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.     

Nuclear accumulation of p21Cip1 at the onset of mitosis: a role at the G2/M-phase transition

Cell cycle arrest in G1 in response to ionizing radiation or senescence is believed to be provoked by inactivation of G1 cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21(Cip1/Waf1/Sdi1). We provide evidence that in addition to exerting negative control of the G1/S phase transition, p21 may play a role at the onset of mitosis. In nontransformed fibroblasts, p21 transiently reaccumulates in the nucleus near the G2/M-phase boundary, concomitant with cyclin B1 nuclear translocation, and associates with a fraction of cyclin A-Cdk and cyclin B1-Cdk complexes. Premitotic nuclear accumulation of cyclin B1 is not detectable in cells with low p21 levels, such as fibroblasts expressing the viral human papillomavirus type 16 E6 oncoprotein, which functionally inactivates p53, or in tumor-derived cells. Moreover, synchronized E6-expressing fibroblasts show accelerated entry into mitosis compared to wild-type cells and exhibit higher cyclin A- and cyclin B1-associated kinase activities. Finally, primary embryonic fibroblasts derived from p21-/- mice have significantly reduced numbers of premitotic cells with nuclear cyclin B1. These data suggest that p21 promotes a transient pause late in G2 that may contribute to the implementation of late cell cycle checkpoint controls.     

Comparative clinical evaluation of the 13C-mixed triglyceride breath test as an indirect pancreatic function test

Three DNA damage-responsive cell cycle checkpoints can be shown to operate in diploid human fibroblasts. One checkpoint arrests growth in G1, another inhibits replicon initiation in S phase cells, and the third delays progression from G2 into mitosis. Progression from G2 into M is controlled in part by a cyclin-dependent kinase (cyclin B/Cdk1) that is regulated by tyrosine phosphorylation. Phosphorylation of Tyr15 on Cdk1 is inhibitory for kinase activity. Activation of cyclin B/Cdk1 at the onset of mitosis is accomplished by a phosphatase, Cdc25C, that interacts with cyclin B/Cdk1 in an autocatalytic feedback loop to remove the inhibitory phosphate at Tyr15 and activate kinase activity. DNA damage triggers G2 delay by inhibiting formation of the autocatalytic feedback loop so that dephosphorylation of Tyr15 does not occur. This suppression of activation of cyclin B/Cdk1 appears to account for the failure of damaged G2 cells to progress into mitosis. Once the damage to DNA is repaired, cells resume progression into mitosis as the cycle is re-engaged. The isoflavone genistein inhibits tyrosine kinases, including one that phosphorylates Cdk1 on Tyr15. This kinase, p56/p53lyn is rapidly induced by treatments that trigger cell cycle checkpoints (ionizing radiation, cytosine arabinoside), suggesting that this kinase may actively delay the onset of mitosis by phosphorylating Tyr15 on Cdk1. Genistein also inhibits type II DNA topoisomerase to produce a form of DNA damage that triggers all of the DNA damage-responsive cell cycle checkpoints. A brief 10 min incubation with the topoisomerase poison amsacrine was sufficient to trigger the S phase checkpoint response and inhibit replicon initiation. Inhibition of replicon initiation by 1 microM amsacrine was maximal 20-30 min after drug treatment and by 120 min, the checkpoint response had decayed to allow near control rates of replicon initiation. Topoisomerase II poisons also are powerful clastogens inducing lethal and carcinogenic chromosomal aberrations. Type II topoisomerase can break DNA in a region of chromosome 11q23 that contains the ataxia telangiectasia gene (ATM). The ATM gene controls all of the DNA damage-responsive cell cycle checkpoints. Chromosomal aberrations in 11q23 are frequently seen in acute myeloid leukemia that develops as a consequence of etoposide chemotherapy. Thus, topoisomerase poisons such as genistein may trigger chromatid breakage to inactivate AT gene function, disable cell cycle control, and induce genetic instability.     

