Abstract
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-diaphorase 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 5′ 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 nuclear factor-κB (NF-κB) site was induced with a similar time course. Both competitor and supershift experiments indicated that a heterodimer of the NF-κB 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 5′ region upstream of a reporter gene, chloramphenicol acetyltransferase. Treatment with mitomycin C resulted in a 5.5-fold increase in the expression of a chloramphenicol acetyltransferase 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-diaphorase promoter region, positions −346 to −588 (containing the AP-1 element) and positions −785 to −890 (containing the NF-κB element) are required for the full expression of the mitomycin C response. The specific involvement of these binding elements was confirmed using mutational analysis. The results demonstrate that mutation of either element alone or of both diminishes the response, indicating an additive interaction between the elements at a minimum. However, inducibility characterizes a promoter fragment as small as 78 base-pairs from the transcription start site. Treatment of cells with mitomycin C induced binding to a 38-base-pair region (−40 to −78) devoid of known transcription factor binding elements. These data suggest that mitomycin C induces the overexpression of DT-diaphorase through a mechanism involving both the AP-1 and NF-κB response elements and that inducibility depends on a novel factor binding element.
Footnotes
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Send reprint requests to: Dr. Peter J. O’Dwyer, Kimmel Cancer Institute, Thomas Jefferson University, Bluemle Life Sciences Building, 233 S. 10th Street, Suite 502, Philadelphia, PA 19107. E-mail: p odwyer{at}lac.jci.tju.edu
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↵1 K.-S. Yao and P. J. O’Dwyer, unpublished observations.
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↵2 K.-S. Yao and P. J. O’Dwyer, unpublished observations.
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This work was supported in part by National Institutes of Health Grant ROI-CA49820 and an appropriation from the Commonwealth of Pennsylvania.
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K.-S. Y and A. H. contributed equally to this work.
- Abbreviations:
- MMC
- mitomycin C
- EMSA
- electrophoretic mobility shift assay
- bp
- base-pair(s)
- CAT
- chloramphenicol acetyltransferase
- AP-1
- activator protein-1
- NF-κB
- nuclear factor-κB
- PBS
- phosphate-buffered saline
- PCR
- polymerase chain reaction
- SDS
- sodium dodecyl sulfate
- SSC
- standard saline citrate
- Received May 29, 1996.
- Accepted November 25, 1996.
- The American Society for Pharmacology and Experimental Therapeutics
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