Intestinal cell cycle regulations. Interactions of cyclin D1, Cdk4, and p21Cip1

OBJECTIVE: The p21Cip1 protein is a potent stoichiometric inhibitor of cyclin-dependent kinase activity, and p21Cip1 mRNA expression is localized to the nonproliferative compartment of the intestinal villus, suggesting an in vivo growth-inhibitory role in the gut. The authors determined whether nontransformed rat intestinal epithelial cells (IECs) underwent reversible cell cycle arrest by contact inhibition, and determined whether increases in the relative amount of p21 associated with cyclin D/Cdk4 protein complexes were associated with cell growth arrest. METHODS: Density arrest was achieved by prolonged culture IEC-6 in confluent conditions (5 or more days). Release from density arrest was achieved by detaching the cells from the culture plate and reseeding them at a 1:4 ratio. The DNA synthesis was estimated by [3H]-thymidine incorporation and expressed as mean plus or minus standard error of the mean (n = 4). Cyclin D1, Cdk4, and p21 mRNA and protein levels were determined by standard Northern and Western blot analyses, respectively. Cyclin D1, Cdk4, and p21 protein complex formation was analyzed by immunoprecipitating the complexes from cell lysates with an antibody to one of the constituents, followed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the precipitated complexes using antibodies to the other proteins. The kinase activity of the immunoprecipitated Cdk4 was determined using recombinant Rb as substrate. RESULTS: The IEC-6[3H]-thymidine incorporation was decreased 7.5-fold from day 1 confluence to day 7 of confluence. Twenty-four hours after release from density arrest, there was a 43-fold increase in [3H]-thymidine incorporation. Cyclin D1 and Cdk4 mRNA levels remained relatively constant during contact inhibition, whereas immunoblotting showed that the levels of cyclin D1 and Cdk4 proteins decreased by 70.9% and 68.7%, respectively, comparing day 3 with day 9 during density arrest. The levels of cyclin D1 increased 5.8-fold and Cdk4 increased by 4.4-fold by 24 hours after reseeding the day 9 density-arrested cultures, coincident with the increase in DNA synthesis. The amount of p21 associated with the cyclin D1 and Cdk4 complex in the density-arrested cells was 170% of that observed in the reseeded, proliferating cells. More important, the p21::Cdk4 ratio was 6.4-fold higher in the density-arrested (quiescent) cells as compared with rapidly proliferating cells by 24 hours after release from growth arrest. Recovery of Cdk4-dependent kinase activity occurred by 4 hours after release from growth arrest, coincident with decreased binding of p21 to the complex. CONCLUSIONS: Intestinal epithelial cells in culture can undergo density-dependent growth arrest. This process involves downregulation of cyclin D1 and Cdk4 at the level of protein expression, whereas the mRNA levels remain relatively unchanged. Further, during contact inhibition, there is more p21 associated with cyclin D1/Cdk4, which further contributes to the inhibition of the kinase complex. The authors also have shown that the process of contact inhibition is reversible, which may explain partly the ability of the intestinal epithelium to increase proliferative activity in response to injury.     

Prevention of bacterial endocarditis

Alterations in the normal cell cycle lead to abnormal cell proliferation and to tumor development. To explore the role of the cyclin D/Cdk4 complex and the retinoblastoma protein (pRb) in the growth and spread of osteoblastic osteosarcoma (OS), 40 tumor samples were selected. In 17 of these cases, lung metastases occurred during follow-up. Expression of pRb, cyclin D1 and its catalytic subunit, Cdk4, was studied by immunohistochemistry and immunoblotting. As controls, non-neoplastic tissues surrounding the tumor were used. The expression level and pattern were compared to clinical outcome. Cdk4 was over-expressed in 80% of OS, independently of clinical outcome, and showed an intense and uniform distribution in tumor cells compared to normal cells. However, co-immunoprecipitation of Cdk4 with cyclin D1 revealed low levels of cyclin D/Cdk4 complex; 20 of 40 OS examined had a negative or minimal immunostaining for active pRb. The probability of relapse was significantly higher in pRb-negative than in the -positive patients (p < 0.05). The ratio of unphosphorylated/hyperphosphorylated pRb was lower in relapsed patients than in patients with no evident disease, though the difference was not statistically significant. High levels of pRb/cyclin D1 were found in all samples exhibiting functional pRb expression. Our results show that G1 phase deregulation is involved in formation and development of OS. The expression levels of both pRb and cyclin D1 had a clear correlation with clinical outcome, suggesting that these parameters could be used as prognostic markers.     

