Figure 5.

Activation of G₁ Cdks is not sufficient for inducing the anchorage-independent cell cycle start. The NRK cell clone overexpressing Cdk4 3-fold (M5) (13, 15) were arrested in G₁ by serum depletion and contact inhibition and stimulated for 3, 5, or 7 hr with FCS. Then, cells were trypsinized and incubated in semisolid DMEM containing 1.17% methylcellulose, 5% FCS, and 25 ng/ml colcemid. As a control (denoted as E+T), NRK cells were stimulated in methylcellulose DMEM containing 5% FCS and EGF plus TGF-β. At 9 hr poststimulation, cells were harvested and lysates were prepared. The activities of Cdk4 and Cdk2 were assayed as described in Materials and Methods. p27 expression and Rb phosphorylation were analyzed as in Fig. 2. At 24 hr poststimulation, the cells were collected and their cell cycle progression was analyzed by flow cytometry. For Cdk6 kinase assay, the NRK cell clone overexpressing Cdk6 5-fold (clone K6–52) was arrested in G₁ and stimulated similarly. The activity of Cdk6 was assayed as described. At 24 hr poststimulation, cells were collected similarly and analyzed by flow cytometry. The Cdk6 overexpressor was indistinguishable from the Cdk4 overexpressor in the flow cytometry patterns at each time point (data not shown).