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Cultured rat liver epithelial cells (RLE) transformed with repeated treatments of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) demonstrate many features of the common biochemical phenotype of multidrug resistance (MDR) seen in vivo in 'resistant hepatocytes'. The cells have increased glutathione-S-transferase placental subunit (GST-Yp), gamma-glutamyltranspeptidase (GGT), glutathione (GSH) and glutathione peroxidase and are resistant to MNNG. Phenotypically identical RLE cells spontaneously transformed by selective culture conditions showed low levels of GGT and GST and were not resistant to MNNG. Both chemical and spontaneous transformants are cross resistant to doxorubicin although resistance is consistently greater in chemical transformants. No direct correlation was found between the degree of resistance to doxorubicin and MDR gene expression in either of the chemically or spontaneously transformed RLE cells. These observations suggest that in chemical carcinogenesis, other mechanisms of drug detoxification are involved and that MDR expression is not a consistent feature.

Gap junctions are intercellular channels that are formed from members of a family of proteins, the connexins (Cxs). Gap junctions play an important role in vital functions, including the regulation of cell growth and cell differentiation. Here, we examined the expression of Cx43, a major Cx in breast tissue, in 32 surgical specimens obtained from breast cancer patients who underwent a primary surgical resection prior to chemotherapy or radiotherapy treatments. The expression of Cx43 gap junctions was compared to the levels of estrogen, progesterone, and erbB2 tyrosine kinase receptors. In addition, a panel of breast cancer cell lines and a series of normal rat mammary tissues and rat mammary tumors induced in vivo by dimethylbenz(a)anthracene were studied. We demonstrated that the lack of Cx43 gap junctions is a common feature of human mammary cancer tissues compared to nonneoplastic breast tissues surrounding primary tumors. Cx43 gap junctions were not observed in ductal carcinomas in situ, infiltrating ductal carcinomas, and infiltrating lobular carcinomas, and they seem to be independent of estrogen, progesterone, and erbB2 receptor status. In breast cancer cell lines and rodent mammary carcinoma tissues, down-regulation of Cx43 occurs at the mRNA level, suggesting a transcriptional mechanism for the decrease of Cx43 protein in breast cancer. In summary, this study provides evidence of decreased expression of Cx43 gap junctions in breast cancer at various stages of progression as well as breast cancer cell lines and raises the possibility that Cx43 may be a useful marker for detecting early oncogenesis in the breast. Because Cx43 gap junctions are lacking in breast cancer and restoration of Cx43 has been shown to reverse the malignant phenotype in vitro, pharmacological up-regulation of Cx43 may prove beneficial in cancer therapeutics.

Notwithstanding ongoing progress in anticancer therapeutics development, the persistent problem remains to selectively target tumors while sparing normal tissues. This is confounding largely because the differences between normal and tumor cells are often subtle and part of a gradient, where a gene product may be more or less expressed in tumor compared with the host normal tissue, but seldom expressed (or turned off) in tumors. The role of glutathione (GSH) and related enzymes in cellular resistance to xenobiotics, including chemotherapy is well established. This study is among those attempting to modulate GSH to therapeutic advantage. The authors briefly describe the experience with the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine, and then in greater detail outline recent evidence for a potentially more selective approach using the cysteine prodrug L-2-oxothiazolidine-4-carboxylate. This has led to a detailed study of the activating enzyme 5-oxo-L-prolinase, including enzymatic and immunocharacterization, as well as in vitro study of the effect of its modulators on anticancer drug toxicity. Using high affinity antibodies the authors have generated interesting information on the distribution of this enzyme in tumor versus normal human tissues. Finally, the authors have been studying the potential for modulating gap junctions as a part of anti-cancer therapeutics, since they transport GSH between cells and are generally deficient in tumor cells. Preliminary studies suggest that gap junction induction may dramatically deplete GSH concentration in tumor cells and sensitize them to a variety of treatments.

