Increased DT-diaphorase expression and cross-resistance to mitomycin C in a series of cisplatin-resistant human ovarian cancer cell lines

Abstract

In a series of ovarian carcinoma cell lines selected in vitro for resistance to cisplatin by continuous exposure to increasing drug concentrations, the level of resistance is proportional to the expression of γ-glutamylcysteine synthetase (γ-GCS). To determine if other detoxicating genes are coordinately expressed, we measured the activity of DT-diaphorase and cytochrome P450 reductase. The specific activity of DT-diaphorase, but not that of cytochrome P450 reductase, increased with increasing resistance to cisplatin. Steady-state mRNA levels for DT-diaphorase correlated with enzyme activity and hence with cisplatin resistance. Since the activity of DT-diaphorase has been associated with sensitivity to quinones, we studied the cytotoxicity of mitomycin C under oxic conditions. Unexpectedly, resistance to mitomycin C increased proportionally with that to cisplatin ( r = 0.997). Pretreatment with buthionine sulfoximine, which inhibits glutathione (GSH) synthesis, failed to sensitize either the sensitive or the resistant lines to mitomycin C. Thus, the basis for collateral resistance to mitomycin C in the cisplatin-resistant lines under oxic conditions is unrelated to overproduction of GSH. Under hypoxia, the toxicity of mitomycin C to the most sensitive (A2780) cell line was unchanged. However, the most resistant (C200) line was 2-fold more resistant to mitomycin C under hypoxic conditions. The coordinate overexpression of DT-diaphorase and γ-GCS in the resistant cell lines is thus associated with hypoxic cell resistance, and supports the involvement of shared mechanisms of gene regulation in the observed resistant phenotype.