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There is a growing need for short-term and cost-effective bioassay to assess the efficacy of potential chemo-preventive agents. We report that the induction of glutathione (GSH) S-transferase pi (mGSTP1-1) by a chemo-preventive agent can be used as a reliable marker to assess its efficacy in retarding chemical carcinogenesis induced by benzo(a)pyrene (BP), which is a widespread environmental pollutant and believed to be a risk factor in human chemical carcinogenesis. This conclusion is based on 1) the relative contribution of mGSTP1-1 of the liver and forestomach of female A/J mice in the detoxification of the ultimate carcinogenic metabolite of BP, (+)-anti-7,8-dihydroxy-9, 10-oxy-7,8,9, 10-tetrahydrobenzo(a)pyrene [(+)-anti-BPDE]; and 2) a positive correlation between the induction of hepatic and forestomach mGSTP1-1 by 5 naturally occurring organosulfides (OSCs) from garlic (diallyl sulfide, diallyl disulfide, diallyl trisulfide, dipropyl sulfide and dipropyl disulfide) and their effectiveness in preventing BP-induced forestomach neoplasia in mice. In the liver, the combined contribution of other GSTs in the detoxification of (+)-anti-BPDE was far less than the contribution of mGSTP1-1 alone. Likewise, in the forestomach, the contribution of mGSTP1-1 far exceeded the combined contribution of other GSTs. Studies on the effects of OSCs against BP-induced forestomach neoplasia revealed a good correlation between their chemo-preventive efficacy and their ability to induce mGSTP1-1 expression in the liver (r = -0.89; p < 0.05) as well as in the forestomach (r = -0.97; p < 0.05). Our results suggest that the induction of mGSTP1-1 may be a reliable marker for evaluating the efficacy of potential inhibitors of BP-induced cancer in a murine model.

The mechanism of increased sensitivity to etoposide (VP-16) in a human bladder cancer cell line (J82/MMC-2), which is >9-fold more resistant to mitomycin C (MMC) compared with parental cells (J82/WT), was investigated. Colony formation assays, following 1 hr drug exposure, revealed that about a 2.2-fold higher concentration of VP-16 was required to kill 50% of the J82/WT cell line compared with J82/MMC-2. The MTT assays, following continuous drug exposure, also showed that the J82/MMC-2 cell line was significantly more sensitive to VP-16 compared with J82/WT. Accumulation of VP-16 was significantly higher in the J82/MMC-2 cell line compared with J82/WT at every drug concentration tested. Likewise, intracellular VP-16 retention was significantly higher in the J82/MMC-2 cell line compared with J82/WT when drug uptake was measured as a function of varying incubation time and at a fixed VP-16 concentration. The efflux of VP-16 from the J82/MMC-2 cell line was equivalent to that from J82/WT. In agreement with the results of drug uptake studies, the levels of VP-16-induced protein-DNA complexes were markedly higher in the J82/MMC-2 cell line compared with J82/WT. The catalytic activity of topoisomerase II (topo II) in 0.35 M NaCl nuclear extract of J82/WT cells was equivalent to that of J82/MMC-2. The levels of topo II mRNA were also comparable in these cells. Our results suggest that the mechanism responsible for the collateral sensitivity of the J82/MMC-2 cell line to VP-16 may be attributable to a relatively higher drug accumulation in this cell line compared with parental cells.

This study describes characteristics of a mitomycin C (MMC)-resistant human bladder cancer cell line, J82/MMC-2, which was established by repeated in vitro exposures of a 6-fold MMC-resistant variant (J82/MMC) to 18 nM MMC. A 9.6-fold higher concentration of MMC was required to kill 50% of the J82/MMC-2 sub-line compared with parental cells (J82/WT). NADPH cytochrome P450 reductase and DT-diaphorase activities were significantly lower in J82/MMC-2 cells compared with J82/WT, suggesting that reduced sensitivity of J82/MMC-2 cells to MMC resulted from impaired drug activation. Consistent with this hypothesis, the formation of MMC-alkylating metabolites was significantly lower in J82/MMC-2 cells compared with J82/WT. Furthermore, DT-diaphorase activity in J82/MMC-2 cells was significantly lower compared with the 6-fold MMC-resistant variant. Glutathione (GSH) levels were comparable in all 3 cell lines. Although GSH transferase (GST) activity was significantly higher in the J82/MMC-2 cells compared with J82/WT, this enzyme activity did not differ between 6- and 9.6-fold MMC-resistant variants. Whereas DNA polymerase alpha mRNA expression was comparable in these cell lines, levels of DNA ligase I mRNA were slightly lower in both MMC-resistant variants relative to J82/WT. However, the DNA polymerase beta mRNA level was markedly higher in the J82/MMC-2 cell line compared with either J82/WT or J82/MMC. Thus, emergence of a higher level of resistance to MMC in J82/MMC-2 cells compared with J82/MMC may be attributed to (i) impaired drug activation through further reduction in DT-diaphorase activity and (ii) enhanced DNA repair through over-expression of DNA polymerase beta.

This study was undertaken to elucidate the mechanism(s) of potentiation of cisplatin (CDDP) cytotoxicity by interferon alpha-2a (IFN alpha-2a) in human squamous carcinoma cell lines SCC-25 and SCC-4. IFN alpha-2a treatment significantly increased the cytotoxicity of CDDP in both cell lines in a dose-dependent manner. In SCC-25 cells, the cytotoxicity of CDDP was increased by about 2- and 4-fold, respectively, by treating the cells with 400 and 800 IU/ml IFN alpha-2a. Sensitivity of SCC-4 cells to CDDP was increased by about 3- and 7-fold, respectively, by 400 and 800 IU/ml IFN alpha-2a treatment. Drug uptake experiments revealed approximately 1.4- to 5-fold higher platinum accumulation in IFN alpha-2a-treated cells as compared to respective controls. Cellular levels of glutathione (GSH) and GSH transferase, which have been suggested to be important determinants of tumor cell sensitivity to CDDP, were not altered by IFN alpha-2a treatment in either of the cell lines. Northern blot analysis showed a moderate increase (about 30-40%) in the level of MT-IIA mRNA by IFN alpha-2a treatment in these cells. Our results suggest that IFN alpha-2a-mediated sensitization of SCC-25 and SCC-4 cell lines to CDDP in vitro may be due to an increase in intracellular platinum accumulation.