Diminished G1 checkpoint after gamma-irradiation and altered cell cycle regulation by insulin-like growth factor II overexpression.
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High levels of insulin-like growth factor II (IGFII) mRNA expression are detected in many human tumors of different origins including rhabdomyosarcoma, a tumor of skeletal muscle origin. To investigate the role of IGFII in tumorigenesis, we have compared the mouse myoblast cell line C2C12-2.7, which was stably transfected with human IGFII cDNA and expressed high and constant amounts of IGFII, to a control cell line C2C12-1.1. A rhabdomyosarcoma cell line, RH30, which expresses high levels of IGFII and contains mutated p53, was also used in these studies. IGFII overexpression in mouse myoblast C2C12 cells causes a reduced cycling time and higher growth rate. After gamma-irradiation treatment, C2C12-1.1 cells were arrested mainly in G0/G1 phase. However, C2C12-2.7 and RH30 cells went through a very short G1 phase and then were arrested in an extended G2/M phase. To verify further the effect of IGFII on the cell cycle, we developed a Chinese hamster ovary (CHO) cell line with tetracycline-controlled IGFII expression. We found that CHO cells with high expression of IGFII have a shortened cycling time and a diminished G1 checkpoint after treatment with methylmethane sulfonate (MMS), a DNA base-damaging agent, when compared with CHO cells with very low IGFII expression. It was also found that IGFII overexpression in C2C12 cells was associated with increases in cyclin D1, p21, and p53 protein levels, as well as mitogen-activated protein kinase activity. These studies suggest that IGFII overexpression shortens cell cycling time and diminishes the G1 checkpoint after DNA damage despite an intact p53/p21 induction. In addition, IGFII overexpression is also associated with multiple changes in the levels and activities of cell cycle regulatory components following gamma-irradiation. Taken together, these changes may contribute to the high growth rate and genetic alterations that occur during tumorigenesis. (+info)
Inhibition of transcription by the trimeric cyclin-dependent kinase 7 complex.
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Cyclin-dependent kinase 7 (CDK7) can be isolated as a subunit of a trimeric kinase complex functional in activation of the mitotic promoting factor. In this study, we demonstrate that the trimeric cdk-activating kinase (CAK) acts as a transcriptional repressor of class II promoters and show that repression results from CAK impeding the entry of RNA polymerase II and basal transcription factor IIF into a competent preinitiation complex. This repression is independent of CDK7 kinase activity. We find that the p36/MAT1 subunit of CAK is required for transcriptional repression and the repression is independent of the promoter used. Our results demonstrate a central role for CAK in regulation of messenger RNA synthesis by either inhibition of RNA polymerase II-catalyzed transcription or stimulation of transcription through association with basal transcription repair factor IIH. (+info)
Cyclin E in human cancers.
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Regulators of the cell cycle such as cyclin E play an important part in neoplasia. The cyclin E protein forms a partnership with a specific protein kinase. This complex phosphorylates key substrates to initiate DNA synthesis. Cyclin-dependent kinase inhibitors (CKIs) are able to suppress the activity of cyclin E. Various substances (including proteins produced by oncogenic viruses) affect cyclin E directly or indirectly through an interaction with CKIs. These interactions are important in elucidating the mechanisms of neoplasia. They may also provide prognostic information in a wide range of common cancers. Cyclin E may even be a target for treatment of cancers in the future. (+info)
Cdc2 activation in fission yeast depends on Mcs6 and Csk1, two partially redundant Cdk-activating kinases (CAKs).
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Cyclin-dependent kinases (Cdks) are fully active only when phosphorylated by a Cdk-activating kinase (CAK) [1]. Metazoan CAK is itself a Cdk, Cdk7, whereas the CAK of Saccharomyces cerevisiae is a distinct enzyme unrelated to Cdks [1]. The Mcs6-Mcs2 complex of Schizosaccharomyces pombe is a putative CAK related to the metazoan enzyme [2] [3]. Although the loss of Mcs6 is lethal, it results in a phenotype that is inconsistent with a failure to activate Cdc2, the major Cdk in S. pombe [3]. We therefore tested the ability of Csk1, a putative regulator of Mcs6 [4], to activate Cdk-cyclin complexes in vitro. Csk1 activated both the monomeric and the Mcs2-bound forms of Mcs6. Surprisingly, Csk1 also activated Cdc2 in complexes with either Cdc13 or Cig2 cyclins. When a double mutant carrying a csk1 deletion and a temperature-sensitive mcs6 allele was incubated at the restrictive temperature, Cdc2 was not activated and the cells underwent a cell division arrest prior to mitosis. Cdc2-cyclin complexes isolated from the arrested cells could be activated in vitro by recombinant CAK, whereas complexes from wild-type cells or either of the single mutants were refractory to activation. Thus, fission yeast contains two partially redundant CAKs: the Mcs6-Mcs2 complex and Csk1. Inactivation of both CAKs is necessary and sufficient to prevent Cdc2 activation and cause a cell-cycle arrest. Mcs6, which is essential, may therefore have required functions other than Cdk activation. (+info)
Cyclin-dependent kinase 2 (Cdk2) is required for centrosome duplication in mammalian cells.
