Alternative splicing and differential expression of DT-diaphorase transcripts in human colon tumors and in peripheral mononuclear cells in response to mitomycin C treatment

Abstract

The two-electron bioreductive enzyme DT-diaphorase catalyzes the metabolism of quinones. The existence of several distinct sizes of DT-diaphorase mRNA transcripts has been observed in human tissues. One of these, an alternatively spliced mRNA that lacks exon 4, has been recently found to be expressed at levels comparable to those of the full-length mRNA. The protein encoded by the mRNA lacking exon 4 has minimal catalytic activity, consistent with the elimination of the quinone-binding site coded for by this exon. We have pursued a number of approaches to examine the significance of this splice variant. We identified a similar truncated transcript in a human HepG2 cDNA library. To determine the frequency of expression of this form of DT-diaphorase in the general population, we examined mRNA obtained from the peripheral mononuclear cells of 16 patients and found substantial interindividual variability in the patterns of transcript expression. Following treatment of these 16 patients with 20 mg/m2 mitomycin C (MMC), the induction of DT-diaphorase transcripts was demonstrated. In most patients, expression of the variant transcript (lacking exon 4) remained constant, while that of the full-length mRNA was elevated. The extent of induction also showed interindividual variability. In one patient, while both transcripts were present at baseline, expression of the variant transcript disappeared almost completely after MMC treatment. To analyze these events under more controlled conditions, we examined the effects of MMC treatment on two human colon tumor cell lines. MMC treatment induced expression of the full-length mRNA but did not influence the abundance of the variant transcript. We then performed single-strand conformational polymorphism analysis of genomic DNA from the 16 patients to investigate the potential role of cis-acting factors in the variable splicing responses. Two patients demonstrated sequence differences in the region spanning exon 4, but in neither was the change in a region critical to splicing regulation. These data demonstrate that the expression of DT-diaphorase in hyman cells is polymorphic, and that the levels of individual transcripts can be regulated by exogenous factors. The findings support a role for alternative splicing in the control of DT-diaphorase gene expression.