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207 records · Page 12Linked to original sources
PubMed2006

Replication-dependent DNA damage response triggered by roscovitine induces an uncoupling of DNA replication proteins.

The cyclin-dependent kinase (CDK) inhibitor roscovitine is under evaluation in clinical trials for its antiproliferative properties. Roscovitine arrests cell cycle progression in G(1) and in G(2) phase by inhibiting CDK2 and CDK1, and possibly CDK7 and CDK9. However, the effects of CDK2 inhibition in S-phase cells have been not fully investigated. Here, we show that a short-term treatment with roscovitine is sufficient to inhibit DNA synthesis, and to activate a DNA damage checkpoint response, as indicated by phosphorylation of p53-Ser15, replication protein A, and histone H2AX. Analysis of DNA replication proteins loaded onto DNA during S phase showed that the amount of proliferating cell nuclear antigen (PCNA), a cofactor of DNA replication enzymes, was significantly reduced by roscovitine. In contrast, chromatin-bound levels of DNA polymerase delta, DNA ligase I and CDK2, were stabilized. Checkpoint inhibition with caffeine could rescue PCNA disassembly only partially, pointing to additional effects due to CDK2 inhibition and the presence of replication stress. These results suggest that in S-phase cells, roscovitine induces checkpoint-dependent and -independent effects, leading to stabilization of replication forks and an uncoupling between PCNA and PCNA-interacting proteins.

Cell Proliferation↗
PubMed2000

The disappearance of cyclins A and B and the increase in activity of the G(2)/M-phase cellular kinase cdc2 in herpes simplex virus 1-infected cells require expression of the alpha22/U(S)1.5 and U(L)13 viral genes.

In uninfected cells the G(2)/M transition is regulated by cyclin kinase complex containing cdc2 and, initially, cyclin A, followed by cyclin B. cdc2 is downregulated through phosphorylation by wee-1 and myt-1 and upregulated by cdc-25C phosphatase. We have examined the accumulation and activities of these proteins in cells infected with wild type and mutants of herpes simplex virus 1. The results were as follows. (i) Cyclin A and B levels were reduced beginning 4 h after infection and were undetectable at 12 to 16 h after infection. (ii) cdc2 protein also decreased in amount but was detectable at all times after infection. In addition, a fraction of cdc2 protein from infected cells exhibited altered electrophoretic mobility in denaturing gels. (iii) The levels of cdk7 or myt-1 proteins remained relatively constant throughout infection, whereas the level of wee-1 was significantly decreased. (iv) cdc-25C formed novel bands characterized by slower electrophoretic mobility that disappeared after treatment with phosphatase. In addition, one phosphatase-sensitive band reacted with MPM-2 antibody that recognizes a phosphoepitope phosphorylated exclusively in M phase. (v) cdc2 accumulating in infected cells exhibited kinase activity. The activity of cdc2 was higher in infected cell lysates than those of corresponding proteins present in lysates of mock-infected cells even though cyclins A and B were not detectable in lysates of infected cells. (vi) The decrease in the levels of cyclins A and B, the increase in activity of cdc2, and the hyperphosphorylation of cdc-25C were mediated by U(L)13 and alpha22/U(S)1.5 gene products. In light of its normal functions, the activated cdc2 kinase may play a role in the changes in the morphology of the infected cell. These results are consistent with the accruing evidence that herpes simplex virus scavenges the cell for useful cell cycle proteins and subverts them for its own use.

CDC2 Protein Kinase↗
PubMed2000

Screening a human population sample for DNA repair gene deficiencies utilizing the protein truncation test.

A significant fraction of human cancers are thought to have a genetic component and several lines of evidence suggest that deficiencies in DNA repair may be a contributing factor. Little is known, however, about the frequency and distribution of variants of DNA repair genes in the general human population. The protein truncation test (PTT) was used to screen 136 healthy volunteers for protein-truncating variants of 10 DNA repair genes: APE, CDK7, ERCC1, WAF1, HOGG1, MGMT, POLB, UNG, HAAG, and CCNH. This sample consisted of 41males (30%) and 95 females (70%) with an average age of 25.3 years, ranging from 17 to 60 years of age. No truncating mutations were found in the 10 genes examined in any of the subjects. The 95% confidence interval for a proportion of 0 over the 272 alleles examined per locus is 0-0.01. The calculated frequency of truncating mutations in each of these genes, among the general population, is thus less than 1%. Among the 10 genes tested in 136 people, a single sample had no PCR product for HAAG, even though PCR products were obtained on all other genes. Total RNA dot hybridization confirmed the presence of HAAG mRNA transcripts in this sample. Despite identification of this single DNA repair variant, these results indicate a low frequency of truncating mutations in DNA repair genes in the general population.

