Rb/Cdk2/Cdk4 triple mutant mice elicit an alternative mechanism for regulation of the G₁/S transition

The G₁/S-phase transition is a well-toned switch in the mammalian cell cycle. Cdk2, Cdk4, and the rate-limiting tumor suppressor retinoblastoma protein (Rb) have been studied in separate animal models, but interactions between the kinases and Rb in vivo have yet to be investigated. To further dissect the regulation of the G₁ to S-phase progression, we generated Cdk2^-/-Cdk4^-/-Rb^-/- (TKO) mutant mice. TKO mice died at midgestation with major defects in the circulatory systems and displayed combined phenotypes of Rb^-/- and Cdk2 ^-/-Cdk4^-/- mutants. However, TKO mouse embryonic fibroblasts were not only resistant to senescence and became immortal but displayed enhanced S-phase entry and proliferation rates similar to wild type. These effects were more remarkable in hypoxic compared with normoxic conditions. Interestingly, depletion of the pocket proteins by HPV-E7 or p107/p130 shRNA in the absence of Cdk2/Cdk4 elicited a mechanism for the G₁/S regulation with increased levels of p27^Kip1 binding to Cdk1/cyclin E complexes. Our work indicates that the G₁/S transition can be controlled in different ways depending on the situation, resembling a regulatory network.