To investigate the role of kinase-mediated mechanisms in regulating mammalian cell proliferation, we determined the effects of the general protein kinase inhibitor staurosporine on the proliferation ofa series ofnontransformed and transformed cultured rodent and human cells. Levels of staurosporine as low as 1 ng/ml prevented nontransformed cells from entering S phase (i.e., induced G1 arrest), indicating that kinase-mediated processes are essential for commitment to DNA replication in normal cells. At higher concentrations of staurosporine (50-75 ng/ml), nontransformed mammalian cells were arrested in both GI and G2. The period of sensitivity of nontransformed human diploid fibroblasts to low levels of the drug commenced 3 hr later than the Go/G, boundary and extended through the GI/S boundary. Interference with activity of the Gl-essential kinase(s) caused nontransformed human cells traversing mid-to-late GI at the time of staurosporine addition to be "set back" to the initial staurosporine block point, suggesting the existence ofa kinase-dependent " GI clock" mechanism that must function continuously throughout the early cycle in normal cells. The initial staurosporine block point at 3 hr into GI corresponds to neither the serum nor the amino acid restriction point. In marked contrast to the behavior of nontransformed cells, neither low nor high concentrations of staurosporine affected G1 progression in transformed cultures; high drug concentrations caused transformed cells to be arrested solely in G2. These results indicate that kinasemediated regulation of DNA replication is lost as the result of neoplastic transformation, but the G2-arrest mechanism remains intact.A large body of evidence suggests that protein phosphorylation plays a central role in the regulation of cell growth, differentiation, and proliferation. Control of cell proliferation involves the temporal activation of a series of interrelated primary and secondary kinases that phosphorylate an array of essential cellular proteins. Cell cycle studies (1-8) indicate that basic cellular processes such as the commitment to DNA replication and the initiation of mitosis are regulated by kinase-mediated mechanisms. However, whereas eukaryotes ranging in complexity from yeasts to primates employ a highly homologous p34cdc2 kinase-dependent mechanism for regulating the onset of mitosis (9-12), there is less compelling evidence for the existence in mammalian cells of a similar kinase-specific mechanism for regulating commitment to genome replication.To obtain information on the role of kinase-mediated mechanisms in commitment to mammalian DNA replication, we examined the effects on cell proliferation of inhibition of protein kinase activity by the drug staurosporine. This drug was selected for these studies because it exhibits inhibitory activity against a wide range of protein kinases (13-16). Our results show that kinase-mediated processes are essential both for progression through most of G1 and for initiation of DNA synthesis, but only in non...

Abstract. The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34 '~c2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wildtype FM3A cell line each possess at least three distinct histone HI kinases. HI kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34 r and the human cyclins A and B1. The results show that p34 r forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the nonpermissive temperature in vitro and in vivo. A third HI kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-1ike 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34 ~2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34 ~c~c~ is essential only during the G2-to-M transition in FT210 cells.