CDC25A phosphatase is a target of E2F and is required for efficient E2F-induced S phase.
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Functional inactivation of the pRB pathway is a very frequent event in human cancer, resulting in deregulated activity of the E2F transcription factors. To understand the functional role of the E2Fs in cell proliferation, we have developed cell lines expressing E2F-1, E2F-2, and E2F-3 fused to the estrogen receptor ligand binding domain (ER). In this study, we demonstrated that activation of all three E2Fs could relieve the mitogen requirement for entry into S phase in Rat1 fibroblasts and that E2F activity leads to a shortening of the G(0)-G(1) phase of the cell cycle by 6 to 7 h. In contrast to the current assumption that E2F-1 is the only E2F capable of inducing apoptosis, we showed that deregulated E2F-2 and E2F-3 activities also result in apoptosis. Using the ERE2F-expressing cell lines, we demonstrated that several genes containing E2F DNA binding sites are efficiently induced by the E2Fs in the absence of protein synthesis. Furthermore, CDC25A is defined as a novel E2F target whose expression can be directly regulated by E2F-1. Data showing that CDC25A is an essential target for E2F-1, since its activity is required for efficient induction of S phase by E2F-1, are provided. Finally, our results show that expression of two E2F target genes, namely CDC25A and cyclin E, is sufficient to induce entry into S phase in quiescent fibroblasts. Taken together, our results provide an important step in defining how E2F activity leads to deregulated proliferation. (+info)
Translational control of the cdc25 cell cycle phosphatase: a molecular mechanism coupling mitosis to cell growth.
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The eukaryotic translation initiation factor 4A (eIF4A) is an RNA helicase required for translation initiation of eukaryotic mRNAs. By engineering fission yeast mutants with diminished eIF4A activity, we have found that translation of cdc25 mRNAs (a dosage-dependent activator of mitosis in all eukaryotic cells) is particularly sensitive to limitations of protein synthesis mediated by limited eIF4A activity. Genetic and biochemical analysis indicated that a rate-limited translation initiation of cdc25 mRNAs, exerted throughout its unusual 5' untranslated leader, acts as a molecular sensor to ensure that a minimum cell mass (protein synthesis) is attained before mitosis occurs. The Cdc13 cyclin B is also among the limited pool of proteins whose translation is sensitive to reduced translation initiation activity. Interestingly, the 5' leader sequences of cdc25 and cdc13 mRNAs have conserved features which are unusual in other yeast mRNAs, suggesting that common mechanisms operate in the expression of these two key mitotic activators at the translational level. (+info)
Benzamide riboside induces apoptosis independent of Cdc25A expression in human ovarian carcinoma N.1 cells.
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One of the mechanisms of action of a new oncolytic agent, benzamide riboside (BR) is by inhibiting inosine 5'-monophosphate dehydrogenase (IMPDH) which catalyzes the formation of xanthine 5'-monophosphate from inosine 5'-monophosphate and nicotinamide adenine dinucleotide, thereby restricting the biosynthesis of guanylates. In the present study BR (10 - 20 microM) induced apoptosis in a human ovarian carcinoma N.1 cell line (a monoclonal derivative of its heterogenous parent line HOC-7). This was ascertained by DNA fragmentation, TUNEL assay, [poly(ADP)ribose polymerase]-cleavage and alteration in cell morphology. Apoptosis was accompanied by sustained c-Myc expression, concurrent down-regulation of cdc25A mRNA and protein, and by inhibition of Cdk2 activity. Both Cdk2 and cdc25A are G1 phase specific genes and Cdk2 is the target of Cdc25A. These studies demonstrate that BR exhibits dual mechanisms of action, first by inhibiting IMPDH, and second by inducing apoptosis, which is associated with repression of components of the cell cycle that are downstream of constitutive c-Myc expression. (+info)
Induction of mammary gland hyperplasia in transgenic mice over-expressing human Cdc25B.
(36/759)
Cdc25 A and B are dual-specificity phosphatases which have been implicated in neoplastic transformation. Although Cdc25A and Cdc25B have been found to be over-expressed in many cancer cell lines and primary tumors, the physiological roles of Cdc25A and B in vivo are largely undefined. To investigate the roles of these proteins in the oncogenic transformation of the mammary gland we used the mouse mammary tumor virus (MMTV) promoter to target over-expression of the Cdc25B transgene in the mammary glands of transgenic mouse lines. Here we report that the over-expression of Cdc25B enhances the proliferation of mammary epithelial cells resulting in the formation of precocious alveolar hyperplasia. At the molecular level, marked increases in cyclin D1 protein have been found in transgenic mammary epithelial cells. The accelerated growth rate of the mammary epithelial cells could also be attributed to the increased levels of cyclin E/cdk2 activity. In addition, a pronounced decrease in apoptosis was also observed during the involution of mammary gland. The reduction of apoptosis during involution correlated well with the reduced expression of c-myc and p53, both of which have been implicated in apoptosis. Taken together, our results clearly indicate that the deregulated expression of Cdc25B generates mammary gland hyperplasia. (+info)
Interferon-alpha inhibits proliferation in human T lymphocytes by abrogation of interleukin 2-induced changes in cell cycle-regulatory proteins.