Cloning and characterization of the Xenopus cyclin-dependent kinase inhibitor p27XIC1

We have isolated a gene encoding Xic-1, a 27-kDa cyclin-dependent kinase (Cdk) inhibitor from Xenopus ovary that shares significant homology with both mammalian CIP1 and Kip1/Kip2. The N- and C-terminal halves of Xic-1 are sufficient for interacting with Cdks and proliferating cell nuclear antigen, respectively. Recombinant Xic-1 inhibits Xenopus cyclin E/Cdk2, cyclin A/Cdk2 and cyclin B/Cdc2 activities, although with quite different IC50 values. Truncation of the N terminus of Xic-1 increases the IC50 value for cyclin A/Cdk2 50-fold with no effect on the inhibition of cyclin E/Cdk2 or cyclin B/Cdc2.Xic-1 inhibits both single-stranded and nuclear DNA synthesis in egg extracts, an effect reversed by proliferating cell nuclear antigen or cyclin E/Cdk2, respectively. These results suggest a function for Xic-1 in the control of DNA synthesis by cyclin E/Cdk2.     

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.     

Cdk2 activity is dispensable for the onset of DNA replication during the first mitotic cycles of the sea urchin early embryo

Earlier work reported the important role of Cdk2 as a regulator of DNA replication in somatic cells and in Xenopus extracts. In the present report we analyze in vivo the involvement of Cdk2 in DNA replication during early embryogenesis using the first mitotic cycles of sea urchin embryos. Unfertilized Sphaerechinus granularis eggs are arrested after the second meiotic cytokinesis. Fertilization resumes the block and induces DNA replication after a short lag period, making sea urchin early embryo a good model for studying in vivo the onset of DNA replication. We show that Cdk2 as well as its potential partner cyclin A are present in the nucleus in G1 and S phase and therefore available for DNA replication. In accordance with data obtained in Xenopus egg extracts we observed that Cdk2 kinase activity is low and stable during the entire cycle. However, in contrast with this in vitro system in which Cdk2 activity is required for the onset of DNA replication, the specific inhibition of Cdk2 kinase by microinjection of the catalytically inactive Cdk2-K33R or the inhibitor p21(Cip1) does not prevent DNA replication. Because olomoucine, DMAP, and emetine treatments did not preclude DNA synthesis, neither cyclin A/Cdk1 nor cyclin B/Cdk1 kinase activities are necessary to replace the absence of Cdk2 kinase in promoting DNA replication. These data suggest that during early embryogenesis Cdks activities, in particular Cdk2, are dispensable in vivo for the initiation step of DNA replication. However, the specific localization of Cdk2 in the nucleus from the beginning of M phase to the end of S phase suggests its involvement in other mechanisms regulating DNA replication such as inhibition of DNA re-replication and/or that its regulating role is achieved through a pathway independent of the kinase activity. We further demonstrate that even after inhibition of Cdk activities, the permeabilization of the nuclear membrane is required to allow a second round of DNA replication. However, in contrast to Xenopus egg extracts, re-replication can take place in the absence of DMAP-sensitive kinase.     

Effect of septal perforations on measures of nasal resistance

It has been shown that p53- human colorectal cancer cells arrest after DNA damage in a G2-like state and may then undergo DNA synthesis without intervening mitosis (Waldman et al., Nature 381, 713-716, 1996). To further clarify the role of p53 in the regulation of the G2/M-phase checkpoint, we have studied cells of three closely related human lymphoblastoid cell lines (TK6, WTK1 and TK6E6, an HPV16 E6-transfected TK6 line) with differing p53 status. The cells were irradiated with 1.5-12 Gy gamma rays with or without 2 mM caffeine. There was no evidence of uncoupling of DNA synthesis and mitosis after irradiation in the p53- cell lines, WTK1 and TK6E6, suggesting that this uncoupling may not be a universal phenomenon. The apparent formation of tetraploid cells after irradiation of cells of the p53- WTK1 line was due to the occurrence of a G2-phase block in a pre-existing tetraploid population. These results support the conclusion that control of the G2/M-phase checkpoint after irradiation may differ among different cell types.     

The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases

The cyclin-dependent kinase Cdk2 associates with cyclins A, D, and E and has been implicated in the control of the G1 to S phase transition in mammals. To identify potential Cdk2 regulators, we have employed an improved two-hybrid system to isolate human genes encoding Cdk-interacting proteins (Cips). CIP1 encodes a novel 21 kd protein that is found in cyclin A, cyclin D1, cyclin E, and Cdk2 immunoprecipitates. p21CIP1 is a potent, tight-binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by cyclin A-Cdk2, cyclin E-Cdk2, cyclin D1-Cdk4, and cyclin D2-Cdk4 complexes. Cotransfection experiments indicate that CIP1 and SV40 T antigen function in a mutually antagonistic manner to control cell cycle progression.     

