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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

2'-Fluoro-5-methyl-beta-L-arabinofuranosyl uracil (L-FMAU) was discovered to have potent antiviral activity against hepatitis B virus (HBV). L-FMAU was more potent than its D-enantiomer and produced dose-dependent inhibition of the viral DNA replication in 2.2.15 cells (human HepG2 cells with the HBV genome), with a 50% inhibitory concentration of 0.1 microM. There was no inhibitory effect on HBV transcription or protein synthesis. In the 2.2.15 cell system, L-FMAU did not show any toxicity up to 200 microM, whereas the D-enantiomer was toxic, with a 50% inhibitory concentration of 50 microM. Repeated treatments of HepG2 cells with L-FMAU at a 1 microM concentration for 9 days did not result in any decrease in the total mitochondrial DNA content, suggesting that a mode of toxicity similar to that produced by 2',3'-dideoxycytidine is unlikely. Also at concentrations as high as 200 microM, L-FMAU did not adversely affect mitochondrial function as determined by lactic acid production by L-FMAU-treated hepatoma cells. L-FMAU was metabolized in the cells to its mono-, di-, and triphosphates, A dose-dependent inhibition of HBV DNA synthesis by L-FMAU triphosphate was observed in the DNA polymerase assays with isolated HBV particles, suggesting that the mode of action of this compound could involve viral polymerase. However, L-FMAU was not incorporated into the cellular DNA. Considering the potent inhibition of the viral DNA synthesis and the nontoxicity of L-FMAU towards the host DNA synthetic machinery, this compound should be further explored for development as asn anti-HBV drug.
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PMID:Inhibition of hepatitis B virus by a novel L-nucleoside, 2'-fluoro-5-methyl-beta-L-arabinofuranosyl uracil. 883 84

Reverse transcription polymerase chain reaction (RT-PCR) has been used to detect hepatitis C virus (HCV) sequences in liver tissue. However, RT-PCR has a variable detection sensitivity, especially on routinely processed formalin-fixed, paraffin-embedded (FFPE) specimens. RNA-RNA and RNA-protein cross-links formed during formalin fixation is the major limiting factor preventing reverse trans criptase from extending the primers. To overcome this problem, we applied the ligation-dependent PCR (LD-PCR) for the detection of HCV RNA in FFPE liver tissue. This method uses two capture probes for RNA isolation and two hemiprobes for the subsequent PCR. Despite cross-links, the capture probes and the hemiprobes are able to form hybrids with HCV RNAs released from the FFPE tissue. The hybrids are isolated through binding of the capture probes to paramagnetic beads. The hemiprobes are then ligated by a T4 DNA ligase to form a full probe that serves as a template for the Taq DNA polymerase. A total of 22 FFPE liver specimens, 21 with hepatocellular carcinoma (HCC) and 1 with biliary cirrhosis secondary to bile duct atresia were selected for this study, of which 13 patients were HCV seropositive and 9 seronegative. HCV RNA was detectable by ID-PCR from all 13 HCV-seropositive HCCs and from 5 of 8 HCV-seronegative HCCs but not from the HCV-seronegative liver with biliary atresia. By contrast, RT-PCR detected HCV sequences in only 5 of the HCV-sero-positive and in 1 of the HCV-seronegative HCCs. To resolve the discordance between the LD-PCR and RT-PCR results, RT-PCR was performed on frozen liver tissue of the discrepant specimens, which confirmed the LD-PCR positive results. In conclusion, LD-PCR is a more sensitive method than RT-PCR for the detection of HCV sequences in routinely processed liver tissues. A high rate of HCV infection (86%) is found in HCC specimens, indicating a previously underestimated role of HCV in HCC pathogenesis.
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PMID:Detection of hepatitis C virus RNA using ligation-dependent polymerase chain reaction in formalin-fixed, paraffin-embedded liver tissues. 890 38

