Biochemical characterization of a mitomycin C-resistant human bladder cancer cell line.

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.

Modulation of cisplatin sensitivity and accumulation by interferon alpha-2A in human squamous carcinoma cell lines.

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.

Mechanism of cross-resistance to cisplatin in a mitomycin C-resistant human bladder cancer cell line.

This study was undertaken to elucidate the mechanism(s) of cross-resistance to cisplatin (CDDP) in a mitomycin C (MMC)-resistant human bladder cancer cell line, J82/MMC. The J82/MMC cell line displayed 2- to 3-fold cross-resistance to CDDP and carboplatin when compared to the parental J82/WT cells. Drug uptake studies revealed that cross-resistance to CDDP in the J82/MMC cell line was independent of reduced platinum accumulation. The J82/MMC cell line exhibited approximately a 1.5-fold resistance to cadmium chloride, an indicator for increased metallothionein (MT) content, when compared to the J82/WT cells. Northern blot analysis showed a 2.7-fold higher level of MT-IIA mRNA in the J82/MMC cell line compared with J82/WT. We have reported previously that, whereas glutathione (GSH) level is comparable in these cells, GSH transferase (GST) activity is significantly higher in the J82/MMC cell line compared with J82/WT. Results of the present study showed that the elevated GST activity in the J82/MMC cell line was due to an over-expression of pi-type GST protein. Although buthionine-S,R-sulfoximine (BSO)-induced GSH depletion significantly enhanced CDDP cytotoxicity in both cell lines, the magnitude of potentiation was markedly higher in J82/MMC cells (about 2.1-fold) relative to J82/WT (about 1.6-fold). Our results suggest that cross-resistance to CDDP in the J82/MMC cell line may be due to alterations in cellular thiols.