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)

DNA mismatch repair (MMR) corrects DNA polymerase insertion errors that have escaped proofreading in order to avoid the accumulation of deleterious mutations. While the role of MMR in the correction of replication errors is well established, its involvement in the processing of DNA damage induced by chemical and physical agents is less clear. A role for some of the MMR proteins, such as MSH2, in the repair of double strand break (DSBs) through recombination has also been envisaged. Why MMR- deficient cells are sensitive to agents causing replication fork stalling and thus DSBs remains unclear. To verify a possible role of MSH2 in homologous recombinational repair, we have treated cells from knockout mice for the MSH2 gene and mouse colorectal carcinoma cells also defective for MSH2 with different doses of camptothecin, an agent known to interfere with DNA replication. In the absence of MSH2, we found a reduced survival rate accompanied by higher levels of chromosomal damage and SCE induction. Furthermore, MSH2(-/-) cells displayed an elevated spontaneous RAD51 focus-forming activity and a higher induction of RAD51 foci following camptothecin treatment. Thus, the absence of MSH2 could result in both spontaneous DNA damage and uncontrolled recombination events leading to the observed higher yield of chromosomal damage and the higher induction of RAD51 foci following CPT treatment. Therefore, our results suggest an involvement of MSH2 in the early events leading to correct RAD51 relocalization after the formation of DSBs specifically produced at the blocked replication fork.
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PMID:Hypersensitivity to camptothecin in MSH2 deficient cells is correlated with a role for MSH2 protein in recombinational repair. 1169 39

A 1.7-kb cDNA clone, pGEM-cDP, was isolated from a cDNA library of IUdR-induced p3HR1 cells. It contains the upstream nucleotide sequence of the Epstein-Barr virus (EBV) DNA polymerase gene from 156,859 to 155,088, and was subcloned into expression vector pET3cp* by the polymerase chain reaction, giving the plasmid pDP1. Using a T7 RNA polymerase expression system, a 77-kD polypeptide was produced from pDP1 in Escherichia coli and specific hyperimmune serum was generated in mice. The truncated EBV DNA polymerase was shown to possess the authentic antigenicity by an indirect immunofluorescence assay and by immunoblotting using EBV-containing cells as antigens. Serum from nasopharyngeal carcinoma (NPC) patients and healthy donors was examined for antibodies against the 77-kD polypeptide by Western blot analyses and ELISAs. About 70% NPC patients were positive, while less than 15% of healthy persons showed weak reactivities in ELISAs. Copyright 1994 S. Karger AG, Basel
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PMID:Serological Responses of Patients with Nasopharyngeal Carcinoma to an N-Terminal Epstein-Barr Virus DNA Polymerase Protein Expressed in Prokaryotic Cells. 1172 14

The complementary-strand transcripts from Epstein-Barr virus BamHI-A-I fragments (also called the BARF0 RNAs) were first found in a nasopharyngeal carcinoma passaged in nude mice, c15, and soon proved to exist in various EBV-associated tumors and virus-carrying B cell lines. The data from previous studies revealed that the BARF0 transcripts have a highly spliced structure, whereas the evidence about their in vivo translational products remains few. The BARF0 RNA is also somehow considered as a new member of the viral latent transcripts. In order to know further about its transcriptional profile, we investigated its transcription in several EBV-carrying B cell lines during different viral stages. Our results show that (1) at least some of the BARF0 transcripts are transcribed during the viral lytic stage in the tested inducible lines; (2) the major lytic BARF0 mRNA appears heterogeneous in size (2.5 to 4.2 kb) among different lines; (3) in P3HR1 and Akata lines, the major BARF0 transcript is highly spliced and sensitive to both an inhibitor of the viral DNA polymerase and of protein synthesis. The identification of such lytic BARF0 RNAs may help us to understand further about their complex gene expression.
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PMID:Expression of the complementary-strand transcripts from BamHI-A region of the Epstein-Barr virus genome in various induced virus-carrying B cell lines. 1183 98