5-Oxo-L-prolinase (5-OPase) is an enzyme of the gamma-glutamyl cycle involved in the synthesis and metabolism of glutathione (GSH), which is known to protect cells from the cytotoxic effects of chemotherapy and radiation. Previous studies on rats have shown that administration of the cysteine prodrug L-2-oxothiazolidine-4-carboxylate, a 5-oxo-L-proline analogue that is metabolized by 5-OPase, preferentially increases the GSH content of normal tissues while paradoxically decreasing it in the tumor and results in an enhanced in vivo tumor response to the anticancer drug melphalan. These observations initiated the present study of 5-OPase in experimental models and clinical specimens to investigate the potential role of this enzyme in the selective modulation of GSH in normal and tumor tissues. First, 5-OPase activity was measured in tissues of tumor-bearing rats, in the peripheral mononuclear cells of normal human subjects, and in surgically resected tumor and the adjacent normal tissues from patients. We found that the activity of 5-OPase in human kidney, liver, and lung is significantly lower than that found in rats. Second, we have raised a polyclonal IgG anti-5-OPase antibody by immunizing rabbits with purified 5-OPase from rat kidney. This antibody has very high affinity (shown by immunoprecipitation) and specificity (shown by Western blot) and cross-reacts with human 5-OPase (shown by Western blot and immunohistochemistry). It was then used to examine the distribution of 5-OPase in paired normal and neoplastic human specimens using Western blot and immunohistochemistry. Examination of paired normal and neoplastic tissues of stomach and lung revealed a significantly lower level of 5-OPase in tumor tissues than in the paired normal tissues. In colon tissues, there is no significant difference in 5-OPase level between the normal and tumor tissues. These findings could have implications for both carcinogenesis and therapy.

Glutathione transferase (GSTs) have been shown to be overexpressed in a number of tumor cell lines selected for resistance to chemotherapeutic drugs and have been implicated in some studies of clinical specimens. In tumor cell lines selected for resistance to chemicals that alkylate DNA, the isoform most frequently overexpressed is GST-Yc, a member of the alpha class GSTs. To date, two variations of the cDNA designated Yc1 with subtle differences have been described, and Yc2 is shown to be clearly distinct. Transfection of a Yc1 cDNA constitutively expressed in rat liver into rat mammary cancer cells confers resistance to alkylators, however, to a lesser extent than is observed in the cells selected for resistance. It has therefore been widely suggested that the GST that is overexpressed in selected resistant cells represents a distinct and novel isoform. We have previously described a rat mammary carcinoma cell line (MLNr) that is resistant to alkylating agents, and overexpresses a GST with characteristics similar to GST-Yc1 and not Yc2. It has many features common to the several other GST-Yc overexpressing alkylator resistant cell lines. We have cloned the specific Yc cDNA overexpressed in MLNr and analyzed it in detail and found that it is identical to one of the previously reported Yc1 cDNAs, suggesting that there is no additional Yc gene specifically induced by nitrogen mustards. Another hypothesis to explain the difference in the level of resistance in selected versus GST-Yc transfected cells is the lack of concurrent increased glutathione (GSH) in the transfectants, which is a common feature in the selected resistant cells. Experiments in which we modulated GSH levels suggest that this is not likely. These studies add to our speculation that other mechanisms may be involved in alkylator resistance.

Glutathione (GSH) is known to play a role in cellular sensitivity to some chemotherapeutic agents and to radiation. Depletion of cellular glutathione increases toxicity of these drugs, and this approach is being explored in the clinic as a form of biochemical modulation using the drug buthionine sulfoximine. The fact that some drug-resistant cell lines have increased GSH levels, and that enhancing glutathione concentrations in animal tissues protects against a variety of xenobiotic agents, suggests a different potential approach to improve anticancer therapy. We previously showed a selective enhancement by the cysteine "pro-drug," L-2-oxothiazolidine-4-carboxylate (OTZ), of GSH concentration in some normal tissues of tumor-bearing rats, whereas there is a paradoxic GSH depletion in tumor. OTZ has been shown to protect animals from a variety of toxins, and in vitro studies showed a selective increase in GSH in normal cells that results in reduced sensitivity to some chemotherapy drugs. This report describes evidence that OTZ provides this effect in an in vivo rat mammary tumor model. We have examined the OTZ "activating" enzyme, 5-oxoprolinase, in these tumors and found it to be 4-fold lower than that of normal rat liver. This may explain at least the lack of increased GSH in tumor in response to OTZ. A limited number of human breast cancer samples show similar activity.

Ifosfamide (IFF) is a nitrogen mustard with significant activity against a number of tumors. Since it is a chiral molecule, it has been suggested that enantioselective metabolism could result in different efficacy and toxicity profiles for (R)- and (S)-ifosfamide. Both experimental animal and clinical data suggest that N-dechloroethyl metabolites of (S)-IFF are more significantly associated with neurological toxicity, which may limit therapeutic use of IFF. We have used purified ifosfamide enantiomers to examine the pharmacokinetics; spectrum of toxicity including lethality, weight loss, and myelosuppression; and antitumor effects of the mixture compared to each of the purified enantiomers. In the MatB mammary carcinoma grown in female Fischer rats we demonstrated that the antitumor efficacy appears to be the same for (R)-IFF and (S)-IFF, while the (R)-IFF has greater myelotoxicity. Pharmacokinetic analysis of plasma concentration-time confirms that the (R)-IFF is metabolized to a greater extent than (S)-IFF via the activation pathway. These data suggest that purified (R)-IFF may be an effective way to delivery active cytotoxic drug while limiting the generation of neurotoxic metabolites.