p21(Waf1/Cip1) inhibition of cyclin E/Cdk2 activity prevents endoreduplication after mitotic spindle disruption

During a normal cell cycle, entry into S phase is dependent on completion of mitosis and subsequent activation of cyclin-dependent kinases (Cdks) in G1. These events are monitored by checkpoint pathways. Recent studies and data presented herein show that after treatment with microtubule inhibitors (MTIs), cells deficient in the Cdk inhibitor p21(Waf1/Cip1) enter S phase with a >/=4N DNA content, a process known as endoreduplication, which results in polyploidy. To determine how p21 prevents MTI-induced endoreduplication, the G1/S and G2/M checkpoint pathways were examined in two isogenic cell systems: HCT116 p21(+/+) and p21(-/-) cells and H1299 cells containing an inducible p21 expression vector (HIp21). Both HCT116 p21(-/-) cells and noninduced HIp21 cells endoreduplicated after MTI treatment. Analysis of G1-phase Cdk activities demonstrated that the induction of p21 inhibited endoreduplication through direct cyclin E/Cdk2 regulation. The kinetics of p21 inhibition of cyclin E/Cdk2 activity and binding to proliferating-cell nuclear antigen in HCT116 p21(+/+) cells paralleled the onset of endoreduplication in HCT116 p21(-/-) cells. In contrast, loss of p21 did not lead to deregulated cyclin D1-dependent kinase activities, nor did p21 directly regulate cyclin B1/Cdc2 activity. Furthermore, we show that MTI-induced endoreduplication in p53-deficient HIp21 cells was due to levels of p21 protein below a threshold required for negative regulation of cyclin E/Cdk2, since ectopic expression of p21 restored cyclin E/Cdk2 regulation and prevented endoreduplication. Based on these findings, we propose that p21 plays an integral role in the checkpoint pathways that restrain normal cells from entering S phase after aberrant mitotic exit due to defects in microtubule dynamics.     

Calmodulin is essential for cyclin-dependent kinase 4 (Cdk4) activity and nuclear accumulation of cyclin D1-Cdk4 during G1

Although it is known that calmodulin is involved in G1 progression, the calmodulin-dependent G1 events are not well understood. We have analyzed here the role of calmodulin in the activity, the expression, and the intracellular location of proteins involved in G1 progression. The addition of anti-calmodulin drugs to normal rat kidney cells in early G1 inhibited cyclin-dependent kinase 4 (Cdk4) and Cdk2 activities, as well as retinoblastoma protein phosphorylation. Protein levels of cdk4, cyclin D1, cyclin D2, cyclin E, p21, and p27 were not affected after CaM inhibition, whereas decreases in the amount of cyclin A and Cdc2 were observed. The decrease of Cdk4 activity was due neither to changes in its association to cyclin D1 nor to changes in the amount of p21 or p27 bound to cyclin D1-Cdk4 complexes. Calmodulin inhibition also produced a translocation of nuclear cyclin D1 and Cdk4 to the cytoplasm. This translocation could be responsible for the decreased Cdk4 activity upon calmodulin inhibition. Immunoprecipitation, calmodulin affinity chromatography, and direct binding experiments indicated that calmodulin associates with Cdk4 and cyclin D1 through a calmodulin-binding protein. The facts that Hsp90 interacts with Cdk4 and that its inhibition induced Cdk4 and cyclin D1 translocation to the cytoplasm point to Hsp90 as a good candidate for being the calmodulin-binding protein involved in the nuclear accumulation of Cdk4 and cyclin D1.     

