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Glutathione S-transferase in chemotherapy resistance and in carcinogenesisl

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ROBYNL. SCHECTER AND MOULAY A. ALAOUI-JAMALI Department of Medicine, Montreal General Hospital Research Institute - McGill University, Montreal, Que., Canada H3G IA4 AND

GERALDBATIST~ Department of Medicine, Montrkal General Hospital Research Institute - McGill University, Montrkal, Que., Canada H3G IA4 and Department of Oncology, McGill University, Montreal, Que., Canada Received September 27, 1991 SCHECTER, R., ALAOUI-JAMALI, M. A., and BATIST,G. 1992. Glutathione S-transferase in chemotherapy resistance and in carcinogenesis. Biochem. Cell Biol. 70: 349-353. Cytosolic glutathione S-transferases are composed of two monomeric subunits. These monomers are the products of different gene families designated alpha, mu, and pi. Dimerization yields either homodimeric or heterodimeric holoenzymes within the same family. The members of this complex group of proteins have been linked to the detoxification of environmentalchemicals and carcinogens, and have been shown to be overexpressed in normal and tumor cells following exposure to cytotoxic drugs. They also are overexpressed in carcinogen-induced rat liver preneoplastic nodules in rat liver. In all of these cases, the changes in exprssion of glutathione S-transferases are paralleled by increased resistance to cytotoxic chemicals. The degree of resistance is related to the substrate specificity of the isozyme. The relationship of the glutathione S-transferase genes to drug resistance has been directly demonstrated by gene transfer studies, where cDNAs encoding the various subunits of glutathione S-transferase have been transfected into a variety of cell types. This review discusses the results of numerous studies that associate resistance to alkylating agents with overexpression of protective detoxifying glutathione S-transferase enzymes. Key words: glutathione S-transferase, chemotherapy, carcinogenesis, alkylating agents, DNA damage. SCHECTER, R., ALAOUI-JAMALI, M. A., et BATIST,G. 1992. Glutathione S-transferase in chemotherapy resistance and in carcinogenesis. Biochem. Cell Biol. 70 : 349-353. Les glutathion S-transfkrases cytosoliques sont formkes de deux sous-unitks monomtres. Ces monomtres sont les produits de diffkrentes familles de gtnes appelks alpha, mu et pi. La dimkrisation donne des holoenzymes homodimtres ou hktkrodimkres dans la m2me farnille. Les membres de ce groupe complexe de protkines sont relies ii la dktoxication des agents chimiques et des carcinogtnes environnementaux et ils sont surexprimks dans les cellules normales et les cellules tumorales aprts exposition ildes agents cytotoxiques. 11s sont kgalement surexprimks dans les nodules prknkoplasiques de foie de rat induits par des carcinogtnes dans le foie de rat. Dans tous ces cas, les changements d'expression des glutathion S-transfkrases s'accompagnent d'une rksistance accrue aux agents chimiques cytotoxiques. Le degrk de resistance est relik ii la spkcificitk du substrat de l'isozyme. La relation entre les gtnes des glutathion S-transferases et la rksistance aux agents est directement dkmontrke par les ktudes de transfert gknique ou les cDNA codant pour les diverses sous-unitks de la glutathion S-transfkrase sont transfectks dans diffkrents types cellulaires. Cette revue discute des rksultats de plusieurs ktudes qui associent la resistance aux agents alkylants ii la surexpression des enzymes glutathion S-transfkrases relikes ildktoxication. Mots cles : glutathion S-transfkrase, chimiothbapie, canckrogknbe, agents alkylants, dommage au DNA. [Traduit par la rkdaction] Glutathione S-transferases represent a family of multifunctional cellular proteins composed of dimers with a molecular mass of 45-50 kDa. These enzymes catalyze conjugation reactions between GSH and a variety of organic molecules. They are also capable of binding a number of lypophilic substances including heme, bilirubin, and steroids. Some members of the family, in addition, possess significant organic peroxidase activity. GSTs are coded for by three gene families named alpha, mu, and pi. In the rat, where GSTs have been most extensively studied, members of the alpha family are designated Ya and Yc; members of the mu family are Ybl, Yb2, and Yb3; and there appears t o be a single pi gene whose product is designated Yp. ABBREVIATIONS: kDa, kilodalton(s); GSH, reduced form of glutathione; GST, glutathione S-transferase; CLL, chronic lymphocytic leukemia; MLN, melphalan; MLNr, melphalan-resistant MatB subline; WT, wild type; MTT, 3-(4,s-dimethylthiazol-2-y1)-2,sdiphenyltetrazolium bromide; PBS, phosphate-buffered saline; BSO, buthionine sulfoximine. h he substance of this paper was presented at the symposium "Resistance to antineoplastic agents" of the 34th Annual Meeting of the Canadian Federation of Biological Societies in June 1991, at Queen's University, Kingston, Ont. ' ~ u t h o rto whom all correspondence should be sent to the following address: Montreal General Hospital Research Institute, 1650 Cedar Avenue, Montrkal, Que., Canada H3G 1A4. Prioted in Canada / ImprimC au Canada

