We have shown that nontransformed mammalian cells arrest early in the G1 phase of the cell cycle when treated with exceedingly low concentrations of the nonspecific kinase inhibitor staurosporine, whereas transformed cels continue to progress through the cell cycle. We have now treated normal or transformed human skin fibroblasts with four other kinase inhibitors. Three of these inhibitors are highly specific: KT5720 inhibits cAMP-dependent protein kinme, KT5823 inhibits cGMP dependent protein kinase, and KT5926 Inhibits myosin light-chain kinase. The fourth inhibitor K252b has a moderate specificity for protein kinase C but also inhibits the three kinaes just mentioned. We have found that these inhibitors reversibly arrest normal human skin fibroblasts at different times in the G1 phase but do not affect the cell cycle poession of nformed cells. The times of arrest within the G1 phase can be divided Into two categories. Two of the inhibitors, KT5926 and K252b, act at an early time that is -4 h after the transition from Go to G1. The cAMP-and cGMPdependent protein kinase inhibitors rest cells at a later tue that is =6 h after the Go/G1 boundary.These data indite that there are multiple kinase-medated phosphorylitions of different substrates that are essential for the pgression of normal cells, but not transformed cells, through the G1 phase. These inhibitors provide us with a set of biochemical probes that should be invaluable in the study ofthe function of kinases during GI phase progression of normal cells.

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...

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A G1-phase delay after exposure to alpha particles has not been report ed previously, perhaps because immortalized cell lines or cell lines from tumor cells were used in past studies. Therefore, we compared the effects of alpha particles (0.19 or 0.57 Gy) and approximately equitoxic doses of gamma rays (2 or 4 Gy) on progression of cells through the cell cycle in normal human skin fibroblasts. Cell cycle analyses were performed using flow cytometry by measuring incorporation of bromodeoxyuridine (BrdUrd) in each phase of the cell cycle up to 44 h after irradiation. We observed an alpha-particle-induced G1-phase delay in human skin fibroblasts even at the lowest dose, 0.19 Gy. At equitoxic doses, more pronounced and persistent G1-phase delays and arrests were observed in gamma-irradiated cultures in that increased fractions of the G1-phase cells remained BrdUrd- over the course of the study after gamma-ray exposure compared to cells exposed to alpha particles. In addition, G1-phase cells that became BrdUrd+ after gamma irradiation re-arrested in G1 phase, whereas BrdUrd+ G1-phase cells in alpha-particle-irradiated cultures continued cycling. In contrast, comparable percentages of cells were delayed in G2 phase after either alpha-particle or gamma irradiation. Both gamma and alpha-particle irradiation caused increases in cellular p53 and p2lCip1 shortly after the exposures, which suggests that the G1-phase delay that occurs in response to alpha-particle irradiation is dependent on p53 like the initial G1-phase delay induced by gamma rays.

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