G1 cyclin-dependent kinases are sufficient to initiate DNA synthesis in quiescent human fibroblasts

Abstract

Mammalian fibroblasts require mitogens in order to exit from G0 (quiescence) and progress through the G1 phase of the cell cycle, although once they pass the restriction point late in G1 they can enter S phase and complete the cell cycle without mitogens [1]. Mitogenic signals are integrated through the GTPase Ras, which regulates the levels of cyclin D1 [2-5], a component of the cell cycle machinery that operates during G1 phase by activating cyclin-dependent kinase 4 (Cdk4). The accumulation of active cyclin E-Cdk2 complexes also requires Ras [6]. These two G1 cyclin-Cdk complexes act on a family of E2F-associated transcriptional repressors typified by the retinoblastoma protein (Rb) to bring about a transcriptional program that promotes passage through S phase [7-9], but can also activate DNA replication independently of Rb-E2F [10-12]. Although G1 cyclin-Cdk complexes are required for S-phase entry and can shorten G1 phase when overexpressed [13-15], it is not known whether they are sufficient for this transition. Here, we report that serum-starved (G0) diploid human fibroblasts initiate DNA synthesis upon microinjection of active G1 cyclin-Cdk complexes, but not upon microinjection of an S-phase cyclin-Cdk complex. These data indicate that G1 Cdk activation is rate-limiting for S-phase entry, and that Cdk activation is likely to be the primary function of growth factor signalling pathways that lead to DNA synthesis.