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Alterations in specific isoenzyme forms of GST have been implicated in drug resistance observed in the setting of chemotherapy exposure in established tumors, as well as the broad cross resistance described in some models of chemical carcinogenesis. We have previously reported a human breast cancer cell line that was selected for resistance to doxorubicin and had cross resistance to a number of other antineoplastic drugs (Batist et al. 1986). There was a specific pattern of biochemical changes observed in these cells that appeared to be quite analogous to those observed in resistant hepatocyte nodules that are generated by chemical carcinogen treatment of rats in vivo (Cowan et al. 1986). The cells in these nodules are said to be preneoplastic and highly likely to go on to form tumors (Farber 1984). They are resistant to the initiating drug and cross-resistant to a variety of other drugs including doxorubicin. In both of these models, there is evidence of decreased drug accumulation, increased expression of the MDR-1 gene, decreased activity or inducibility of phase I enzymes (primarily the cytochrome P-450 system, and increased activities of phase I1 enzymes. In particular, the Yp form of GST is highly overexpressed in both the human breast cancer cell line and the preneoplastic nodule cells that exhibit this broad cross-resistance (Farber et al. 1979; Batist et al. 1986). In a subsequent study, it was shown that transformation of normal rat liver epithelial cells by Hras oncogene resulted in specific elevation of the pi GST gene and MDR-I gene expression as well (Burt et al. 1988). These data suggest that in the process of carcinogenesisthere may be a selection pressure, resulting in a tumor that is resistant to therapeutic drugs. Furthermore, certain forms of GST may play a principal role in this phenomenon. A number of factors have strongly supported a direct role of GST forms in chemotherapy resistance. The data are strongest with regard to alkylating agents. A number of alkylator-resistant sublines have been shown to overexpress GST alpha activity (Buller et al. 1987; Evans et al. 1987; Lewis et al. 1988; Schecter et al. 1991). A recent report demonstrated that certain members of the GST alpha family can be selectively induced by alkylating agents (Clapper et al. 1991). Alkyl chlorides and aziridinium ions, spontaneously forming reactive intermediates of alkylating agents, react with GSH to form conjugates and these reactions can be catalyzed by GSTs (Bolton et al. 1991). Resistant cell lines that revert to a more sensitive phenotype lose their elevated GST activity (Hansson et al. 1991; Tew et al. 1988). GST inhibitors are capable of overcoming resistance in these cell lines (Tew et al. 1988; Clapper et al. 1990). There has been a limited examination of clinical specimens, but in CLL cells (Schisselbauer et al. 1990) and in specimens from ovarian and neuroblastoma cancer patients whose tumors were clinically resistant to chemotheraphy, there is elevated GST activity (Wolf et al. 1987; Kuroda et al. 1991). Finally, there are recent publications and reports of transfection of cDNAs encoding alpha GST subunits (Black et al. 1989; Pulchalski and Fahl 1990; Leyland-Jones et al. 1991). In many but not all target cells, they confer resistance to alkylating agents. The rat Yc GST has never been studied in this fashion. Although the evidence is still too limited to be absolutely certain, there is a general pattern of GST isoenzymes specificity with regard to resistance to individual classes of drugs. As stated above, the alpha class is most likely to be associated with alkylating agents, and in particular, nitrogen

