Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.22 (cdc2)
 8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although a number of transfection experiments have suggested potential targets for the action of the E2F1 transcription factor, as is the case for many transcriptional regulatory proteins, the actual targets in their normal chromosomal environment have not been demonstrated. We have made use of a recombinant adenovirus containing the E2F1 cDNA to infect quiescent cells and then measure the activation of endogenous cellular genes as a consequence of E2F1 production. We find that many of the genes encoding S-phase-acting proteins previously suspected to be E2F targets, including DNA polymerase alpha, thymidylate synthase, proliferating cell nuclear antigen, and ribonucleotide reductase, are indeed induced by E2F1. Several other candidates, including the dihydrofolate reductase and thymidine kinase genes, were only minimally induced by E2F1. In addition to the S-phase genes, we also find that several genes believed to play regulatory roles in cell cycle progression, such as the cdc2, cyclin A, and B-myb genes, are also induced by E2F1. Moreover, the cyclin E gene is strongly induced by E2F1, thus defining an autoregulatory circuit since cyclin E-dependent kinase activity can stimulate E2F1 transcription, likely through the phosphorylation and inactivation of Rb and Rb family members. Finally, we also demonstrate that a G1 arrest brought about by gamma irradiation is overcome by the overexpression of E2F1 and that this coincides with the enhanced activation of key target genes, including the cyclin A and cyclin E genes.
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PMID:Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. 762 16

The molecular karyotype of a murine isolate of Encephalitozoon cuniculi, a microsporidian with a wide range of mammalian hosts, comprises eleven chromosomes ranging in size between 217 and 315 kb. To determine specific chromosomal markers, a partial genomic library was constructed and cloned DNA fragments were hybridized to chromosomal bands separated by pulsed-field gel electrophoresis. Most probes were assigned to single chromosomes, indicating prevalence of low-copy number nucleotide sequences within the very small genome of E. cuniculi (2.9 Mb). A few probes were shown to hybridize to all chromosomes. These repetitive DNA fragments corresponded to either rRNA genes or some non-coding regions whose sequences were characterized by short micro- and minisatellites. The chromosomal locations of beta-tubulin genes and six newly identified protein-encoding genes were determined. Genes encoding dihydrofolate reductase, thymidylate synthase, serine hydroxymethyl transferase, a cdc2 kinase-like protein and helicase ERCC6-like protein were each located on a single chromosome whereas genes for both beta-tubulin and aminopeptidase were on two different chromosomes. The mapping will serve as a reference for further analysis of intraspecific karyotype polymorphism in different isolates from different host species.
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PMID:Mapping of repetitive and non-repetitive DNA probes to chromosomes of the microsporidian Encephalitozoon cuniculi. 921 May 86

Tomudex (ZD1694) is a specific antifolate-based thymidylate synthase inhibitor active in a variety of solid tumor malignancies. Studies were carried out in vitro to evaluate downstream molecular alterations induced as a consequence of the potent and sustained inhibition of thymidylate synthase by Tomudex. Twenty-four hours following the initial 2-h treatment with Tomudex, human A253 head and neck squamous carcinoma cells, not expressing p53 and p21(WAF1), were accumulated with DNA content characteristic of early S phase of the cell cycle with a concomitant reduction of cells in G1 and G2/M phases. The changes in cyclin and cdk protein expression and their kinase activities were examined in control and drug-treated A253 cells. Tomudex treatment resulted in the decrease in p27(kip1) expression, with an increase in cyclin E and cdk2 protein expression and kinase activities 24 h after a 2-h exposure. Although cyclin A protein expression was markedly increased, cyclin A kinase activity was only slightly increased. Cyclin D1, cyclin B, cdk4, and cdc2 protein expression and kinase activities remain constant. Lack of activation of cyclin A- and B-cdc2 was associated with a reduced proportion of cells in G2/M phases. Increased cyclin E-cdk2 protein expression was accompanied by the inhibition of DNA synthesis, with a decrease in E2F-1 expression. These results propose that cyclin E-cdk2 kinase can negatively regulate DNA replication. The studies with dThyd rescue from cyclin E-cdk2 protein overexpression and growth inhibition by Tomudex indicate that increased cyclin E-cdk2 protein expression is associated with effective inhibition of thymidylate synthase and resultant dNTP pool imbalance. Provision of dThyd more than 24 h after exposure to Tomudex allowed cells to replicate DNA for a single cycle back to G1, but did not prevent the profound growth-inhibitory effect manifested in the following 5 days. Tomudex treatment resulted in a time-dependent induction of the megabase DNA fragments, followed by secondary 50- to 300-kb DNA fragmentation. The 50- to 300-kb DNA fragmentation may be derived from the inhibition of DNA synthesis associated with cyclin E-cdk2 activation. These results suggest that the megabase DNA fragmentation is induced as a consequence of inhibition of thymidylate synthase by Tomudex and kilobase DNA fragmentation may correlate with the reduction of p27(kip1) expression and the increase in cyclin E and cdk2 kinase activities. Activation of cyclin E and cdk2 kinases allows cells to transit from G1 to S phase accompanied by the inhibition of DNA synthesis. The changes in cell cycle regulatory proteins associated with growth inhibition and DNA damage by Tomudex are not p53 dependent.
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PMID:Cyclin E-cdk2 activation is associated with cell cycle arrest and inhibition of DNA replication induced by the thymidylate synthase inhibitor Tomudex. 1004 61

