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 cellular responses. At sufficiently high dosage, cisplatin blocks cell proliferation and finally kills cells; this effect is the basis for its widespread use as an anticancer drug. Cisplatin-treated cells arrest in the G1 phase of the cell cycle, most likely due to a signal generated by the stabilization of p53 and the subsequent induction of p21WAF-1/Cip1. We show here that cisplatin-treated mammalian cells accumulate normal levels of cyclin D1 and cyclin E but fail to produce cyclin A. The block to cyclin A gene expression occurs at the level of transcription and is mediated by an E2F binding site in the cyclin A promoter. It is shown here that, upon cisplatin treatment, transcriptionally active free E2F becomes limiting, coincident with the accumulation of hypophosphorylated species of the retinoblastoma protein family. Immunoprecipitation experiments suggest that the loss of free E2F results, at least in part, from the sequestration of E2F-4/DP-1 heterodimers by p107. A role for the kinase inhibitor p21WAF-1/Cip1 in repression of the cyclin A promoter is supported by our finding that ectopic expression of p21WAF-1/Cip1 is sufficient to inhibit transcription from 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 signaling pathway leading to G1 arrest in response to DNA damage by cisplatin and potentially other genotoxic agents.
Spitkovsky
D
D
Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Heidelberg, Germany.
Schulze
A
A
Boye
B
B
Jansen-Dürr
P
P
D016475:3T3 Cells / Q000737:chemistry / Q000187:drug effects; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D015152:Blotting, Northern; D015153:Blotting, Western; D002352:Carrier Proteins; D002453:Cell Cycle / Q000187:drug effects* / Q000235:genetics; D018797:Cell Cycle Proteins; D002945:Cisplatin / Q000494:pharmacology*; D019938:Cyclin D1; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism* / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation; D050684:E2F Transcription Factors; D050704:E2F4 Transcription Factor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000737:chemistry / Q000187:drug effects; D016193:G1 Phase / Q000187:drug effects / Q000235:genetics; D006801:Humans; D051379:Mice; D015513:Oncogene Proteins / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D000970:Antineoplastic Agents; C533431:Arid4a protein, mouse; C493956:CDKN1A protein, human; D002352:Carrier Proteins; C493957:Cdkn1a protein, mouse; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050704:E2F4 Transcription Factor; C493762:E2F4 protein, human; C493763:E2f4 protein, mouse; D004791:Enzyme Inhibitors; D015513:Oncogene Proteins; D012333:RNA, Messenger; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D019938:Cyclin D1; D002945:Cisplatin