A major limitation to successful cancer treatment is the existence of drug resistance. While several mechanisms of drug resistance have now been well characterized, mechanisms of resistance to alkylating drugs have remained less well defined. Several experimental models of alkylator resistance have implicated isoforms of glutathione S-transferase (GST) but transfection experiments using cloned isoforms of GST have yielded conflicting results. While there are several plausible explanations for these apparently contradictory findings, the issue that clonal variability might potentially confound the results of conventional transfection experiments has been raised. To address this issue properly, we have studied rat GST-Yc expression and drug sensitivity to alkylating drugs in populations of mouse NIH 3T3 fibroblasts following either transfection or transduction with an N2-based retrovirus vector. In comparison with cells treated with an antisense vector, Yc-transfected and Yc-transduced populations of NIH 3T3 cells expressed increased levels of GST-Yc mRNA (Northern blot), increased levels of immunodetectable GST-Yc (Western blot), and, respectively, 1.4- and 1.9-fold increases in total GST activity and 6.1- and 8.3-fold increases in glutathione peroxidase activity (associated with the Yc subunit). Yc-transfected and Yc-transduced cell populations were, respectively, 5.8- (P0.001) and 2.4-fold (P0.05) resistant to chlorambucil and 10.8- (P0.01) and 5.4-fold (P0.001) resistant to mechlorethamine. The range of resistance of clonal isolates from either population was 1.8-6.0-fold for chlorambucil and 4.6-6.1-fold for mechlorethamine (P0.05). In contrast, these cells showed unaltered sensitivity to the antimetabolite methotrexate, a nonalkylating drug. These results clearly demonstrate that the rat GTS-Yc is able to confer alkylating drug resistance in mouse fibroblasts. The ability to confer alkylating drug resistance following retrovirus-mediated gene transfer also raises the possibility of using GST-Yc somatic gene transfer to confer protection to the hematopoietic system in a gene therapy strategy applicable to cancer.

The role of glutathione-S-transferase (GST) in alkylator drug resistance has been studied in MatB rat mammary carcinoma cells. A series of GST transfectant cell lines was established by using an expression vector containing the complementary DNA for the rat GST Yc gene under regulation of the SV40 early region promoter and the antibiotic resistance plasmid pSV2neo. Transfectant cell lines expressing up to 4-fold higher total GST activity than in the parental wild type cell line were identified. Southern blot analysis confirmed a DNA fragment corresponding in size to the transfected GST Yc complementary DNA. Wild type MatB cells contain very low levels of Yc protein, whereas the Yc+ clones showed greatly increased amounts of the Yc subunit. The effect of increased GST Yc activity on the sensitivity of the transfected clones to various cytotoxic agents was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival assay. The clones expressing recombinant GST Yc were more resistant to melphalan (6- to 12-fold), mechlorethamine (10- to 16-fold), and chlorambucil (7- to 30-fold). In late passage populations of the GST Yc+ clones that had been grown over a period of 14 months under continuous selection in G418, GST activity was decreased and it was paralleled by a decrease in Yc protein. These late passage clones with diminished GST Yc content also demonstrate a partial reversion toward the wild type phenotype as determined by cytotoxicity assays using melphalan, mustargen, and chlorambucil. Interstrand DNA cross-links induced by mechlorethamine were significantly lower at 0, 2, and 20 h posttreatment in one of the GST Yc+ clones when compared to wild type MatB cells. These studies indicate that GST Yc overexpression can confer resistance to alkylating agents and that this correlates with inhibition of DNA cross-link formation.

The effectiveness of a calcium antagonist analog Ro11-2933 to modulate doxorubicin (DOX) response in DOX-sensitive (WT) and -resistant (DOXr, 200-fold) cell lines was investigated and compared to verapamil (VP) in vitro and in vivo in rats bearing mammary carcinoma using equivalent nontoxic doses. In vitro exposure to a nontoxic concentration of Ro11-2933 (2 microM) normalizes the DOX accumulation defect observed in DOXr cells, increases DOX-induced DNA single-strand breaks and effectively sensitizes DOXr cells to DOX. Ten microM VP was required to obtain an effect equivalent to that seen with 2 microM Ro11-2933. Intravenous administration of DOX at 5 mg/kg to the rat bearing the DOXr tumors has no significant therapeutic effect on tumor growth (P.5), whereas it was found effective in inhibiting the growth of WT tumors (P.05). Ro11-2933 or VP administered alone has no significant effect on tumor growth as compared to a saline-treated group (P.1). Combination of Ro11-2933 with DOX effectively inhibits DOXr tumor growth as compared to DOX alone. Combination of DOX with VP was found less effective than Ro11-2933 and the results were not statistically significant from DOX treatment alone (P.5). Our data demonstrate that Ro11-2933 is well tolerated after i.v. administration and an effective modulator of DOX resistance in a solid tumor model.

