (LAK) or activated macrophages . In order to check whether this effect was due to different expression of glycoprotein P170 between the two LoVo sublines (30% vs . 90% of positive cells), a pharmacological and genetic modulation of P170 was carried out in LoVo cells . Treatment of LoVo/Dx with the calcium channel blocker verpamil (VRP), strongly impaired P170 function as evaluated by reduced Dx resistance, without affecting the lysability of LoVo/Dx cells by LAKs . Moreover, the significant inhibition of P170 expression resulting from the treatment of LoVo/Dx with mdr1 anti-sense olideoxynucleotide also failed to change the high lysability of LoVo/Dx by LAKs . These results, therefore, indicate that molecules other than P170 are involved in the increased lysis of LoVo/Dx subline by immune effectors and that down-regulation of the P170 expression or function will not reduce the potential effectiveness of cancer chemo-immunotherapy. Cancer Res, 1990 Oct 1, 50(19), 6222 - 8 Expression of rat microsomal epoxide hydrolase gene during liver chemical carcinogenesis; Kondo S et al.; A complementary DNA library was constructed from mRNA of rat liver induced by an initiating dose of a chemical carcinogen, diethylnitrosamine . Using a differential hybridization technique, a complementary DNA clone which is induced more than 10-fold by an acute single dose of diethylnitrosamine was identified . The DNA sequence of this clone was matched with rat microsomal epoxide hydrolase . This gene may be of great interest, since it was found to be highly expressed in neoplastic nodules and primary hepatocellular carcinomas induced by different carcinogenic regimes . The inducible high level expression of this gene becomes constitutive during the process of hepatocarcinogenesis . The gene was also found to be inducibly expressed during partial hepatectomy in a similar manner as a multidrug-resistant gene (mdr-I) . No change in the transcriptional initiation site was observed in the gene expression between induced and uninduced rat livers . The 5' upstream region of the gene was characterized and some potential controlling elements for gene regulation, such as Sp-1, AP-2, and Hepatitis B virus enhancer, were found . Based on our own and published results, we hypothesize that the altered expression of this xenobiotic enzyme in nodules and cancer cells could be a result of constitutive internal stimuli which might be associated with cell growth. No To Shinkei, 1990 Oct, 42(10), 965 - 70 {Immunohistochemical study of placental form of glutathione S-transferase in human brain tumors and fetal brains}; Nakamura M et al.; The activity of glutathione S-transferase placental form (GST-pi) was examined in 100 cases including various histologic subtypes and grading of human brain tumors and 10 cases of fetal brains by immunohistochemical studies . The 69% of cases with brain tumors were shown to be positive for GST-pi . This activity in neuroepithelial tumors tended to increase in order to tumor grading, however, medulloblastoma and primitive neuroectodermal tumor (PNET) were not immunoreactive with GST-pi . Embryonal carcinoma showed strong staining, although fetal brains were negative . The metastatic brain tumors showed the same reactivity with GST-pi as those of original carcinomas . Moreover, the difference of GST-pi activity was investigated on some brain tumors treated with or without antitumor drug, such as 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) . The 85% of recurrent cases showed strong staining with GST-pi, and GST-pi activity seemed to be increased after treated with ACNU . The present study indicated that GST-pi might be a useful marker for human brain tumor, as the same conclusion was applicable to other neoplastic lesions examined previously . It is suggested that the increased GST-pi activity with malignancy of tumor may indicate the tendency to recurrence . The presence of such activity in tumor cells may also imply their acquired multidrug resistance . Our findings suggest that the evaluation of GST-pi activity in brain tumors will offer a predictive value for eventual behavior of the tumor. Nippon Hinyokika Gakkai Zasshi, 1990 Oct, 81(10), 1487 - 93 {In vitro effects of epidermal growth factor (EGF) on growth of urological malignant tumor cells}; Hattori T et al.; It is well known that Epidermal Growth Factor (EGF) is a cell-regulating factor for variety of tissues in vitro including normal and malignant cells . Furthermore, Takano et al reported that a decreased expression of EGF receptor in clones of human cancer KB cell line might be one of the pleiotropic properties of multidrug-resistant cells . However, both the influence of EGF on human urological cancer cell lines and the relation between EGF receptors and sensitivities of antitumor drugs on these cell lines have not been fully described . We have studied the effects of EGF on growth of 4 transitional carcinoma cell lines of bladder (TCCaB), 1 squamous cell carcinoma cell line of bladder (SCCaB), 5 renal cell carcinoma cell lines (RCC) and 3 prostatic carcinoma cell lines (CaP), as well as the relationship between the number of EGF receptors and drug sensitivities of these cell lines in vitro against methotrexate, vinblastine, adriamycin, cisplatin and etoposide (VP16) . The present results determined by the in vitro colony forming efficiency method showed that exogenous addition of EGF to cell cultures at 0.1 ng/ml stimulated the growth of SCCaB by 169.0%, and at 1 ng/ml inhibited that of RCC by 2.9%-79.0%, relative to control . The more EGF receptors by 125I-EGF binding assay, the higher inhibition of VP16 on the growth of these cell lines . These results suggested that EGF stimulated the growth of SCCaB and inhibited the growth of RCC in vitro, and we found that these phenomena were correlated with neither the number of EGF receptors nor affinities of that receptors.(ABSTRACT TRUNCATED AT 250 WORDS) Hematol Oncol Clin North Am, 1990 Oct, 4(5), 871 - 94 Pharmacology and drug resistance in childhood lymphoblastic leukemia; Adamson PC et al.; In the first part of this article, the pharmacology of the chemotherapeutic agents used in the treatment of childhood lymphoblastic leukemia is reviewed, detailing their mechanisms of action, pharmacokinetics, and toxicities . A section on central nervous system pharmacology discusses advances made in the treatment of meningeal leukemia . Mechanisms of drug resistance are discussed in the second section of the article, outlining the biochemical basis for and clinical implications of both agent-specific resistance and multidrug resistance. J Clin Pathol, 1990 Oct, 43(10), 833 - 9 Tissue preparation for simultaneous flow cytometric quantitation of tumour associated antigens and DNA in solid tumours; van Dam PA et al.; A multiparameter flow cytometric assay for the simultaneous study of tumour associated antigens (TAA) and DNA in fresh solid tumours was devised . Cell suspensions were prepared by disaggregating unfixed solid tumour samples mechanically over a stainless steel mesh . Indirect immunofluorescence was used to identify the TAA, and DNA was stained with propidium iodide . Cell morphology was well preserved, cell clumping was negligible, and high quality indirect immunofluorescence quality indirect immunofluorescence and DNA staining were obtained . The technique is simple, rapid, and reproducible . Multiparameter assays can be developed to study prognostic indicators such as membrane oncoproteins, receptors, and multidrug resistance in solid tumours . With a suitable panel of antibodies the technique might become an aid in the differential diagnosis and biochemical diagnosis of some solid tumours. Gan To Kagaku Ryoho, 1990 Oct, 17(10), 1975 - 81 {Regulation of the multidrug resistance (MDR)1 gene expression}; Kohno K et al.; MDR1 gene encodes a gp-170 membrane protein which acts as a energy-dependent pump to transport anticancer agents out of the cells . In this article, we briefly summarize the MDR gene family, gene amplification, gene expression by differentiation and gene expression in clinical tumors . We also describe the characterization of the promotor and tissue specific enhancer of the MDR1 gene and our recent study of the regulatory mechanism of this gene expression. Mol Cell Biol, 1990 Oct, 10(10), 5541 - 7 Increased expression and DNA-binding activity of transcription factor Sp1 in doxorubicin-resistant HL-60 leukemia cells; Borellini F et al.; The processes responsible for the multidrug-resistant (Mdr) phenotype in Adriamycin (doxorubicin)-resistant HL-60 leukemia cells (HL-60/AR) are not defined . Since enhanced transcription of resistance-related proteins is associated with Mdr cells, we sought to determine whether changes in the expression of specific transcription factors were a feature characteristic of the Mdr process . Nuclear extracts were prepared from wild-type and resistant cells and compared for their ability to bind DNA consensus sequences for the transcription factors Sp1 and NF kappa B contained in the 5' long terminal repeat region of human immunodeficiency virus type 1 . Southwestern (DNA-protein) blots showed a family of DNA-binding proteins of 105 kilodaltons (kDa) that were present only in HL-60/AR cells . Competitive gel shift assays indicated that these factors were related to transcription factor Sp1, and immunoblotting with an Sp1 antibody identified this factor as Sp1 . DNase footprinting of the promoter region in the human immunodeficiency virus type 1 5' long terminal repeat showed that protection occurred at two Sp1 sites as well as two NF kappa B sites and the trans-acting region with nuclear extracts only from resistant cells . Preliminary evidence also suggests that phosphorylation may play a negative regulatory role in the activity of Sp1, since calf intestine alkaline phosphatase stimulated the DNA-binding activity of Sp1 in vitro . These results indicate that HL-60/AR cells contain an abundance of DNA-binding proteins, particularly Sp1, which probably interact with other cis-acting regulatory proteins in a cooperative manner. Leukemia, 1990 Oct, 4(10), 695 - 9 The role of the MDR-1/P-170 mechanism in the development of multidrug resistance in chronic myeloid leukemia; Weide R et al.; We studied blood and bone marrow cells from 42 patients with Ph-chromosome positive chronic myeloid leukemia (CML) and 20 normal subjects for amplification of the multidrug resistance gene (MDR-1) by Southern blotting and for overexpression of P-glycoprotein (P-170) by immunocytochemistry on intact cells with the monoclonal antibody C219 . No P-170 could be detected in normal bone marrow or buffy coat . Overexpression of P-170 without amplification of MDR-1 was found in four of 11 patients with chronic phase CML at diagnosis, seven of 16 patients treated with busulfan or hydroxyurea in chronic phase and four of 15 patients in blast crisis . The P-170 overexpression involved only cells of the granulocyte lineage and varied from weak to strong in individual patients . It did not correlate with duration of or response to treatment during chronic phase . In transformation P-170 expression was seen in differentiated cells of the granulocyte lineage but not in blast cells, although three patients had been treated intensively with lipophilic and other cytotoxic drugs to which they had become resistant . We conclude that resistance to busulfan and hydroxyurea in chronic phase and resistance of blast cells to other cytotoxic drugs in transformation are not mediated primarily through the MDR-1/P-170 pathway. Br J Haematol, 1990 Oct, 76(2), 226 - 30 Expression of the multiple drug resistance gene (mdr-1) and epitope masking in chronic lymphatic leukaemia; Cumber PM et al.; Resistance to cytotoxic agents is a common clinical problem in the treatment of chronic lymphatic leukaemia (CLL) . The multidrug resistant (MDR) phenotype characterized by increased levels of a specific cell membrane p-glycoprotein, confers cross resistance to a wide range of structurally dissimilar antineoplastic drugs . We have studied the expression of this p-glycoprotein in chronic lymphatic leukaemia measured by immunofluorescence using a monoclonal antibody MRK 16 by flow cytometry . Initial results showed that only 12% of lymphocyte samples from CLL patients showed increased p-glycoprotein, conflicting with a previous observation that 53% of CLL patients had an increased level of mdr-1 mRNA . Treatment of the cells with neuraminidase to remove sialic acid residues increased the proportion of patients showing increased p-glycoprotein to 52% . This suggest that in a subset of CLL patients post translational modification of the protein occurs masking the epitope recognized by MRK 16 . Abnormal sialylation patterns associated with malignancy are a well-recognized phenomenon. Biochem Biophys Res Commun, 1990 Sep 28, 171(3), 1002 - 7 Human immunodeficiency virus I-induced expression of P-glycoprotein; Gollapudi S et al.; Because prolonged treatment of HIV infection with 3'-azido-3'-deoxythymidine (AZT) is associated with in vitro resistance to AZT, we examined whether HIV could induce/amplify the expression of p-glycoprotein in infected cells resulting in reduced drug accumulation leading to reduced sensitivity to AZT . We show that both H9 (T cell line) and U937 (monocytic cell line) cells, upon infection with HIV, expressed increased levels of P-glycoprotein and accumulated significantly less AZT and daunorubicin as compared to uninfected cells . Sodium azide increased intracellular accumulation of daunorubicin in infected cells, suggesting a metabolically active drug efflux mechanism . Addition of cyclosporin A partially corrected intracellular drug accumulation in HIV infected cells . In addition, similar to multidrug resistant tumor cells, HIV-infected cells show depolarization of plasma membrane . Taken together, these data suggest that HIV-induced increased P-glycoprotein expression could be one of the mechanisms for reduced intracellular accumulation