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mustards. On the contrary, the mu form of GST has been shown to be elevated in a nitrosourea-resistant subline, and there is direct evidence that it catalyzes a denitrosation reaction that detoxifies this class of drugs (Smith et al. 1989). GST pi is elevated in a number of cell lines selected for resistance to doxorubicin (Batist et al. 1986; Deffie et al. 1988), and transfection of the GST pi full-length cDNA conferred low-level resistance in a particular dose range to doxorubicin selectively (Nakagawa et al. 1990). This laboratory has recently described a novel experimental model for studying chemotherapy resistance in tumor cell lines in both in vitro and in vivo conditions (Schecter et al. 1991). MatB 13762 is a mammary carcinoma cell line established in Fischer female rats. The tumor cells grow in vitro with good cloning efficiency. When injected subcutaneously, they grow as solid masses that develop a vascular supply and metastasize to regional lymph nodes. The in vivo characteristics of the tumor are reminiscent of clinical breast cancer. The tumors grown both in vitro and in vivo are estrogen-receptor negative, and histologically appear to be poorly differentiated adenocarcinoma. A number of drugresistant sublines have been developed in vitro by selecting cells and slowly increasing concentrations of drug. A melphalan-resistant MatB subline (MLNr) is 16-fold resistant to melphalan, and is cross-resistant to nitrosoureas, cisplatin, and other nitrogen mustards, as well as to a small but consistent degree to Adriamycin and vincristine. These cells are also resistant to radiation therapy relative to the wild-type parental cells (WT). The melphalan-resistant cells have a significant increase in their cellular glutathione level and also in GST activity. Using Western immunoblotting, we have previously demonstrated that the Yc form of GST is minimally expressed in the WT cells and significantly expressed in the melphalan-resistant cells, whether they are grown in vitro or as solid tumors in vivo (Schecter et al. 1991). One of the important features of this experimental model is that animals bearing either the WT or the resistant tumor cell lines can be treated intravenously to determine the in vivo resistance of the cells to any particular drug and the effect of a biochemical modulator on sensitivity to the drug. Thus, we have shown that the melphalan-resistant tumor grown as a subcutaneous mass is resistant to doses of melphalan administered intravenously that significantly inhibit growth of the WT tumor and, in fact, cause tumor disappearance. It requires approximat~lyfour times the dose of melphalan administered intravenously to result in the same degree of tumor response seen with the WT tumor. To determine the mechanism of resistance to melphalan in the MLNr cells, we have examined the interaction of the drug with DNA. For nitrogen mustard-type drugs, there is a significant amount of evidence correlating cytotoxicity to the formation of DNA-DNA interstrand cross-links (Ross et al. 1978; Zwelling et al. 1981). Using alkaline elution, we have demonstrated that in the WT cells melphalan induces the formation of cross-links which slowly develop to a peak at approximately 8 h and thereafter slowly decline as the cross-links are removed. However, in the melphalanresistant cells we have demonstrated virtually no cross-link formation (Alaoui-Jamali et al. 1991). This work recapitulates data described using another cell line (Robson et al. 1987). Studies of the cellular pharmacokinetics of melphalan in the MLNr cells have demonstrated a 35% decrease in the drug accumulation. However, metabolic ~

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FIG. 1. Immunoblotting of GST isozymes. Cytosolic preparations (20 pg) were resolved on 12% polyacrylamide gels by electrophoresis. The proteins were transferred overnight to nitrocellulose membranes and subsequently reacted with a monoclonal antibody prepared in mouse against the GST Yc subunit purified from normal rat liver. A second antibody conjugated to horseradish peroxidase was used as the control. Samples shown are from wildtype rat mammary carcinoma MatB cells (WT), 16-fold melphalanresistant cells (MLNr), WT cells transfected with pSV2 neo plasmid (WT/neo), or with a GST Yc expression vector (Yc clones R38, R47, and R49).