Identification of a new coiled body component

The cyclin-dependent kinase (Cdk) inhibitor p27 interrupts progression of the cell cycle by inhibiting various cyclin/Cdk activities. Since the protein level of p27 does not correlate with its mRNA level or protein synthesis rate in most cases, it is suggested that degradation of the protein may be regulated via an unidentified mechanism(s) involving a post-translational modification(s). We present evidence here that p27 phosphorylation is cell cycle-dependent and peaks in the late G1 phase and that the level of p27 protein is inversely correlated with its phosphorylation. Although both cyclin D1- and cyclin-E-dependent kinases are active in the late G1 phase in human fibroblasts, cyclin E/Cdk2 specifically phosphorylates p27 on threonine-187 in vitro. Interestingly, ectopic expression of T187A revealed that it was far more stable in vivo than wild type p27. Thus, phosphorylation of p27 by cyclin E/ Cdk2 may affect the stability of its protein and play a role in how the protein functions.     

The risk of persistent paresthesia is not increased with repeated axillary block

We have identified the human papillomavirus (HPV) DNA replication initiation protein E1 as a tight-binding substrate of cyclin E/cyclin-dependent kinase (Cdk) complexes by using expression cloning. E1, a DNA helicase, collaborates with the HPV E2 protein in ori-dependent replication. E1 formed complexes with cyclin E in insect and mammalian cells, independent of Cdks and E2. Additional cyclins, including A-, B-, and F-type (but not D-type), interacted with the E1/E2 complex, and A- and E-type cyclin kinases were capable of phosphorylating E1 and E2 in vitro. Association with cyclins and efficient phosphorylation of E1 required the presence of a cyclin interaction motif (the RXL motif). E1 lacking the RXL motif displayed defects in E2-dependent HPV ori replication in vivo. Consistent with a role for Cdk-mediated phosphorylation in E1 function, an E1 protein lacking all four candidate Cdk phosphorylation sites still associated with E2 and cyclin E but was impaired in HPV replication in vitro and in vivo. Our data reveal a link between cyclin/Cdk function and activation of HPV DNA replication through targeting of Cdk complexes to the E1 replication-initiation protein and suggest a functional role for E1 phosphorylation by Cdks. The use of cyclin-binding RXL motifs is now emerging as a major mechanism by which cyclins are targeted to key substrates.     

Role of cyclin E and cyclin E-dependent kinase in mitogenic stimulation by cementum-derived growth factor in human fibroblasts

Cementum-derived growth factor (CGF) is a 14 kDa polypeptide sequestered in tooth cementum. It is an IGF-I like molecule that is weakly mitogenic to fibroblasts, but its mitogenic action is synergistically potentiated in the presence of epidermal growth factor (EGF) or serum. We have examined whether the CGF affects cyclin E levels and the activity of cyclin-dependent kinase (Cdk) associated with this cyclin, and whether these changes contribute to the synergism in mitogenic activity between CGF and EGF. Optimal DNA synthesis by serum-starved human gingival fibroblasts required the presence of CGF for 0-12 h and EGF for 0-3 h. Therefore, cells were serum starved for 48 h and then exposed to CGF, EGF, or CGF + EGF. Cells incubated with 10% fetal bovine serum (FBS) served as positive controls. At various time points after the addition of growth factors, cyclin E levels were examined by Western analysis. Cdk associated with cyclin E was immunoprecipitated with anti-cyclin E antibody and kinase activity was measured using H1 histone as substrate. Cyclin E and the H1 kinase activity levels increased after 8-12 h in cells exposed to CGF and in positive controls exposed to 10% FBS. They returned to basal level 4 h later in cells exposed to CGF alone, whereas in the presence of CGF + EGF and FBS they remained elevated for up to 20 h. The cyclin E levels did not increase in the presence of EGF alone. Cyclin-dependent kinase inhibitors p21cip1 and p27kip1 were barely detectable in these cells. Fibroblasts transfected with LXSN-cyclin E, a retroviral vector containing cyclin E cDNA, overexpressed cyclin E and their steady-state cyclin E-Cdk activity was higher than control cells. DNA synthesis by cyclin E overexpressing cells was higher, but optimal DNA synthesis by these cells required the presence of CGF and EGF. These results show that CGF action involves an increase in the levels of cyclin E and E-Cdk activity and that the higher levels are maintained in the presence of both CGF and EGF. They also indicate that sustained high cyclin E levels and Cdk2 activity during G1 phase are necessary, but not sufficient, for optimal mitogenic response in human fibroblasts.     