of antiviral agents and resistance to AZT and perhaps to other anti-retroviral agents. J Biol Chem, 1990 Sep 25, 265(27), 16509 - 13 Competitive interaction of cyclosporins with the Vinca alkaloid-binding site of P-glycoprotein in multidrug-resistant cells; Tamai I et al.; The mechanism of reversal of resistance to Vinca alkaloids by cyclosporins is unclear . We investigated the molecular mechanism of reversal of Vinca alkaloid resistance by cyclosporin A (CsA) and its nonimmunosuppressive analog O-acetyl C9(1) CsA (SDZ 33-243) in multidrug resistant DC-3F/VCRd-5L Chinese hamster cells . CsA at 3 microM increased vincristine (VCR) sensitivity and almost totally reversed VCR resistance . SDZ 33-243 at 1 microM reduced the IC50 for VCR in resistant cells from 62.0 to 0.00062 microM . CsA and SDZ 33-243 at 10 microM increased {3H}vinblastine (VBL) accumulation in DC-3F/VCRd-5L cells by 27- and 22-fold, respectively . At 10 microM, these compounds also increased {3H}VCR accumulation by 3.5- and 4.0-fold, respectively . {3H}VCR uptake by membrane vesicles from DC-3F/VCRd-5L cells showed high and low affinity components with Michaelis-Menten kinetics, and apparent Km values were 0.140 +/- 0.0523 and 24.8 +/- 6.67 microM, respectively . Kinetic analysis of {3H}VCR uptake in membrane vesicles in the presence of 0.2 microM CsA revealed that CsA competitively inhibited the high affinity {3H}VCR uptake with an apparent inhibition constant (Ki) of 0.126 +/- 0.0173 microM . In addition, CsA and SDZ 33-243 inhibited VBL photoaffinity labeling of P-glycoprotein in a dose-dependent manner, with half-maximum inhibition at 0.5 and 0.4 microM, respectively, compared with that of VBL at 0.6 microM . These data confirm that cyclosporins modulate Vinca alkaloid resistance at least partially through interaction with P-glycoprotein. J Natl Cancer Inst, 1990 Sep 19, 82(18), 1493 - 6 Expression of multidrug-resistance (MDR1) gene in normal epithelia and in invasive carcinomas of the uterine cervix; Riou GF et al.; Total RNA from 92 invasive cervical cancers was analyzed for the presence of multidrug-resistance (MDR1) gene (also known as PGY1) transcripts . A 4.5-kilobase MDR1 transcript band was detected in 40 (43%) of 92 invasive cervical carcinomas and in 15 (68%) of 22 normal cervices . MDR1 levels were low {mean, 2.5 arbitrary units (U)} except in one liver metastasis (50 U) treated with a drug regimen including vincristine . Of eight carcinomas treated by radiotherapy and/or chemotherapy, seven (88%) exhibited MDR1 transcripts as compared with 24 (35%) of 69 untreated carcinomas (Fisher's exact test; P = .01) . In conclusion, our data suggest that the MDR1 gene plays a role in drug resistance of certain cervical cancers, but also that other mechanisms may be involved. Int J Cancer, 1990 Sep 15, 46(3), 546 - 51 The use of clonogenic assays in assessing the response of human lung cancer cell lines to alpha and gamma interferons alone or in combination with adriamycin; Jabbar SA et al.; The antiproliferative and cytotoxic effects of purified IFN-alpha and recombinant IFN-gamma were investigated using both direct cell counting and a clonogenic assay on a panel of 5 established human lung cancer cell lines and for 2 of them also on their multidrug-resistant counterparts . There was considerable heterogeneity in the response of the cell lines to the IFNs in terms of growth inhibition . Clonogenic assay of IFN-treated cells indicated that, where a cell line had responded markedly to an IFN, only a small fraction of the cells remaining after IFN treatment were clonogenically viable . When cells were placed into the clonogenic assay in the presence of IFNs, the time course of colony formation was different from that seen in the control cultures for most of the cell lines . The measured "surviving fraction" was greatly dependent upon the time of colony counting . When the effects of IFNs in combination with ADM were studied, conclusions regarding the interaction of the effects of the agents also depended upon the time at which colonies were counted. Cancer Res, 1990 Sep 15, 50(18), 6100 - 6 Membrane vesicle formation due to acquired mitoxantrone resistance in human gastric carcinoma cell line EPG85-257; Dietel M et al.; A newly established gastric carcinoma cell line (EPG85-257P) exhibited a high sensitivity to mitoxantrone (DHAD) as determined by a monolayer proliferation assay . The concentration to inhibit cell growth to 50% of controls (IC50) was 0.0022 micrograms/ml culture medium . The cells were continuously incubated for more than 4 months in the presence of stepwise increased concentrations of DHAD, and the IC50 was increased to 0.41 micrograms/ml, i.e., 186.4-fold . This resistant variant was named EPG85-257RNOV . The EPG85-257RNOV cells became cross-resistant to Adriamycin with enhanced IC50 by 10.5-fold and to daunomycin with enhanced IC50 by 3.9-fold . No distinct resistance was observed to vinblastine, vincristine, and colchicine . Verapamil (10(-6), 4 X 10(-6) and 10(-5) M) and cyclosporin A (10(-6), 3 X 10(-6) and 10(-5) M) did not reverse DHAD resistance . As shown by immunocytochemistry (monoclonal antibodies: C219 and JSB-1) and Northern blot analysis, DHAD resistance was not associated with the appearance of the multidrug resistance (MDR)-associated (Mr 170,000) P-glycoprotein or the overexpression of P-glycoprotein mRNA . The data indicate a chemoresistance pattern unlike typical MDR (often called "atypical" MDR) . The phenotypes of parent and resistant EPG85-257 cells were compared using interference contrast microscopy, electron microscopy, and immunocytochemistry . After DHAD application the following structural characteristics were found to be associated with emergence of resistance: (a) intensive formation of surface vesicles in the resistant variant . Such vesicles were almost absent in sensitive cells; (b) the vesicles contained the selecting DHAD which was visualized by its blue color; and (c) in electron microscopy the vesicles were formed by an inner and an outer double membrane, presumably derived from the plasmalemma . These observations suggest a complex cellular mechanism responsible for DHAD resistance which includes formation of membrane vesicles, vesicular drug binding, and drug compartmentalization. Cancer Res, 1990 Sep 15, 50(18), 5931 - 6 Human tumor cell line resistance to chemotherapeutic agents does not predict resistance to natural killer or lymphokine-activated killer cell-mediated cytolysis; Harker WG et al.; Cancer cells selected for resistance to natural product chemotherapeutic agents typically display cross-resistance to a variety of structurally and mechanistically diverse agents, a phenomenon known as multidrug resistance . Preliminary studies involving cells selected for multidrug resistance in vitro have suggested that the development of resistance to these agents might simultaneously confer resistance to some forms of immunotherapy . Using human tumor cell line models, we have investigated the relationship between either intrinsic or selected multidrug resistance and sensitivity to natural killer (NK) or lymphokine-activated killer (LAK) cell-mediated cytolysis . We compared the NK and LAK cell susceptibility of three human tumor cell lines displaying distinct mechanisms of selected drug resistance with that of the parental drug-sensitive lines . We also evaluated the NK and LAK susceptibility of five established renal cell carcinoma lines, all of which were found to be intrinsically resistant to doxorubicin and vinblastine . The drug-resistant cell lines were variably sensitive to NK-mediated lysis . In contrast, all drug-resistant cell lines tested were LAK cell sensitive . The NK and LAK cell-mediated cytolytic sensitivities of the drug-resistant cell lines correlated well with those of the drug-sensitive parental lines, suggesting that susceptibility to lysis was related intrinsically to each tumor type, and not to the resistance phenotype . We attempted to correlated the NK sensitivity of these cells with the cell surface expression of Class I or II histocompatibility antigens, or the presence or absence of the membrane inhibitor of complement-mediated reactive lysis . None of these phenotypic markers were found to predict NK resistance . We therefore conclude that these cells, which are either spontaneously resistant to commonly utilized antitumor agents or are multidrug resistant as a result of drug exposure in vitro, remain sensitive to LAK cell-mediated cytolysis . Our studies suggest that interleukin 2-induced LAK cells may be useful in the therapy of some chemotherapy-resistant cancers. Biochim Biophys Acta, 1990 Sep 7, 1027(3), 225 - 8 Dimerization of the P-glycoprotein in membranes; Boscoboinik D et al.; Plasma membranes from a CHO cell line, CHRC5, which exhibits multidrug resistance was studied using radiation inactivation analysis . The P-glycoprotein content of the membrane was determined by Western blots . Irradiation resulted in the loss of P-glycoprotein . The dependence of this loss on radiation dose corresponded to a target size of 250 kDa which is the molecular mass of a dimer of the P-glycoprotein . This is strong evidence to indicate that the P-glycoprotein self associates in the membrane. Exp Hematol, 1990 Sep, 18(8), 940 - 4 Assessment of purging with multidrug resistance (MDR) modulators and VP-16: results of long-term marrow culture; Aihara M 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 bone marrow cells . VP-16 was toxic to normal bone marrow at concentrations greater than 50 microM, resulting in no granulocyte-macrophage colony-forming units (CFU-GM) in short-term methylcellulose cultures . However, in long-term marrow cultures (LTMC) treatment with VP-16 without the addition of MDR modulators resulted in only a twofold decrease in total cell number at a VP-16 concentration of 50 microM, compared to media alone in the adherent cell layer, and approximately 20% recovery of CFU-GM . The addition of MDR modulators did not result in excessive cytotoxicity, reducing the total CFU-GM by two- to threefold even at the higher VP-16 concentration . Therefore, these modulators in conjunction with VP-16 can be safely used on normal bone marrow cells and may provide an effective method to purge MDR-tumor cells. Exp Cell Res, 1990 Sep, 190(1), 69 - 75 Retention of vital dyes correlates inversely with the multidrug-resistant phenotype of adriamycin-selected murine fibrosarcoma variants; Bucana CD et al.; Retention of the vital dyes rhodamine 123 (R-123) and hydroethidine (HET) correlates inversely with the multidrug resistant phenotypes of the adriamycin (ADM)-selected variants of a uv-induced murine fibrosarcoma cell line (UV-2237M) . The differential affinity of these dyes for specific cellular organelles makes them unique compounds for studies of cellular transport . HET enters viable cells freely, is dehydrogenated to ethidium bromide (EtBr), and is subsequently accumulated in the nucleus . Viable cells are impermeable to extracellular EtBr, facilitating kinetic analysis of the efflux of intracellular EtBr . We found that the metabolite EtBr was rapidly cleared by ADM-resistant but not by ADM-sensitive cells . R-123 has a high affinity to mitochondria . Our results show that ADM-sensitive cells retain R-123 whereas the ADM-resistant cells do not . The clearance of both R-123 and EtBr from these cells was inhibited by verapamil . Therefore, R-123 and HET may be considered MDR-associated compounds useful in studying the MDR phenotype of cancer cells . Previously we reported a direct correlation between the level of activity of the calcium- and phospholipid-dependent protein kinase (protein kinases C) and ADM resistance in UV-2237M variant lines . In this report, we demonstrate a direct correlation between cellular calcium and MDR in these cells . Although chelation of extracellular calcium by EDTA did not alter the fluorescence profile of R-123 of the various cell lines, treating the ADM-resistant variants with verapamil restored cellular calcium to the same level as that of the parental cells and, at the same time, retarded the facilitated efflux of R-123 and EtBr and partially reversed cancer cell resistance to ADM. Br J Cancer, 1990 Sep, 62(3), 378 - 84 P388 leukaemia cells resistant to the anthracycline menogaril lack multidrug resistant phenotype; Badiner GJ et al.; Menogaril is an anthracycline presently in Phase II clinical trials . Menogaril-resistant mouse leukaemia P388 cells were developed in vitro by 4 months of exposure to step-wise increasing concentrations of menogaril after which resistant cells (P388/MEN) were cloned in 320 ng ml-1 menogaril . P388/MEN cells were 40-fold more resistant to menogaril in vitro compared to P388/O and were also resistant in vivo . Resistance to menogaril was stable for at least 2 months in the absence of the drug . The results indicate that P388/MEN, although resistant to an anthracycline, did not display the typical multidrug resistant phenotype . It was not cross-resistant to several structurally unrelated drugs such as actinomycin D, cisplatin, or vinblastine, but it was cross-resistant to the anthracycline, adriamycin . Uptake and efflux of menogaril was similar in sensitive and resistant cell lines . Also, resistance was not reversed by verapamil . No major karyotypic difference was noted between P388/O and P388/MEN . There was no significant amplification or overexpression of the mdr gene in P388/MEN compared to P388/O . In contrast to P388/MEN, P388 cells resistant to adriamycin displayed the typical multidrug resistant phenotype . Glutathione content of P388/MEN cells was similar to that of P388/O and depletion of glutathione did not potentiate menogaril cytotoxicity . Therefore, we conclude that glutathione is not likely to be involved in menogaril resistance to P388/MEN cells. Hiroshima J Med Sci, 1990 Sep, 39(3), 71 - 7 Expression of the multidrug resistance gene in human tumors; Kim R; The expression of MDR1 gene was investigated