Repair of alkylated bases in DNA is performed by O6-methylguanine-DNA methyltransferase (MGMT) and a set of enzymes of the base excision repair pathway involving N-methylpurine-DNA glycosylase (MPG), apurinic endonuclease (APE), DNA polymerase beta (Pol beta) and DNA ligase. The level of expression of these enzymes may exert a profound effect on resistance of cells towards alkylating drugs. We have comparatively analyzed the expression of MGMT and the different base excision repair genes in rat hepatoma cells (line H4IIE) after exposure to alkylating agents, X-rays and the glucocorticoid hormone dexamethasone. Furthermore, the effect of these agents on the activity of the cloned human MGMT promoter was assayed. Exposure of cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or ionizing radiation increased MGMT mRNA levels up to 4.5-fold. Under the same conditions of treatment, exerting only a weak toxic effect, MPG and DNA ligase I mRNA levels were not enhanced, whereas the amounts of APE and Pol beta mRNA transiently increased by approximately 2-fold after X-ray and MNNG treatment, respectively. Dexamethasone induced both MGMT, APE and Pol beta mRNA and the induction paralleled the increase in mRNA of the glucocorticoid-dependent gene tyrosine aminotransferase. The observed increase in MGMT mRNA was due to promoter activation, which was shown in transient transfection assays with MGMT promoter-CAT reporter constructs in H4IIE cells. In these assays, the human MGMT promoter was found to be induced by methylating agents (MNNG and methyl methanesulfonate), ionizing radiation and dexamethasone. Weak induction of the promoter was observed after UV irradiation. Treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate was ineffective in promoter activation. The transfected MGMT promoter was not inducible by mutagens in HeLa S3 cells, which do not respond with induction of the endogenous MGMT gene. This is the first report showing hormone induction of a DNA repair gene (MGMT). The induction of MGMT and other genes encoding enzymes involved in DNA alkylation damage repair may be relevant in cancer therapy by causing resistance of tumor cells to alkylating drugs.
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PMID:Induction of the alkyltransferase (MGMT) gene by DNA damaging agents and the glucocorticoid dexamethasone and comparison with the response of base excision repair genes. 896 45

The effects of coculture and conditioned medium of rat hepatoma Reuber H-35 cells on the subsequent in vitro development and hatching of mouse 2-cell embryos were examined. The hatching of embryos obtained from CD-1 mice was accelerated by coculture with Reuber H-35 cells in the presence of 3 mg/ml BSA. The promoting effect on complete hatching from zona pellucida was evident even in cell-conditioned medium containing 60 micrograms/ml BSA. In the presence of 60 micrograms/ml BSA, more than 20% of embryos completely hatched, whereas none hatched in the control culture. The promoting activity was also found in both the M(r) < 10,000 and the M(r) > 10,000 subfractions of the conditioned medium separated by ultrafiltration. The cell number per blastocyst was increased to 1.1- to 1.3 times the control by culturing embryos from the 2-cell stage with the conditioned medium or its subfractions. The effective target of promoting factors for complete hatching was after the morula stage, and blastocysts hatched completely even when incubated in conditioned medium for 6 h. Inhibitors of DNA polymerase alpha, protein synthesis, and protein kinase partially reduced (40-90% inhibition) the promoting effect of the conditioned medium. On the other hand, protease inhibitors showed no effect. In a caseinolytic assay, protease activity was undetectable in the conditioned medium. Incubating the 125I-labeled proteins derived from the M(r) > 10,000 fraction with blastocysts revealed that at least 9 proteins with apparent molecular masses of 76, 60, 49, 38, 34, 31, 24, 22, and 18 kDa specifically bound to or accumulated in the embryos. Moreover, reverse-transcriptase polymerase chain reaction showed that Reuber H-35 cells expressed mRNAs for epidermal growth factor, transforming growth factors alpha and beta 1, and stem cell factor. These results indicated that embryonic development and the process of zona hatching was accelerated by factors synthesized by Reuber H-35 cells. This and other studies demonstrated that Reuber H-35 cells exert positive (later than 2-cell stage) and negative (at 2-cell stage) effects upon the development of mouse embryos at different embryonic stages. These factors will serve as valuable tools to clarify the proliferating and differentiating mechanisms of the preimplantation embryo.
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PMID:Rat hepatoma Reuber H-35 cells produce factors that promote the hatching of mouse embryos cultured in vitro. 909 89

