Involvement of Activator Protein-1 and Nuclear Factor-kB Transcription Factors in the Control of the DT-Diaphorase Expression Induced by Mitomycin C Treatment

The antitumor antibiotic mitomycin C is activated by several bioreductive enzymes, including DT-diaphorase. In HT29 cells, mitomycin C treatment results in the induction of DT-diapho-rase as reflected in elevated steady state DT-diaphorase mRNA levels. An increase in the transcriptional rate was demonstrated by nuclear run-on assay. To investigate the molecular basis of the change in transcriptional activity caused by mitomycin C treatment, electrophoretic mobility shift assays were used to demonstrate the induction of nuclear factor binding to elements in the 59 flanking region of the DT-diaphorase gene. Treatment of HT29 cells with mitomycin C resulted in the dose-dependent induction of binding activity directed to the activator protein-1 (AP-1) binding element with a time course similar to that of mRNA elevation. Supershift assays using specific antibodies to Jun and Fos demonstrated the participation of both proteins in the binding activities generated. A binding activity for the nu-clear factor-kB (NF-kB) site was induced with a similar time course. Both competitor and supershift experiments indicated that a heterodimer of the NF-kB proteins p50 and p65 was contained in the bound complex. To further investigate the functional consequences of such binding, we transfected HT29 cells with a plasmid containing 3 kb of the DT-diaphorase 59 region upstream of a reporter gene, chloramphenicol acetyl-transferase. Treatment with mitomycin C resulted in a 5.5-fold increase in the expression of a chloramphenicol acetyltrans-ferase construct containing 3 kb of DT-diaphorase promoter sequence. Using a series of deletion mutations based on this full-length construct, we found that two regions of the DT