analysis demonstrates that melphalan does accumulate in the cells and is metabolized at an equivalent rate. We do not consider it likely that this degree of difference in accumulation can account for the dramatic loss or dramatic decrease in formation of cross-links. To determine the role of glutathione and GST in this mechanism of resistance, we have examined the potential for inhibitors of GSH synthesis and of GST activity to affect the DNA cross-link formation. In both cases, there was an increase in cross-link formation, suggesting that GSH and GST are playing a role in inhibiting the formation of these cross-links (Alaoui-Jamali et al. 1991). We are examining the role of GST in melphalan resistance more directly. In most models, it is the Yc subunit that is overexpressed in alkylator resistance. We have, therefore, transfected a fulllength cDNA complementary to the GST Yc into MatB WT cells. The cDNA is contained in a pUC8 expression vector with a SV40 promoter enhancer fragment and a SV40 late poly(A) fragment. This vector is cotransfected with a PSV2 neo vector using the calcium phosphate technique, and cells are selected for G418 resistance. Clones expressing increased levels of GST activity were harvested and grown for further analysis. Figure 1 demonstrates Western blot analysis of preparations from WT and MLNr cells, as well as WT cells transfected with only the PSV2 neo plasmid, and three Yc transfectant clones are shown. The irnmunoblot is developed with monoclonal antibody that detects the Yc GST in rat liver cytosol seen in the extreme left lane. As shown, the Yc transfectant clone with the highest level of Yc expression is R49. The level of expression, however, is still significantly less than that seen in the melphalan-resistant cells. Using a colorimetric cytotoxicity assay, we obtained the results shown in Fig. 2 for the cell survival curve of the WT/neo

FIG. 2. Dose-response of drug-sensitive and -resistant MatB sublines. The level of resistance toward melphalan was examined using the MTT assay. Briefly, cells were cultured for 24 h in 96-well culture plates containing 100 pL of media before drug exposure. After 100 pL of various concentrations of melphalan were added to the cells (initial plating cell density of 2 x lo4 cells/well) for 72 h. The incubation time and the initial cell density were adjusted so that untreated cells were in the exponential growth phase at the time of the evaluation. For the MTT assay, 0.1 mg of MTT (25 p1 of 2 mg/mL in PBS) was added to each well and incubated at 37OC for an additional 6 h, after which 100 pL of isopropanol-HCl(4:l) was added to each well and mixed vigorously. Absorbance at a wavelength of 570 nm was estimated using a Microplate reader. Results are expressed as the percent survival of treated versus control wells, where cells were incubated in the absence of drug.

control cells, Yc clone R49, and MLNr cells after exposure to a range of melphalan concentrations. The melphalanresistant cells are highly resistant and the R49 cells are resistant, but to a lesser degree. These data suggest a Yc doseresponse curve; there is a relationship between the concentration of the Yc subunit and the level of sensitivity or resistance to melphalan. The concentration of melphalan that kills 50% of cells of the R49 clone is approximately fivefold higher than for the control cells. A previous study performed by Pulchalski and Fahl(1990) demonstrated that Ya GST transfection into COS cells resulting in a 1.8-fold increase in GST could confer a 1.5-fold level of resistance to cisplatin and 2.9-fold level of resistance to chlorambucil, a nitrogen mustard related to melphalan. The MatB melphalan-resistant cells selected in vitro for resistance to melphalan when grown in vivo have a 2.7-fold increase in GST activity relative to the WT tumor grown in vivo. Sensitivity to intravenous melphalan in in vivo grown tumors is approximately one quarter (i.e., fourfold resistant) that of WT cells. Although these levels of modulation biochemically and biologically appear to be small, particularly with regard to the most common in vitro models of cell lines selected with different classes of drugs (e.g., anthracyclines), they are fairly consistent with the findings both in vitro and in vivo of alkylator resistance. In general, this represents a level of resistance not significantly greater than 10-fold. Clinical specimens from a patient with ovarian cancer that was clinically resistant was compared with specimens from the same patient obtained at the start of therapy demonstrated at 2.1-fold increase in GST level and a 3-fold level of resistance to cisplatin and to chlorambucil, both of the latter measured in vitro from the cells harvested from the patient. Similarly, in a larger series of

