Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have studied changes in cyclin A- and B1-dependent kinases during apoptosis induced in human promyelocytic leukemia (HL60) cells treated with the topoisomerase I inhibitor camptothecin. We found that cyclin B1/Cdc2 kinase activity transiently increases within 30 min after camptothecin treatment. This increase is followed by a rapid inactivation of the cyclin B1/Cdc2 kinase that is associated with Cdc2 tyrosine phosphorylation without any change in Cdc2 or cyclin B1 protein levels. The DNA polymerase inhibitor aphidicolin abrogates camptothecin-induced changes in cyclin B1/Cdc2 kinase activity, indicating that DNA replication-induced DNA damage is essential for both Cdc2 alterations and apoptosis activation. Apoptosis and the initial cyclin B1/Cdc2 kinase activation were amplified using synchronized S-phase cells, and cyclin A/cdk2 kinase did not change under these conditions. The same transient activation and subsequent inactivation of cyclin B1/Cdc2 kinase were observed after DNA damage by etoposide or bis-(2-chloroethyl)methylamine hydrochloride. These observations suggest that DNA damage promotes the transient and unscheduled stimulation of cyclin B1/Cdc2 kinase activity in HL60 cells prior to apoptosis.
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PMID:Unscheduled activation of cyclin B1/Cdc2 kinase in human promyelocytic leukemia cell line HL60 cells undergoing apoptosis induced by DNA damage. 781 49

Cyclins A and B1 activate cyclin-dependent kinases CDK2 and CDC2, which regulate cell progression through S and G2. Expression of these cyclins is generally measured in populations of synchronized cells, by immunoblotting. Such studies neither provide information regarding intercellular variability in cyclin expression nor yield precise data on a time relationship between initiation and termination of DNA replication in relation to cyclin expression. Furthermore, cell synchronization by DNA polymerase inhibitors or excess of thymidine induces cell growth imbalance and alters expression of cyclins, thereby introducing an experimental bias. Using a novel flow cytometric method of detection of incorporated bromodeoxyuridine (BrdUrd) in the present study, we have been able to correlate expression of immunocytochemically discerned cyclins A and B1 with incorporation of BrdUrd and the cell cycle position of individual MOLT-4 cells. On the basis of differences in amount of incorporated BrdUrd and DNA content, the following cohorts of cells in narrow windows of the cell cycle were identified: (a) cells initiating and (b) terminating DNA replication during a 1-h pulse of BrdUrd; (c) cells replicating DNA throughout the duration of BrdUrd pulse; (d) G1 cells; and (e) G2 cells that remained in G2 for at least 1 h after exiting S phase. These populations were characterized with respect to expression of cyclins A and B1. Expression of cyclin A was an early event of S phase, and 84% of cells entering S phase during 1 h of exposure to BrdUrd were already cyclin A positive. More than 95% of S-phase cells, as well as the cells exiting S during BrdUrd pulse, were also cyclin A positive. The maximal rate of accumulation of cyclin A was seen during the first hour of progression through S phase. In contrast, the maximal accumulation rate of cyclin B1 showed cells during the first hour of progression through G2. A strong correlation between expression of cyclin A and the rate of DNA replication, estimated by the degree of BrdUrd incorporation (r = 0.99), was observed.
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PMID:Correlation between DNA replication and expression of cyclins A and B1 in individual MOLT-4 cells. 904 Nov 74

We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G(2)/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes.
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PMID:Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins. 1200

Most DNA synthesis in HeLa cell nucleus is concentrated in discrete foci. These synthetic sites can be identified by electron microscopy after allowing permeabilized cells to elongate nascent DNA in the presence of biotin-dUTP. Biotin incorporated into nascent DNA can be then immunolabeled with gold particles. Two types of DNA synthetic sites/replication factories can be distinguished at ultrastructural level: (1) electron-dense structures--replication bodies (RB), and (2) focal replication sites with no distinct underlying structure--replication foci (RF). The protein composition of these synthetic sites was studied using double immunogold labeling. We have found that both structures contain (a) proteins involved in DNA replication (DNA polymerase alpha, PCNA), (b) regulators of the cell cycle (cyclin A, cdk2), and (c) RNA processing components like Sm and SS-B/La auto antigens, p80-coilin, hnRNPs A1 and C1/C2. However, at least four regulatory and structural proteins (Cdk1, cyclin B1, PML and lamin B1) differ in their presence in RB and RF. Moreover, in contrast to RF, RB have structural organization. For example, while DNA polymerase alpha, PCNA and hnRNP A1 were diffusely spread throughout RB, hnRNP C1/C2 was found only at the very outside. Surprisingly, RB contained only small amounts of DNA. In conclusion, synthetic sites of both types contain similar but not the same sets of proteins. RB, however, have more developed microarchitecture, apparently with specific functional zones. This data suggest possible differences in genome regions replicated by these two types of replication factories.
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PMID:The microarchitecture of DNA replication domains. 1624 14