Mechanisms of resistance to Tomudex include increased thymidylate synthase activity, as well as reduced intracellular drug uptake and polyglutamation. However, little is known about other mechanisms of resistance, such as a possible protection against Tomudex-induced apoptosis mediated by bcl-2. We transfected the MDA-MB-435 human breast cancer cell line, which is characterized by a mutated p53 gene, with cDNA of the bcl-2 gene and generated two clones (MDA-bcl4 and MDA-bcl7) characterized by bcl-2 expression twofold and fourfold that observed in the control cell clone (MDAneo). A concomitant overexpression of p21wafl was also detected in the MDA-bcl7 clone. The MDA-bcl4 clone was three times more resistant to a 24-h Tomudex exposure than the MDAneo clone, whereas the MDA-bcl7 clone was as sensitive to Tomudex as the control cell clone. A lower sensitivity of the MDA-bcl4 clone than MDAneo and MDA-bcl7 clones to 5-fluorouracil and gemcitabine was also observed. No significant difference was noted in the susceptibility of clones to fludarabine and methothrexate. Basal levels of thymidylate synthase activity were superimposable in the three clones. Tomudex induced a marked accumulation of cells in the S phase in all the clones. However, an apoptotic hypodiploid DNA peak and the characteristic nuclear morphology of apoptosis were observed only in the MDA-bcl7 clone after exposure to Tomudex. No difference in the treatment-induced modulation of proteins involved in cell cycle progression (cyclin A, cdk2, pRB, E2F-1) and apoptosis (bcl-2, bax) was observed in the three clones. The only exception was that the expression of p21wafl in the MDA-bcl4 clone was inducible at a Tomudex concentration much higher than that required to induce the protein in the other clones. Overall, the results indicate that bcl-2 and p21wafl proteins concur in determining the cellular profile of sensitivity/resistance to Tomudex.
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PMID:Involvement of bcl-2 and p21waf1 proteins in response of human breast cancer cell clones to Tomudex. 1049 50

