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At least 127 records · Page 7Linked to original sources
PubMed1995

Concurrent amplification of cyclin E and CDK2 genes in colorectal carcinomas.

The genetic changes of cyclin A, DI, E and CDK2 were examined in human colorectal carcinomas by Southern-blot analysis. Gene amplification of cyclin E was detected in 5 of 53 (9.4%) primary colorectal carcinoma tissues. Interestingly, in 3 of 5 tumors showing cyclin E gene amplification, the CDK2 gene was amplified simultaneously with rearrangements. No obvious correlation was detected between gene amplification and clinicopathological features of colorectal carcinomas. Out of 7 colon carcinoma cell lines, 2 showed gene amplification of cyclin E without gene amplification of CDK2. No amplification of cyclin A or DI gene was found in any of the colorectal carcinoma tissues or colon carcinoma cell lines. Our results suggest that the concurrent amplification of cyclin E and CDK2 genes may play a role in colorectal carcinogenesis.

Aged↗
PubMed1995

Crystallization and preliminary crystallographic study of human CksHs1: a cell cycle regulatory protein.

The cell cycle regulatory protein CksHs1 has been crystallized in a form suitable for X-ray studies. CksHs1 crystals were grown in the presence of vanadate, a phosphatase inhibitor, but were also obtained with phosphate or tungstate as a cofactor. They belong to the hexagonal space group P6(1)22 with unit cell dimensions: a = b = 94 A, c = 131.6 A, and gamma = 120 degrees. The crystals grown in the presence of vanadate diffract X-rays to at least 2.8 A. Molecular replacement results from the homologous human CksHs2 structure reveal that a dimer forms the crystal habit, giving the unusual Vm value of 4.4 A3/Da or a solvent content of 72%.

CDC2-CDC28 Kinases↗
PubMed1995

Overexpression of cyclin D1 blocks proliferation of normal diploid fibroblasts.

Human cyclin D1 forms complexes with several Cdks, with proliferating cell nuclear antigen, and with a recently discovered 21-kDa inhibitor of Cdk activity. Substrates for cyclin D1/Cdks have not been identified in vivo, but it has been proposed that the D class of cyclins might play a role in regulating the activity of the retinoblastoma gene product p105Rb. Here we report that normal human diploid fibroblasts that endogenously or ectopically express high levels of cyclin D1 are unable to enter S phase in response to normally mitogenic stimuli. Fibroblasts that have reached the end of their in vitro life span (senescent cells) express five-fold higher levels of cyclin D1 protein than low-passage cells and individual cells in mass culture that fail to initiate DNA synthesis in response to serum addition have severalfold higher levels of this cyclin than proliferation-competent cells. These observations provide evidence that cyclin D1 is involved with the regulation of cell proliferation by more than one mechanism.

Base Sequence↗
PubMed1995

Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells.

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication initiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase alpha as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase alpha are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.

Base Sequence↗
PubMed1995

Association of Cdk-activating kinase subunits with transcription factor TFIIH.

The RNA polymerase II large subunit contains an essential carboxy-terminal domain (CTD) believed to be involved in the response to regulators during transcription initiation. The CTD is phosphorylated on a portion of RNA polymerase II molecules in vivo and it can be phosphorylated by the general transcription factor TFIIH in vitro. A highly purified TFIIH from rat liver has been described; this, like human and yeast TFIIH, contains associated CTD kinase and helicase activities. We report here that two polypeptides of the purified mammalian TFIIH are the MO15/Cdk7 kinase and cyclin H subunits of the Cdk-activating kinase Cak, previously identified as a positive regulator of Cdc2 and Cdk2. TFIIH and Cak preparations are each capable of phosphorylating recombinant CTD and recombinant Cdk2 proteins. The presence of Cak in TFIIH indicates that Cak may have roles in transcriptional regulation and in cell-cycle control.

Animals↗
PubMed1992

Human cyclin-dependent kinase 2 is activated during the S and G2 phases of the cell cycle and associates with cyclin A.

We have analyzed the cell cycle regulation of human cyclin-dependent kinase 2 (CDK2), a protein closely related to the cell cycle-regulatory protein kinase CDC2. We find that CDK2 activity, like that of CDC2, oscillates during the cell cycle in cultured mammalian fibroblasts. Unlike CDC2 activity (which peaks during mitosis), CDK2 activity rises in late G1 or early S phase and declines during mitosis. Active S-phase CDK2 migrates in multiple large complexes on gel filtration, and CDK2 in one of these complexes is associated with cyclin A. These findings suggest that CDK2 and CDC2, in association with distinct cyclins, regulate separate functions in the mammalian cell cycle.

Amino Acid Sequence↗
PubMed1995

Deregulated expression of cyclin D1 and other cell cycle-related genes in carcinogen-induced rat mammary tumors.