in human solid tumors with respect to adriamycin resistance . Forty fresh human surgical specimens were analyzed by RNA dot blot assay for their expression of the human MDR1 gene and by immunohistological staining using a monoclonal antibody against P-glycoprotein (MDR1 gene product) . The MDR1 mRNA level was increased in 11 cases of 40 cancer patients, including three rectal cancers, two breast cancers, two gastric cancers, one colon cancer, one renal cell carcinoma, one gall bladder cancer and one malignant lymphoma of stomach . However, considerable variation of the MDR1 mRNA level was noted among cancers of a specific type . Immunohistochemical studies with the monoclonal antibody were shown to be positive in 18 tumors . In all tumors tested, the MDR1 mRNA level and the immunohistochemical analysis showed a significant correlation . However, two of five tumors which resisted adriamycin treatment were found to be negative in MDR1 transcript, but positive in immunohistological analysis . These results indicate that immunohistochemical analysis would be more sensitive for detecting P-glycoprotein-expression, and that resistance to adriamycin, being multifactorial, can be associated at least, in part with the increased amount of MDR1 gene product. Anticancer Res, 1990 Sep-Oct, 10(5A), 1297 - 302 Clinical significance of multiple drug resistance in human cancers; Benard J et al.; This review summarizes the basic aspects of multidrug resistance (MDR) . We first propose a brief reappraisal of the occurrence of MDR encoded P-glycoprotein, (P-g-P) in various normal and tumor tissues . We then present a series of studies undertaken to estimate the MDR1 gene expression or the presence of P-g-P in various human cancers . Finally, we conclude on the potential use of P-g-P measurements in some cancers as a useful if not powerful means of reversing clinical multidrug resistance. Jpn J Cancer Res, 1990 Sep, 81(9), 949 - 55 In situ localization of the human multidrug-resistance gene mRNA using thymine-thymine dimerized single-stranded cDNA; Sugawara I et al.; In order to detect the mRNA transcribed from the multidrug-resistance gene (MDR1), thymine-thymine (T-T) dimerized single-stranded DNA probes have been utilized for hybridization with mRNA either on nitrocellulose filters or in cells and tissues . S1 nuclease digestion rather than sonication was used to obtain short T-T dimerized single-stranded DNA (300-400 bases) so that they could penetrate well into the cytoplasm . The hybridized T-T DNA was detected immunohistochemically using rabbit anti-T-T DNA antibody (Ab) and peroxidase-labeled goat anti-rabbit IgG Ab . Employing this system, MDR1 mRNA could be localized clearly in the human multidrug-resistant cell lines K562/ADM, CEM/VLB, 2780AD, and KBC4 cells as well as in human fetal kidney and gastric carcinoma . Furthermore, our system successfully detected the expression of MDR1 mRNA in cell lines of increasing resistance . These results paralleled results obtained at the protein level by immunohistochemistry . The analysis of MDR1 RNA expression by this in situ hybridization technique should be useful in the study of normal human tissues and tumor samples expressing the MDR1 gene. Proc Natl Acad Sci U S A, 1990 Sep, 87(18), 7225 - 9 Molecular basis of preferential resistance to colchicine in multidrug-resistant human cells conferred by Gly-185----Val-185 substitution in P-glycoprotein; Safa AR et al.; Expression of P-glycoprotein, encoded by the human MDR1 gene, results in cross-resistance to many lipophilic cytotoxic drugs (multidrug resistance) . P-glycoprotein is believed to function as an energy-dependent efflux pump that is responsible for decreased drug accumulation in multidrug-resistant cells . Previous work showed that preferential resistance to colchicine in a colchicine-selected multidrug-resistant cell line was caused by spontaneous mutations in the MDR1 gene that resulted in a Gly-185----Val-185 substitution in P-glycoprotein . We have now compared transfectant cell lines expressing either the wild-type Gly-185 or the mutant Val-185 P-glycoprotein with regard to their levels of resistance to and accumulation and binding of different drugs . In cells expressing the mutant protein, increased resistance to colchicine and decreased resistance to vinblastine correlated with a decreased accumulation of colchicine and increased accumulation of vinblastine . Expression of the mutant P-glycoprotein also resulted in significantly increased resistance to epipodophyllotoxin and decreased resistance to vincristine and actinomycin D; smaller changes in resistance were observed for several other drugs . Unexpectedly, the mutant P-glycoprotein showed increased binding of photoactive analogs of vinblastine and verapamil and the photoactive compound azidopine and decreased binding of a photoactive colchicine analog . These results suggest that the Gly-185----Val-185 substitution affects not the initial drug-binding site of P-glycoprotein but another site, associated with the release of P-glycoprotein-bound drugs to the outside of the cell. Proc Natl Acad Sci U S A, 1990 Sep, 87(18), 7160 - 4 Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction; Noonan KE et al.; The resistance of tumor cells to chemotherapeutic drugs is a major obstacle to successful cancer chemotherapy . In human cells, expression of the MDR1 gene, encoding a transmembrane efflux pump (P-glycoprotein), leads to decreased intracellular accumulation and resistance to a variety of lipophilic drugs (multidrug resistance; MDR) . The levels of MDR in cell lines selected in vitro have been shown to correlate with the steady-state levels of MDR1 mRNA and P-glycoprotein . In cells with a severalfold increase in cellular drug resistance, MDR1 expression levels are close to the limits of detection by conventional assays . MDR1 expression has been frequently observed in human tumors after chemotherapy and in some but not all types of clinically refractory tumors untreated with chemotherapeutic drugs . We have devised a highly sensitive, specific, and quantitative protocol for measuring the levels of MDR1 mRNA in clinical samples, based on the polymerase chain reaction . We have used this assay to measure MDR1 gene expression in MDR cell lines and greater than 300 normal tissues, tumor-derived cell lines, and clinical specimens of untreated tumors of the types in which MDR1 expression was rarely observed by standard assays . Low levels of MDR1 expression were found by polymerase chain reaction in most solid tumors and leukemias tested . The frequency of samples without detectable MDR1 expression varied among different types of tumors; MDR1-negative samples were most common among tumor types known to be relatively responsive to chemotherapy. Hum Pathol, 1990 Sep, 21(9), 949 - 58 ABO blood type predicts the cytolocalization of anti-P-glycoprotein monoclonal antibody reactivity in human colon and ureter; Weinstein RS et al.; Classic multidrug resistance is mediated by a P-glycoprotein . Using monoclonal antibody C219 (MAb C219) in an immunohistochemical study, we found high levels of putative Golgi P-glycoprotein in normal columnar and transitional epithelium in subpopulations of patients with specific blood types . For example, Golgi staining was present in blood type A patients in 46% of normal colon samples (N = 21) and 88% of normal ureter samples (N = 17) . In comparison, Golgi staining was present in blood group O patients in only 6% of normal colon samples (N = 34) and in 0% of normal ureter samples (N = 19) . The association of MAb C219 Golgi staining with blood type A and lack of Golgi staining with blood type O was statistically significant in normal colon (P = .001) and normal ureter (P less than .0001) . Inappropriate hyperexpression of P-glycoprotein was frequently found in colon carcinomas . Additional evidence that Golgi MAb C219 reactivity represents P-glycoprotein is presented . This includes (1) immunostaining of Golgi with two anti-P-glycoprotein MAbs, C219 and JSB-1, and (2) experiments in which Mab C219 Golgi reactivity was blocked by preincubation of MAb C219 with a specific P-glycoprotein epitope-containing peptide . The high degree of association of Golgi P-glycoprotein with blood type A may suggest a role for P-glycoprotein in processing or trafficking of specific blood group antigens. J Urol, 1990 Sep, 144(3), 755 - 9 Mechanisms and modulation of multidrug resistance in primary human renal cell carcinoma; Mickisch GH et al.; Human renal cell carcinomas show a high degree of intrinsic multidrug resistance . In experimental cell lines, the membrane bound P-170 glycoprotein and the glutathione redox cycle seem to contribute to this phenomenon . P-170 may be inactivated by calcium antagonists; the glutathione redox cycle by buthionine sulfoximine . We studied the resistance patterns of 35 human renal cell carcinomas against vinblastine, doxorubicin and carboplatinum in a tetrazolium-based microculture assay . Concomitantly, P-170 expression was traced immunohistochemically using moab C219 and the glutathione content was determined enzymatically . Reversal of multidrug resistance was examined by applying the R-stereoisomer of verapamil and/or by addition of buthionine sulfoximine . A high degree of chemoresistance was seen in 27 tumors against vinblastine, in 30 tumors against doxorubicin and in 31 tumors against carboplatinum . Chemoresponse was found in eight, five or four cases respectively . P-170 was detected in 70% of highly vinblastine resistant and in 63% of highly doxorubicin resistant tumors, but in none of the less resistant cases . Resistance against carboplatinum and doxorubicin was significantly associated with elevated glutathione levels as compared to less resistant renal cell carcinomas . R-verapamil lead to a strong reversal of vinblastine resistance and to a distinct circumvention of doxorubicin resistance, but revealed no effect in carboplatinum resistance . Buthionine sulfoximine overcame carboplatinum resistance and modified doxorubicin resistance, but had no influence on vinblastine resistance . The combined application of R-verapamil and buthionine sulfoximine reversed doxorubicin resistance but did not act synergistically in vinblastine or carboplatinum resistance . Both mechanisms, P-170 and glutathione, occurred independently of each other and may well explain multidrug resistance of human renal cell carcinomas. J Histochem Cytochem, 1990 Sep, 38(9), 1277 - 87 Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues; Cordon-Cardo C et al.; We have characterized the normal human tissue distribution and tumor expression of the human multidrug resistance gene (MDR1) product P-glycoprotein (Pgp) by immunohistochemical staining of frozen tissue sections of human normal and tumor tissues, using three mouse monoclonal antibodies (MAb) which recognize at least two different epitopes of Pgp . Pgp expression on normal human tissues was detected in specialized epithelial cells with secretory/excretory functions, trophoblasts in the placenta, and on endothelial cells of capillary blood vessels at blood-tissue barrier sites . There were significant differences in the staining patterns of these MAb . Mouse MAb HYB-241 and HYB-612 each recognize an extracellular epitope of Pgp, whereas mouse MAb C219 detects a carboxy terminal intracellular epitope and has recently been reported to crossreact with the MDR3 gene product . HYB-241 and HYB-612 strongly stain endothelial cells and trophoblasts, whereas C219 is weakly positive or unreactive on these cells . Likewise, C219 strongly stains the biliary pole of hepatocytes, skeletal and heart muscle fibers, whereas HYB-241 and HYB-612 are unreactive on these cells . Immunopathological studies were performed on a wide variety of human tumors . Pgp expression on human tumors was most commonly detected in colon . renal, and adrenal carcinomas; rarely in lung and gastric carcinomas and certain germ cell tumors; and was undetectable in breast and endometrial carcinomas tested . Few sarcomas and none of the melanomas, neuroblastomas, gliomas, and pheochromocytomas had detectable Pgp expression . Intensity and pattern of staining varied among different cases of a given tumor type; although homogeneous immunoreactivity was observed, heterogeneity of expression in a single histological section was more common . The finding of Pgp expression in a variety of normal tissues with diverse physiological functions suggests that the role of Pgp may not be limited to excretion of xenobiotics . Pgp expression in capillaries of the brain and testis may explain the failure of drugs such as vincristine and actinomycin-D to penetrate into these tissues, allowing them to remain as pharmacological sanctuaries for malignant cells . Although Pgp expression can now be detected in a variety of human tumors, further studies are needed to establish the possible significance of this finding. Cancer Res, 1990 Sep 1, 50(17), 5392 - 8 Non-P-glycoprotein mediated mechanism for multidrug resistance precedes P-glycoprotein expression during in vitro selection for doxorubicin resistance in a human lung cancer cell line; Baas F et al.; Two different mechanisms that contribute to multidrug resistance (MDR) were found in derivatives of the human squamous lung cancer cell line SW-1573 . The parental cell line has a low amount of mdr1 P-glycoprotein mRNA . In three independent selections for doxorubicin resistance, MDR variants arose in which mdr1 P-glycoprotein mRNA and protein was not detectable . Selection on higher doxorubicin concentrations gave rise to variants containing high levels of mdr1 mRNA, due to transcriptional activation of the mdr1 gene . Upon continued selection for higher levels of doxorubicin resistance, the mdr1 gene became amplified, resulting in an additional increase in the level of mdr1 mRNA . The cross-resistance pattern of the sublines that lack mdr1 P-glycoprotein expression is different from that seen in the mdr1 overexpressing cells . Both