Figure 3.

E2F1 induces S phase in the absence of cdk activity. (A) E2F-mediated S phase induction in p21-arrested cells. REF52 cells were held at density arrest for 24 hr and infected with either Ad-Con (control virus with empty expression cassette) at a moi of 125 ffu per cell, Ad-E2F1 or Ad-E2F1/VP16 (AVP16′′) at an moi of 25 ffu per cell together with Ad-p21 (moi of 50, 100, or 200 ffu per cell as indicated) or Ad-Np21 (moi of 100 ffu per cell), and then replated at subconfluent densities. Cells were labeled with BrdUrd from 12 to 24 hr postinfection, and BrdUrd was detected by using indirect immunofluorescence. At least 300 nuclei were scored for BrdUrd immunofluorescence. (B) E2F induces DNA replication. REF52 cells were held at density arrest for 24 hr before infection with either Ad-Con (moi of 125 ffu per cell), Ad-p21 (moi of 100 ffu per cell), or Ad-p21 and Ad-E2F1 or Ad-E2F2 (moi of 100 and 25 ffu per cell, respectively). Cells were then replated at subconfluent densities, harvested at the indicated times postinfection, and processed for flow cytometry. The horizontal axis reflects relative DNA content and the vertical axis represents cell number. (C) E2F-mediated S phase induction in the absence of cyclin D- or cyclin E-associated kinase activity. Cells treated as in A were lysed in IP buffer, and the lysates were immunoprecipitated with control IgG antibodies (Ig) or antibodies against either cyclin D1 or cyclin E. The sample designated A represents lysates made from density-arrested cells immunoprecipitated with the corresponding specific antibodies. The kinase assays were performed as described (42) except glutathione S-transferase–Rb was used as the substrate. (D) E2F-mediated S phase induction in the absence of cdk2- or cyclin A-associated kinase activity. Cells treated as in A were lysed in IP buffer and the lysates immunoprecipitated with control IgG antibodies (Ig) or antibodies against either cdk2, cyclin A, or cyclin B1. The samples designated A represent lysates made from density-arrested cells immunoprecipitated with the corresponding specific antibodies. The kinase assays were performed as in C. (E) E2F-mediated S phase and cyclin B1-associated kinase induction. Density-arrested REF52 cells were infected with either Ad-Con (moi of 125 ffu per cell; open bar), Ad-p21 (moi of 100 ffu per cell, gray bar), or Ad-p21 and Ad-E2F1 (moi of 100 ffu per cell and 125 ffu per cell, respectively, black bar) and replated at subconfluent densities. Cells were either labeled with BrdUrd for 1 hr before fixation at the indicated times and processed as in A (Upper) or lysed in IP buffer at the indicated times and assayed for cyclin B1-associated kinase activity as in D (Lower). (F) Assay for Rb phosphorylation. Representative cell samples treated as in A were harvested in SDS/PAGE loading buffer, and Rb was detected by using Western blotting. To better visualize the Rb product in lane 2, we show a 3-fold longer exposure of this sample in lane 1.