DT-DIAPHORASE ACTIVITY AND MITOMYCIN-C SENSITIVITY IN NONTRANSFORMED CELL STRAINS DERIVED FROM MEMBERS OF A CANCER-PRONE FAMILY

Non-transformed skin fibroblasts derived from five members of a cancer-prone family and three unrelated healthy volunteers were assayed for their levels of activity of the quinone reductase DT-diaphorase and for their sensitivity to the antitumor quinone mitomycin C (MMC). Previous studies of skin fibroblasts derived from one afflicted member of this family (3437T) demonstrated increased resistance to MMC under aerobic exposure conditions and a reduced level of DT-diaphorase. In the present study 3437T cells and a cell strain derived from another afflicted member of the cancer-prone family were found to be hyperresistant to the cytotoxic effects of MMC, and demonstrated negligible DT-diaphorase activity (30 +/- 10 nmol/min/mg protein). Cell strains derived from the three other family members demonstrated intermediate DT-diaphorase activity (400-800 nmol/min/mg protein). Enzyme activities of 1800-6000 nmol/min/mg protein were measured in the three control cell strains. A protein that was reactive with a rabbit polyclonal antibody raised against rat DT-diaphorase and corresponded to the known mol. wt of DT-diaphorase was clearly evident in the three control cell strains, but absent in the two MMC-hyperresistant cell strains. This protein was present in intermediate amounts in the remaining members of the cancer-prone family. Southern analysis of DNA isolated from all eight cell strains and restricted with EcoRI demonstrated the presence of a DNA sequence of approximately 15 kb which hybridized to a rat DT-diaphorase cDNA probe. Northern analysis revealed the presence of an RNA species approximately 1200 bp in size, consistent with that for a human DT-diaphorase mRNA, in all cell strains derived from family members. A post-transcriptional defect would, therefore, appear to be responsible for the decreased enzyme activity observed in the resistant cell strains. These results suggest a role for DT-diaphorase in MMC bioactivation and that reduced levels of the protein may be causally related to the cancer-prone tendency of this family.