Insensitivity to growth inhibition by TGF-beta1 correlates with a lack of inhibition of the CDK2 activity in prostate carcinoma cells

TGF-beta is a potent growth inhibitor of epithelial cells. However, many transformed cells have lost their sensitivity to this growth inhibitory effect. The molecular mechanism of such insensitivity is not yet understood. Here, we have studied the TGF-beta1 effect on normal human prostate and carcinoma cells. Our results showed that normal cells were sensitive to growth inhibition, whereas tumor cells were not or only minimally inhibited regardless of the concentration of TGF-beta1 (20 to 80 pM) or time of exposure (1-5 days). p21WAF1/Cip1/Sdi1 and p15INK4B but not p27KIP1 were detectable by Western blotting in normal and tumor cells. TGF-beta1 treatment increased the association of p21WAF1/Cip1/Sdi1 with the Cdk2/cyclin E complex in both normal and prostate tumor cells. However, there was no increase in the association of p15INK4B nor p27Kip1 with the Cdk/cyclin complexes. In normal cells, the increase in the association of p21WAF1/Cip1/Sdi1. With the Cdk2/cyclin E complex resulted in inhibition of the Cdk2 activity. In contrast, although there was an increase in the association of p21WAF1/Cip1/Sdi1 with the Cdk2/cyclin E complex in tumor cells, there was no inhibition of the Cdk2 activity. These results indicate that a lack of inhibition of the Cdk2 activity correlates with insensitivity to TGF-beta1 in prostate tumor cells.     

Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1

The proliferation and terminal differentiation of granulosa cells are critical for normal follicular growth, ovulation, and luteinization. Therefore, the in situ localization and hormonal regulation of cell cycle activators (cyclin D1, D2, and D3) and cell cycle inhibitors (p27Kip1 and p21Cip1) were analyzed in ovaries of mice and rats at defined stages of follicular growth and differentiation. Cyclin D2 mRNA was specifically localized to granulosa cells of growing follicles, while cyclin D1 and cyclin D3 were restricted to theca cells. In hypophysectomized (H) rats, cyclin D2 mRNA and protein were increased in granulosa cells by treatment with estradiol or FSH and were increased maximally by treatment with both hormones. In serum-free cultures of rat granulosa cells, cyclin D2 mRNA was rapidly elevated in response to FSH, forskolin, and estradiol, indicating that estradiol as well as cAMP can act directly and independently to increase cyclin D2 expression. The levels of p27Kip1 protein were not increased in response to estradiol or FSH. In contrast, when ovulatory doses of human CG (LH) were administered to hormonally primed H rats to stimulate luteinization, cyclin D2 mRNA and protein were rapidly decreased and undetectable within 4 h, specifically in granulosa cells of large follicles. Also in response to LH, the expression of the cell cycle inhibitor p27Kip1 was induced between 12 and 24 h (p21Cip1 was induced within 4 h) and remained elevated specifically in luteal tissue. A critical role for cyclin D2 in the hormone-dependent phase of follicular growth is illustrated by the ovarian follicles of cyclin D2-/- mice, which do not undergo rapid growth in response to hormones, but do express markers of FSH/LH action, cell cycle exit, and terminal differentiation. Collectively, these data indicate that FSH and estradiol regulate granulosa cell proliferation during the development of preovulatory follicles by increasing levels of cyclin D2 relative to p27Kip1 and that LH terminates follicular growth by down-regulating cyclin D2 concurrent with up-regulation of p27Kip1 and p21Cip1.     