types of MDR cell lines are resistant to doxorubicin, daunorubicin, etoposide, colchicine, gramicidin D, and vincristine . However, in the non-P-glycoprotein-mediated MDR cell lines, resistance levels are lower and a preferential resistance for etoposide is seen. Tumori, 1990 Aug 31, 76(4), 353 - 9 Clinical relevance of immunocytochemical detection of multidrug-resistance-associated P-glycoprotein in hematologic malignancies; Musto P et al.; P-glycoprotein (P-170) is the phenotypic marker of tumoral cells that show the phenomenon of multidrug resistance (MDR) . Using an immunocytochemical approach, we employed the monoclonal antibody C219 (which recognizes an epitope of such a glycoprotein) to evaluate in cytologic samples the expression of P-170 on neoplastic cells from 52 patients affected by different hematologic malignancies and its eventual correlation to clinical outcome . Longitudinal studies were also performed in 14 patients . Results obtained demonstrated that a) the so-called "MDR phenotype" may be heterogeneously represented (from less than 1 to 100% of positive cells) in hemopoietic tumors at diagnosis (without exposure to pharmacologic agents), as well as during the course of the disease, although a more substantial presence of P-170 occurred in treated patients . There was no correlation between neoplastic kinetic activity (such as expression of Ki 67 recognized nuclear proliferation-associated antigen) and P-170-positive cells . b) Percentage of positive cells as well as intensity of staining seemed to be important in determining MDR; in general, there was a strong correlation between expression of P-170 in more than 20% of neoplastic cells and a lack of response to chemotherapy . However, some false-positive and false-negative cases were observed . c) The detection of scattered P-170-positive cells may predict a pharmacologic selection of intrinsic or mutant-resistant clones. Eur J Biochem, 1990 Aug 28, 192(1), 69 - 74 Interaction of a new photosensitive derivative of vinblastine, NAPAVIN, with tubulin and microtubules in vitro; Nasioulas G et al.; We have synthesized a new photoreactive vinblastine derivative, 3-{{2-amino(4-azido-2-nitrophenyl)ethyl}-amino)-carbonyl)-O4-deceatyl -3-de (methoxycarbonyl)-vincaleukoblastine (NAPAVIN), which can be photoactivated with light in the 455-nm region as well as with ultraviolet irradiation . Previous studies had shown that photoactivated NAPAVIN is much more effective than vinblastine in inhibiting cell proliferation of multidrug resistant cell lines . The experiments reported here demonstrate that the unirradiated derivative is very similar to vinblastine in its interactions with brain tubulin and microtubules, regarding inhibition of in vitro assembly, binding, aggregation, and production of protofilament spirals . Irradiation of {3H}NAPAVIN in the presence of tubulin led to covalent binding of the drug to both subunits of the protein . Labeling also occurred when NAPAVIN was first irradiated, then incubated with tubulin in the dark, indicating the production of a fairly stable reactive species with a half-life of about 400 min . We conclude that labeling by this compound, under some conditions, occurs not by a nitrene but by an electrophilic photoproduct. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 230 - 4 Isolation and characterization of the structural gene encoding elongation factor 3; Sandbaken M et al.; The unique yeast translational factor EF-3 participates in the elongation cycle by stimulating the function of EF-1 alpha in binding aminoacyl-tRNA to the ribosome . We have isolated the structural gene encoding EF-3 from the yeast Saccharomyces cerevisiae . The YEF3 gene is found in one copy per haploid genome and is essential for vegetative growth . DNA sequence analysis reveals that the YEF3 gene contains an open reading frame of 1044 codons . The deduced amino acid sequence has two repeats of a nucleotide-binding motif . Each of these repeats shows similarity to the nucleotide-binding motif of hydrophilic, membrane-associated ATPases including human multidrug resistant protein MDR . Factor 3 manifests ribosome-dependent ATP hydrolysis . Introduction of the YEF3 gene on a high copy number plasmid into yeast strains increases the ribosome-dependent ATPase activity and EF-3 protein levels by 3-5-fold . Yeast strains containing elevated EF-3 protein levels also exhibit increased sensitivity to the aminoglycoside antibiotics hygromycin and paromomycin . These drugs are known to increase translational errors . These observations suggest that EF-3 may affect translational accuracy. Cancer Res, 1990 Aug 15, 50(16), 4946 - 50 Flow cytometric characterization of antifolate resistance in cultured mammalian cells using fluoresceinated methotrexate and daunorubicin; Sherwood SW et al.; We have studied antifolate-resistant rodent cell lines with respect to dihydrofolate reductase gene expression and expression of the "classic" multidrug resistance phenotype by flow cytometry . Using a series of antifolate-resistant and colchicine-resistant Chinese hamster ovary cell lines obtained by single-step and stepwise selection protocols, we show that viable cell staining with fluoresceinated methotrexate and daunorubicin correlates well with drug resistance and expression of dihydrofolate reductase protein and the "classic" MDR phenotype in these cell lines . We show that flow cytometric analysis makes it possible to rapidly assess the potentially complex drug resistance phenotype(s) of cells selected with hydrophilic and lipophilic antifolates. J Biol Chem, 1990 Aug 5, 265(22), 13137 - 42 Identification of a membrane glycoprotein overexpressed in murine lymphoma sublines resistant to cis-diamminedichloroplatinum(II); Kawai K et al.; A series of murine thymic lymphoma cell sublines was selected in vitro for resistance to cis-diamminedichloroplatinum(II) (CDDP) . The level of CDDP resistance correlated with reduced drug accumulation in these cells . A rabbit antiserum was raised against the plasma membrane of a CDDP-resistant subline and used in Western blot analyses . Increased expression of a surface antigen of approximately 200 kDa was observed and found to correlate with the degree of resistance . Further biochemical and immunological studies demonstrated that this is a plasma membrane glycoprotein . However, it is different from the multidrug resistance-associated P-glycoprotein with a molecular weight of about 170,000 . We have called this unique CDDP resistance-associated membrane protein CPR-200. Blood, 1990 Aug 1, 76(3), 489 - 94 Philadelphia chromosome positive childhood acute lymphoblastic leukemia: clinical and cytogenetic characteristics and treatment outcome . A Pediatric Oncology Group study; Crist W et al.; Among 3,638 children with acute lymphoblastic leukemia (ALL) entered on Pediatric Oncology Group (POG) protocols between June 1981 and April 1989, successful cytogenetic studies were available for 2,519, 58 (2.3%) of which had the Philadelphia (Ph) chromosome detected . Features associated with the presence of the Ph chromosome were high leukocyte count (median, 33 x 10(9)/L), older age median, 9.6 years), a higher proportion of French-American-British L2 morphology, and a lower frequency of mediastinal mass . Immunologic marker studies at diagnosis in 56 Ph+ cases identified early pre-B ALL in 42 cases (75%), pre-B-cell in 9 (16%), and T-cell in 5 (9%) . This distribution is similar to that found in Ph+ ALL . Intensive multiagent chemotherapy induced complete remissions in only 78% of eligible Ph+ patients compared with 96% of those without an identified Ph chromosome (P less than .001) . Of 44 eligible Ph+ patients treated on POG frontline protocols for children with non-T, non-B-cell ALL, 27 have failed therapy, compared with 520 of 1,892 without an identified Ph chromosome (logrank P less than .001) . Ph+ ALL is an aggressive form of acute leukemia that frequently presents in older children with a high leukocyte count, FAB L2 morphology, and a pseudodiploid karyotype, and becomes multidrug-resistant early . Thus, Ph+ cases require early identification to permit treatment with intensive induction regimens and experimental approaches such as bone marrow transplantation. J Natl Cancer Inst, 1990 Aug 1, 82(15), 1260 - 3 Expression of a human complementary DNA for the multidrug resistance gene in murine hematopoietic precursor cells with the use of retroviral gene transfer; McLachlin JR et al.; In multidrug resistance, cells become simultaneously resistant to anthracyclines, vinca alkaloids, epipodophyllotoxins, and certain other natural product cytotoxic drugs . Resistance results from synthesis of a multidrug transporter (P-glycoprotein) encoded by the MDR1 gene (also known as the PGY1 gene) . In the present study, a retrovirus vector containing a complementary DNA for the human multidrug resistance gene HaMDR1/A was used to transfer the multidrug resistance phenotype to bone marrow cells of the DBA/2J mouse . A high proportion of transduced bone marrow cells showed resistance to both colchicine and vinblastine, as determined by in vitro colony formation of hematopoietic precursor cells . In addition, brief culturing of the cells in a cytotoxic drug following exposure to the retrovirus vector could be used to increase the proportion of bone marrow cell colonies that were resistant . These results may serve as a model for the generation and selection of bone marrow cells resistant to the toxic effects of chemotherapeutic agents in vivo. Exp Parasitol, 1990 Aug, 71(2), 169 - 75 Entamoeba histolytica: physiology of multidrug resistance; Ayala P et al.; Cross-resistance to unrelated drugs has been previously observed in multidrug-resistant carcinoma cells and the goal of this work was to determine whether a similar mechanism existed in Entamoeba histolytica . An emetine and a colchicine-resistant clone, C2(90) (IC50 = 62 microM, and 1.5 mM, respectively), and the parental clone, A (IC50 = 5 microM and 1 mM, respectively), were analyzed for resistance to other drugs and for the effect of verapamil . Both clones, C2(90) and A, exhibited similar resistance to both daunomycin (IC50 = 50 microM) and actinomycin D (IC50 = 13 nM) . In the presence of verapamil, the IC50 for emetine was reduced to 0.5 microM, while the IC50 for colchicine was reduced to 0.3 mM . These results demonstrate that verapamil reverses both emetine and colchicine resistance in the mutant C2(90) . In uptake experiments with {3H}emetine, drug accumulation was lower in resistant trophozoites . However, in the presence of verapamil, drug accumulation was increased in clone C2(90) to a level close to that of the parental strain, clone A . These results are consistent with observations made using malaria and multidrug-resistant tumor cells and suggest that a P-glycoprotein-like molecule may play a role in drug resistance in E . histolytica. Planta Med, 1990 Aug, 56(4), 368 - 70 Antimalarial activity of Tanzanian medicinal plants; Weenen H et al.; Tanzanian medicinal plants were extracted and tested for in vitro antimalarial activity, using the multidrug resistant K1 strain of Plasmodium falciparum . Of 49 plants investigated, extracts of three plants were found to have an IC50 between 5-10 micrograms/ml, extracts of 18 other plants showed an IC50 between 10 and 50 micrograms/ml, all others were less active . The three most active extracts were obtained from the tubers of Cyperus rotundus L . (Cyperaceae), the rootbark of Hoslundia opposita Vahl . (Labiatae), and the rootbark of Lantana camara L . (Verbenaceae). J Bioenerg Biomembr, 1990 Aug, 22(4), 571 - 92 Binding protein-dependent transport systems; Higgins CF et al.; Bacterial binding protein-dependent transport systems are the best characterized members of a superfamily of transporters which are structurally, functionally, and evolutionary related to each other . These transporters are not only found in bacteria but also in yeasts, plants, and animals including man, and include both import and export systems . Although any single system is relatively specific, different systems handle very different substrates which can be inorganic ions, amino acids, sugars, large polysaccharides, or even proteins . Some are of considerable medical importance, including Mdr, the protein responsible for multidrug resistance in human tumors, and the product of the cystic fibrosis locus . In this article we review the current state of knowledge on the structure and function of the protein components of these transporters, the mechanism by which transport is mediated, and the role of ATP in the transport process. Cancer Res, 1990 Aug 1, 50(15), 4698 - 701 3'-Deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin conquers multidrug resistance by rapid influx following higher frequency of formation of DNA single- and double-strand breaks; Horichi N et al.; The mechanism of action of 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin (MX2) was examined in a human leukemia cell line (K562) and its Adriamycin (ADM)-resistant subline (K562/ADM) . ADM and MX2 showed an equivalent antitumor effect against K562 . K562/ADM was highly resistant to ADM . In cellular pharmacokinetic studies, MX2 showed faster and greater influx than did ADM in both K562 and K562/ADM . The efflux of ADM was rapid in K562/ADM but not in K562 . On the other hand, the efflux of MX2 was rapid in both cell lines . The formation of DNA single-strand breaks and double-strand breaks by ADM was significantly lower in K562/ADM than K562 . On the other hand, formation of those breaks by MX2 was not decreased . Although some of the DNA breaks induced by MX2 were resealed, there was no difference in the degree of resealing in K562 and K562/ADM cells . On the other hand, most of the small number of DNA breaks in K562/ADM induced by ADM were resealed . The topoisomerase II activity in K562 and K562/ADM was not significantly different . It is concluded that MX2 conquers multidrug resistance by rapid influx