Acyclovir (ACV) triphosphate and azidothymidine (AZT) triphosphate inhibit the DNA polymerase of human hepatitis B virus (HBV) by 50% at submicromolar concentrations, but no effects of ACV or AZT treatment have been noted on the clinical manifestations of hepatitis B. We synthesized 1-O-octadecyl-sn-glycero-3-phospho-acyclovir (ODG-P-ACV), 1-O-hexadecylpropanediol-3-phospho-acyclovir (HDP-P-ACV), and 1-O-octadecyl-sn-glycero-3-phospho-azidothymidine (ODG-P-AZT), and evaluated their antiviral activity in human hepatoma cells that constitutively produce HBV (2.2.15 cells). ACV and AZT up to 100 microM caused only slight inhibition of HBV replication in 2.2.15 cells. However, HDP-P-ACV and ODG-P-ACV inhibited viral replication by 50% at 0.5 and 6.8 microM, respectively. ODG-P-AZT also showed increased antiviral activity, with a 50% reduction in HBV replication at 2.1 microM. Based on the EC50, HDP-P-ACV, ODG-P-ACV, and ODG-P-AZT were > 200, > 14.7, and > 48 times more active than their free nucleosides in reducing HBV replication in 2.2.15 cells. To evaluate the biochemical basis for the increased antiviral activity, we studied the uptake and metabolism of 1-O-octadecyl-sn-glycero-3-phospho-[3H]acyclovir (ODG-P-[3H]ACV) in HepG2 cells. Cellular uptake of ODG-P-[3H]ACV was found to be substantially greater than that of [3H]ACV, and cellular levels of ACV-mono-, -di-, and -triphosphate were much higher with ODG-P-ACV. ODG-P-[3H]ACV was well absorbed orally. Based on urinary recovery of tritium after oral or parenteral administration of the radiolabeled compounds, oral absorption of ODG-P-ACV in mice was 100% versus 37% for ACV. ODG-P-ACV plasma area under the curve was more than 7-fold greater than that of ACV. Lipid prodrugs of this type may be useful orally in treating viral diseases.
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PMID:Enhanced oral absorption and antiviral activity of 1-O-octadecyl-sn-glycero-3-phospho-acyclovir and related compounds in hepatitis B virus infection, in vitro. 925 56

To investigate whether or not DNA polymerases alpha, delta, and epsilon from tumor cells have acquired properties that might be responsible for mutations found in tumor development, we investigated copying fidelities of DNA polymerases alpha, delta, and epsilon from the highly malignant Novikoff hepatoma cells and compared them to the corresponding enzymes from normal rat liver. DNA polymerases were purified more than 300-fold by three chromatographic steps. Copying fidelity was studied using steady-state kinetics and an 18-mer oligonucleotide primed with a 12-mer (13-mer for extension experiments) as DNA primer-template. Three experimental approaches were chosen: i) extension of DNA primers with mismatched 3'-OH ends opposite dGMP, ii) DNA insertion of nucleotides opposite m6G in the template and iii) extension of DNA primers with mismatched 3'-OH ends opposite m6G. i) Extension of DNA primers with mismatched 3'-OH ends opposite dGMP. DNA primer templates containing G:T and G:A mispairs at the 3'-OH position of the primer were easily extended by DNA polymerases alpha, delta and epsilon from both normal rat liver and Novikoff hepatoma cells. The G:G mismatch was elongated with low efficiency. Notably, DNA polymerase alpha from Novikoff hepatoma cells extended G:A and G:G mismatches significantly faster than the enzyme from normal cells. ii) Insertion of nucleotides opposite m6G. DNA polymerases alpha, delta, and epsilon from normal rat liver preferably catalyzed incorporation of dAMP opposite m6G at dNTP concentrations < 100 microM. When dNTP concentrations were raised to > or = 100 microM, dCMP (DNA polymerases delta and epsilon) and dTMP (DNA polymerase alpha) were also incorporated. The same insertion characteristics were found for the enzymes from Novikoff cells, however, insertion efficiencies of dAMP and dCMP were significantly higher for polymerases delta and epsilon. iii) Extension of primers with mismatched 3'-OH ends opposite m6G. Only m6G:dAMP and m6G:dCMP mismatches were extended by DNA polymerases alpha, delta and epsilon from both sources. No differences in extension efficiency were observed between the enzymes from normal and hepatoma cells. Taken together, our results suggest that DNA polymerases alpha, delta, and epsilon from Novikoff cells catalyzed incorporation of the wrong nucleotides more readily and extended mismatches more easily. These results may provide a rationale why numerous mutations accumulate during tumor development.
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PMID:Evidence for reduced copying fidelity of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells. 962 Feb 26