Regulation of cyclin-dependent kinase 4 during adipogenesis involves switching of cyclin D subunits and concurrent binding of p18INK4c and p27Kip1

Terminal differentiation of many cell lineages involves an exit from the mitotic cycle and entry into, and maintenance of, a permanent state of G1 arrest. We found that during terminal differentiation of mouse 3T3-L1 preadipocytes, the level of cyclin-dependent kinase 4 (CDK4) remained constant, but the subunit composition of the CDK4 complex underwent a dynamic rearrangement. As 3T3-L1 cells differentiated, the levels of cyclin D1 and cyclin D1-CDK4 complexes declined to negligible levels. Meanwhile, cyclins D2 and D3 levels and their associations with CDK4 increased transiently and persistently, respectively, with cyclin D3 becoming the predominant cyclin partner of CDK4 in mature adipocytes. At least five CDK inhibitors are expressed during the differentiation program of 3T3-L1 cells. Both p15INK4b and p16INK4a continuously declined to undetectable levels immediately after differentiation induction. p21 was transiently expressed during the exit of 3T3-L1 cells from mitotic clonal expansion and then decreased to undetectable levels in mature adipocytes. The level of p27KiP1 and p27-CDK4 complexes remain high during differentiation and in mature adipocytes. Distinctly, there is a remarkable induction of p18INK4c mRNA and protein that was not seen in the closely related nondifferentiating 3T3-C2 cell line, suggesting that p18 induction in 3T3-L1 cells is related to cell differentiation, not cell cycle arrest. The pRb kinase activity of cyclin D3 and CDK4 was not detected in quiescent 3T3-L1 cells and was then induced as the cells entered the mitotic clonal expansion phase. Unexpectedly, cyclin D3 and CDK4 pRb kinase activity remained high after 3T3-L1 cells completed their mitotic division and was still readily detectable in mature adipocytes. Our study reveals an active regulation, rather than passive inhibition, of CDK4 activity during adipocyte differentiation. Two central features of this complex regulation are switching of activating cyclin D subunits and concurrent binding by the p18 and p27 CDK inhibitors.     

TGF-beta 1 induces the cyclin-dependent kinase inhibitor p27Kip1 mRNA and protein in murine B cells

TGF-beta1 inhibits the cell cycle progression of many types of cells by arresting them in the G1 phase. This cell cycle arrest has been attributed to the regulatory effects of TGF-beta1 on both the levels and the activities of the G1 cyclins and their kinase partners. The activities of these kinases are negatively regulated by a number of proteins, such as p15INK4b, p21WAF1/Cip1, and p27Kip1, that physically associate with cyclins, cyclin-dependent kinases (Cdk), or cyclin-Cdk complexes. In epithelial cell lines, TGF-beta1 was previously shown to inhibit cell cycle progression through down-regulation of Cdk4 and/or up-regulation of p15INK4b and/or p21WAF1/Cip1. However, TGF-beta1 had little or no effect on the p27Kip1 mRNA and protein levels. In this report, we show that, in contrast to observations in epithelial cell lines, TGF-beta1 increased the p27Kip1 mRNA and protein levels in the murine B cell lines CH31 and WEHI231. This TGF-beta1-mediated induction of p27Kip1 also resulted in an increased association of p27Kip1 with Cdk2 and a decreased Cdk2 kinase activity. In contrast to epithelial cells, however, TGF-beta1 had little or no effect on the Cdk4 and p21WAF1/Cip1 protein levels in these B cells. Finally, although several studies suggested a direct role of p53 in TGF-beta1-mediated cell cycle arrest in epithelial cells, TGF-beta1 inhibited cell cycle progression in CH31 even in the absence of wild-type p53. Taken together, these results suggest that TGF-beta1 induces G1 arrest in B cells primarily through a p53-independent up-regulation of p27Kip1 protein.     

p27Kip1: a key mediator of retinoic acid induced growth arrest in the SMS-KCNR human neuroblastoma cell line