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patients with chronic lymphocytic leukemia, it has been found that patients resistant to nitrogen mustards have approximately a twofold increase in GST activity (Schisselbauer et al. 1990). Earlier in this article, it was noted that there appear to be analogous biochemical changes in some established tumors selected for resistance to chemotherapeutic drugs and in chemical carcinogen transformed cells. We have previously hypothesized that chemical carcinogenesis selects a biochemical phenotype that results in multidrug resistance (Cowan et al. 1986). Clinically, some human tumors exhibit the phenotype of de novo resistance to most anticancer drugs. Many of these tumors are also probably induced by environmental chemical carcinogens (e.g., lung cancer, colon cancer). We have, therefore, wondered whether or not the process of carcinogenesis in these tumors is in fact determining the chemosensitivity of the resulting tumors by selecting a biochemical phenotype. In a study of 17 patient samples, comparing the biochemical phenotype of colon carcinoma with adjacent normal mucosa, we have demonstrated significant increases in GSH and GSH-related enzymes, particularly GST. Moreover, we demonstrated that almost all of the time the GST Yp subunit is significantly increased in tumor versus normal adjacent mucosa (MekhailIshak et al. 1989). More recently, we have observed that not only is the Yp GST increased, but that using a cDNA probe for the GST Yb subunit, there is quite consistently a significant decrease in tumor versus normal mucosa Yb GST (Batist et al. 1989). Very little is known about the regulation of expression of the various GST isoenzymes, but we have previously reported this phenomenon of inverse levels of expression of Yp versus Yb. In rat liver epithelial cells growing in culture, we noted that the Yp form is most highly expressed in proliferating cells and that the expression decreases as the cells reach confluence (Batist et al. 1989). Precisely the opposite is observed for the Yb form, which is most highly expressed in confluent cells and decreased in proliferating cells. We have on the basis of these findings, hypothesized the potential role of GST in colon carcinogenesis. Since some GST forms detoxify carcinogens and Yb is particularly efficient with carcinogenic epoxides, and since specific GST expression may be related to the proliferative status of the cell, it is possible that human colon cancers, which are thought to arise in the proliferating crypt cells, have a decreased detoxification capacity by virtue of the proliferative effect on Yb expression. We suggest that the GST profile observed in the established colon tumor versus normal mucosa may be reflective of the target cells that were transformed, i.e., rapidly proliferating crypt cells with high GST Yp and low GST Yb. Immunohistochemical studies done by Ranganathan and Tew (1991) have demonstrated distribution of these specific isoenzyme forms, i.e., Yp in crypts and Yb closer to the surface of the villi. An alternative hypothesis is that the decreased GST Yb is itself a feature of transformation or even of tumor progression. We have not yet directly demonstrated that the GST Yb is functionally important in colon carcinogens per se. Having demonstrated alterations in the expression of specific forms of GSTs in clinical disease and in experimental models, work is ongoing to try to modulate these changes to therapeutic benefit. For example, the well-known diuretic drug ethacrynic acid is a competitive inhibitor with some specificity for the Yc and Yp forms of GST. Phase I studies

combining ethacrynic acid with nitrogen mustard agents have been undertaken at two centers to determine optimal scheduling and dosing (Schilder et al. 1990; Hantel et al. 1991). Phase I1 studies in specific diseases such as CLL, where GST has been shown to be elevated in resistant specimens, are scheduled to begin shortly. Phase I studies with BSO, a selective inhibitor of the rate-limiting GSH synthetic enzyme, are underway to determine the toxicity in optimal dosing to significantly deplete GSH. One phase I study combining BSO with radiotherapy is active at McGill University. In the laboratory, a number of groups are actively looking for agents that can overcome resistance to alkylating agents. In summary, a fair amount of definition is required to be certain of the precise and specific role of GSTs in both carcinogenesis and drug resistance. Work on a number of fronts appears to be yielding results that may have some significant scientific and clinical impact in the near future.