The main objective of this study to analyze which of 31 cellular factors (resistance proteins, proliferative factors, apoptotic factors, angiogenic factors, proto-oncogenes) most accurately predict the resistance of non-small cell lung carcinomas. To this purpose, we used a short-term in vitro test that measures changes in the rate at which radioactive nucleic acid precursors are incorporated into tumor cells after the addition of doxorubicin to determine the response to doxorubicin in 94 non-small cell lung carcinomas. The results obtained by the short-term test were related to the various cellular factors which were in turn determined by immunohistochemistry and flow cytometry. A significant correlation was found between the data obtained by the short-term test and the expression of P-glycoprotein 170 (P = 0.00004), glutathione-S-transferase-pi (P = 0.0002), metallothionein (P = 0.0008), thymidylate synthase (P = 0.002), O6-methylguanine-DNA-methyltransferase (P = 0.008) and lung resistance-related protein (LRP, P = 0.03). There was only a weak correlation between heat shock proteins (HSP70) and no correlation between the expression of topoisomerase II or catalase and the short-term test results. To measure the proliferative activity, the following were determined: PCNA, cyclin A, cyclin D and cdk2. Only a weak relationship was found between the expression of cdk2 (P = 0.04) and PCNA (P = 0.05) and the doxorubicin response in vitro. Of the investigated pro-apoptotic factors (Fas/CD95, Fas ligand, caspase-3), only Fas/CD95 is significantly associated with the drug response (P = 0.007). The apoptotic index also reveals a significant correlation (P = 0.03). Angiogenesis, as measured by the microvessel density and the angiogenic factors, is inversely correlated to the resistance of non-small cell lung cancer. Platelet-derived endothelial cell growth factor (PD-ECGF) and vascular endothelial growth factor (VEGF) exhibit a significant relationship to the drug resistance (P = 0.0006 and P = 0.004, respectively). Of the investigated proto-oncogenes (Fos, Jun, ErbB-1, ErbB-2, Myc, Ras), only ErbB-2 is weakly associated with the in vitro short term test. In order to determine whether combining factors can result in improved predictive information, combinations of the factors (pairs, triplets) were analyzed. The systematic investigation of these combinations yields an improvement in the predictive information. With one factor up to 76.6% of the tumors, with two factors up to 85.4% and with three factors up to 89.5% of the tumors could be correctly diagnosed.
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PMID:Cellular predictive factors for the drug response of lung cancer. 1113 47

Aryl hydrocarbon receptor (AhR) agonists inhibit 17beta-estradiol (E2) induced growth of MCF-7 human breast cancer cells in vitro and rodent mammary tumor growth in vivo. Genes associated with inhibitory AhR-estrogen receptor (ER) crosstalk were investigated in MCF-7 human breast cancer cells using poly(A)(+)RNA from cells treated with either 1 nM E2 (target) or E2 plus 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (reference) or 25 microM diindolylmethane (DIM) as AhR agonists in MCF-7 cells. Suppression subtractive hybridization (SSH) was subsequently used to identify 33 genes with sequence homology to known human genes that are induced by E2 and inhibited by AhR agonists in MCF-7 cells; two unknown genes were also identified. Many of these genes are involved in cell proliferation and these include cell cycle regulators (cdc28/cdc2-associated protein), nucleotide synthases (thymidylate synthase), early intermediate genes (early growth response alpha, EGRalpha) and other proteins involved in signaling pathways (calmodulin, ATP synthase alpha subunit). Thus SSH has identified a diverse spectrum of new genes that are affected by inhibitory AhR-ER crosstalk and among this group are a subset of genes that may be critical for the in vivo antitumorigenic effects of AhR agonists.
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PMID:Identification of estrogen-induced genes downregulated by AhR agonists in MCF-7 breast cancer cells using suppression subtractive hybridization. 1117 85

NB1011, a phosphoramidate derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine, is a novel anti-cancer agent that selectively targets tumor cells expressing high levels of thymidylate synthase (TS), an enzyme required for DNA biosynthesis. NB1011 treatment of high-TS-expressing breast carcinoma cells (MCF7TDX) results in the induction of p53 and p21 protein levels, whereas no p53 or p21 induction is observed in the low-TS-expressing MCF7 tumor cells. Furthermore, MCF7TDX cells accumulate in the G(2)/M phase of the cell cycle in response to NB1011. In this study, the effect of NB1011 on the phosphorylation status of p53 was analyzed. We demonstrate that NB1011 treatment of various tumor cell lines expressing high TS results in the phosphorylation of p53 on Ser15, whereas this p53 phosphorylation is not observed in low-TS-expressing tumor cells. Also, we examined the role of several key cell cycle regulators in the growth inhibition observed in response to NB1011. Our results show that the mRNA and protein levels of the G(2)/M regulators cdc2, cyclin B1 and cdc25C are down-regulated in MCF7TDX cells, while unaffected in MCF7 cells. The mRNA and protein levels of 14-3-3sigma, also a direct transcriptional target of p53, are up-regulated in MCF7TDX cells following NB1011 treatment, while unchanged in MCF7 cells. Taken together, our data indicate that the growth inhibition caused by NB1011 in MCF7TDX cells is mediated through phosphorylation of p53 and activation of the G(2)/M checkpoint.
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PMID:NB1011 induces Ser15 phosphorylation of p53 and activates the G2/M checkpoint. 1285 88