Negative regulation of Wee1 expression and Cdc2 phosphorylation during p53-mediated growth arrest and apoptosis

The G2 cell cycle checkpoint protects cells from potentially lethal mitotic entry after DNA damage. This checkpoint involves inhibitory phosphorylation of Cdc2 at the tyrosine-15 (Y15) position, mediated in part by the Wee1 protein kinase. Recent evidence suggests that p53 may accelerate mitotic entry after DNA damage and that the override of the G2 checkpoint may play a role in the induction of apoptosis by p53. To determine the biochemical mechanism by which p53 inactivates the G2 checkpoint, the effects of p53 activation on Wee1 expression, Cdc2-Y15 phosphorylation, and cyclin B1-associated Cdc2 kinase activity were examined. Under conditions of either growth arrest or apoptosis, p53 activation resulted in the down-regulation of Wee1 expression and dephosphorylation of Cdc2. A parallel increase in cyclin B1/Cdc2 kinase activity was observed during p53-mediated apoptosis. Negative regulation of the Wee1 expression and Cdc2 phosphorylation by p53 was also evident in thymus tissue from p53+/+ mice but not from p53-/- mice. Inactivation of the G2 checkpoint may contribute to the tumor suppressor activity of p53.     

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.     

Cyclin E2, a novel human G1 cyclin and activating partner of CDK2 and CDK3, is induced by viral oncoproteins

G1 cyclin E controls the initiation of DNA synthesis by activating CDK2, and abnormally high levels of cyclin E expression have frequently been observed in human cancers. We have isolated a novel human cyclin, cyclin E2, that contains significant homology to cyclin E. Cyclin E2 specifically interacts with CDK inhibitors of the CIP/KIP family and activates both CDK2 and CDK3. The expression of cyclin E2 mRNA oscillates periodically throughout the cell cycle, peaking at the G1/S transition, and exhibits a pattern of tissue specificity distinct from that of cyclin E1. Cyclin E2 encodes a short lived protein whose turnover is most likely governed by the proteasome pathway and is regulated by phosphorylation on a conserved Thr-392 residue. Expression of the viral E6 oncoprotein in normal human fibroblasts increases the steady state level of cyclin E2, but not cyclin E1, while expression of the E7 oncoprotein upregulates both. These data suggest that the expression of these two G1 E-type cyclins may be similarly regulated by the pRb function, but distinctly by the p53 activity.     

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.     

Activation of cyclin-dependent kinase 2 (Cdk2) in growth-stimulated rat astrocytes. Geranylgeranylated Rho small GTPase(s) are essential for the induction of cyclin E gene expression

The role of the mevalonate cascade in the control of cell cycle progression in astrocytes has been investigated. Serum stimulation of rat astrocytes in primary culture induces the expression of cyclin E followed by the activation of cyclin-dependent kinase 2 (Cdk2) during G1/S transition. The expression of p27, cyclin D1, and the activities of Cdk4 and Cdk-activating kinase (CAK), composed of Cdk7 and cyclin H, were not affected. Serum did, however, stimulate the expression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA at mid-G1 phase. Moreover, an inhibitor of HMG-CoA reductase, pravastatin, reduced cyclin E expression and Cdk2 activation and caused G1 arrest in the astrocytes. In contrast, mevalonate and its metabolite, geranylgeranylpyrophosphate (GGPP) but not farnesylpyrophosphate (FPP), reversed the inhibitory effects of pravastatin on cyclin E expression and Cdk2 activation and allowed G1/S transition. Rho small GTPase(s) were geranylgeranylated and translocated to membranes in the presence of GGPP during G1/S transition. The effect of GGPP on cyclin E expression was abolished by botulinum C3 exoenzyme, which specifically inactivates Rho. These data indicate that geranylgeranylated Rho small GTPase(s) are essential for the induction of cyclin E expression, Cdk2 activation, and G1/S transition in rat astrocytes.     