Dysregulation of cyclin expression has been reported for several human malignancies, including breast cancer. To further investigate the role of cyclin genes in mammary tumorigenesis we analyzed the expression of cyclins D1, E and A and other cell cycle-related proteins in a series of nine N-methyl-N-nitrosourea-induced primary rat mammary tumors. Western blot analysis revealed a 10- to 15-fold increase in the level of cyclin D1 protein in most (7/9) of the tumors, when compared with normal rat mammary gland. The two tumors that did not show this increase also displayed negligible levels of the retinoblastoma protein. A moderate increase, 1.5- to 2-fold, in the level of cyclin E was observed in four tumors and three tumors displayed abnormal low molecular weight cyclin E-related proteins. None of the tumors showed amplification of the cyclin D1 or E genes when studied by Southern blot analysis. All nine tumors showed a 2- to 6-fold increase in the level of cyclin A protein. Most of the tumors also displayed a marked increase in levels of the CDK2 and CDK4 proteins. These changes did not appear to be simply a consequence of increased cell proliferation, as assessed by proliferating cell nuclear antigen analysis. Thus, aberrant expression of cyclins and other cyclin-related genes occurs frequently in mammary tumorigenesis in both rodents and humans.

Animals↗
PubMed1995

Proteolytic cleavage of initiation factor eIF-4 gamma in the reticulocyte lysate inhibits translation of capped mRNAs but enhances that of uncapped mRNAs.

Infection of cells with the foot-and-mouth-disease virus, a member of the picornavirus family, results in the shut-off of host protein synthesis. A major contributory mechanism is the proteolytic destruction of the gamma subunit of the complex eIF-4, which functions in translation to promote the binding of the 43S ribosomal preinitiation complex to the 5' end of the cellular mRNA molecules bearing a 5' terminal cap structure. Picornavirus RNA molecules, which are uncapped, use a distinct mechanism for translational initiation, which can operate in the absence, or at low levels, of eIF-4. The proteolysis of eIF-4 gamma in cells infected by foot-and-mouth-disease virus results from expression of a virus-encoded cysteine proteinase known as Leader (or L) protease. We have used a transcription plasmid encoding this protease as a tool to deplete in vitro translation systems of eIF-4 gamma in order to elucidate in more detail the role of this polypeptide in the control of translation. Using in vitro transcribed mRNAs we have observed a marked contrast between capped and uncapped transcripts in the response of their translation to the proteolysis of eIF-4 gamma. Translation of capped mRNAs is, as expected, severely impaired, and is restored by addition of eIF-4 complex containing the intact gamma-subunit. On the other hand, translation of uncapped transcripts, normally inefficient, is substantially enhanced. The data suggest that the translation of uncapped mRNAs may be stimulated in this system by one or more of the proteolytic degradation products of eIF-4 gamma.

3C Viral Proteases↗
PubMed1995

Cardiac myocyte terminal differentiation. Potential for cardiac regeneration.

The exact mechanism of terminal differentiation in cardiac myocytes is currently unknown. Studies in the skeletal muscle system provided a model where muscle lineage termination gene directly interacts with Rb to produce and maintain the terminally differentiated state. This interaction provided the critical components for the lock in cell cycle arrest in skeletal muscle cell. Cardiac muscle appears on the surface very similar to skeletal muscle especially since they share large numbers of structural and contractile proteins. However, it is clear that cardiac muscle cells are distinct biologically at the regulatory level. First and foremost, differentiation and capacity for hyperplasia (mitosis) is not mutually exclusive, in that the heart being the first functional organ embryologically is able to grow via cell division until shortly after birth. Thereafter further growth is provided by hypertrophy. In skeletal muscle, these two processes, differentiation and ability to undergo mitosis, appear to be mutually exclusive. Second, cardiac muscles have not been shown to express any of the skeletal muscle determination basic helix loop helix factors like myoD or any proteins that are functionally similar. Third, heterokaryons of cardiac myocytes and fibroblasts reveal a lack of dominance of the cardiac muscle phenotype. This is distinctly different in skeletal muscle, whose phenotype is dominant which provided a platform to identify the skeletal muscle determination gene, myoD. Although various basic helix loop helix proteins and homeobox genes have been identified in cardiac myocytes, their function remains to be elucidated. At this time no cardiac determination gene has been identified. Despite these differences, we have shown that the biology of pocket proteins Rb and P107 is similar in skeletal and cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

tsJT16, a cell cycle ts mutant of rat fibroblast defective in early G0/G1 transition, fails to induce G1-cyclin and cdk2 genes after serum stimulation at the nonpermissive temperature.

tsJT16 is a cell-cycle temperature-sensitive (ts) mutant derived from rat fibroblasts whose functional defect appears soon after the growth stimulation from G0 phase. In addition to c-fos, c-myc and ornithine decarboxylase gene, 7 primarily inducible genes, c-jun, KC, JE, 2F1, 2A9, egr-1, and egr-2, were further shown to be expressed after serum stimulation at both permissive and nonpermissive temperatures. However, expression of secondarily inducible genes, cyclin D1 and D3 and cdk2, was ts and was cycloheximide sensitive. Expression of cyclin C was not inhibited by cycloheximide but it was ts. Failure in expression of G1 cyclins and Cdk2 is suggested to be a causal event for inability of growth induction of tsJT16 at the nonpermissive temperature.