Base Sequence↗
PubMedDate not supplied

Cyclins that don't cycle--cyclin T/cyclin-dependent kinase-9 determines cardiac muscle cell size.

A subset of cyclin-dependent protein kinases--Cdk7, Cdk8, and Cdk9--participates directly, in complex ways, with the fundamental machinery for gene transcription, as elements of general transcription factors whose substrate is the C-terminal domain (CTD) of RNA polymerase II. Here, we review recent data implicating the CTD kinase Cdk9 as a critical determinant of cardiac hypertrophy, in vitro and in vivo. Diverse trophic signals that increase cardiac mass all activated Cdk9 (work load, the small G-protein Gaq, and the calcium-dependent phosphatase calcineurin in mouse myocardium; endothelin-1, a hypertrophic agonist, in cultured cardiomyocytes). Little or no change occurred in levels of the kinase or its activator, cyclin T. Instead, in all four hypertrophic models, Cdk9 activation involves the dissociation of 7SK small nuclear RNA (snRNA), an endogenous inhibitor. In culture, dominant-negative Cdk9 blocked ET-1-induced hypertrophy, whereas an anti-sense "knockdown" of 7SK snRNA provoked spontaneous cell growth. In trans-genie mice, concordant with these results, activation of Cdk9 activity via cardiac-specific overexpression of cyclin Tl suffices to provoke hypertrophy. Together, these findings implicate Cdk9 activity as a pivotal regulator of pathophysiological heart growth. Because hypertrophy, in turn, is a cardinal risk factor for developing cardiac pump failure, these results support the logic of examining Cdk9 as a potential drug target in heart disease.

Animals↗
PubMed2003

[Analysis of transient G1/S arrest in E1A+E1B-19kDa transformed cells after ionizing radiation].

Expression of human adenovirus type 5 E1A oncogene in normal rodent cells leads to disruption of the G1/S cell cycle arrest realization in response to DNA damage. It has been shown here that rat embryo fibroblasts transformed by E1Aad5 oncogene in complementation with E1B-19 kDa gene realize the irradiation-induced transient G1/S arrest, which depends on selective suppression of CyclinE-Cdk2 activity despite functional inactivation of p21Waf1 inhibitor. Inhibitor p21Waf1 is not revealed in complexes with cyclins E and A in E1A + E1B-19 kDa transformants, however, it is not due to p21Waf1 interaction with E1A oncoproteins, because the E1A-p21Waf1 complex formation in E1A + cHa-ras transformants does not prevent the high level of CycIE, A-p21Waf1 association. In the case of p21Waf1 inactivation, the main way of cyclin-kinase activity regulation in E1A + E1B-19 kDa cells may be Cdk2 phosphorylation. However, irradiation of E1A + E1B-19 kDa transformed cells induces no changes in CAK (Cdk7-associated) kinase activity and in the protein level of Cdc25A phosphatase, which are responsible for activating Thr160 phosphoralation and Tyr15 dephosphorylation on Cdk2. Using phospho-Tyr15-Cdk2 specific antibodies, no increase of phosphorylation at Tyr15 position on immunoprecipitated Cdk2 was detected after irradiation. It seems likely that in the case of inactivated inhibitor p21Waf1 the transient G1/S block after irradiation in E1A + E1B-19 kDa transformants depends on suppression of Cycl-E-Cdk2 activity caused by inhibition of Thr160 Cdk2 phosphorylation, but his occurs with the involvement of other kinases rather than CAK.

Adenovirus E1A Proteins↗
PubMed1995

[Cyclin dependent kinases. From molecular biology to pathology].

Cyclin-dependent kinases (cdks) is a family of serine-threonine kinases whose principal role is the promotion of the cell transition through the regulatory points of the cell cycle (G1 and G2/M). The best known human cdks are: cdk1-cdk7 and p58-GTA. The latter one, contrarily to the other cdks, is supposed to act as a antiproliferative factor. Most cdks may be involved in the development of neoplastic disorders. This hypothesis is based on their biological features (interactions with viral oncoproteins), their hyperexpression in some malignancies and frequent deletions of cdk inhibitory genes in cancer cells. cdk5, which displays the maximal kinase activity in non-proliferating brain neurons may participate in the pathogeny of the Alzheimer's disease.