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Centrosome duplication is indispensable for the formation of the bipolar mitotic spindle. Surprisingly, even if DNA replication or mitosis is inhibited, centrosome duplication can still occur [1] [2] [3] [4] [5]. Thus, it remains unknown how centrosome duplication is coordinated with the cell cycle. Here, we show that centrosome duplication requires cyclin-dependent kinase 2 (Cdk2) in mammalian cells. We have found that in Chinese hamster ovary (CHO) cells, whereas centrosome duplication is not inhibited by hydroxyurea (HU) treatment, which arrests the cells in S phase, it is inhibited by mimosine treatment, which arrests the cells in late G1 phase. Cdk2 activity was higher in HU-treated cells than in mimosine-treated cells. Remarkably, inhibition of the Cdk2 activity in HU-treated cells with butyrolactone I or roscovitine [6], or by expression of the Cdk inhibitor p21(Waf1/Cip1), blocked the continued centrosome duplication. Moreover, overexpression of Cdk2 reversed the inhibition of centrosome duplication by mimosine treatment. These results indicate a requirement of Cdk2 activity for centrosome duplication and therefore suggest an underlying mechanism for the coordination of centrosome duplication with the cell cycle. (+info)
Anchorage dependence of mitogen-induced G1 to S transition in primary T lymphocytes.
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Anchorage dependence defines the cellular requirement for integrin-mediated adhesion to substrate to initiate DNA replication in response to growth factors. In this study we investigated whether normal T cells, which spend extended periods in a nonadherent state, show similar requirements for cell cycle progression in response to TCR stimulation. Resting primary T lymphocytes were induced to enter the cell cycle by TCR triggering, and leukocyte integrins were either engaged using purified ICAM-1 or inhibited with function-blocking mAbs. Our data indicate that leukocyte integrins complement TCR-driven mitogenic signals not as a result of their direct clustering but, rather, via integrin-dependent organization of the actin cytoskeleton. Leukocyte integrin-dependent reorganization of the actin cytoskeleton cooperates with the TCR to effect mitogen-activated protein kinase activation, but also represents a required late (4-8 h poststimulation) component in the mitogenic response of normal T cells. Prolonged leukocyte integrin-dependent spreading, in the context of intercellular contact, is a requisite for the production of the mitogenic cytokine IL-2, which, in turn, is involved in the induction of D3 cyclin and is primarily responsible for the decrease in the cyclin-dependent kinase inhibitor p27kip, resulting in retinoblastoma protein inactivation and S phase entry. Thus, T lymphocytes represent a peculiar case of anchorage dependence, in which signals conveyed by integrins act sequentially with the activating stimulus to effect a sustained production of the essential mitogenic cytokine. (+info)
HLA class I-mediated induction of cell proliferation involves cyclin E-mediated inactivation of Rb function and induction of E2F activity.
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Chronic rejection of transplanted organs is manifested as atherosclerosis of the blood vessels of the allograft. HLA class I Ags have been implicated to play a major role in this process, since signaling via HLA class I molecules can induce the proliferation of aortic endothelial as well as smooth muscle cells. In this study, we show that HLA class I-mediated induction of cell proliferation correlates with inactivation of the Rb protein in the T cell line Jurkat as well as human aortic endothelial cells. HLA class I-mediated inactivation of Rb can be inhibited specifically by neutralizing Abs to basic fibroblast growth factor (bFGF), suggesting a role for FGF receptors in the signaling process. Signaling through HLA class I molecules induced cyclin E-associated kinase activity within 4 h in quiescent endothelial cells, and appeared to mediate the inactivation of Rb. A cdk2 inhibitor, Olomoucine, as well as a dominant-negative cdk2 construct prevented HLA class I-mediated inactivation of Rb; in contrast, dominant-negative cdk4 and cdk6 constructs had no effect. Furthermore, there was no increase in cyclin D-associated kinase activity upon HLA class I ligation, suggesting that cyclin E-dependent kinase activity mediates Rb inactivation, leading to E2F activation and cell proliferation. (+info)
Limiting amounts of p27Kip1 correlates with constitutive activation of cyclin E-CDK2 complex in HTLV-I-transformed T-cells.
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Human T-cells immortalized (interleukin-2 [IL-2] dependent) by the human T-cell lymphotropic/leukemia virus type I (HTLV-I), in time, become transformed (IL-2 independent). To understand the biochemical basis of this transition, we have used the sibling HTLV-I-infected T-cell lines, N1186 (IL-2 dependent) and N1186-94 (IL-2 independent), as models to assess the responses to antiproliferative signals. In N1186 cells arrested in G1 after serum/interleukin-2 (IL-2) deprivation, downregulation of the cyclin E-CDK2 kinase activity correlated with decreased phosphorylation of CDK2 and accumulation of p27Kip1 bound to the cyclin E-CDK2 complex, as seen in normal activated PBMCs (peripheral blood mononuclear cells). In contrast, N1186-94 cells failed to arrest in G1 upon serum starvation, displayed constitutive cyclin E-associated kinase activity, and, although CDK2 was partially dephosphorylated, the amount of p27Kip1 bound to the complex did not increase. This observation, extended to two other IL-2-dependent as well as to three IL-2-independent HTLV-I-infected T-cell lines, suggests that the lack of cyclin E-CDK2 kinase downregulation found in the late phase of HTLV-I transformation may correlate with insufficient amounts of p27Kip1 associated with the cyclin E-CDK2 complex. Reconstitution experiments demonstrated that the addition of p27Kip1 to lysates from N1186-94 starved cells resulted in the downregulation of cyclin E-associated kinase activity supporting the notion that the unresponsiveness of the cyclin E-CDK2 complex to growth inhibitory signals may be due to inadequate amounts of p27Kip1 assembled with the complex in HTLV-I-transformed T-cells. In fact, the amount of p27Kip1 protein was lower in most HTLV-I-transformed (IL-2-independent) than in the immortalized (IL-2-dependent) HTLV-I-infected T-cells. Furthermore, specific inhibitors of the phosphatidylinositol 3-kinase (P13K) induced an increase of p27Kip1 protein levels, which correlated with G1 arrest, in both IL-2-dependent and IL-2-independent HTLV-I-infected T-cells. Altogether, these results suggest that maintaining a low level of expression of p27Kip1 is a key event in HTLV-I transformation. (+info)