We asked whether the constitutive level of DNA strand breaks (SBs) in four human squamous carcinoma cell lines is associated with their radiosensitivity, measured by the clonogenic assay. Because impairment in DNA replication and the action of endogenous deoxyribonucleases are two major sources of DNA strand breaks under normal cell metabolism, we also analyzed DNA polymerase and DNA ligase activities as well as the functional status of Poly(ADP-ribose) polymerase (PARP) and nucleolytic degradation of genomic DNA. We showed that the two relatively radioresistant cell lines, UM-SCC-1 and UT-SCC-5, had a statistically significant lower constitutive level of DNA SBs, as measured by DNA precipitation technique, compared with the two relatively radiosensitive cell lines, UM-SCC-14A and UT-SCC-9. We found that cell lines with a higher level of broken DNA tended to have a higher constitutive level of DNA polymerase alpha activity, measured by incorporation of [(3)H]dTTP in DNase I-activated DNA. UM-SCC-1, UT-SCC-5, and UM-SCC-14A did not show any difference in DNA ligase activity when a nicked oligonucleotide was used as substrate. The most radiosensitive cell line, UT-SCC-9, had a significantly lower ligation efficiency compared to the other three cell lines. The functional status of the PARP was the same in the four cell lines. Although none of the four cell lines showed a characteristic apoptotic or necrotic degradation of genomic DNA, when tested with the "plasmid rejoining assay," a significant degradation of the plasmid DNA in UT-SCC-9 was detected. We conclude that the high fraction of DNA SBs for UT-SCC-9, the most radiosensitive cell line, is most likely a consequence of low ligation efficiency combined with a relatively high DNA polymerase alpha activity and the nuclease degradation of DNA.
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PMID:Radiosensitivity of human squamous carcinoma cell lines is associated with amount of spontaneous DNA strand breaks. 1199 85

Single nucleotide instability (SNI), an increase in spontaneous point mutation rates (MRs) without involvement of microsatellite instability, is present in rat mammary carcinoma cell lines and human breast cancer cell lines. A:T to C:G transversions, which are generally rare, were frequently observed in two rat mammary carcinoma cell lines and in their primary carcinomas, and were considered to be related to the molecular mechanism of SNI. In this study, two known molecular mechanisms that cause increases of A:T to C:G transversions, inactivation of the MutT mammalian homologue (Mth1) gene and overexpression of the DNA polymerase k (Pol k) gene, were analyzed in two rat mammary carcinoma cell lines and 11 rat primary carcinomas. PCR-SSCP analysis revealed no mutations in the entire Mth1 coding region. Quantitative real-time RT-PCR analysis showed that Mth1 mRNA expression was slightly, but significantly, increased in the primary carcinomas (P = 0.001 using GAPDH for normalization, and P = 0.002 using histone H4, t-test), contrary to our expectation, and was decreased to 1 / 2 in the cell lines. The expression of Pol k, which is known to be error-prone with frequent A:T to C:G transversions, was rather decreased in the cell lines and primary carcinomas. Inactivation of Mth1 and overexpression of Pol k were unlikely to have caused SNI in the two rat mammary carcinoma cell lines with a high frequency of A:T to C:G transversions, and searching for other unknown molecular mechanisms is important.
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PMID:The absence of Mth1 inactivation and DNA polymerase kappa overexpression in rat mammary carcinomas with frequent A:T to C:G transversions. 1203 45

