Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In a previous study, we found that treatment of HCT-8 cells with ZD1694, a specific antifolate-based thymidylate synthase inhibitor, resulted in DNA fragmentation. In this study, we have demonstrated the dose- and time-dependent induction of DNA fragmentation accompanied by elevation of p53 and WAF1 protein expression by ZD1694. WAF1 mRNA showed a time-dependent increase, whereas p53 mRNA was not found to be significantly overexpressed. The initial increase in WAF1 mRNA was detected at 4 hr, but increased WAF1 protein expression was detected 8-24 hr after a 2-hr exposure. The amount of total and hypophosphorylated pRb seems to be rising greatly after ZD1694 exposure. The effects of ZD1694 on the expression of E2F1 and formation of the E2F1-Rb complex were investigated after a 2-hr drug exposure (IC90). The results showed a time-dependent decrease in E2F1 mRNA and protein expression; an increase in the abundance of the E2F-Rb complex could be demonstrated beginning 4 hr after drug exposure by a gel shift assay. Kinetic analysis showed increased availability of hypophosphorylated pRb for inhibition of E2F, which could indirectly result from WAF1-induced inhibition cyclin-dependent kinase activity. Whereas thymidylate synthase inhibition by ZD1694 was rapid in onset and maintained for at least 24 hr after drug treatment, drug-induced cellular growth inhibition was significant 24 hr after drug exposure. The increased abundance of hypophosphorylated pRb and binding to transcription factor E2F-1 is consistent with ZD1694-induced cell growth inhibition in HCT-8 cells. Therefore, the observed effect on downstream events after effective inhibition of thymidylate synthase may offer the critical determinants of response to ZD1694.
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PMID:p53 and WAF1 are induced and Rb protein is hypophosphorylated during cell growth inhibition by the thymidylate synthase inhibitor ZD1694 (Tomudex). 910 28

The p16 (MTS1) tumour-suppressor gene is a cyclin-dependent kinase (cdk) inhibitor that decelerates the cell cycle by inactivating the cdks that phosphorylate the retinoblastoma tumour-suppressor gene (Rb) protein (pRb). In cervical cancers, pRb is inactivated by the HPV E7 oncoprotein or by mutations. The hypothesis of earlier reports was that the disruption of the p16/cdk-cyclin/Rb cascade is essential for malignant cervical transformation/carcinogenesis. We previously established in vitro model systems of cervical cancer representing four steps of oncogenic progression initiated by the two most common oncogenic HPVs in ectocervical and endocervical epithelial cells. This report used these systems to investigate the role of p16 in cervical cancers. A dramatic enhancement of the p16 RNA level was observed after immortalization by HPV 16 or 18. Furthermore, the p16 protein was newly observed following immortalization. However, no further changes were found for RNA or protein levels after serum selection or malignant transformation. For three cervical carcinoma cell lines, similar high levels of p16 expression were seen. Point mutations or homozygous deletions of p16 were not observed in the in vitro systems or in clinical specimens. These results suggest that the inactivation of the p16/cdk-cyclin/Rb cascade does not occur during malignant transformation but occurs during the immortalization by HPV in HPV-harbouring premalignant lesions, the in situ equivalent of immortalized cells. Also suggested is that p16 has no role in the specific malignant transformation step from immortal premalignant lesions during the carcinogenesis of HPV-initiated cervical cancers.
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PMID:Induction of p16 during immortalization by HPV 16 and 18 and not during malignant transformation. 916 31

Viral transformation of mouse and human fibroblasts has very different effects on the composition of cyclin-dependent kinase (Cdk) complexes. In human cells transformed by the large T-antigen of simian virus 40 (SV40 T-Ag) and human tumour cell lines that lack a functional retinoblastoma gene product (pRb) no cyclin D1-Cdk4 complexes can be detected because all the available Cdk4 is associated with the Cdk-inhibitor p16INK4a. In contrast, SV40-transformed mouse cells and fibroblasts from Rh1-nullizygous mouse embryos contain normal levels of cyclin D1-Cdk4 complexes. To investigate this species difference, we have compared the biochemical properties and expression of mouse p16INK4a with that of its human counterpart. There is a marked increase in p16 RNA and protein levels as primary embryo fibroblasts approach their finite lifespan in culture, but mouse p16 expression does not appear to be influenced by the status of pRb. Transformed or spontaneously immortalized mouse cells therefore do not achieve the very high levels of p16 characteristic of pRb-negative human cell lines. We suggest that these differences may be related to the different frequencies with which mouse and human cells can be immortalized in culture.
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PMID:Accumulation of p16INK4a in mouse fibroblasts as a function of replicative senescence and not of retinoblastoma gene status. 924 3

