LOSS OF HETEROZYGOSITY AT THE NAD(P)H-QUINONE OXIDOREDUCTASE LOCUS ASSOCIATED WITH INCREASED RESISTANCE AGAINST MITOMYCIN-C IN A HUMAN BLADDER-CARCINOMA CELL-LINE

The human bladder carcinoma cell line RT112 and the mitomycin C-resistant cell line RT112MMC, derived from RT112 cells, were examined for their expression of NAD(P)H:quinone oxidoreductase (NQOR) and glutathione S-transferases (GSTs). RT112 cells were 40-fold more sensitive towards mitomycin C than RT112MMC cells. The NQOR mRNA level in RT112MMC cells was decreased to 15% as compared to RT112 cells. NQOR enzyme activity was 391 +/- 140 mU/mg protein in RT112 cells, whereas NQOR activity in RT112MMC cells was not measurable. As shown by a fast PCR-based assay and DNA-sequencing, the cell line RT112 is heterozygous, whereas RT112MMC is homozygous for a null allele of the NQOR gene without enzymatic activity. Accordingly, both wild-type and null allele mRNAs were present in RT112 cells, whereas only null allele mRNA was found in RT112MMC. The lack of NQOR enzyme activity in RT112MMC cells was thus associated with loss of heterozygosity at the NQOR locus. By a PCR-RFLP assay, three kidney carcinoma patients without measurable NQOR enzyme activity were shown to be homozygous for the null allele. The PCR assay described here is useful for examination of large numbers of samples. The relative amount of GST-PI mRNA was decreased by 30% in RT112MMC as compared to RT112, contributing to a diminished level of GST enzyme activity, using CDNB as a substrate, from 95 +/- 62 mU/mg protein in RT112 to 26 +/- 6 mU/mg protein in RT112MMC.