following a higher frequency of formation of DNA single- and double-strand breaks in K562/ADM cells. Mol Pharmacol, 1990 Aug, 38(2), 198 - 206 Characterization of auromomycin-resistant hamster cell mutants that display a multidrug resistance phenotype; Rauscher FJ 3rd et al.; We have selected and characterized Chinese hamster ovary (CHO) cells resistant to auromomycin (AUR), an antitumor antibiotic composed of a protein moiety and a nonpeptide chromophore . AUR is cytotoxic as a consequence of DNA strand-scission activity associated with the chromophore . Initial single-step selections for clones resistant to AUR detected a subpopulation of phenotypically resistant colonies, but nearly all such clones failed to display heritable resistance . One isolate that did show somewhat increased resistance was selected further and yielded a clone designated AURR-R1 that exhibits stable 10-fold increased resistance to AUR . The R1 line is also resistant to the AUR chromophore and cross-resistant to the closely related agent neocarzinostatin (NCS) and to the NCS chromophore . For AUR-treated whole cells, resistance to AUR cytotoxicity was inversely correlated with DNA damage as measured by filter elution; by contrast, isolated nuclei from sensitive and resistant cells displayed similar levels of AUR-induced DNA damage . The R1 cell line was found to be cross-resistant to colchicine, Adriamycin, Daunomycin, and vinblastine . The resistance phenotype is expressed with incomplete dominance in cell hybrids and appears similar to the "classic" multidrug resistance of CHO cells selected with other agents . Indeed, we found the multidrug-resistant CHO line CCHR-C5 to be about 5-fold cross-resistant to AUR and to NCS . We ascertained that AUR-resistant (AURR) isolates express elevated levels of the molecular weight 170,000 P-glycoprotein often associated with multidrug resistance and also contain amplified DNA sequences that contain the gene for P-glycoprotein . When multiple-step enrichment selections were carried out as an alternative approach for isolating AURR mutants, each of nine clonal isolates showed phenotypes resembling the AURR-R1 line . Thus, our findings imply that increased cellular resistance to AUR may frequently be associated with P-glycoprotein-mediated multidrug resistance. Jpn J Cancer Res, 1990 Aug, 81(8), 834 - 41 Synergistic effect of cyclosporin A and verapamil in overcoming vincristine resistance of multidrug-resistant cultured human leukemia cells; Ishida Y et al.; Reversal of vincristine (VCR) resistance by cyclosporin A (CyA) or the combination of CyA and verapamil (VER) was investigated by using four P-glycoprotein (P-gp)-associated human multidrug-resistant (MDR) cell lines (K562/ADM, KYO-1, HEL and CMK) . Drug sensitivity was expressed as 50% inhibitory concentration (IC50) . The degree of reversal of resistance was expressed as x-fold decrease by dividing the IC50 value without modifier(s) by that with modifier(s) . CyA overcame P-gp-associated MDR significantly in all four MDR cell lines . Reversal of VCR resistance by CyA appeared to be dose-dependent . In the case of low-grade MDR cell lines (KYO-1, HEL and CMK), CyA at the low concentration of 0.5 microgram/ml was still effective . The degree of reversal of VCR resistance in this condition was greater (6.3- to 16-fold decrease) in the low-grade MDR cell lines than in a high-grade MDR cell line (K562/ADM) (2.9-fold decrease) . At a high concentration (5 micrograms/ml) of CyA, however, it was greater (240-fold decrease) in the high-grade MDR cell lines than in the low-grade MDR cell line (20- to 100-fold decrease) . This indicates that concentration of CyA required for overcoming drug resistance in MDR cells was dependent on the degree of drug resistance . CyA overcame VCR resistance more efficiently than VER . The combination of CyA and VER enhanced reversal of VCR resistance in a supra-additive or at least an additive manner and overcame VCR resistance at low concentrations of both modifiers that are clinically achievable with safety. Cancer Res, 1990 Aug 1, 50(15), 4692 - 7 Decreased NADPH:cytochrome P-450 reductase activity and impaired drug activation in a mammalian cell line resistant to mitomycin C under aerobic but not hypoxic conditions; Hoban PR et al.; Mitomycin C (MMC) is regarded as the prototype bioreductive alkylating agent in clinical use . To elucidate the biochemical basis of MMC resistance, we isolated a drug resistant derivative (designated CHO-MMC) of a Chinese hamster ovary cell line (CHO-K1) by exposure to progressively higher concentrations of MMC . CHO-MMC cells exhibited a 17-fold increase in resistance to MMC and were 33-fold cross-resistant to the monofunctional derivative, decarbamoyl mitomycin C . In contrast, CHO-MMC cells showed only a 2-fold level of resistance to BMY 25282, a more easily activated analogue of MMC, and exhibited parental sensitivity to MMC under radiobiologically hypoxic conditions . CHO-MMC cells showed no increased resistance to a range of DNA damaging agents including several other alkylating agents (e.g., melphalan and methyl methanesulfonate) . Cross-resistance to drugs associated with the multidrug resistant phenotype (e.g., Adriamycin and vincristine) was present only at very low levels . Using a specific high performance liquid chromatography technique, we examined the rates of reduction of MMC and BMY 25282 in cell extracts from CHO-K1 and CHO-MMC cells under both aerobic (air) and hypoxic (N2) conditions . Reduction rates for both drugs were at least 30-fold faster under nitrogen than in air . Metabolism of MMC was undetectable in air but was readily detectable under nitrogen and was 2- 3-fold slower in CHO-MMC cell extracts than in CHO-K1 cell extracts . Although BMY 25282 was more readily reduced under nitrogen, no difference was detected between extracts from CHO-K1 or CHO-MMC cells in the rate of reduction of BMY 25282 under either air or nitrogen . The activity of NADPH:cytochrome P-450 (cytochrome c) reductase, an enzyme implicated in the bioreductive activation of MMC, was 3-4-fold lower in CHO-MMC cells than in the parental line . These findings suggest that the resistance of CHO-MMC cells to MMC under aerobic conditions may be due to impaired metabolic activation of the drug as a result of a decrease in NADPH:cytochrome P-450 reductase activity . This supports the view that decreased bioreductive enzyme activity may be a significant mechanism for acquired resistance to MMC in tumor cells in vivo and that more readily activated analogues may be potentially useful in overcoming this specific form of resistance. Br J Cancer, 1990 Aug, 62(2), 177 - 82 Immunohistochemical detection of multidrug resistance associated P-glycoprotein in tumour and stromal cells of human cancers; Schlaifer D et al.; The distribution of Gp 170, a multidrug resistance (MDR) associated glycoprotein, also called P-glycoprotein (P-gp), was examined by immunohistochemistry, using C219 and MRK16 monoclonal antibodies . Sixty-five tumour tissues were studied which included 40 non-lymphoid tumours, 15 chemoresistant non-Hodgkin's lymphomas and 10 Hodgkin's disease . The study was performed on both cryostat and special fixation processed and paraplast embedded (ModAMeX) sections . The latter method preserves fixation-sensitive antigens such as P-gp and allows a more precise morphological identification of neoplastic and non-neoplastic cell populations in contrast to cryostat sections . Immunohistochemical expression of P-gp was expected and confirmed in many non-lymphoid tumours, but stromal macrophages and endothelial cells were also frequently stained in these cases . In non-Hodgkin's lymphomas, cells that were stained with both C219 and MRK16 monoclonal antibodies on cryostat sections were identified as macrophages and endothelial cells and not neoplastic lymphoid cells, by the ModAMeX technique . These findings suggest that the quantitative assessment of MDR RNA by Northern blotting performed on fresh homogenates overestimates the MDR content of neoplastic cells in a number of lymphoid and non-lymphoid tumours . In addition, the mechanism of chemoresistance in non-Hodgkin's lymphomas is less likely to be associated with P-gp expression. Biotechniques, 1990 Aug, 9(2), 206 - 11 A simple polymerase chain reaction method for detection and cloning of low-abundance transcripts; Fuqua SA et al.; A simplified polymerase chain reaction (PCR) technique for the detection, semiquantitation and cloning of low-abundance RNAs is described . This assay involves first-strand cDNA synthesis by reverse transcription of mRNA with specific oligonucleotide primers, followed by second-strand synthesis and PCR amplification, in the same tube with only the addition of TaqI DNA polymerase . The assay is sufficiently sensitive to detect target RNA from as little as 1 ng of total RNA . The beta-actin transcripts may also be simultaneously reverse transcribed, amplified and used as an internal standard to determine relative expression of specific RNAs . Using this simple technique, expression of the multidrug resistance (mdr 1) gene can easily be detected in human breast tumors . The technique is also applicable for the cloning of rare transcripts. Cancer Res, 1990 Jul 15, 50(14), 4291 - 4 In vitro bone marrow purging of multidrug-resistant cells with a mouse monoclonal antibody directed against Mr 170,000 glycoprotein and a saporin-conjugated anti-mouse antibody; Dinota A et al.; Selective elimination of multidrug resistance-positive cells (LoVo/Dx) was obtained by using the monoclonal antibody MRK 16, which recognizes a surface epitope of the Mr 170,000 glycoprotein, and a sheep anti-mouse immunoglobulin antibody, conjugated to the ribosome-inactivating protein saporin 6 . The killing was greatly decreased or even abolished by adding the monoclonal antibody at a 100-fold concentration . Both the MRK 16 and anti-mouse saporin 6 conjugate did not show any killing activity when they were used separately . In cell suspensions composed of 90% normal bone marrow cells and 10% multidrug resistance-positive cells, the monoclonal antibody MRK 16 followed by the anti-mouse immunotoxin caused the elimination of 99% multidrug resistance-positive cells, as revealed by immunofluorescence and immunocytochemistry as well as by a clonal assay . Human normal hematopoietic precursors (granulomonocytic colony-forming units, erythroid burst-forming units, and multipotent granulomonocytic, erythroid, and megakaryocytic-forming units) were not affected by the MRK 16 plus immunotoxin treatment . This technique might be suitable for ex vivo bone purging in an appropriate clinical setting, such as autologous bone marrow transplantation. J Natl Cancer Inst, 1990 Jul 4, 82(13), 1133 - 40 Expression of mdr1 and mdr3 multidrug-resistance genes in human acute and chronic leukemias and association with stimulation of drug accumulation by cyclosporine; Herweijer H et al.; We determined the expression levels of the mdr1 and mdr3 multidrug-resistance genes (also known as PGY1 and PGY3, respectively) in peripheral blood cells from 69 adult patients with acute and chronic leukemias, using an RNase protection assay . Expression of mdr1 was found in samples from patients with acute nonlymphocytic leukemia (13 of 17), chronic myelocytic leukemia (CML, chronic phase, 10 of 10; blast crisis, three of four), acute lymphocytic leukemia (ALL, eight of 11), B-cell chronic lymphocytic leukemia (B-CLL, 17 of 17), hairy cell leukemia (HCL, one of two), and T-cell prolymphocytic leukemia (one of one), but not in B-cell prolymphocytic leukemia (B-PLL, 0 of seven) . Expression of mdr3 was only detected in samples from B-cell lymphocytic leukemias: CML, lymphoid blast crisis (one of one), B-cell ALL (two of two), B-CLL (17 of 17), B-PLL (seven of seven), and HCL (two of two) . In vitro drug uptake studies by on-line flow cytometry showed that in leukemia cells expressing either mdr1 or mdr3, the steady-state accumulation of daunorubicin could be significantly increased by addition of cyclosporine and, to a lesser extent, by verapamil . Because cyclosporine and verapamil are known as inhibitors of the mdr1-encoded P-glycoprotein drug-efflux pump, and because the mdr1 and mdr3 genes are highly homologous, our data suggest that the mdr3 gene encodes a functional drug pump in B-cell lymphocytic leukemias . The results of this study may have implications for clinical therapy for acute or chronic leukemias expressing the mdr1 or mdr3 gene, in particular, treatment with combinations of cytotoxic drugs plus agents that reverse multidrug resistance . Since mdr1 and mdr3 are frequently expressed in untreated as well as treated leukemia, such combination therapy should be considered for untreated patients as well as treated patients. Br J Cancer, 1990 Jul, 62(1), 85 - 8 Cyclosporin A and verapamil have different effects on energy metabolism in multidrug-resistant tumour cells; Broxterman HJ et al.; Cyclosporin A (Sandimmune) rapidly induced an increase in daunorubicin accumulation in multidrug-resistant human ovarian carcinoma cells (2780AD) and was more potent than verapamil . Steady-state 3H-cyclosporin A accumulation at 37 degrees C in 2780AD cells was 60-70% of that in the sensitive A2780 cells . A rapid increase of ATP consumption and lactate production was induced in 2780AD cells by verapamil, but not by cyclosporin A . These results suggest that the interactions of cyclosporin A and verapamil with P-glycoprotein, which leads to inhibition of drug transport, have a different mechanistic basis. Anticancer Res, 1990 Jul-Aug, 10(4), 1075 - 8 In vitro drug sensitivity testing with agar-containing glass capillaries; Lathan B et al.; Capillary cloning has been shown to have advantages over conventional cloning of human tumor cells in Petri dishes . We have recently published in this Journal an optimization of the capillary method towards homogeneous colony distribution and high cloning efficiency . In the present study this modified