Alzheimer Disease↗
PubMed1996

Human glioblastomas with no alterations of the CDKN2A (p16INK4A, MTS1) and CDK4 genes have frequent mutations of the retinoblastoma gene.

A series of 195 human gliomas were studied as to the status of their CDKN2A, CDK4 and RB1 genes. Among 120 glioblastomas, 40% had no wild-type CDKN2A gene, 12% amplified the CDK4 gene, and 14% had no wild-type RBI gene. With two exceptions, each tumour had only one of these abnormalities. Thus the majority of the glioblastomas (64%) had distinct genetic aberrations which would obviously disrupt the control of transition from G1 to the S-phase of the cell cycle. A further 30% had loss of one allele of the CDKN2A and/or RBI genes. Only seven (6%) glioblastomas had no abnormalities of these genes. Anaplastic astrocytomas showed similar changes to the glioblastomas but at lower frequencies-34% showing no aberrations of the genes analysed. The astrocytomas showed solely loss of one allele of the RBI gene in 28% of tumours, with retention of one wild-type copy. In the glioblastomas with no alterations of CDKN2A, CDK4 or RB1, several other genes (CCND1, CCND2, CCND3, CDK6, E2F, CDK7, MYC and MYCN) whose products take part in cell cycle regulation were examined. No abnormalities were detected. Thus some aberration of the CDKN2A, CDK4 and RB1 genes appears to be almost obligatory in glioblastomas.

Astrocytoma↗
PubMed1997

Phosphorylation of the carboxy-terminal repeat domain in RNA polymerase II by cyclin-dependent kinases is sufficient to inhibit transcription.

Cdc2 kinase triggers the entry of mammalian cells into mitosis, the only cell cycle phase in which transcription is globally repressed. We show here that Cdc2 kinase phosphorylates components of the RNA polymerase II transcription machinery including the RNA polymerase II carboxy-terminal repeat domain (CTD). To test specifically the effect of CTD phosphorylation by Cdc2 kinase, we used a yeast in vitro transcription extract that is dependent on exogenous RNA polymerase II that contains a CTD. Phosphorylation was carried out using immobilized Cdc2 so that the kinase could be removed from the phosphorylated polymerase. ATP gamma S and Cdc2 kinase were used to produce an RNA polymerase IIO that was not detectably dephosphorylated in the transcription extract. RNA polymerase IIO produced in this way was defective in promoter-dependent transcription, suggesting that phosphorylation of the CTD by Cdc2 kinase can mediate transcription repression during mitosis. In addition, we show that phosphorylation of pol II with the human TFIIH-associated kinase Cdk7 also decreases transcription activity despite a different pattern of CTD phosphorylation by this kinase. These results extend previous findings that RNA polymerase IIO is defective in preinitiation complex formation. Here we demonstrate that phosphorylation of the CTD by cyclin-dependent kinases with different phosphoryl acceptor specificities can inhibit transcription in a CTD-dependent transcription system.

Cyclin-Dependent Kinases↗
PubMed1997

Contact inhibition-induced inactivation of the cyclin D-dependent kinase in rat fibroblast cell line, 3Y1.

Inhibitory proteins for Cdks (CKIs) are involved in cell cycle arrest induced by anti-mitotic factors, chemicals, or DNA damage in mammalian cells. High cell density also induces cell cycle arrest with unreplicated genomic DNA even in the presence of mitotic dose of the growth factors, termed contact inhibition. Although rat fibroblast cell line, 3Y1, arrested in quiescence by contact inhibition, Cdk4 bound its regulatory subunit, cyclin D1 or D3. However, these complexes were enzymatically inactive. Phosphorylation of the cyclin D1-bound Cdk4 by Cdk4-activating kinase, composed of cyclin H and MO15 (alias Cdk7), which was reconstituted in Spodoptera frugiperda cells (Sf9) could convert inactive cyclin D1-Cdk4 complex into active form in vitro, suggesting that threonine 172 in the Cdk4, whose phosphorylation is required for its activation, was in part unphosphorylated in the contact-inhibited 3Y1. Although MO15 was active in cell extracts prepared from the contact-inhibited 3Y1, activation of bacterially produced Cdk4 in the cell extracts was inhibited. Removing p27kip1 from the cell extracts allowed MO15 holoenzyme to phosphorylate the Cdk4 and to activate it, indicating that an access of MO15 to Cdk4 was inhibited by p27kip1 in the contact-inhibited 3Y1.

Animals↗