The control of cell cycle progression is orchestrated by an extraordinary diverse and dynamic in function group of proteins. Critical in the progression are the actions of the E2F family of transcription factors which regulate the expression of genes necessary for the G 1 /S transition and the WAF/CIP/KIP family of cdk inhibitors which can inhibit cell cycle progression. In this report, we have identi®ed E2F binding sites in both the human and mouse p21 promoters that bind E2F protein complexes from nuclear extracts in a cell cycle-dependent manner. In ectopic expression experiments we determined that E2F1, but not E2F4, can strongly transactivate the human p21 gene through these E2F binding sites which are located in the 7215/+1 region of the p21 gene. The transactivation of the p21 gene through regulatory elements within the 7215/+1 region of the promoter was correlated with increased levels of endogenous E2F1 and p21 proteins at the G 1 /S boundary. The signi®cance of transactivation of the p21 gene by E2F is that p21 function is important in cell cycle progression as well as for cell cycle arrest. Indeed, E2F-induced levels of p21 protein during the G 1 / S transition is consistent with the recent ®ndings demonstrating that p21 acts as an assembly factor for kinase active cyclin/cdk/p21 complexes.

Cyclin D1 plays a key regulatory role during the G 1 phase of the cell cycle and its gene is ampli®ed and overexpressed in many cancers. To address the relationship between cyclin D1 and other cell cycle regulatory proteins, we established human glioma and rodent ®broblast cell lines in which cyclin D1 expression could be regulated ectopically with tetracycline. In both of these cell lines, we found that ectopic expression of cyclin D1 in asynchronously growing cells was accompanied by increased levels of the p53 tumor suppressor protein and the cyclin/cdk inhibitor p21. Despite the induction of these cell cycle inhibitory proteins, cyclin D1-associated cdk kinase remained activated and the cells grew essentially like that of the parent cells. Although growth parameters were unchanged in these cells, morphological changes were clearly identi®able and anchorage independent growth was observed in NIH3T3 cells. In a ®rst step toward elaborating the mechanism for cyclin D1-mediated induction of p21 gene expression we show that co-expression of E2F-1 and DP-1 can speci®cally transactivate the p21 promoter. In support of these ®ndings and a direct e ect of E2F on induction of p21 gene expression a putative E2F binding site was identi®ed within the p21 promoter. In summary, our results demonstrate that ectopic expression of cyclin D1 can induce gene expression of the cdk inhibitor p21 through an E2F mechanism the consequences of which are not to growth arrest cells but possibly to stabilize cyclin D1/cdk function.

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