capillary cloning system was investigated for its feasibility for drug sensitivity testing . For the human breast cancer cell line MDA-231 and the drug sensitive Chinese Hamster Ovary cell line CHO-AB as well as for its multidrug resistant mutant CHO-C5 a similar linear dose response effect was obtained with the capillary cloning system and with the Petri dish system . The capillary cloning system, however, was 2 to 4 fold more sensitive for the detection of cytotoxic drug effects . It was concluded that the optimized capillary cloning system is well suited for drug sensitivity testing. J Med Chem, 1990 Jul, 33(7), 1914 - 9 Structure-activity relationships of antineoplastic agents in multidrug resistance; Selassie CD et al.; Clinical resistance to many antineoplastic agents is a major cause of treatment failure . The well-documented phenomenon addressed as multidrug resistance (MDR) allows cells to withstand exposure to lethal doses of drugs with dissimilar chemical structures, modes of action, and physicochemical properties . In one of the earliest studies on MDR, Biedler and Riehm in an attempt to explain the cross-resistance profile of actinomycin D resistant Chinese hamster cells suggested that molecular weight was an important determinant . Our statistical analysis of their data validates their claim and indeed strongly demonstrates that cross resistance is enhanced by the increased size and hydrophobicity of the antitumor agent . Our preliminary studies with methotrexate-resistant L1210 cells indicates that cross resistance is increased in the case of moderate-sized, hydrophilic drugs . These two studies and others suggest that current chemotherapy regimens may be improved by treating resistant cells with antineoplastic agents displaying physicochemical characteristics opposite to that of the original inducing agent. Cancer Res, 1990 Jul 1, 50(13), 3997 - 4002 Reversal of multidrug resistance by lipophilic drugs; Hofsli E et al.; The phenomenon of multidrug resistance implies that a wide spectrum of structurally and functionally unrelated chemotherapeutic drugs are recognized and processed by the molecular system which protects multidrug-resistant (MDR) cells against lipophilic cytotoxic drugs . This suggests that lipophilic agents with low toxicity may also be recognized and processed by this molecular system . At high concentrations these agents might saturate the system, thereby reversing multidrug resistance . In support of this hypothesis, 19 (73%) of 26 arbitrarily chosen lipophilic drugs were in this study found to increase the accumulation of actinomycin D in MDR WEHI 164 cells . The most potent of these drugs were also shown to sensitize these cells to the cytotoxic effect of actinomycin D and doxorubicin . There was a good correlation between the ability of the lipophilic drugs to induce an increased accumulation of actinomycin D in MDR cells and their ability to sensitize these cells to the cytotoxic effect of chemotherapeutic drugs . The ability to reverse drug resistance appeared to be additive, since increased accumulation of actinomycin D was also obtained by combining low concentrations of various lipophilic drugs . This may be a way to reduce the in vivo toxic effect of the lipophilic drugs yet still obtain a reversal of drug resistance . When MDR cells were exposed to lipophilic drugs which reversed drug resistance, the synergistic cytotoxic effect of actinomycin D and tumor necrosis factor was obtained at reduced actinomycin D concentrations. Int J Parasitol, 1990 Jul, 20(4), 503 - 13 Chemotherapy and drug resistance in malaria; Cowman AF et al.; Over recent years many antimalarial drugs have been rendered useless by the development of resistance by the malaria parasite . New antimalarials are rapidly suffering the same fate as the traditional therapies and yet a biological understanding of the mechanisms of resistance has, until recently, not been described . This review describes recent work which has identified the mechanism of resistance to the dihydrofolate reductase (DHFR) inhibitors as being due to point mutations within the DHFR gene that render the enzyme less susceptible to inhibition by the drugs . The relationship between chloroquine resistance and the recently described multidrug resistance gene is explored and the possibility that this is the main cause of chloroquine resistance by the parasite is discussed . Parasites have developed resistance against many of the quinine-like antimalarials over the past three decades and the possibility that this is linked to the appearance of chloroquine resistance must be considered. Cancer Res, 1990 Jul 1, 50(13), 3921 - 7 Elevated pentose cycle and glucuronyltransferase in daunorubicin-resistant P388 cells; Gessner T et al.; Anthracycline resistance of P388 daunorubicin-resistant cells cannot be accounted for merely by differences in drug uptake and retention; protection against intracellular drug was also indicated . Cytotoxicity of daunorubicin may be partially due to the formation of free radicals and reactive oxygen species (hydrogen peroxide, hydroxyl radical, singlet oxygen, and superoxide anion radical) . Protection against free radicals and peroxides is largely dependent upon the availability of reduced glutathione, which in turn requires NADPH for its continual regeneration . Pentose phosphate cycle (also called hexose monophosphate shunt) is known to provide NADPH for maintenance of glutathione . Activities of the two NADPH-producing dehydrogenases of the cycle, glucose-6-phosphate and 6-phosphogluconate dehydrogenase, were 40% higher (P less than 0.05) and activity of the cycle in intact cells was 2-fold higher in the resistant than the sensitive cells . The cycle was as active in these cells as it is known to be in macrophages, indicating a very effective protection against oxidative stress, free radicals, and alkylating electrophiles . Elevated activity of the pentose phosphate pathway in drug-resistant cells can represent a mechanism of resistance against multiple structurally unrelated drugs . Efflux of daunorubicin may be aided by further metabolism to glucuronides . Daunorubicinol, a known active metabolite of daunorubicin, can be metabolized to a glucuronide by the cells and eliminated into the surrounding medium . Glucuronidation of daunorubicinol was evidenced by (a) release of daunorubicinol following glucuronidase hydrolysis of media from cell incubations with 1.8 microM daunorubicin and (b) production of radioactive glucuronide when cell homogenates were incubated with UDP-{14C}glucuronic acid plus daunorubicinol . Glucuronyltransferase activity with a broad substrate specificity was found in the cells . Using model substrates, 1-naphthol and o-aminophenol, it was determined that glucuronyltransferase activity was 4 times higher in daunorubicin-resistant than -sensitive P388 cells . Elevated glucuronyltransferase could contribute to daunorubicin and multidrug resistance. Haematologica, 1990 Jul-Aug, 75(4), 363 - 74 Multidrug resistance: focus in hematology; Carulli G et al.; The appearance of chemoresistance is the most relevant limitation of chemotherapy . It has been shown that multidrug resistance (MDR) is frequently related to the expression of a membrane glycoprotein (P-170) . This protein is able to bind ATP and leads to decreased accumulation of structurally unrelated antineoplastic drugs extensively used in the management of hematological patients . The availability of monoclonal antibodies and probes allowed extensive studies both "in vitro" and "in vivo" of the protein structure and of its mechanism of action . The P 170 activity may be antagonized by drugs able to compete with chemotherapic agents for the binding or by calcium antagonists that inhibit the expulsion activity of the protein . P 170 has been found in variable percentages of several hematological malignancies such as leukemia, myelodysplastic syndromes, myeloma and lymphoma . The reported data seem to indicate that the patients carrying P 170-positive neoplastic cells should be treated with drugs that are not bound by the protein . However, the possibility of inhibiting the protein function and the recent reports suggesting the use of P 170 as a target for immunotoxins could be the basis for new therapeutic protocols. Br J Cancer, 1990 Jul, 62(1), 89 - 95 Chemosensitisation by verapamil and cyclosporin A in mouse tumour cells expressing different levels of P-glycoprotein and CP22 (sorcin); Twentyman PR et al.; The relationships between resistance to adriamycin, vincristine, colchicine and etopside, expression of P-glycoprotein and CP22 (sorcin), and resistance modification by verapamil and cyclosporin A have been studied in a panel of multidrug-resistant (MDR) mouse tumour cell lines . Whereas there was a generally good correlation between the degree of resistance and the amount of P-glycoprotein, no relationship between resistance and CP22 expression was seen . At 3.3 microM verapamil, the sensitisation of the MDR cell lines was no greater than that of the parent line . At 6.6 microM verapamil, however, sensitisation of the MDR lines generally exceeded that of the parent line, although the line CR 2.0, expressing very high levels of P-glycoprotein was an exception . Little sensitisation to etoposide was seen in any of the lines . When cyclosporin A was used as the sensitiser at either 2.1 or 4.2 microM, there was a greater effect in lines expressing moderate to high levels of P-glycoprotein than in the parent line, although this tendency was less for adriamycin than for the other cytotoxics . Sensitisation to etoposide was much greater with cyclosporin A than with verapamil . At low levels (less than 1 microM) of CsA, however, sensitisation to colchicine was greater in the parent line than in cell line CR 2.0 . These studies indicate that chemosensitisation by verapamil and cyclosporin A is extremely complex, depending upon sensitiser dose, the particular cytotoxic and the cell line . At low doses of the sensitisers, the sensitisation may be greater in lines expressing low levels of P-glycoprotein than in lines showing high levels. Br J Cancer, 1990 Jul, 62(1), 37 - 41 Transport of the multidrug resistance modulators verapamil and azidopine in wild type and daunorubicin resistant Ehrlich ascites tumour cells; Sehested M et al.; Verapamil has been proposed to modulate the multidrug resistance phenotype by competitive inhibition of an energy dependent efflux of cytotoxic drug . However, the accumulation of both 14C-verapamil and 3H-verapamil was similar in wild type EHR2 and multidrug resistant EHR2/DNR+ Ehrlich ascites cells, and was much less in both cell lines in energy deprived medium than in medium containing glucose . Azidopine accumulation was also similar in both EHR2 and EHR2/DNR+ cells but, in contrast to verapamil, did not differ significantly with changes in cellular energy levels . Azidopine photolabelled a 170 kDa protein in EHR2/DHR+ plasma membrane vesicles which was immunoprecipitated by monoclonal antibody towards P-glycoprotein . Azidopine increased daunorubicin accumulation and modulated vincristine resistance in EHR2/DNR+ cells in a similar fashion to verapamil . Azidopine photolabelling was inhibited by vincristine and verapamil, but not by daunorubicin . Vincristine, but not daunorubicin, was able to increase both azidopine and verapamil accumulation in EHR2/DNR+ cells only . Finally, though both verapamil and azidopine are a substrate for P-glycoprotein in EHR2/DNR+ cells, they do not themselves appear to be transported by the multidrug resistance efflux mechanism to any significant extent in these cells. Anticancer Res, 1990 Jul-Aug, 10(4), 897 - 902 Expression of multiple drug resistance gene, MDR1, and N-myc oncogene in an Italian population of human neuroblastoma patients; Corrias MV et al.; Thirty-four patients of an Italian population affected by neuroblastoma (NB) were evaluated at diagnosis for multidrug resistance gene (MDR1) and N-myc oncogene amplification . No patients showed MDR1 amplification, while extra copies of the N-myc gene were found in 9 out of 34 patients (26%) . N-myc amplification was correlated (p = 0.008) with a shorter progression-free survival . RNA was purified from fresh tumor biopsies and analysed in 29 NB samples . MDR1 gene expression was found to be increased in 5 out of 29 tumor samples at onset (17%) and in 1 out of 3 at relapse, but none of them expressed both MDR1 and N-myc genes simultaneously . No correlation was found between MDR1 or N-myc genes expression and tumor progression . MDR1 mRNA transcription may occur spontaneously after onset, suggesting that certain NB tumors could be resistant to antineoplastic drugs at onset . All 5 patients showing MDR1 mRNA transcription achieved complete or partial clinical remission after polychemotherapy . This was presumably due to inclusion in the therapeutic protocol of a high dose of Cisplatin, a drug not susceptible to the effects of the MDR1 gene product . Our findings show that cells which actively transcribe for the MDR1 gene are present in several untreated NB patients . No gene amplification was detected and probably the MDR1 gene expression is regulated at the transcriptional level. Am J Obstet Gynecol, 1990 Jul, 163(1 Pt 1), 69 - 73 Expression of P-glycoprotein in epithelial ovarian cancer: evaluation as a marker of multidrug resistance; Rubin SC et al.; The multidrug-resistance gene, MDR1, encodes a plasma membrane glycoprotein termed P-glycoprotein that mediates active cellular efflux of certain chemotherapeutic agents . P-Glycoprotein expression was evaluated in 98 frozen tumor specimens from 57 patients with epithelial ovarian cancer by the indirect immunoperoxidase technique with monoclonal antibodies C219 and JSB-1 used for detection . Tumor specimens were further characterized antigenically with a panel of monoclonal antibodies representing a variety of epithelial cell antigens . Included were 57 specimens from 33 previously untreated patients; 11 specimens