The histone H4 gene promoter provides a paradigm for defining transcriptional control operative at the G1/S phase transition point in the cell cycle. Transcription of the cell cycle-dependent histone H4 gene is upregulated at the onset of S phase, and the cell cycle control element that mediates this activation has been functionally mapped to a proximal promoter domain designated Site II. Activity of Site II is regulated by an E2F-independent mechanism involving binding of the oncoprotein IRF2 and the multisubunit protein HiNF-D, which contains the homeodomain CDP/cut, CDC2, cyclin A, and the tumor suppressor pRb. To address mechanisms that define interactions of Site II regulatory factors with this cell cycle control element, we have investigated these determinants of transcriptional regulation at the G1/S phase transition in FDC-P1 hematopoietic progenitor cells. The representation and activities of histone gene regulatory factors were examined as a function of FDC-P1 growth stimulation. We find striking differences in expression of the pRb-related growth regulatory proteins (pRb/p105, pRb2/p130, and p107) following the onset of proliferation. pRb2/p130 is present at elevated levels in quiescent cells and declines following growth stimulation. By contrast, pRb and p107 are minimally represented in quiescent FDC-P1 cells but are upregulated at the G1/S phase transition point. We also observe a dramatic upregulation of the cellular levels of pRb2/p130-associated protein kinase activity when S phase is initiated. Selective interactions of pRb and p107 with CDP/cut are observed during the FDC-P1 cell cycle and suggest functional linkage to competency for DNA binding and/or transcriptional activity. These results are particularly significant in the context of hematopoietic differentiation where stringent control of the cell cycle program is requisite for expanding the stem cell population during development and tissue renewal.
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PMID:Cell cycle-dependent modifications in activities of pRb-related tumor suppressors and proliferation-specific CDP/cut homeodomain factors in murine hematopoietic progenitor cells. 928 29

The herpes simplex virus 1 (HSV-1) infected-cell protein 0 (ICP0) has the characteristics of a promiscuous transactivator of genes introduced into cells by infection or transfection. To identify cellular proteins interacting with ICP0, we used a domain of exon II of ICP0 that is known to be crucial for regulatory function of the protein as bait in the yeast two-hybrid screen. Our results were as follows. (i) A cDNA in a positive yeast colony was found to encode cyclin D3, a cell cycle regulator of G1 phase. (ii) A purified chimeric protein consisting of glutathione S-transferase (GST) fused to cyclin D3 specifically formed complexes with ICP0 contained in HSV-1-infected cell lysate. (iii) To enhance the expression of cyclin D3, the gene was inserted into the viral genome and overexpressed in infected cells. The overexpressed cyclin D3 colocalized with ICP0 in nuclear structures characteristic of ND10 and which earlier have been reported to contain ICP0. (iv) The accumulation of cyclin D3 protein in Vero cells infected with an alpha0 deletion mutant was reduced relative to that of cells infected with wild-type virus or a recombinant virus in which the deleted alpha0 sequences were restored. (v) Lysates of Spodoptera frugiperda Sf9 cells doubly infected with baculoviruses genetically engineered to express cyclin D3 and cyclin-dependent kinase 4 (CDK4) phosphorylated GST fused to retinoblastoma protein (GST-pRb) but did not phosphorylate the GST-alpha0(20-241) or GST-alpha0(543-768) fusion protein or immunoprecipitated ICP0 proteins. Moreover, the chimeric GST-ICP0(exon II) protein shown to bind cyclin D3 had no effect on the activity of the kinase on GST-pRb when added to mixtures of lysates of Sf9 cells which coexpressed cyclin D3 and CDK4. These results indicate that ICP0 interacts with, colocalizes with, and stabilizes the cyclin D3 cell cycle regulator and does not affect its interaction with the cyclin-dependent kinase.
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PMID:Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3. 931 10

Retroviral expression of the cyclin-dependent kinase (CDK) inhibitor p16(INK4a) in rodent fibroblasts induces dephosphorylation of pRb, p107 and p130 and leads to G1 arrest. Prior expression of cyclin E allows S-phase entry and long-term proliferation in the presence of p16. Cyclin E prevents neither the dephosphorylation of pRb family proteins, nor their association with E2F proteins in response to p16. Thus, cyclin E can bypass the p16/pRb growth-inhibitory pathway downstream of pRb activation. Retroviruses expressing E2F-1, -2 or -3 also prevent p16-induced growth arrest but are ineffective against the cyclin E-CDK2 inhibitor p27(Kip1), suggesting that E2F cannot substitute for cyclin E activity. Thus, cyclin E possesses an E2F-independent function required to enter S-phase. However, cyclin E may not simply bypass E2F function in the presence of p16, since it restores expression of E2F-regulated genes such as cyclin A or CDC2. Finally, c-Myc bypasses the p16/pRb pathway with effects indistinguishable from those of cyclin E. We suggest that this effect of Myc is mediated by its action upstream of cyclin E-CDK2, and occurs via the neutralization of p27(Kip1) family proteins, rather than induction of Cdc25A. Our data imply that oncogenic activation of c-Myc, and possibly also of cyclin E, mimics loss of the p16/pRb pathway during oncogenesis.
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PMID:Cyclin E and c-Myc promote cell proliferation in the presence of p16INK4a and of hypophosphorylated retinoblastoma family proteins. 931 92