Lethal lesions after ionizing radiation are thought to be mainly unrepaired or misrepaired DNA double-strand breaks, ultimately leading to lethal chromosome aberrations. However, studies with radioprotectors and repair inhibitors indicate that single-strand breaks, damaged nucleotides or abasic sites can also influence cell survival. This paper reports on studies to further define the role of base damage and base excision repair on the radiosensitivity of human cells. We retrovirally transduced human tumor cells with a dominant negative form of DNA polymerase beta, comprising the 14 kDa DNA-binding domain of DNA polymerase beta but lacking polymerase function. Radiosensitization of two human carcinoma cell lines, A549 and SQD9, was observed, achieving dose enhancement factors of 1.5-1.7. Sensitization was dependent on expression level of the dominant negative and was seen in both single cell clones and in unselected virally transduced populations. Sensitization was not due to changes in cell cycle distribution. Little or no sensitization was seen in G(1)-enriched populations, indicating cell cycle specificity for the observed sensitization. These results contrast with the lack of effect seen in DNA polymerase beta knockout cells, suggesting that polDN also inhibits the long patch, DNA polymerase beta-independent repair pathway. These data demonstrate an important role for BER in determining sensitivity to ionizing radiation and might help identify targets for radiosensitizing tumor cells.
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PMID:The role of DNA polymerase beta in determining sensitivity to ionizing radiation in human tumor cells. 1208 86

The sensitive detection of human cells in immunodeficient rodents is a prerequisite for the monitoring of micrometastasis of solid tumours, dissemination of leukaemic cells, or engraftment of haematological cells. We developed a universally applicable polymerase chain reaction method for the detection of a human-specific 850-bp fragment of the alpha-satellite DNA on human chromosome 17. The method allows the detection of one human cell in 10(6) murine cells and could be established as both, a conventional DNA polymerase chain reaction-assay for routine screening, and a quantitative real-time polymerase chain reaction-assay using TaqMan-methodology. It was applied to the following xenotransplantation systems in SCID and NOD/SCID mice: (1) In a limiting dilution assay, cells of the MDA-MB 435 breast carcinoma were injected into the mammary fat pad of NOD/SCID mice. It could be shown that 10 cells mouse(-1) were sufficient to induce a positive polymerase chain reaction signal in liver and lung tissue 30 days after transplantation as an indicator for micrometastasis. At this time a palpable tumour was not yet detectable in the mammary fat pad region. (2) Cells of a newly established human acute lymphatic leukaemia were administered intraperitoneally to SCID mice. These cells apparently disseminated and were detectable as early as day 50 in the peripheral blood of living mice, while the leukaemia manifestation was delayed by day 140. (3) In a transplantation experiment using mature human lymphocytes we wanted to standardise conditions for a successful survival of these cells in NOD/SCID mice. It was established that at least 5 x 10(7) cells given intravenously were necessary and that the mice had to be conditioned by 2 Gy body irradiation to get positive polymerase chain reaction bands in several organs. (4) Engraftment studies with blood stem cells originating from cytapheresis samples of tumour patients or from cord blood were undertaken in NOD/SCID mice in order to define conditions of successful engraftment and to use this model for further optimisation strategies. The polymerase chain reaction method presented allowed a reliable prediction of positive engraftment and agreed well with the results of immunohistochemical or FACS analysis. All together, the polymerase chain reaction method developed allows a sensitive and reliable detection of low numbers of human cells in immunodeficient hosts. In combination with real-time (TaqMan) technique it allows an exact quantification of human cells. As this method can be performed with accessible material of living animals, follow up studies for the monitoring of therapeutic interventions are possible in which the survival time of mice as evaluation criteria can be omitted.
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PMID:Sensitive PCR method for the detection and real-time quantification of human cells in xenotransplantation systems. 1243 25

