A study of DT-diaphorase as a prodrug-activating enzyme

grantor: University of TorontoDT-diaphorase (DTD) is an enzyme that acts on its substrates in a single-step two electron transfer reaction. It has a dual role both as a prodrug-activating enzyme and as a protector against carcinogenesis mediated by xenobiotics. A frequently occurring base change, a C to T transition in exon six of the DTD gene, has been associated with low to undetectable DTD activity. Also, tumor cells vary in their DTD levels, which poses a limitation for the use of drugs that rely on DTD for their chemotherapeutic effects. To test directly the effect of the base change on enzyme activity, mammalian cells were transfected with expression vectors containing DTD cDNA with a C or T nucleotide at position 609. Results indicated that this base change is a mutation in the DTD gene which results in impaired activity and reduced protein stability. The relationship between the status of the DTD nucleotide 609 allele and enzymatic activity was assessed in a panel of human fibroblast cell strains. DTD activity was, on average, 2-fold higher in cells that were homozygous for the wild-type allele compared to heterozygotes. Cells that were homozygous for the variant allele displayed low to undetectable DTD activity and trace levels of DTD protein. A unique cell strain that is heterozygous yet has low to undetectable DTD activity and protein was shown to express DTD mRNA exclusively from the variant allele. These results demonstrate that the status of the nucleotide 609 allele can, to a first approximation, predict for DTD activity and function. The possibility of using DTD in gene therapy was investigated by developing adenoviral vectors that deliver DTD to sensitize cells that are deficient in this enzyme. Infection of mitomycin C-resistant cells with an adenoviral vector that carries a minigene for wild-type DTD rendered them sensitive to this drug. The degree of sensitization depended on multiplicity of infection. These studies demonstrate the role of the C to T mutation in affecting DTD activity and function and provide "proof of principle" data for the use of DTD as an enzyme for virus-directed enzyme-prodrug therapy.Ph.D