(37/759)
IFN-alpha exerts prominent regulatory functions on the immune system. One such effect is the inhibition of proliferation of in vitro stimulated T lymphocytes. The exact physiological function of this activity is not known, but it has been implicated in the antiviral effects of IFN, its antitumor action in T-cell malignancies, and the regulation of the in vivo T-cell response. Here, we have investigated the mechanism underlying the IFN-alpha-mediated growth inhibition of normal human PHA- and IL-2-stimulated T lymphocytes by an analysis of how IFN-alpha treatment influences known molecular events that normally accompany the transition from quiescence to proliferation in these cells. IFN-alpha treatment was found to profoundly block S-phase entry of stimulated T lymphocytes. This correlated with a strong inhibition of IL-2-induced changes in G1-regulatory proteins, including the prevented up-regulation of G1 cyclins and cyclin-dependent kinases as well as an abrogation of mitogen-induced reduction of p27Kip1 levels. This latter effect was due to a maintained stability of the p27Kip1 protein in the IFN-alpha-treated cells. In line with these findings, phosphorylation of the pocket proteins was abrogated in IFN-alpha-treated cells. Furthermore, our data indicate that IFN-alpha has selective effects on the pathways that emerge from the IL-2 receptor because IFN-alpha treatment does not block IL-2-induced up-regulation of c-myc or Cdc25A. (+info)
Clostridium botulinum C2 toxin delays entry into mitosis and activation of p34cdc2 kinase and cdc25-C phosphatase in HeLa cells.
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The Clostridium botulinum C2 toxin ADP-ribosylates monomeric actin, thereby inducing disassembly of actin filaments, alteration of focal adhesions, and rounding of cells. After treatment with C2 toxin, cells stop to proliferate but remain viable for about 2 days. In view of reported correlations between the structure of the actin cytoskeleton and cell cycle transition, the effects of C2 toxin on the G(2)/M phase transition of the cell division cycle were studied. Since C2 toxin delayed entry into mitosis in HeLa cells, those enzymes which control entry into mitosis, the cyclin-dependent protein kinase mitosis-promoting factor (MPF) and the phosphatase cdc25-C were examined after treatment of synchronized cells with C2 toxin. MPF is composed of the regulatory cyclin B and the enzymatic p34cdc2 kinase subunits. For its activation at the G2/M border, p34cdc2 needs to be associated with cyclin B and additionally dephosphorylated at Tyr-15 by the specific phosphatase cdc25-C. Treatment of synchronized cells in S or G2 phase with C. botulinum C2 toxin prevented p34cdc2 protein kinase activation by inhibiting its tyrosine dephosphorylation at the G2/M border. Furthermore, the activity of cdc25-C phosphatase was decreased after treatment of cells with C2 toxin. Our results suggest that the prevented activation of the mitotic inducers p34cdc2 kinase and cdc25-C phosphatase represents the final downstream events in the action of C2 toxin resulting in a G(2) phase cell cycle delay in synchronized HeLa cells. (+info)
Increased susceptibility to carcinogen-induced mammary tumors in MMTV-Cdc25B transgenic mice.
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Cdc25 phosphatases activate cyclin-dependent kinases (Cdks) by dephosphorylating critical phospho-tyrosine and phospho-threonine residues on these proteins. Several types of studies indicate that Cdc25s can enhance cell proliferation and oncogenesis. Furthermore, overexpression of Cdc25A and/or B have been detected in several types of primary human cancers, including breast cancers. To further assess the oncogenic capacity of Cdc25B in vivo, we have generated transgenic mice that overexpress Cdc25B in the mammary epithelium, driven by the MMTV - LTR promoter. Although these mice are grossly normal for up to 18 months, the ectopic expression of Cdc25B in their mammary glands increases the susceptibility of these mice to induction of mammary tumors by the carcinogen 9,10-dimethyl-1, 2-benzanthracene (DMBA). (+info)
Loss of cell cycle control by deregulation of cyclin-dependent kinase 2 kinase activity in Evi-1 transformed fibroblasts.
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The Evi-1 transcriptional repressor protein has two distinct zinc finger DNA binding domains designated ZF1 and ZF2 and is implicated in the progression of human and murine leukemias, in which it is abnormally expressed. In this report, we show that Evi-1-expressing Rat1 fibroblasts are anchorage independent, have an abbreviated G1 phase of the cell cycle, and have a reduced requirement for serum mitogens for S-phase entry. These biological changes are accompanied by a moderately increased production of cell cycle-regulatory proteins cyclin A and cyclin-dependent kinase (Cdk) 2, a dramatic deregulation of Cdk2 kinase activity, and a corresponding increase in the levels of hyperphosphorylated retinoblastoma protein (pRb). We show that the elevated cyclin A-Cdk2 activity is due to the combination of increased accumulation and stabilization of cyclin A bound to a faster-migrating species of Cdk2 believed to be the active threonine 160 phosphorylated form and a substantial reduction in complexed p27. Cyclin E kinase activity is also elevated due to a reduction in p27. A significant reduction in total cellular p27 protein levels and a moderate reduction in p27 mRNA are observed, but no changes in Cdk regulatory kinases and phosphatases occur. The Evi-1 transcriptional repressor domain and the ZF1 DNA binding domain are required for both cell transformation and induction of Cdk2 catalytic activity. We propose that one consequence of Evi-1 expression is to repress the transcription of target genes, which may include p27, that deregulate the normal control of the G1 phase of the cell cycle, providing a cellular proliferative advantage that contributes to transformation in vitro and leukemogenesis in vivo. (+info)