Assessment of the relationship between genotypic status of a DT-diaphorasepoint mutation and enzymatic activity

DT-diaphorase, a cytosolic reductase, has been implicated as an activator o f chemotherapeutic prodrugs and a detoxifier of certain potentially carcino genic xenobiotics. A common C to T nucleotide 609 substitution in DT-diapho rase cDNA has been associated with protein instability and reduced catalyti c activity. The degree to which the allelic status of the substitution corr elates with enzymatic activity was assessed in 45 normal human skin fibrobl ast strains using a PCR-RFLP assay. Included in this study was the 3437T st rain, which is unique in that it is heterozygous for the polymorphism yet c ontains undetectable enzymatic activity. An allele-specific RT-PCR-RFLP tec hnique attributed this phenomenon to exclusive DT-diaphorase mRNA expressio n from the variant allele. Overlap in activities was observed between indiv idual strains homozygous for the wild-type allele and heterozygotes, but th e former group displayed enzymatic activity that was on average 2-fold high er. Western blot analysis of the two strains in this panel that are homozyg ous for the Variant allele revealed that they express relatively low amount s of DT-diaphorase protein, consistent with the role of the substitution in protein instability This work confirms that genotypic status is a reliable initial estimate of DT-diaphorase activity. (C) 2000 Cancer Research Campa ign.