A specific biochemical phenotype is expressed during chemical hepatocarcinogenesis, which includes increased activity of the various isoenzyme forms of glutathione S-transferase (GST) composed of class alpha (Ya/Yc), class mu (Yb) and class pi (Yp) gene products. In vitro cell lines of normal and chemically transformed rat liver epithelial cells provide an opportunity to examine the regulation of expression of GST isoenzymes. We have studied the effect of the state of proliferation in culture on both the enzymic activity and the isoenzyme-specific mRNA expression. In normal rat liver epithelial cells (WB-F344), basal expression of the Yp subunit decreases, and of the Yb subunit increases, in cells at confluence compared with those in logarithmic-phase growth. In a subline of WB-F344 cells that has been chemically treated in vitro (GN6), there was greater Yp expression; however, the effect of growth status on both subunits was the same as in the nontransformed WB-344 cells, and the Ya subunit was not expressed. Inhibition of RNA synthesis with actinomycin D was limited, demonstrating pronged half-lives of the GST mRNAs, and shows a slightly greater decrease in GST-Yp specific mRNA levels in the confluent cells. Also nuclear run-off experiments demonstrate identical transcription rate in confluent and pre-confluent cells. These data suggest that the increase in Yp steady-state RNA in preconfluent cells is due to increased stability of the GST-Yp mRNA. In tumor cells derived from GN6 cells, the regulatory effects of growth status on the Yb and Yp expressions were absent. However, in these cells Ya subunit was expressed and this was subject to the effects of cell proliferation. We conclude that the growth status of cells in culture exerts a significant regulatory control on the GST isoenzyme gene expression. The effects are probably mediated at independent regulatory sites in each gene. The state of transformation of rat liver epithelial cells may determine responsiveness to this effect.

Many in vitro tumor models have been examined to help understand the precise mechanisms responsible for drug resistance. The importance of these results in vivo remains uncertain. MatB 13762 is a rat mammary adenocarcinoma cell line that can be grown both in vitro and as a solid tumor in Fischer 344 rats, thus permitting the examination of tumor cell drug resistance under both conditions. Two cell lines have been selected in vitro for resistance to Adriamycin (AdrR) and melphalan (MlnR), respectively. Each subline has the following features: AdrR, increased mdr-1 messenger RNA, a high level of cross-resistance to vincristine and atypical low level resistance to melphalan and 1,3-bis(2-chloroethyl)-1-nitrosourea, decreased cellular glutathione content, and increased expression of Yc and Yp glutathione S-transferase isozymes; MlnR, low level drug resistance to melphalan and cross-resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea, Adriamycin, and vincristine; increased cellular concentration of glutathione; elevated glutathione S-transferase activity; and greatly increased messenger RNA specific to the Yc and Yp glutathione-S-transferase subunits. Most of the biochemical and molecular features described above are present but significantly less prominent in tumors grown in vivo. This model provides the opportunity to examine the magnitude of expression and the clinical significance of in vitro resistance in an in vivo model.

The effects of GSH depletion in a human breast cancer cell line and a multi-drug resistant subline (ADRr) were determined in a number of experimental conditions. The ADRr cells contained lower GSH concentration which cannot be explained solely on the basis of differences in cell kinetics, and yet the rate-limiting synthetic enzyme gamma-glutamylcysteine synthetase was increased 2-fold. Inhibition of GSH synthesis by BSO resulted in more rapid and more pronounced GSH depletion in ADRr compared to the wild-type cells, suggesting that enhanced GSH utilization and efflux in the resistant cells account for the lowered basal concentration. In addition, the gamma-glutamyl moiety salvage enzyme gamma-glutamyltranspeptidase was reduced markedly in the ADRr cell line. Since these cells have overexpression of the efflux pump protein P-glycoprotein, we examined the effects on cellular GSH of inhibition of the pump's function by verapamil. We found that verapamil significantly depleted cellular GSH. In a rat mammary carcinoma cell line selected in Adriamycin for multi-drug resistance, a similar molecular phenotype has been described including diminished cellular GSH concentration. Verapamil treatment of these cells also resulted in significant depletion of cellular GSH. These results are consistent with the recent report that combined treatment of BSO and verapamil has an additive effect on cytotoxicity. It is likely that decreased basal GSH concentration is due to oxidation and conjugation of it in reactions catalyzed by the enhanced peroxidase and GST found in these cells.