were also available from eight patients in this group after chemotherapy . An additional 30 specimens were studied from 24 other patients after chemotherapy . In only four of the 57 patients with ovarian cancer (7%) did one or more of the specimens express P-glycoprotein . Two of these patients had tumors that were considered clinically drug resistant . No increase in P-glycoprotein expression was noted after exposure to chemotherapy, including the eight individuals for whom specimens were available both before and after treatment . Although drug resistance is a major problem in treatment of ovarian cancer, resistance to the drugs most active against these tumors probably occurs through a mechanism other than expression of the MDR1 gene product. Mol Cell Biol, 1990 Jul, 10(7), 3596 - 606 Structural analysis of the mouse mdr1a (P-glycoprotein) promoter reveals the basis for differential transcript heterogeneity in multidrug-resistant J774.2 cells; Hsu SI et al.; In multidrug-resistant mouse J774.2 cells, the differential overproduction of functionally distinct phosphoglycoprotein isoforms reflects the amplification or transcriptional activation or both of two mdr gene family members, mdr1a and mdr1b . The mdr1a gene is a complex transcriptional unit whose expression is associated with multiple transcript sizes . Independently selected multidrug-resistant J774.2 cell lines differentially overexpress either 4.6- and 5.0-kilobase (kb) or 4.7- and 5.1-kb mdr1a transcripts . However, abundant overproduction of the mdr1a gene product was observed only in cell lines which overexpressed the 4.6- and 5.0-kb mRNAs . In order to determine the basis for mdr1a transcript heterogeneity and the relationship between transcript size and steady-state mdr1a protein levels, genomic and cDNA sequence analyses of the 5' and 3' ends of the mdr1a gene were carried out . Promoter sequence analysis and primer extension mapping indicated that mdr1a transcripts were differentially initiated from two putative promoters to generate either 5.1- and 4.7-kb or 5.0- and 4.6-kb transcripts in four multidrug-resistant J774.2 cell lines . Sequence analysis of 3' cDNA variants and a 3' genomic fragment revealed that the 5.1- and 5.0-kb mRNAs had identical 3'-untranslated regions which differed from those of the 4.7- and 4.6-kb mRNAs as a result of the utilization of a more downstream alternative poly(A) addition signal . Transcript initiation from the putative upstream promoter correlated with a 70 to 85% decrease in steady-state mdr1a protein levels relative to transcript levels . In addition, the identification of putative AP-1 and AP-2 promoter elements suggests a possible role for protein kinase A and protein kinase C in the regulation of mdr1a . The implications of these findings for mdr gene expression and regulation are discussed. Exp Cell Res, 1990 Jul, 189(1), 133 - 41 Transfection of a mutant regulatory subunit gene of cAMP-dependent protein kinase causes increased drug sensitivity and decreased expression of P-glycoprotein; Abraham I et al.; Wild-type Chinese hamster ovary (CHO) cells were transfected with a DNA clone (MT-REV, site A) carrying a mouse gene for a dominant mutant regulatory subunit (RI) gene of cAMP-dependent protein kinase (PKA) from S49 cells along with a marker for G418 resistance . G418-resistant transfectant clone R-2D1 was resistant to 8-Br-cAMP-induced growth inhibition and morphological changes . The cells also did not phosphorylate a 50-kDa protein after cAMP stimulation and had decreased PKA activity, both characteristics of PKA mutants . Northern blot analysis indicated that clone R-2D1 was actively transcribing the MT-REV (site A)-specific RNA . We also tested clone R-2D1 for sensitivity to certain natural product hydrophobic drugs and found increased sensitivity to several drugs including adriamycin . Hypersensitivity to these drugs has previously been shown by us to be a characteristic of a CHO PKA mutant cell line . Expression of the mutant RI gene is also associated with a decrease in expression of the multidrug resistance associated P-glycoprotein (gp170) mRNA and protein . These results show that the PKA mutant RI gene from S49 cells acts as a dominant mutation to reduce the total PKA activity in the CHO transfectants as it does in mouse S49 cells . This study also confirms that reduced PKA activity modulates the basal multidrug resistance of these cells, apparently by causing decreased expression of the mdr gene at the protein and mRNA level. Biochem Biophys Res Commun, 1990 Jun 29, 169(3), 986 - 90 Cross-resistance to anti-HIV nucleoside analogs in multidrug-resistant human cells; Yusa K et al.; Human multidrug-resistant K562/ADM cells showed 12-fold and 31-fold resistance to AZT (3'-azido-2', 3'dideoxythymidine) and DDC (2', 3'-dideoxycytidine), respectively . Other multidrug-resistant human cells CEM/VLB100 and AdrRMCF-7 also showed resistance to these nucleoside analogs . However, verapamil (10 microM) failed to reverse the resistance to the nucleoside analogs . Accumulation of {3H}AZT in human multidrug-resistant K562/ADM, CEM/VLB100 and AdrRMCF-7 cells decreased by 23, 35, and 42% respectively, as compared to their parental cells . These results suggest that anti-HIV nucleoside analogs including AZT, DDC could be transported by outward drug-transport system in the multidrug-resistant cells. J Biol Chem, 1990 Jun 25, 265(18), 10282 - 8 Differential transport properties of two mdr gene products are distinguished by progesterone; Yang CP et al.; P-glycoprotein is an integral membrane protein that is overproduced in multidrug-resistant cells . It is likely to function as an energy-dependent drug efflux pump to maintain intracellular drug concentrations below cytotoxic levels . Individually isolated multidrug-resistant murine cell lines, J7.V1-1 and J7.V3-1, overproduce P-glycoproteins encoded by the mdr1b and mdr1a genes, respectively . The transport properties of these cell lines and the drug binding characteristics of their P-glycoproteins have been compared . It is concluded that 1) the mdr1a gene product is a more efficient efflux pump than the mdr1b gene product, and 2) whereas a single class of vinblastine binding sites is present in J7.V1-1 membrane vesicles, there appears to be two classes of such sites in J7.V3-1 membrane vesicles . The effects of verapamil and progesterone, two compounds that are known to interact with P-glycoprotein, have been analyzed in the two cell lines . Progesterone inhibited drug binding and efflux and increased drug sensitivity to vinblastine with more potency in J7.V1-1 cells than in J7.V3-1 cells . It is concluded that progesterone, but not verapamil, can be used to differentiate the two mdr gene products in the mouse. Biochem Biophys Res Commun, 1990 Jun 15, 169(2), 383 - 90 Information from combined 1H and 31P NMR studies of cell extracts: differences in metabolism between drug-sensitive and drug-resistant MCF-7 human breast cancer cells; Kaplan O et al.; Combined analysis of both 1H and 31P NMR spectra of extracts of drug-sensitive and multidrug-resistant MCF-7 human breast cancer cells enabled quantitative comparisons between the concentrations of major metabolites, as well as of their precursors . In resistant cells high energy phosphorus compounds, phosphocreatine and ATP, and also their precursors, creatine and ADP, were elevated compared to the sensitive cells . In phospholipid metabolism, the sensitive cells showed higher phosphocholine and phosphoethanolamine concentrations than the resistant cells, but choline levels were similar . These results delineated the differences in control of metabolic pathways between drug-sensitive and drug-resistant cells. Cancer Res, 1990 Jun 15, 50(12), 3670 - 4 Effects of calcium antagonists in multidrug resistant primary human renal cell carcinomas; Mickisch GH et al.; Human renal cell carcinomas display a characteristically high degree of intrinsic chemoresistance to a multitude of chemotherapeutic agents . It was suggested previously, that P-170 glycoprotein contributes to this phenomenon in renal cell carcinoma indicated by elevated MDR-1 gene mRNA levels and by the expression of this specific resistance characteristic . The P-170-related efflux mechanism can be inactivated by certain calcium antagonists . P-170 was traced immunohistochemically using monoclonal antibody C 219 . Concomitantly, we studied the enhancement of vinblastine cytotoxicity with 4 major classes of calcium-blocking agents in a microculture tetrazolium assay . Seven different calcium antagonists were selected: verapamil (VPM, racemic form), its R-stereoisomer (R-VPM), diltiazem, flunarizine, nifedipine, and its derivatives nimodipine and nitrendipine . Verapamil or R-verapamil causes a significant decrease of viable tumor cells as compared to vinblastine alone (P less than 0.001) . Similar effects were found with diltiazem, nifedipine, and its derivatives reaching approximately 70% of the VPM/R-VPM activity . Flunarizine showed only minor enhancement of cytotoxicity . P-170 expression was demonstrated in 18 of 32 tumors, and a relation to chemoresistance was evident . None of the chemoresponders, but 18 of 25 (72%) of the highly resistant tumors, revealed this resistance factor . It was concluded that certain calcium antagonists in combination with chemotherapy may well offer therapeutic options in renal cell carcinoma as they apparently inactivate the underlying mechanism conferring resistance . The new stereoisomer R-VPM, in particular, may be used in clinical trials since it combines strong enhancement of vinblastine drug responsiveness with a 10-fold lower cardiovascular activity as compared to racemic VPM, thus allowing higher concentrations to be applied. Cancer Res, 1990 Jun 15, 50(12), 3619 - 26 Enhancement of murine tumor cell sensitivity to adriamycin by presentation of the drug in phosphatidylcholine-phosphatidylserine liposomes; Fan D et al.; In vitro incubation of mouse UV-2237M fibrosarcoma cells with liposomes containing Adriamycin (ADR) produced significant cytotoxicity in drug-sensitive cells and in multidrug-resistant variants of this tumor . ADR was encapsulated in the aqueous space of multilamellar liposomes composed of phosphatidylcholine and phosphatidylserine . The preparation was stable in medium at 37 degrees C for up to 7 days . Free unencapsulated ADR produced cytostasis in parental ADR-sensitive cells but not in variant lines selected for resistance to the drug . In contrast, ADR encapsulated in multilamellar liposomes (MLV) produced high levels of cytostasis in both ADR-sensitive and ADR-resistant cells . The phospholipid composition of the MLV influenced the outcome of ADR-mediated cytostasis . ADR encapsulated in MLV consisting of only phosphatidylcholine did not produce cytostasis . Increasing the proportion of phosphatidylserine in the MLV increased the level of ADR-mediated cytotoxicity in cells resistant to free ADR . This effect was not due to simple modification of tumor cell surface by liposomes since ADR added to resistant cells together with liposomes containing buffer produced less cytostasis . The cytostasis of resistant cells by ADR in liposomes was not due to appreciable changes in the intracellular ADR concentration or localization within the cells because ADR-induced DNA cleavage was not found in ADR-resistant cells treated with cytostatic amounts of liposomal ADR . Whether the enhanced sensitivity of tumor cells to ADR was due to localized damage to the plasma membrane through a phosphatidylserine-mediated release of the drug to the cell surface is now under active investigation. Cancer Res, 1990 Jun 15, 50(12), 3600 - 4 Isolation and characterization of a rat liver epithelial cell line resistant to the antiproliferative effects of transforming growth factor beta (type 1); Chapekar MS et al.; Rat liver epithelial cells resistant to the growth-inhibitory effects of transforming growth factor beta 1 (TGF-beta 1) were isolated after 3 h exposure to 1.5 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine followed by continuous treatment with 1 ng/ml TGF-beta 1 for 6 weeks . In comparison to the parental or N-methyl-N'-nitro-N-nitrosoguanidine-exposed rat liver epithelial cells (concentration causing 50% inhibition of the rate of DNA synthesis, 0.25 ng/ml), these cells were 10-fold more resistant to the antiproliferative effects of TGF-beta 1 and exhibited resistance to growth inhibition by a highly purified liver-derived growth inhibitor, recombinant human tumor necrosis factor, and transforming growth factor beta 2 . Single cell cloning of these resistant cells led to the isolation of a nontransformed clonal cell population (clone 11) which maintained stable resistance in the absence of TGF-beta 1 treatment . Binding of 125I-labeled TGF-beta 1 to rat liver epithelial cells and clone 11 cells was similar . Clone 11 cells exhibited a 5-10-fold resistance to the cytotoxins Adriamycin and vinblastine as assessed by a clonogenic assay . This drug resistance was accompanied by an increase in the steady state levels of the mRNAs for multidrug resistance gene (MDR-1), glutathione S-transferase-P, TGF-beta 1, and c-myc genes . The data presented here suggest an association between resistance to the growth-inhibitory effects of TGF-beta 1- and MDR-1-mediated multidrug resistance. Biochem Biophys Res Commun, 1990 Jun 15, 169(2), 796 - 802 mdr1/P-glycoprotein gene segments analyzed from various human leukemic cell lines exhibiting different multidrug resistance profiles; Gekeler V et al.; Three high-level multidrug-resistant sublines of the human T-lymphoblastoid cell line CCRF-CEM were selected independently with either actinomycin D, vincristine or adriamycin . They exhibited distinct quantitative differences of cross-resistance profiles, and showed amplification and marked expression of the mdrl/P-glycoprotein gene . DNA and RNA were prepared from the cell lines, and additionally from three cell samples of patients suffering from acute