DOWN-REGULATION OF CYCLIN-A GENE-EXPRESSION UPON GENOTOXIC STRESS CORRELATES WITH REDUCED BINDING OF FREE E2F TO THE PROMOTER

Treatment of mammalian cells by DNA-damaging agents leads to various c ellular responses. At sufficiently high dosage, cisplatin blocks cell proliferation and finally kills cells; this effect is the basis for it s widespread use as an anticancer drug. Cisplatin-treated cells arrest in the G(1) phase of the cell cycle, most likely due to a signal gene rated by the stabilization of p53 and the subsequent induction of p21( WAF-1/Clp1). We show here that cisplatin-treated mammalian cells accum ulate normal levels of cyclin D1 and cyclin E but fail to produce cycl in A. The block to cyclin A gene expression occurs at the level of tra nscription and is mediated by an E2F binding site in the cyclin A prom oter. It is shown here that, upon cisplatin treatment, transcriptional ly active free E2F becomes limiting, coincident with the accumulation of hypophosphorylated species of the retinoblastoma protein family. Im munoprecipitation experiments suggest that the loss of free E2F result s, at least in part, from the sequestration of E2F-4/DP-1 heterodimers by p107, A role for the kinase inhibitor p21(WAF-1/Clp1) in repressio n of the cyclin A promoter is supported by our finding that ectopic ex pression of p21(WAF-1/Clp1) is sufficient to inhibit transcription fro m the cyclin A gene, dependent on the E2F site. The data establish the E2F site in the human cyclin A promoter as a key target for the signa ling pathway leading to G(1) arrest in response to DNA damage by cispl atin and potentially other genotoxic agents.