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Transcriptional Repression of the Cyclin-dependent Kinase Inhibitor p21^WAF1/CIP1 Gene Mediated by cis Elements Present in the 3'-Untranslated Region¹

Department of Medicine, Division of Oncology and Greenebaum Cancer Center [A. K. R., C. K. A. H., X-S. L., A. H., J. A. F.], and Department of Biochemistry [T. M. G.], University of Maryland School of Medicine, and Veterans Affairs Medical Center [J. A. F.], Baltimore, Maryland 21201

The cyclin-dependent kinase inhibitor p21^WAF1/CIP1 plays a major role in the induction of G₁ cell cycle arrest following DNA damage and is known to be regulated by p53-dependent and -independent pathways. Here, we show that p21^WAF1/CIP1 transcription is also regulated, independently of p53, by the cis elements that are located downstream of the transcription start site. A cDNA fragment of 180 bp, located 260 bases 3' to the translation termination codon of p21^WAF1/CIP1 cDNA, was cloned in both the sense and antisense orientations downstream of the CMV promoter, upstream of the SV40 promoter, and both upstream and downstream of the p21^WAF1/CIP1 promoter in the plasmids carrying the luciferase reporter gene. The constructs were transiently transfected in human breast carcinoma cells MCF-7 and MDA-MB-468 and a Syrian hamster smooth muscle cell line DDT₁MF₂ and were found to elicit 2–3-fold or higher repression of luciferase activities. By using overlapping deletions of the above 180-bp fragment, we identified a 48-bp subfragment that contains putative cis element(s) that participate in the transcriptional repression of the p21^WAF1/CIP1 gene. The overlapping subfragments bind, in vitro, to specific proteins present in the nuclear extracts of MDA-MB-468 and DDT₁MF₂ cells. We, therefore, propose that additional mechanism(s) exist that regulate expression of the cellular p21^WAF1/CIP1 and may contribute to p21^WAF1/CIP1-dependent control of the cell cycle.

¹ This work was supported in part by the Medical Research Services of the Department of Veterans Affairs, by NIH Grant CA-63335 (to J. A. F.), by a University of Maryland Institutional Research Award (to A. K. R.), and V. A. Cancer Development Award (to A. H.).

² To whom requests for reprints should be addressed, at Greenebaum Cancer Center, University of Maryland, Bressler Research Building, Room 9-031, 655 West Baltimore Street, Baltimore, MD 21201.

Received 6/ 5/97. Accepted 9/19/97.

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