Here we examined the role of cellular vitamin C in genotoxicity of carcinogenic chromium(VI) that requires reduction to induce DNA damage. In the presence of ascorbate (Asc), low 0.2-2 microM doses of Cr(VI) caused 10-15 times more chromosomal breakage in primary human bronchial epithelial cells or lung fibroblasts. DNA double-strand breaks (DSB) were preferentially generated in G2 phase as detected by colocalization of H2AX and 53BP1 foci in cyclin B1-expressing cells. Asc dramatically increased the formation of centromere-negative micronuclei, demonstrating that induced DSB were inefficiently repaired. DSB in G2 cells were caused by aberrant mismatch repair of Cr damage in replicated DNA, as DNA polymerase inhibitor aphidicolin and silencing of MSH2 or MLH1 by shRNA suppressed induction of H2AX and micronuclei. Cr(VI) was also up to 10 times more mutagenic in cells containing Asc. Increasing Asc concentrations generated progressively more mutations and DSB, revealing the genotoxic potential of otherwise nontoxic Cr(VI) doses. Asc amplified genotoxicity of Cr(VI) by altering the spectrum of DNA damage, as total Cr-DNA binding was unchanged and post-Cr loading of Asc exhibited no effects. Collectively, these studies demonstrated that Asc-dependent metabolism is the main source of genotoxic and mutagenic damage in Cr(VI)-exposed cells.
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PMID:Ascorbate acts as a highly potent inducer of chromate mutagenesis and clastogenesis: linkage to DNA breaks in G2 phase by mismatch repair. 1716 90

Curcumin has been shown to inhibit the growth of various types of cancer cells; however, at concentrations much above the clinically achievable levels in humans. The concentration of curcumin achieved in the plasma after oral administration in humans was estimated to be around 1.8 microM. Here, we report that treatment of BxPC-3 human pancreatic cancer cells with a low and single exposure of 2.5 microM curcumin for 24 h causes significant arrest of cells in the G2/M phase and induces significant apoptosis. Immunoblot studies revealed increased phosphorylation of H2A.X at Ser-139 and Chk1 at Ser-280 and a decrease in DNA polymerase-beta level in curcumin-treated cells. Phosphorylation of H2A.X and Chk1 proteins are an indicator of DNA damage whereas DNA polymerase-beta plays a role in the repair of DNA strand breaks. Normal immortalised human pancreatic ductal epithelial (HPDE-6) cells remained unaffected by curcumin treatment. In addition, we also observed a significant increase in the phosphorylation of Chk1 at Ser-345, Cdc25C at Ser-216 and a subtle increase in ATM phosphorylation at Ser-1981. Concomitant decrease in the expressions of cyclin B1 and Cdk1 were seen in curcumin-treated cells. Further, curcumin treatment caused significant cleavage of caspase-3 and PARP in BxPC-3 but not in HPDE-6 cells. Silencing ATM/Chk1 expression by transfecting BxPC-3 cells with ATM or Chk1-specific SiRNA blocked the phosphorylation of ATM, Chk1 and Cdc25C and protected the cells from curcumin-mediated G2/M arrest and apoptosis. This study reflects the critical role of ATM/Chk1 in curcumin-mediated G2/M cell cycle arrest and apoptosis in pancreatic cancer cells.
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PMID:Activation of ATM/Chk1 by curcumin causes cell cycle arrest and apoptosis in human pancreatic cancer cells. 1940 1

Varicella-zoster virus (VZV) replicates in quiescent T cells, neurons, and skin cells. In cultured fibroblasts (HFFs), VZV induces host cyclin expression and cyclin-dependent kinase (CDK) activity without causing cell cycle progression. CDK1/cyclin B1 phosphorylates the major viral transactivator, and the CDK inhibitor roscovitine prevents VZV mRNA transcription. We investigated the antiviral effects of additional compounds that target CDKs or other cell cycle enzymes in culture, ex vivo, and in vivo. Cytotoxicity and cell growth arrest doses were determined by Neutral Red assay. Antiviral effects were evaluated in HFFs by plaque assay, genome copy number, and bioluminescence. Positive controls were acyclovir (400 microM) and phosphonoacetic acid (PAA, 1 mM). Test compounds were roscovitine, aloisine A, and purvalanol A (CDK inhibitors), aphidicolin (inhibits human and herpesvirus DNA polymerase), l-mimosine (indirectly inhibits human DNA polymerase), and DRB (inhibits casein kinase 2). All had antiviral effects below the concentrations required for cell growth arrest. Compounds were tested in skin organ culture at EC(99) doses; all prevented VZV replication in skin, except for aloisine A and purvalanol A. In SCID mice with skin xenografts, roscovitine (0.7 mg/kg/day) was as effective as PAA (36 mg/kg/day). The screening systems described here are useful models for evaluating novel antiviral drugs for VZV.
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PMID:Compounds that target host cell proteins prevent varicella-zoster virus replication in culture, ex vivo, and in SCID-Hu mice. 2030 80