Retinoic acid (RA) treatment of SMS-KCNR neuroblastoma (NB) cells leads to G1 growth arrest and neuronal differentiation. To investigate the molecular mechanisms by which RA alters cell growth, we analysed the expression and activity of components of the cell cycle machinery after culture in RA. Within 2 days of RA treatment and prior to the arrest of NB cells in the G1 phase of the cell cycle, there is a complete downregulation of G1 cyclin/Cdk activities. Protein levels for the G1 cyclin/Cdks were essentially unchanged during this time although there was a decrease in the steady-state levels of p67N-Myc and hyperphosphorylated Rb proteins. The Cdk inhibitors, p21Cip1 and p27Kip1 were constitutively expressed in KCNR while p15INK4B and p16INK4A were not detected. RA induced an increase in the expression of p27Kip1 but not p21Cip1. Furthermore, coincident with the decrease in kinase activity there was an increase in G1 cyclin/Cdk bound p27Kip1. These results indicate that changes in the level of p27Kip1 and its binding to G1 cyclin/Cdks may play a key role in RA induced growth arrest of NB cells.     

G1 arrest of U937 cells by onconase is associated with suppression of cyclin D3 expression, induction of p16INK4A, p21WAF1/CIP1 and p27KIP and decreased pRb phosphorylation

Onconase is a 12 kDa protein homologous to pancreatic RNase A isolated from amphibian oocytes which shows cytostatic and cytotoxic activity in vitro, inhibits growth of tumors in mice and is in phase III clinical trials. The present study was aimed to reveal mechanisms by which onconase perturbs the cell cycle progression. Human histiocytic lymphoma U937 cells were treated with onconase and expression of cyclins D3 and E, as well as of the cyclin-dependent kinase inhibitors (CKIs) p16INK4A, p21WAF1/CIP1 and p27KIP1 (all detected immunocytochemically) was measured by multiparameter flow cytometry, in relation to the cell cycle position. Also monitored was the status of phosphorylation of retinoblastoma protein (pRb) by a novel method utilizing mAb which specifically detects underphosphorylated pRb in individual cells. Cell incubation with 170 nM onconase for 24 h and longer led to their arrest in G1 which was accompanied by a decrease in expression of cyclin D3, no change in cyclin E, and enhanced expression of all three CKIs. pRb was underphosphorylated in the onconase arrested G1 cells but was phosphorylated in the cells that were still progressing through S and G2/M in the presence of onconase. The cytostatic effect of onconase thus appears to be mediated by downregulation of cyclin D3 combined with upregulation of p27KIP1, p16INK4A and p21WAF1/CIP1, the events which may prevent phosphorylation of pRb during G0/1 and result in cell arrest at the restriction point controlled by Cdk4/6 and D type cyclins.     

TGF-beta mediated G1 arrest in a human melanoma cell line lacking p15INK4B: evidence for cooperation between p21Cip1/WAF1 and p27Kip1

We have studied TGF-beta mediated G1 arrest in WM35, an early stage human melanoma cell line. These cells have lost p15INK4B expression through loss of one chromosome 9 and rearrangement of the other. In asynchronously growing WM35, TGF-beta caused reductions in cyclin D1, cyclin A and cdk4 proteins and their associated kinase activities and an increase in both p21Cip1/WAF1 and p27Kip1. These findings were confirmed in cells released from quiescence in the presence of TGF-beta, in which TGF-beta inhibited or delayed the reduction in the cdk inhibitors that normally occurs in late G1. In contrast to observations in other cell types, there was an increased association of both p21Cip1/WAF1 and p27Kip1 with cyclin D1/cdk4 and with cyclin E/cdk2 during TGF-beta mediated arrest of asynchronously growing cells. Upregulation of p21Cip1/WAF1 preceded that of p27Kip1. Furthermore, p21Cip1/WAF1 and p27Kip1 were not present in the same cdk complexes but bound distinct populations of target cdk molecules. Both p21Cip1/WAF1 and p27Kip1 immunoprecipitates from asynchronously growing cells contained active kinase complexes. These KIP-associated kinase activities were reduced in TGF-beta arrested cells. It has been proposed that in TGF-beta arrested epithelial cells, up-regulation of p15INK4B and of p15INK4B binding to cdk4 serves to destabilize the association of p27Kip1 with cyclin D1/cdk4, promoting p27Kip1 binding and inhibition of cyclin E/cdk2. Our findings demonstrate that this is not a universal mechanism of G1 arrest by TGF-beta. In TGF-beta arrested WM35, which lack p15INK4B, the increased p21Cip1/WAF1 may serve a similar function to that of p15INK4B: initiating kinase inhibition and providing an additional mechanism to supplement the effect of p27Kip1 on G1 cyclin/cdks.     