Requirement of cyclin E-Cdk2 inhibition in p16(INK4a)-mediated growth suppression

Loss-of-function mutations of p16(INK4a) have been identified in a large number of human tumors. An established biochemical function of p16 is its ability to specifically inhibit cyclin D-dependent kinases in vitro, and this inhibition is believed to be the cause of the p16-mediated G1 cell cycle arrest after reintroduction of p16 into p16-deficient tumor cells. However, a mutant of Cdk4, Cdk4(N158), designed to specifically inhibit cyclin D-dependent kinases through dominant negative interference, was unable to arrest the cell cycle of the same cells (S. van den Heuvel and E. Harlow, Science 262:2050-2054, 1993). In this study, we determined functional differences between p16 and Cdk4(N158). We show that p16 and Cdk4(N158) inhibit the kinase activity of cellular cyclin D1 complexes through different mechanisms. p16 dissociated cyclin D1-Cdk4 complexes with the release of bound p27(KIP1), while Cdk4(N158) formed complexes with cyclin D1 and p27. In cells induced to overexpress p16, a higher portion of cellular p27 formed complexes with cyclin E-Cdk2, and Cdk2-associated kinase activities were correspondingly inhibited. Cells engineered to express moderately elevated levels of cyclin E became resistant to p16-mediated growth suppression. These results demonstrate that inhibition of cyclin D-dependent kinase activity may not be sufficient to cause G1 arrest in actively proliferating tumor cells. Inhibition of cyclin E-dependent kinases is required in p16-mediated growth suppression.     

Herpes viral cyclin/Cdk6 complexes evade inhibition by CDK inhibitor proteins

The passage of mammalian cells through the restriction point into the S phase of the cell cycle is regulated by the activities of Cdk4 and Cdk6 complexed with the D-type cyclins and by cyclin E/Cdk2. The activities of these holoenzymes are constrained by CDK inhibitory proteins. The importance of the restriction point is illustrated by its deregulation in many tumour cells and upon infection with DNA tumour viruses. Here we describe the properties of cyclins encoded by two herpesviruses, herpesvirus saimiri (HVS) which can transform blood lymphocytes and induce malignancies of lymphoid origin in New World primates, and human herpesvirus 8 (HHV8) implicated as a causative agent of Kaposi's sarcoma and body cavity lymphomas. Both viral cyclins form active kinase complexes with Cdk6 that are resistant to inhibition by the CDK inhibitors p16(Ink4a), p21Cip1 and p27Kip1. Furthermore, ectopic expression of a viral cyclin prevents G1 arrest imposed by each inhibitor and stimulates cell-cycle progression in quiescent fibroblasts. These results suggest a new mechanism for deregulation of the cell cycle and indicate that the viral cyclins may contribute to the oncogenic nature of these viruses.     

Molecular characterization of seven Chinese isolates of infectious bursal disease virus: classical, very virulent, and variant strains

We have previously reported that certain tyrphostins which block EGF-R phosphorylation in cell-free systems fail to do so in intact cells. Nevertheless, we found that this family of tyrphostins inhibits both EGF- and calf serum-induced cell growth and DNA synthesis [Osherov, N.A., Gazit, C., Gilon, and Levitzki, A. (1993). Selective inhibition of the EGF and HER2/Neu receptors by Tyrphostins. J. Biol. Chem. 268, 11134-11142.] Now we show that these tyrphostins exert their inhibitory activity even when added at a time when the cells have already passed their restriction point and receptor activation is no longer necessary. AG555 and AG556 arrest 85% of the cells at late G1, whereas AG490 and AG494 cause cells to arrest at late G1 and during S phase. No arrest occurs during G2 or M phase. Further analysis revealed that these tyrphostins act by inhibiting the activation of the enzyme Cdk2 without affecting its levels or its intrinsic kinase activity. Furthermore, they do not alter the association of Cdk2 to cyclin E or cyclin A or to the inhibitory proteins p21 and p27. These compounds also have no effect on the activating phosphorylation of Cdk2 by Cdk2 activating kinase (CAK) and no effect on the catalytic domain of cdc25 phosphatase. These compounds lead to the accumulation of phosphorylated Cdk2 on tyrosine 15 which is most probably the cause for its inhibition leading to cell cycle arrest at G1/S. A structure-activity relationship study defines a very precise pharmacophore, suggesting a unique molecular target not yet identified and which is most probably involved in the regulation of the tyrosine-phosphorylated state of Cdk2. These compounds represent a new class of cell proliferation blockers whose target is Cdk2 activation.