lymphatic leukemia . Applying the polymerase chain reaction (PCR) for amplification, we cloned and sequenced from these sources segments of the mdrl/P-glycoprotein gene around the codon 185 which codes for an amino acid residue possibly influencing the drug binding function of the P-glycoprotein . Altogether, only 2 single nucleotide differences in an intron were found in 2 out of 40 recombinants each harboring a 209 bp genomic or a 269 bp cDNA fragment of the mdrl/P-glycoprotein gene . Our result does not support the idea of clustered point mutations in this segment of the P-glycoprotein gene as a cause of different multidrug resistance profiles . We additionally examined another segment of the P-glycoprotein gene in its second half, essentially delivering the same negative result, though. J Biol Chem, 1990 Jun 15, 265(17), 10073 - 80 Characterization of adriamycin-resistant human breast cancer cells which display overexpression of a novel resistance-related membrane protein; Chen YN et al.; Development of multidrug resistance due to overexpression of P-glycoprotein (Pgp), a cell membrane drug efflux pump, occurs commonly during in vitro selections with adriamycin (Adr) . Pgp-mediated drug resistance can be overcome by the calcium channel blocker verapamil (Vp), which acts as a competitive inhibitor of drug binding and efflux . In order to identify other mechanisms of Adr resistance, we isolated an Adr-resistant subline by selecting the human breast cancer cell line MCF-7 with incremental increases of Adr in the presence of 10 microgram/ml verapamil . The resultant MCF-7/AdrVp subline is 900-fold resistant to Adr, does not overexpress Pgp, and does not exhibit a decrease in Adr accumulation . It exhibits a unique cross-resistance pattern: high cross-resistance to the potent Adr analogue 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin, lower cross-resistance to the alkylating agent melphalan, and a sensitivity similar to the parental cell line to vinblastine . The levels of glutathione and glutathione S-transferase are similar in the parental line and the Adr-resistant subline . Topoisomerase II-DNA complexes measured by the potassium-sodium dodecyl sulfate precipitation method shows a 2-3 fold decrease in the resistant subline . The MCF-7/AdrVp cells overexpress a novel membrane protein with an apparent molecular mass of 95 kDa . Polyclonal antibodies raised against the P-95 protein demonstrate a correaltion between the level of expression and Adr resistance . Removal of Adr but not verapamil from the selection media results in a decline in P-95 protein levels that parallels a restoration of sensitivity to Adr . Immunohistochemistry demonstrates localization of the P-95 protein on the cell surface . The demonstration of high levels of the protein in clinical samples obtained from patients refractory to Adr suggests that this protein may play a role in clinical drug resistance. Cancer Lett, 1990 Jun 15, 51(3), 193 - 201 Phenotypic and cytogenetic analysis of atypical multidrug resistance in human leukaemic cells selected with methotrexate at high concentration; Kavallaris M et al.; A series of CCRF-CEM sublines selected for extreme resistance to methotrexate has been shown previously to exhibit cross resistance to a number of agents belonging to the multidrug resistance phenotype . The mechanism(s) underlying resistance to vincristine, vinblastine and actinomycin D in the most resistant subline (CEM/MTX R3) has now been investigated . Efflux of {3H}vincristine was more rapid in CEM/MTX R3 than in either CCRF-CEM cells or a methotrexate-resistant subline not refractory to Vinca alkaloids . In addition, verapamil completely reversed resistance to vincristine, vinblastine and actinomycin D in the CEM/MTX R3 cells . While these results are suggestive of P-glycoprotein-mediated multidrug resistance, Northern analysis revealed no detectable expression of the mdr 1/gene in CEM/MTX R3 cells . Likewise, karyotypic analysis of the resistant subline, while revealing certain clonal abnormalities, provided no evidence of alteration in the mdr 1/gene locus on chromosome 7 . The data suggest therefore the operation, in these cells, of a novel mechanism of resistance. Br J Haematol, 1990 Jun, 75(2), 208 - 11 Reversal of drug-resistance by cyclosporin-A in a patient with acute myelocytic leukaemia; Sonneveld P et al.; The emergence of resistant leukaemia in a patient with acute myeloid leukaemia (AML) was evaluated during clinical progression of the disease . At relapse, a decrease of the intracellular accumulation of daunorubicin (DNR) as determined by real time flow cytometry was associated with a relative overexpression of RNA encoding for the multidrug resistance phenotype (MDR1), and by a decreased in vitro sensitivity to DNR of clonogenic AML cells (IC50 0.8-3.4 microM) . Intracellular DNR accumulation and in vitro DNR sensitivity could be completely restored by adding cyclosporin-A (3 microM) to the cells . At progressive relapse the patient was treated with re-induction therapy (DNR 30 mg/m2 x 3, cytarabine 200 mg/m2 x 7) to which cyclosporin-A was added (Cy-A 4 mg/kg twice daily for 3 d . 2.5 mg/kg twice daily for 2 d), which resulted in elimination of the MDR1 positive AML cells with restoration of the original DNR accumulation and in vitro sensitivity . After 12 weeks the resistant clone reappeared in the blood and bone marrow. Cancer Res, 1990 Jun 1, 50(11), 3239 - 44 Antitumor activity of ethyl 5-amino-1,2-dihydro-2-methyl-3-phenyl-pyrido {3,4-b}pyrazin-7-ylcarbamate, 2-hydroxyethanesulfonate, hydrate (NSC 370147) against selected tumor systems in culture and in mice; Waud WR et al.; Ethyl 5-amino-1,2-dihydro-2-methyl-3-phenylpyrido{3,4-b}pyrazin- 7-ylcarbamate, 2-hydroxyethanesulfonate, hydrate (NSC 370147) was evaluated for antitumor activity against a spectrum of tumor systems in culture and in mice . NSC 370147 was cytotoxic to a variety of mouse and human cell lines at nanomolar concentrations . The compound exhibited good in vivo antitumor activity against several murine tumors (P388 and L1210 leukemia, colon 11/A and 36, mammary 16/C, and M5076 sarcoma) . Activity was largely independent of route of administration but favored a prolonged treatment schedule . NSC 370147 was as active against murine leukemia sublines resistant to Adriamycin, amsacrine, vincristine, melphalan, cisplatin, methotrexate, and CI-920 (a topoisomerase II inhibitor) as against the corresponding parental lines . Only the 1-beta-D-arabinofuranosylcytosine-resistant P388 subline exhibited any cross-resistance to NSC 370147 . NSC 370147 has a spectrum of activity similar to that of vincristine and, unlike vincristine, is active against multidrug-resistant cell lines . Therefore, NSC 370147 is a candidate for clinical trial because of its favorable activity compared to vincristine, its effectiveness against multidrug-resistant cells, and its retention of activity for p.o . administration. Cancer Res, 1990 Jun 1, 50(11), 3218 - 25 Biological characterization of multidrug-resistant human colon carcinoma sublines induced/selected by two methods; Yang LY et al.; Two independent multidrug-resistant (MDR) sublines, AdR1.2 and SRA1.2, were developed from the established human colon carcinoma cell line LoVo . AdR1.2 was developed by a long-term continuous exposure of LoVo cells to Adriamycin in stepwise increments of concentration; SRA1.2 was selected/induced by pulse treatments by using a single concentration of Adriamycin . The two resistant sublines were cross-resistant and cross-sensitive to a similar spectrum of cytotoxic agents . However, AdR1.2 was most resistant to Adriamycin among the nine agents tested, and SRA1.2 was most resistant to Vinca alkaloids . Although SRA1.2 had biological characteristics similar to those of LoVo, AdR1.2 had remarkably altered biological properties, including no detectable carcinoembryonic antigen secretion, a smaller proportion of proliferating cells and a lower growth rate, lower fraction of cells in S phase, a lower colony-forming ability, and smaller colonies . In addition, the resistant phenotype of AdR1.2 was reversed when the cells were grown in a drug-free medium, whereas SRA1.2 maintained its resistance for at least 10 months under similar conditions . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the plasma membrane proteins demonstrated overproduction of an Mr 130,000 protein in both the resistant sublines . The Mr 130,000 protein was not immunoreactive with C219 monoclonal antibody against p170, but the absence of Mr 130,000 protein in an AdR1.2 revertant and the parental LoVo suggests that it is an MDR-related plasma membrane protein . The absence of a 46-kDa cytosolic protein and the presence of a Mr 150,000 plasma membrane protein were found in AdR1.2 but not in SRA1.2 . This Mr 150,000 protein immunoreacted with C219 . This protein was also present, although in a reduced amount, in an AdR1.2 revertant that retained three times the MDR of LoVo cells and was thus comparable to SRA1.2 . The two MDR sublines thus may represent two independent subclones which may serve as two different models for the study of multidrug resistance in human colon cancer. Mol Pharmacol, 1990 Jun, 37(6), 840 - 7 Biochemical correlates of the antitumor and antimitochondrial properties of gossypol enantiomers; Benz CC et al.; Racemic gossypol has been shown to have antitumor properties that may be due to its ability to uncouple tumor mitochondria or to its inhibitory effects on a variety of nonmitochondrial enzymes . We have studied the antimitochondrial and enzyme-inhibiting properties of gossypol in human carcinoma cell lines of breast (MCF-7, T47-D), ovarian (OVCAR-3) colon (HCT-8), and pancreatic (MiaPaCa) origin by comparing the effects of its purified (+)- and (-)-enantiomers . (-)-Gossypol shows up to 10-fold greater antiproliferative activity than (+)-gossypol in the cancer cell lines and in normal hematopoietic stem cells grown in vitro, with IC50 values ranging from 1.5 to 4.0 microM for the cancer cells and from 10 to 20 microM for the human marrow stem cells . As well, multidrug-resistant MCF/Adr cells appear more resistant to (-)-gossypol than their parental cell line . Electron microscopy indicates that the earliest ultrastructural change in tumor cells exposed to a cytotoxic (10 microM) concentration of (-)-gossypol is the selective destruction of their mitochondria . Consistent with this observation, 31P magnetic resonance spectroscopy detects pronounced changes in tumor cell high energy phosphate metabolism within 24 hr of (-)-gossypol treatment, manifest by 1.6- to greater than 50-fold differential reductions in the intracellular ratios of ATP/Pi, relative to (+)-gossypol-treated cell lines; the magnitude of these antimitochondrial effects correlates with the antiproliferative activity of (-)-gossypol . Northern blot RNA analyses suggest that treatment with a 5-10 microM dose of (-)-gossypol induces a transient increase in the expression of heat shock gene products, particularly hsp-70 transcripts . The mean 5-fold increase in (-)-gossypol-induced hsp-70 mRNA appears coincident with a comparable heat-stimulated increase in transcript levels, as compared with control or (+)-gossypol-treated cells . The enzyme-inhibiting properties of gossypol enantiomers were compared in cell-free assays measuring glutathione-S-transferase-alpha, -mu, and pi activities, calmodulin stimulation of cyclic nucleotide phosphodiesterase, and protein kinase C activity . Both enantiomers are near equivalent antagonists of calmodulin stimulation and protein kinase C activity, exceeding the potency of known inhibitors such as phenothiazines by as much as 50-fold . In contrast, (-)-gossypol is a 3-fold more potent inhibitor of glutathione-S-transferase-alpha and -pi isozyme activity, resulting in IC50 values of 1.6 and 7.0 microM, respectively, for these two isozymes.(ABSTRACT TRUNCATED AT 400 WORDS) Mol Pharmacol, 1990 Jun, 37(6), 801 - 9 Multidrug resistance in cells transfected with human genes encoding a variant P-glycoprotein and glutathione S-transferase-pi; Fairchild CR et al.; The nucleotide sequence of the mdr1 gene encoding a putative drug efflux pump (P-glycoprotein) is homologous to a class of bacterial membrane-associated transport proteins . These bacterial proteins are part of a multicomponent system that includes soluble periplasmic proteins that bind substrates, channeling them through the membrane in an energy-dependent manner . We have investigated the possibility that a similar multicomponent transport system exists in a multidrug-resistant human MCF-7 breast cancer cell line that was initially selected for resistance to doxorubicin (AdrR MCF-7) . AdrR MCF-7 cells overexpress both the mdr1 gene and the pi class isozyme of glutathione S-transferase (GST-pi) (EC 2.5.1.18) . The latter is one of several isozymes known to have a ligand-binding function in addition to drug-metabolizing capabilities . Although we have recently shown that transfection of a functional GST-pi expression vector is insufficient to confer resistance to doxorubicin in cells that lack P-glycoprotein expression {Mol . Pharmacol . 36:22-28 (1989)}, we examined the possibility that GST-pi interacts with P-glycoprotein to alter multidrug resistance . To do this, we have cloned cDNAs encoding these proteins from AdrR MCF-7 cells, constructed expression vectors containing these two genes, and transfected these vectors sequentially into drug-sensitive MCF-7 cells . The human mdr1 cDNA isolated from AdrR MCF-7 is a variant gene whose sequence differs from that isolated previously from vinblastine-resistant KB cells {Cell 53:519-529 (1989)}, resulting in an amino acid substitution of alanine to serine at position 893 (mdr1/893ala) . Transfection of eukaryotic expression vectors containing the mdr1 gene isolated from AdrR MCF-7 cells produced a multidrug-resistant phenotype in recipient cells, with a cross-resistance pattern similar to that in the AdrR MCF-7 cells . To d