Thyroid hormone induces differentiation of many different tissues in mammals, birds, and amphibians. The different tissues all differentiate from proliferating precursor cells, and the normal cell cycle is suspended while cells undergo differentiation. We have investigated how thyroid hormone affects the expression of the E2F-1 protein, a key transcription factor that controls G1- to S-phase transition. We show that during thyroid hormone-induced differentiation of embryonic carcinoma cells and of oligodendrocyte precursor cells, the levels of E2F-1 mRNA and E2F-1 protein decrease. This is caused by the thyroid hormone receptor (TR) regulating the transcription of the E2F-1 gene. The TR binds directly to a negative thyroid hormone response element, called the Z-element, in the E2F-1 promoter. When bound, the TR activates transcription in the absence of ligand but represses transcription in the presence of ligand. In addition, liganded TR represses transcription of the S-phase-specific DNA polymerase alpha, thymidine kinase, and dihydropholate reductase genes. These results suggest that thyroid hormone-induced withdrawal from the cell cycle takes place through the repression of S-phase genes. We suggest that this is an initial and crucial step in thyroid hormone-induced differentiation of precursor cells.
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PMID:Hormone-dependent repression of the E2F-1 gene by thyroid hormone receptors. 1251 8

Vanada- and niobatricarbadecaboranyl monohalide complexes proved to be potent cytotoxic agents against murine and human leukemia and lymphoma growth as well as HeLa suspended uterine carcinoma. The vanada complex reduced the growth of KB nasopharynx, Hepe liver, HCT-8 ileum and 1-A9 ovary solid carcinomas. A mode of action study in human HL-60 promyelocytic leukemia cells showed that DNA and purine de novo syntheses were significantly inhibited with suppression of the regulatory enzymes activities of DNA polymerase alpha and PRPP-amido transferase. There was moderate inhibition of RNA synthesis and m-RNA polymerase activity. These complexes did not inhibit human topoisomerase I or II activity, although the niobium complex nicked the DNA. The complexes did activate caspases 3, 6 and 9 which are linked to apoptosis programmed cell death. These vanada- and niobatricarbadecaboranyl monohalide complexes appear to be more specific in their effects on leukemia cell metabolism than other sandwich complexes which have broad effects on multiple enzymes.
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PMID:Cytotoxicity and mode of action of vanada- and niobatricarbadecaboranyl monohalide complexes in human HL-60 promyelocytic leukemia cells. 1257 74

p12(CDK2-AP1) (p12) is a growth suppressor isolated from normal keratinocytes. Ectopic expression of p12 in squamous carcinoma cells reversed the malignant phenotype of these cells, in part due an ability of p12 to bind to both DNA polymerase alpha/primase and to cyclin-dependent kinase 2 (CDK2), thereby inhibiting their activities. We report in this article that in normal epithelial cells, transforming growth factor beta1 (TGF-beta1) induces p12 expression transcriptionally, which, in turn, mediates the growth inhibitory activity of TGF-beta1. We created inducible p12 antisense HaCaT cell lines [ip12 (-) HaCaT] and showed that selective reduction of cellular p12 resulted in an increase in: (a) CDK2-associated kinase activity; (b) protein retinoblastoma (pRB) phosphorylation; and (c) [(3)H]thymidine incorporation, and partially reversed TGF-beta1-mediated inhibition of CDK2 kinase activity, pRB phosphorylation, and cell proliferation. Furthermore, we generated p12-deficient mouse oral keratinocytes (MOK(p12-/-)) and compared their growth characteristics and response to TGF-beta1 with that of wild-type mouse oral keratinocytes (MOK(WT)). Under normal culture conditions, the number of MOK(p12-/-) in S phase is 2-fold greater than that of MOK(WT). Concomitantly, fewer cells are in G(2) phase in MOK(p12-/-) than that in MOK(WT). Moreover, response to TGF-beta1-mediated growth suppression is compromised in MOK(p12-/-) cells. Mechanistic studies showed that MOK(p12-/-) have increased CDK2 activity and reduced sensitivity to inhibition by TGF-beta1. Collectively our data suggest that p12 plays a role in TGF-beta1-mediated growth suppression by modulating CDK2 activities and pRB phosphorylation.
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PMID:Role of p12(CDK2-AP1) in transforming growth factor-beta1-mediated growth suppression. 1474 61


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