Vjekoslav Dulić

p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest

gamma-Irradiation of human diploid fibroblasts in the G1 interval caused arrest of the cell cycle prior to S phase. This cell cycle block was correlated with a lack of activation of both cyclin E-Cyclin-dependent kinase 2 (Cdk2) and cyclin A-Cdk2 kinases and depended on wild-type p53. Although the accumulation of cyclin A was strongly inhibited in gamma-irradiated cells, cyclin E accumulated and bound Cdk2 at normal levels but remained in an inactive state. We found that both whole-cell lysates and inactive cyclin E-Cdk2 complexes prepared from irradiated cells contained an activity capable of inactivating cyclin E-Cdk2 complexes. The protein responsible for this activity was shown to be p21CIP1/WAF1, recently described as a p53-inducible Cdk inhibitor. Our data suggest a model in which ionizing radiation confers G1 arrest via the p53-mediated induction of a Cdk inhibitor protein.

Regulation of retinoblastoma protein functions by ectopic expression of human cyclins

The retinoblastoma susceptibility gene (RB) product, the retinoblastoma protein (pRb), functions as a regulator of cell proliferation. Introduction of the RB gene into SAOS-2 osteosarcoma cells, which lack functional pRb, prevents cell cycle progression. Such growth-suppressive functions can be modulated by phosphorylation of pRb, which occurs via cell cycle-regulated kinases. We show that constitutively expressed cyclins A and E can overcome pRb-mediated suppression of proliferation. pRb becomes hyperphosphorylated in cells overexpressing these cyclins, and this phosphorylation is essential for cyclin A- and cyclin E-mediated rescue of pRb-blocked cells. This suggests that G1 and S phase cyclins can act as regulators of pRb function in the cell cycle by promoting pRb phosphorylation.

Isolation of three novel human cyclins by rescue of G1 cyclin (cln) function in yeast

We have isolated a number of cDNAs derived from human mRNAs that are able to substitute for G1 cyclin genes in S. cerevisiae. Several of these encode human cyclins A, B1, and B2. Three novel genes have been identified, which we call cyclins C, D, and E. The novel proteins are sufficiently distantly related to the other members of the cyclin family and to each other as to constitute three new classes of cyclins. Cyclin C and E mRNAs accumulate periodically through the cell cycle, peaking at different times in G1.

G1 control in mammalian cells.

SUMMARY Cyclin-dependent kinases (Cdks) control the major cell cycle transitions in eukaryotic cells. On the basis of a variety of experiments where cyclin function either is impaired or enhanced, D-type cyclins as well as cyclins E and A have been linked to G1 and G1/S phase roles in mammalian cells. We therefore sought to determine if agents that block the G1/S phase transition do so at the level of regulating the Cdk activities associated with these cyclins. A variety of conditions that lead to G1 arrest were found to correlate with accumulation of G1-specific Cdk inhibitors, including treatment of fibroblasts with ionizing radiation, treatment of epithelial cells with TGF-β, treatment of HeLa cells with the drug lovastatin, and removal of essential growth factors from a variety of different cell types. Mechanistically, inhibition of Cdks was found to involve the stoichiometric binding of Cdk inhibitor proteins. p21Waf1/Cip1 was associated with DNA damage induced arrest while p27Kip1/p28Ick1 accumulated under a variety of antiproliferative conditions.

Uncoupling between Phenotypic Senescence and Cell Cycle Arrest in Aging p21-Deficient Fibroblasts

ABSTRACT Irreversible G1 arrest in senescent human fibroblasts is mediated by two inhibitors of cyclin-dependent kinases (Cdks), p21Cip1/SDI1/WAF1 and p16Ink4A. To determine the physiological and molecular events that specifically require p21, we studied senescence in human diploid fibroblasts expressing the human papillomavirus type 16 E6 oncogene, which confers low p21 levels via enhanced p53 degradation. We show that in late-passage E6 cells, high Cdk activity drives the cell cycle, but population expansion is slowed down by crisis-like events, probably owing to defective cell cycle checkpoints. At the end of lifespan, terminal-passage E6 cells exhibited several aspects of the senescent phenotype and accumulated unphosphorylated pRb and p16. However, both replication and cyclin-Cdk2 kinase activity were still not blocked, demonstrating that phenotypic and replicative senescence are uncoupled in the absence of normal p21 levels. At this stage, E6 cells also failed to upregulate p27 and inactivate cyclin-Cdk complexes in response to serum deprivation. Eventually, irreversible G1 arrest occurred coincident with inactivation of cyclin E-Cdk2 owing to association with p21. Similarly, when p21−/− mouse embryo fibroblasts reached the end of their lifespan, they had the appearance of senescent cells yet, in contrast to their wild-type counterparts, they were deficient in downregulating bromodeoxyuridine incorporation, cyclin E- and cyclin A-Cdk2 activity, and inhibiting pRb hyperphosphorylation. These data support the model that the critical event ensuring G1arrest in senescence is p21-dependent Cdk inactivation, while other aspects of senescent phenotype appear to occur independently of p21.

Altered Regulation of Cell Cycle Genes and Proteins in Senescent Human Diploid Fibroblasts

Human diploid fibroblasts have a finite proliferative lifespan at the end of which they are unable to enter S phase in response to mnitogenic stimulation even though they remain alive for many months.1 The G1 arrest state of senescent cells has much in common with the G l arrest state of early passage quiescent cells, because serum stimulation induces the expression of many early to mid-G1 genes in both senescent and quiescent cells. For example, mitogen-stimulated senescent cells are similar to mitogen-stimulated quiescent cells in their expression of c-myc, c-jun and c-H-ras.2,3,4 Nevertheless, mitogen-stimulated senescent cells are unable to enter S phase, whereas mitogen-stiinulated early passage quiescent cells enter S phase approximately 12–18 hours after stimulation. As a means of investigating the molecular basis for the failure to enter S phase in senescent human fibroblasts, we have sought to identify molecules and/or functions that are deficient in the mitogen response pathways in senescent cells. Since the control of cell proliferation in eukaryotes from yeast to man involves the regulated synthesis, activation and degradation of a family of cyclins, which interact with the Cdc2/CDC28 family of cyclin-dependent kinases (Cdk’s), 5,6 we have been analysing the amount and the activity of several cyclins and Cdk’s in senescent and quiescent human fibroblasts.7,8