Anticarcinogenic effect of a flavonoid antioxidant, silymarin, in human breast cancer cells MDA-MB 468: induction of G1 arrest through an increase in Cip1/p21 concomitant with a decrease in kinase activity of cyclin-dependent kinases and associated cyclins

There is an increasing interest in identifying potent cancer preventive and therapeutic agents against breast cancer. Silymarin, a flavonoid antioxidant isolated from milk thistle, exerts exceptionally high to complete anticarcinogenic effects in tumorigenesis models of epithelial origin. In this study, we investigated the anticarcinogenic effect of silymarin and associated molecular mechanisms, using human breast carcinoma cells MDA-MB 468. Silymarin treatment resulted in a significantly high to complete inhibition of both anchorage-dependent and anchorage-independent cell growth in a dose- and time-dependent manner. The inhibitory effects of silymarin on cell growth and proliferation were associated with a G1 arrest in cell cycle progression concomitant with an induction of up to 19-fold in the protein expression of cyclin-dependent kinase (CDK) inhibitor Cip1/p21. Following silymarin treatment of cells, an incremental binding of Cip1/p21 with CDK2 and CDK6 paralleled a significant decrease in CDK2-, CDK6-, cyclin D1-, and cyclin E-associated kinase activity with no change in CDK2 and CDK6 expression but a decrease in G1 cyclins D1 and E. Taken together, these results suggest that silymarin may exert a strong anticarcinogenic effect against breast cancer and that this effect possibly involves an induction of Cip1/p21 by silymarin, which inhibits the threshold kinase activities of CDKs and associated cyclins, leading to a G1 arrest in cell cycle progression.     

Critical role for the 310 helix region of p57(Kip2) in cyclin-dependent kinase 2 inhibition and growth suppression

Although crystal structural analysis of cyclin A/cyclin-dependent kinase 2 (Cdk2)/p27 (Russo, A. A., Jeffrey, P. D., Pattern, A. K., Massague, J., and Pavletich, N. P. (1996) Nature 382, 325-331) has suggested that the 310 helix region in Cdk inhibitors of the Cip/Kip family may be involved in the inhibition of cyclin/Cdk activities, there is no biochemical evidence supporting this hypothesis. In the present study, we demonstrated that cyclin and Cdk binding domains of p57 were necessary but were not sufficient in themselves for the inhibition of cyclin A/Cdk2 and cyclin E/Cdk2, and that the 3(10) helix region of this protein is indispensable for the inhibition of these complexes. In contrast, the 3(10) helix regions of p21 and p27 were not required, and cyclin- and Cdk-binding domains alone were sufficient for the inhibition of all cyclin/Cdk complexes examined. Site-directed mutagenesis identified phenylalanine 79 and tyrosine 80 within the 3(10) helix region of p57 as crucial residues for kinase inhibition, supporting the structural evidence that the 3(10) helix binds deep inside the catalytic cleft of Cdk2, mimicking ATP. Mutations within the 3(10) helix region of the p57 molecule completely abolished the ability to arrest the cell cycle at G1 in vivo. These results indicate that this region is specifically utilized by p57 in selectively inhibiting cyclin A or E/Cdk2+ activities. Thus the 3(10) helix motif may confer a specific regulatory mechanism by which p57 differentially regulates Cdk2 and Cdk4 activities.     