Addition of insulin-like growth factor I (IGF-I) to quiescent breast tumor-derived MCF-7 cells causes stimulation of cyclin D1 synthesis, hyperphosphorylation of the retinoblastoma protein pRb, DNA synthesis, and cell division. All of these effects are independent of the mitogen-activated protein kinase (MAPK) pathway since none of them is blocked by PD098059, the specific inhibitor of the MAPK activating kinase MEK1. This observation is consistent with the finding that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong inducer of MAPK activity in MCF-7 cells, effectively inhibits proliferation. The anti-proliferative effect of TPA in these cells may be accounted for, at least in part, by the MAPK-dependent stimulation of the synthesis of p21(WAF1/CIP1), an inhibitor of cyclin/cyclin-dependent kinase complexes. In contrast, all of the observed stimulatory effects of IGF-I on cell cycle progression, cyclin D1 synthesis, and pRb hyperphosphorylation were blocked by the specific phosphatidylinositol 3-kinase inhibitor LY294002, suggesting that phosphatidylinositol 3-kinase activity but not MAPK activity is required for transduction of the mitogenic IGF-I signal in MCF-7 cells.
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PMID:Mitogenic signaling of insulin-like growth factor I in MCF-7 human breast cancer cells requires phosphatidylinositol 3-kinase and is independent of mitogen-activated protein kinase. 938 70

It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27Kip1 are limited to cell cycle control at the G1/S-phase transition and in the maintenance of cellular quiescence. To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression. The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G1/S transition, contributes to regulation of the G2/M transition. Both G1- and G2-arrested cells were observed in all cell types, with different preponderances. Preponderant G1 arrest in response to p21 expression correlated with the presence of functional pRb. G2 arrest was more prominent in pRb-negative cells. The arrest distribution did not correlate with the p53 status, and proliferating-cell nuclear antigen (PCNA) binding activity of p21 did not appear to be involved, since p27, which lacks a PCNA binding domain, produced similar arrest distributions [corrected], DNA endoreduplication occurred in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in p21 G2-arrested cells. These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G1 arrest as well as to blockade of DNA replication from either G1 or G2 phase is the pRb regulatory system. Finally, the tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway.
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PMID:Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication. 941 9

Cell cycle checkpoints are safeguards that ensure the initiation of downstream events only after completion of upstream processes. The tumor suppressors p53 and pRb prevent initiation of a second round of replication in response to spindle inhibitors, but it has yet to be proven that this is a mitotic checkpoint response. We show that asynchronous human fibroblasts arrest in G1 with 4 N DNA content after nocodazole treatment, whereas isogenic p53- and pRb-deficient fibroblasts rereplicate. Importantly, nocodazole elicits a reversible arrest in G0-G1 synchronized normal human fibroblasts but not in isogenic p53-deficient derivatives. Furthermore, the G1 cyclin-dependent kinase inhibitors p21 and p16 also play critical roles in limiting rereplication. Hence, p53 and pRb are required during G1 to prevent entry into a replicative cycle and appear to provide a connection between the structural integrity of the microtubules and the cell cycle machinery in interphase cells.
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PMID:p53 and pRb prevent rereplication in response to microtubule inhibitors by mediating a reversible G1 arrest. 945 79

Ectopic expression of the c-Myc oncoprotein prevents cell cycle arrest in response to growth-inhibitory signals, differentiation stimuli, or mitogen withdrawal. Moreover, Myc activation in quiescent cells is sufficient to induce cell cycle entry in the absence of growth factors. Thus, Myc transduces a potent mitogenic stimulus but, concomitantly, induces apoptosis in the absence of survival factors. We review here recent progress in our understanding of the molecular mechanisms linking Myc activity to cell cycle control. Myc is a positive regulator of G1-specific cyclin-dependent kinases (CDKs) and, in particular, of cyclin E/CDK2 complexes. Cyclin D/CDK4 and CDK6 may conceivably also be activated by Myc, but the circumstances in which this occurs remain to be explored. Myc acts via at least three distinct pathways which can enhance CDK function: (1) functional inactivation of the CDK inhibitor p27Kip1 and probably also of p21Cip1 and p57Kip2, (2) induction of the CDK-activating phosphatase Cdc25A and (3) - in an ill understood and most likely indirect way - deregulation of cyclin E expression. Constitutive expression of either Myc or cyclin E can prevent growth arrest by p16INK4a (an inhibitor of cyclin D/CDK4, but not of cyclin E/CDK2). In cells, p16INK4a inhibits phosphorylation, and thus induces activation of the Retinoblastoma-family proteins (pRb, p107 and p130). Surprisingly, this effect of p16 is not altered in the presence of Myc or cyclin E. Thus, Myc and cyclin E/CDK2 activity unlink activation of p16 and pRb from growth arrest. Finally, Myc may itself be a functional target of cyclin D/CDK4 through its direct interaction with p107. We discuss how the effects of Myc on cell cycle control may relate to its oncogenic activity, and in particular to its ability to cooperate with activated Ras oncoproteins.
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PMID:Myc and the cell cycle. 946 63


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