Acknowledgements This work was supported by grants from the National Cancer Institute of Canada and the Cancer Research Society Inc. Alaoui-Jamali, M.A., Panasci, L., Schecter, R.L., et al. 1991. Atypical cross-resistance and multiple mechanisms in a melphalan resistant rat mammary carcinoma cell line. Proc. Am. Assoc. Cancer Res. 32: 359. Batist, G., Tulpule, A., Sinha, B.K., et af. 1986. Overexpression of a novel anionic glutathione transferase in multidrug resistant human breast cancer cells. J. Biol. Chem. 261: 15 544 - 15 549. Batist, G., Tsao, M.-S., Mekhail-Ishak, K., and Woo, A. 1989. Inverse regulation of basal expression of GST mu and GST pi mRNA in human colon carcinoma (T) and normal mucosa (n) and in rat liver epithelial cells in culture. Proc. Am. Assoc. Cancer Res. 30: 12. Black, S., Beggs, J.D., and Miles, J.S. 1989. Expression of human glutathione transferases in S. cerevisiae confers resistance to alkylating agents and chlorambucil. Proc. Natl. Acad. Sci. U.S.A. 87: 2443-2447. Bolton, M.G., Colvin, O.M., and Hilton, J. 1991. Specificity of isozymes of murine hepatic glutathione-S-transferase for the conjugation of glutathione with L-phenylalanine mustard. Cancer Res. 51: 2410-2414. Buller, A.L., Clapper, M.L., and Tew, K.D. 1987. GlutathioneS-transferase in nitrogen mustard-resistant and -sensitive cell lines. Mol. Pharmacol. 31: 575-578. Burt, R.K., Garfield, S., Johnson, K., and Thorgeirsson, S.S. 1988. Transformation of rat liver epithelial cells with v-H-ras or v-raf causes expression of MDR-1, glutathione-S-transferase pi and increased resistance to cytotoxic chemicals. Carcinogenesis (London), 9: 2329-2333. Clapper, M.L., Hoffman, S.J., and Tew, K.D. 1990. Sensitization of human colon tumor xenografts to L-phenylalanine mustard using ethacrynic acid. J. Cell. Pharmacol. 1: 71-78. Clapper, M.L., Seestaller, L.M., and Tew, K.D. 1991. Induction of glutathione-S-transferase alpha RNA in tumor cells following exposure to chlorambucil. Proc. Am. Assoc. Cancer Res. 32: 361. Cowan, K.H., Batist, G., Tulpule, A., et al. 1986. Similar biochemical changes associated with multi-drug resistance in human breast cancer cells and carcinogen-induced resistance to xenobiotics in rats. Proc. Natl. Acad. Sci. U.S.A. 83: 9328-9332. Deffie, A.M., Alam, T., Senevirante, C., et al. 1988. Multifactorial resistance to Adriamycin: relationship of DNA repair, glutathione transferase activity, drug efflux, and P-glycoprotein in cloned cell lines of Adriamycin-sensitive and -resistant P388 leukemia. Cancer Res. 48: 3595-3602.