Lack of relationship between CDK activity and G1 cyclin expression in breast cancer cells

The G1 cyclins, cyclin D1 and E, are rate limiting for progression through G1 phase of the cell cycle in breast epithelial cells and are oncogenic when expressed in the mammary epithelium of transgenic mice. These genes are frequently overexpressed in clinical breast cancer where overexpression appears to be associated with specific disease phenotypes, altered responsiveness to therapeutic intervention and patient survival. In order to investigate the functional correlates of cyclin D1 and cyclin E overexpression we employed a panel of normal, immortalized and neoplastic breast epithelial cell lines to examine the relationships between cyclin gene expression, cyclin-CDK complex formation and CDK activity. In agreement with earlier studies cyclin D1 and E expression varied over an approximately tenfold range among the 18 cell lines studied. There was no apparent relationship, however, between cyclin D1 expression and the in vitro activity of its major kinase partner, Cdk4, although MDA-MB-134 cells displayed the highest level of both cyclin D1 expression and Cdk4 activity. Similarly, there was no significant relationship between cyclin E expression and cyclin E-Cdk2 activity. Fractionation of whole cell lysates by gel filtration chromatography revealed that approximately 90% of the cyclin E protein was present in inactive complexes containing the CDK inhibitors p21 and p27. Much of the small fraction of active cyclin E protein was of very high apparent molecular mass, >400 kDa, suggesting that formation of these complexes is a more important determinant of cyclin E-Cdk2 activity than cyclin E abundance. These data suggest that properties of cyclins D1 and E in addition to their ability to activate Cdk4 and Cdk2 may contribute to the effects of overexpression on the breast cancer phenotype.     

Human cytomegalovirus inhibits cellular DNA synthesis and arrests productively infected cells in late G1

Human embryonic lung fibroblasts (LU) can be productively infected with human cytomegalovirus (HCMV). During the course of productive infection, the virus elicits a number of responses that resemble certain aspects of G1 cell cycle progression. The virus activates cyclin E/Cdk2 kinase in both subconfluent, serum-arrested, and density-arrested cultures. Activation of cyclin E-dependent kinase is due, in part, to induction of cyclin E and, in part, to inhibition of the cyclin kinase inhibitors, Cip1 and Kip1. However, G1 progression is incomplete in HCMV-infected cells. Neither cyclin A nor cyclin D is induced, and cellular DNA synthesis does not occur if one takes care to avoid addition of fresh serum to serum-starved cultures. The data indicate that the virus induces a state of late G1 arrest, in which cyclin E/Cdk2 activates nucleotide metabolism and other biosynthetic processes that are necessary for viral replication. Failure to activate host cell DNA synthesis ensures that the virus will have uncompleted access to such precursors.     

Mechanisms of cyclin-dependent kinase inactivation by progestins

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. In breast cancer cells the predominant effect of synthetic progestins is long-term growth inhibition and arrest in G1 phase. Progestin-mediated growth arrest of T-47D breast cancer cells was preceded by inhibition of cyclin D1-Cdk4, cyclin D3-Cdk4, and cyclin E-Cdk2 kinase activities in vitro and reduced phosphorylation of pRB and p107. This was accompanied by decreases in the expression of cyclins D1, D3, and E, decreased abundance of cyclin D1- and cyclin D3-Cdk4 complexes, increased association of the cyclin-dependent kinase (CDK) inhibitor p27 with the remaining Cdk4 complexes, and changes in the molecular masses and compositions of cyclin E complexes. In control cells cyclin E eluted from Superdex 200 as two peaks of approximately 120 and approximately 200 kDa, with the 120-kDa peak displaying greater cyclin E-associated kinase activity. Following progestin treatment, almost all of the cyclin E was in the 200-kDa, low-activity form, which was associated with the CDK inhibitors p21 and p27; this change preceded the inhibition of cell cycle progression. These data suggest preferential formation of this higher-molecular-weight, CDK inhibitor-bound form and a reduced number of cyclin E-Cdk2 complexes as mechanisms for the decreased cyclin E-associated kinase activity following progestin treatment. Ectopic expression of cyclin D1 in progestin-inhibited cells led to the reappearance of the 120-kDa active form of cyclin E-Cdk2 preceding the resumption of cell cycle progression. Thus, decreased cyclin expression and consequent increased CDK inhibitor association are likely to mediate the decreases in CDK activity accompanying progestin-mediated growth inhibition.