We have demonstrated previously that interferon (IFN)-gamma sensitizes human colon carcinoma cell lines to the cytotoxic effects of 5-fluorouracil combined with leucovorin and to the thymidylate synthase inhibitor, ZD9331, dependent on thymineless stress-induced DNA damage, independent of p53. Here we demonstrate that the cyclin-dependent kinase (CDK) inhibitor p21(Cip1) regulates thymineless stress-induced cytotoxicity in these cells. HCT116 wild-type (wt) and p53-/- cells underwent apoptosis and loss in clonogenic survival when exposed to ZD9331, whereas p21Cip1-/- cells were resistant. In contrast, IFN-gamma induced marked cytotoxicity in p21Cip1-/- cells only. ZD9331 induced p21Cip1 up-regulation in all of the cell lines examined, as did thymidine deprivation in thymidylate synthase-deficient (thymidylate synthase-) cells. Furthermore, selective induction of p21Cip1 in RKO was sufficient to induce apoptosis. P21Cip1, cdk1, cdk2, and cyclin E mRNA expression increased coincident with S-phase accumulation in HT29 cells treated with ZD9331 or 5fluorouracil/leucovorin, as demonstrated by cDNA microarray analyses. Cell cycle analyses revealed that HCT116 wt and p21Cip1 -/- cells accumulated in S phase within 24 h of ZD9331 exposure; however, wt cells exited S-phase more rapidly, where apoptosis occurred before mitosis, either in late S or G2. Finally, the CDK inhibitor roscovitine potentiated the cytotoxic activity of ZD9331 in both wt and p21Cip1-/- cells, strongly suggesting a role for p21Cip1-dependent CDK inhibition in cytotoxicity induced by thymidylate synthase inhibition. In summary, p21Cip1 positively regulates the cytotoxic action of thymidylate synthase inhibitors, negatively regulates the cytotoxic action of IFN-gamma, and enhances S-phase exit after thymineless stress, possibly via interaction with CDK-cyclin complexes.
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PMID:P21Cip1 is a critical mediator of the cytotoxic action of thymidylate synthase inhibitors in colorectal carcinoma cells. 1534 18

Transcription factor LSF is required for progression from quiescence through the cell cycle, regulating thymidylate synthase (Tyms) expression at the G(1)/S boundary. Given the constant level of LSF protein from G(0) through S, we investigated whether LSF is regulated by phosphorylation in G(1). In vitro, LSF is phosphorylated by cyclin E/cyclin-dependent kinase 2 (CDK2), cyclin C/CDK2, and cyclin C/CDK3, predominantly on S309. Phosphorylation of LSF on S309 is maximal 1 to 2 h after mitogenic stimulation of quiescent mouse fibroblasts. This phosphorylation is mediated by cyclin C-dependent kinases, as shown by coimmunoprecipitation of LSF and cyclin C in early G(1) and by abrogation of LSF S309 phosphorylation upon suppression of cyclin C with short interfering RNA. Although mouse fibroblasts lack functional CDK3 (the partner of cyclin C in early G(1) in human cells), CDK2 compensates for this absence. By transient transfection assays, phosphorylation at S309, mediated by cyclin C overexpression, inhibits LSF transactivation. Moreover, overexpression of cyclin C and CDK3 inhibits induction of endogenous Tyms expression at the G(1)/S transition. These results identify LSF as only the second known target (in addition to pRb) of cyclin C/CDK activity during progression from quiescence to early G(1). Unexpectedly, this phosphorylation prevents induction of LSF target genes until late G(1).
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PMID:Phosphorylation by cyclin C/cyclin-dependent kinase 2 following mitogenic stimulation of murine fibroblasts inhibits transcriptional activity of LSF during G1 progression. 1923 34