Animals↗
PubMed1994

Selective repression of growth-regulating cdk2, cyclin E and E2F1 genes in human cell senescence.

Normal human diploid TIG-1 fibroblasts underwent replicative senescence around 64-68 population doubling levels (PDL) by the irreversible serum-unresponsive G1-growth arrest. Repression of growth-promoting genes was searched in this study. The RT-PCR and Western blot analyses have shown that in senescent TIG-1 cells at PDL64-67, cdk2 and cyclin E were selectively repressed at the mRNA and protein levels even after serum stimulation, and cdc2 and cyclin A were less repressed than cdk2 and cyclin E. Such a specific lack of cdk2 and cyclin E proteins correlated with unphosphorylation of the retinoblastoma gene product (pRB) in senescent cells. Transcription factor E2F1 was also completely repressed at the mRNA and protein levels in senescent TIG-1 cells. Middle-passage cells exhibited active expressions of all the above genes and pRB phosphorylation. Therefore, the present results have indicated the selective repressions of cdk2, cyclin E and E2F1 in senescent cells.

Base Sequence↗
PubMed1995

A model of the G1 phase of the cell cycle incorporating cyclin E/cdk2 complex and retinoblastoma protein.

A mathematical model of cyclin E, cdk2 and retinoblastoma protein control of the G1 phase of the human cell cycle is proposed. The model includes retinoblastoma (Rb) protein phosphorylation by a cyclin E/cdk2 complex and its subsequent dephosphorylation at the end of the cell cycle. The numerical solutions to this model demonstrates the cyclic behavior of the cyclin E/cdk2 complex, with and without Rb function, cell cycle. This model suggests an inhibition of cyclin E/cdk2 complex formation (or its activation) by hypophosphorylated retinoblastoma protein. The experimental results of cell cycle arrest upon injection of transforming growth factor-beta, alpha-interferon or D-erythro-sphingosine during G1 phase are reproduced. Cell cycle behavior predicted by this model for increasing the concentration of hypophosphorylated retinoblastoma protein during the G1 phase is discussed. Additional results are obtained by numerical simulation.

CDC2-CDC28 Kinases↗
PubMed1995

Stabilization of cyclin E and cdk2 mRNAs at G1/S transition in Rat-1A cells emerging from the G0 state.

mRNAs for cyclin E and Cdk2 have a role in the commitment to DNA replication in the cell cycle, and are induced in Rat-1A cells by serum stimulation. Cyclin E and cdk2 genes are transcribed in quiescent cells, but their transcripts rapidly turn over and levels are kept low. The rate of transcription of the cdk2 gene is slightly increased after serum stimulation, while that of cyclin E is fairly constant. At the G1/S transition of serum-stimulated cells, transient stabilization of the two types of mRNAs occurs, an event which may lead to induction of each mRNA. Artificial expression of an immediate-early protein delta FosB results in proliferation of quiescent Rat-1A cells, and this is accompanied by an efficient induction of cyclin E and cdk2 mRNAs. In delta FosB-expressing cells, two types of mRNAs are stabilized to the same extent seen in serum-stimulated cells. The expression of cyclin E and cdk2 genes is upregulated by stabilization of their transcripts, at least in part. We propose that delta FosB may have a role in regulation of progression of the cell cycle in serum-stimulated Rat-1A cells by triggering stabilization of mRNAs for cyclin E and Cdk2.

Animals↗
PubMed1995

A complex between E2F and the pRb-related protein p130 is specifically targeted by the simian virus 40 large T antigen during cell transformation.

The p130 protein is a recently cloned member of the retinoblastoma protein family. We show here that transformation of NIH3T3-L1 fibroblasts (L1 cells) by the simian virus 40 large T antigen (LTAg) depends on the disruption of DNA binding complexes between transcription factor E2F and p130. LTAg binds to the pocket region of p130 in vivo and disrupts the E2F-p130 complexes. E2F-p130 complexes are present only in quiescent L1 cells and disappear at the G1/S phase boundary concomitantly to induction of DNA synthesis and expression of the E2F-regulated cdc2 gene. p130 is a substrate of cyclin-dependent kinase 2 (Cdk2) in vitro and associates with a Cdk in vivo which is activated upon serum stimulation in late G1. Overexpression of p130 inhibits cdc2 promoter activity and entry of quiescent L1 cells into S phase. The results demonstrate that p130 is negative regulator of cell cycle progression which is specifically targeted by LTAg during cell transformation.

3T3 Cells↗