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Evans, C.G., Bodell, W.J., Tokuda, K., et al. 1987.Glutathione and related enzymes in rat brain tumor cell resistance to 1,3-bis(chloroethy1)-1-nitrosourea and nitrogen mustard. Cancer Res. 47: 2525-2530. Farber, E. 1984. Cellular biochemistry of the stepwise development of cancer with chemicals: G.H.A. Clowes Memorial Lecture. Cancer Res. 44: 5463-5474. Farber, E., Parker, S., and Gruenstein, M. 1979. The resistance of putative premalignant liver cell populations, hyperplastic nodules, to the acute cytotoxic effects of some hepatocarcinogens. Cancer Res. 36: 3879-3887. Hansson, J., Berhane, K., Castro, V.M., et 01. 1991.Sensitization of human melanoma cells to the cytotoxic effect of melphalan by the glutathione transferase inhibitor ethacrynic acid. Cancer Res. 51: 94-98. Hantel, A., Nelson, J., Belknap, S., et al. 1991.Phase I study of IV melphalan and the glutathione S-transferase (GST) inhibitor ethacrynic acid. Proc. Am. Assoc. Cancer Res. 32: 194. Kuroda, H., Sugimoto, T., Ueda, K., et al. 1991. Different drug sensitivity in two neuroblastoma cell lines established from the same patient before and after chemotherapy. Int. J. Cancer 47: 732-737. Lewis, A.D., Hickson, I.D., Robson, C.N., et al. 1988.Amplification and increased expression of alpha class glutathione-stransferase-encoding genes associated with resistance to nitrogen mustards. Proc. Natl. Acad. Sci. U.S.A. 85: 8511-8515. Leyland-Jones, B.R., Townsend, A. J., Tu, C.-P.D., et al. 1991. Antineoplastic drug sensitivity of human MCF-7 breast cancer cells stably transfected with a human alpha class glutathione-stransferase gene. Cancer Res. 51: 587-594. Mekhail-Ishak, K., Hudson, N., Tsao, M.-S., and Batist, G. 1989. Implications for therapy of drug-metabolizing enzymes in human colon cancer. Cancer Res. 49: 48664869. Nakagawa, K., Saijo, N., Tsuchida, S., et al. 1990. GlutathioneS-transferase pi as a determinant of drug resistance in transfectant cell lines. J. Biol. Chem. 265: 42964301. Pulchalski, R.B., and Fahl, W.E. 1990.Expression of recombinant glutathione-S-transferase pi, Ya, Ybl confers resistance to alkylating agents. Proc. Natl. Acad. Sci. U.S.A. 87: 2443-2447.

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Ranganathan, S., and Tew, K.D. 1991. Immunohistochemical localization of glutathione S-transferase I, p, and r in normal tissue and carcinomas from human colon. Carcinogenesis (London), 12: 2383-2387. Robson, C.N., Lewis, A.D., Wolf, C.R., et al. 1987. Reduced levels of drug-induced DNA cross linking in nitrogen mustardresistant Chinese hamster ovary cells expressing elevated glutathione-S-transferaseactivity. Cancer Res. 47: 6022-6027. Ross, W.E., Ewing, R.A.G., and Kohn, K.W. 1978. Differences between melphalan and nitrogen mustard in the formation and removal of DNA crosslinks. Cancer Res. 38: 1502-1506. Schecter, R.L., Woo, A., Duong, M., and Batist, G. 1991.In vivo and in vitro mechanisms of drug resistance in a rat mammary carcinoma model. Cancer Res. 51: 1434-1442. Schilder, R.J., Nash, S., Tew, K.D., et al. 1990. Phase I trial of Thiotepa (TT) in combination with the glutathione transferase (GST) inhibitor ethacrynic acid (EA). Proc. Am. Assoc. Cancer Res. 31: 177. Schisselbauer, J.C., Silber, R., Papadopoulos, E., et al. 1990. Characterization of glutathione S-transferase expression in lymphocytes from chronic lymphocytic leukemia patients. Cancer Res. 50: 3562-3568. Smith, M.T., Evans, C.G., Doane-Setzer, P., et al. 1989. Denitrosation of 1,3-bis(chloroethy1)-1-nitrosourea by class mu glutathione transferases and its role in cellular resistance in rat brain tumor cells. Cancer Res. 49: 2621-2625. Tew, K.D., Bomter, A.M., and Hoffman, S.J. 1988. Effect of ethacrynic acid and piriprost on sensitivity to chlorambucil of human colon carcinoma cell lines. Cancer Res. 48: 3622-3635. Wolf, C.R., Hayward, I.P., Laurie, S.S., et al. 1987.Cellular heterogeneity and drug resistance in two ovarian adenocarcinoma cell lines derived from a single patient. Int. J. Cancer 39: 695-702. Zwelling, L.A., Michaels, S., and Schwartz, H. 1981.DNA crosslinking as an indicator of sensitivity and resistance of mouse L1210 leukemia cells to cis-diamminedichloroplatinum(II) and L-phenylalanine mustard. Cancer Res. 41: 640-649.

Glutathione S-transferase in chemotherapy resistance and in carcinogenesis.

Cytosolic glutathione S-transferases are composed of two monomeric subunits. These monomers are the products of different gene families designated alp...
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