Adriamycin-resistant (AdrR) human breast cancer cells have been selected which exhibit cross-resistance to a wide range of anti-cancer drugs. This multidrug-resistant phenotype is associated with increases in the activities of glutathione peroxidase and glutathione transferase. The 45-fold increase in glutathione transferase activity is associated with the appearance of a new anionic isozyme in AdrR cells which is immunologically related to the anionic glutathione transferase present in human placenta. The increase in transferase and the level of drug resistance is relatively stable during passage of AdrR cells in the absence of adriamycin for over 10 months. A similar anionic glutathione transferase isozyme is also found in rat hyperplastic liver nodules, a preneoplastic state resulting from exposure to carcinogens. A rat cDNA which codes for the anionic glutathione transferase in rat hyperplastic nodules hybridizes to a 1.1-kilobase pair mRNA which is overexpressed in the AdrR MCF-7 cells. The anionic transferase has been purified from the AdrR cells and found to have characteristics which distinguish it from other anionic human glutathione transferases, including high levels of intrinsic peroxidase activity. The overexpression of a similar anionic glutathione transferase in human breast cancer cells selected for multidrug resistance and in rat hyperplastic liver nodules, which develop resistance to various hepatotoxins, suggests a possible role for this drug-conjugating enzyme in the mechanism of resistance in both of these states.

The present study was designed to determine whether alterations in host metabolism associated with progressive tumor growth were a result of the anorexia frequently observed with cancer or could be attributed to other direct tumor effects. Rates of tyrosine flux, oxidation, and incorporation into protein, as well as fractional protein-synthetic rates in nonsecretory liver, muscle, and tumor, were determined in overnight-fasted rats, 5 to 6 (Stage I), 10 to 11 (Stage II), and 15 to 16 (Stage III) days following s.c. implantation of RNC-254 fibrosarcoma. Tumor-bearing rats were allowed to consume a purified diet containing 20% protein ad libitum, and results were compared to non-tumor-bearing rats pair fed quantities of food equivalent to tumor-bearing animals or allowed to consume the diet ad libitum. Results demonstrate that during later stages of tumor growth (Stage III) calorie intake and nontumor body weight gain were reduced in tumor-bearing rats (p less than 0.05). Fifteen and 16 days following implantation, there were significant changes in amino acid kinetics that were not observed after earlier periods of tumor growth and that could not be explained by any reduction in dietary intake. Rates of tyrosine appearance in the plasma and subsequent incorporation into whole-body protein were increased 33 and 34%, respectively (p less than 0.05), when compared to non-tumor-bearing rats fed equivalent quantities of food. Whole-body tyrosine oxidation rates were unchanged. Skeletal protein synthesis, as reflected by gastrocnemius or rectus abdominus muscle, was reduced from 10.5 and 10.1%/day to 7.4 and 6.0%/day, respectively (p less than 0.05), in tumor-bearing compared to pair-fed animals. The findings suggest that significant alterations in protein metabolism occur in advanced stages of experimental neoplastic disease which cannot be explained by reductions in dietary intake and are aimed at providing adequate quantities of endogenous amino acids for net tumor growth.

Clinical and experimental evidence demonstrates that hypertrophied cardiac tissue is more sensitive to ischemic injury than is normal myocardium. Recent studies indicate that cardiac ischemia-reperfusion injury involves the generation of toxic oxygen free radicals. We used the spontaneously hypertensive rat (SHR) model, with its otherwise genetically identical control (the Wistar-Kyoto [WKY] rat), to investigate the potential role of enzymes that generate and detoxify oxygen radicals in the sensitivity of hypertrophied heart to ischemia and reperfusion. Because hypertension develops progressively with age in SHRs, we assayed xanthine oxidase, superoxide dismutase, catalase, and glutathione peroxidase at three different time points and found significant fluctuations at different ages. At age 26 weeks, physiological measurements demonstrated hypertension and increased sensitivity to ischemia and reperfusion, measured as significantly decreased left ventricular recovery after injury. At this age, xanthine oxidase, which may generate oxygen radicals, was significantly increased in SHR compared with WKY rats (p = 0.003). Superoxide dismutase, which is a principal step in oxygen-radical detoxification, was significantly lower (p = 0.044). These data suggest that differences in the constitutive levels of oxygen-radical metabolic pathways are different in hypertrophied myocardium, and it is suggested that this finding may play a role in the response of these hearts to ischemia-reperfusion injury.