It is well known that point mutations exist in oncogenes and tumor suppressor genes of tumor cells, and one of the causes of these mutations may be misincorporation by error-prone DNA polymerases. This hypothesis is supported by the observation of decreased fidelity levels of DNA polymerases in mouse spleen containing tumorigenic cells after infection with Friend virus, and in aged animals that suffer high rates of tumorigenesis. However, this decrease in fidelity is disadvantageous for tumor cells maintained by serial transplantation. Therefore, we measured the fidelity levels of DNA polymerases in tumor cells transplanted through many passages. The fidelity levels of DNA polymerases from Yoshida ascites hepatoma, Rhodamine sarcoma, mouse ascites hepatoma-134, and Ehrlich ascites carcinoma cells derived from rats and mice are very high for in-vitro DNA synthesis on synthetic polynucleotides. These results suggest that many kinds of mutant cells arise during tumorigenesis. Among these mutant cells, cells showing decreased DNA polymerase(s) fidelities are present and these cells may undergo cell death. On the other hand, cells with mutations in various oncogenes and tumor suppressor genes and without mutations in DNA polymerase genes may survive as serially transplantable tumor cells.
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PMID:Fidelity levels of DNA polymerases in tumorigenic state cells and serially transplantable tumor cells. 988 76

The telomere hypothesis postulates stabilization of telomere length and telomerase activation as key events in cellular immortalization and carcinogeneses. Accordingly, telomerase has been suggested as a novel and highly selective target for design of antitumor drugs. Screening of a chemical library including 16 000 synthetic compounds yielded six that strongly inhibited telomerase activity in extracts of cultured human cells, including four isothiazolone derivatives and two unrelated compounds. The most potent inhibitor was 2-[3-(trifluoromethyl)phenyl]isothiazolin-3-one (TMPI), a concentration of 1.0 microM inhibited telomerase activity by 50% according to a telomere repeat amplification protocol (TRAP) assay. Analysis using partially purified telomerase from AH7974 rat hepatoma cells demonstrated noncompetitive inhibition with the telomere-repeat primer and mixed inhibition with the dNTPs; the inhibition constant was 2.5 microM. TMPI did not inhibit eukaryotic DNA polymerase alpha, beta, or human immunodeficiency virus reverse transcriptase (HIV RT). Thus, inhibition by TMPI was highly selective for telomerase. Inhibition by TMPI was quenched by 1 mM of dithiothreitol or glutathione, suggesting that TMPI inhibits telomerase by acting at a cysteine residue. TMPI inhibition of this enzyme may find application as an antineoplastic agent.
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PMID:Isothiazolone derivatives selectively inhibit telomerase from human and rat cancer cells in vitro. 1047 2

Tumor development is characterized by accumulation of mutations. Such mutations, if induced by carcinogens in DNA polymerase genes, would confer mutator properties on the DNA replication machinery, even at later stages of development. To investigate whether DNA polymerase delta can be mutated, we compared these enzymes from highly malignant Novikoff hepatoma cells and from regenerating normal rat liver. We sequenced the DNA polymerase delta cDNA from both sources and investigated the physico-chemical properties, inhibition characteristics, and copying fidelity of the purified enzymes. The cDNA sequences examined included the entire reading frame encoding the catalytic subunit (subunit I) of DNA polymerase delta. First-strand cDNAs were prepared from total RNA of both normal rat liver and Novikoff cells by reverse transcription, and the polymerase delta sequences were amplified by the polymerase chain reaction. cDNA (3325 bp) were sequenced. A single heterozygous mutation (CGG --> CAG) has been detected in nucleotide position 1948 (codon 648) of the polymerase delta gene from Novikoff cells, resulting in an Arg to Gln change. Position 648 lies just proximal to the conserved region VI, which is part of the "fingers" subdomain of alpha-like polymerases. This subdomain is involved in dNTP binding. Upon comparison of biochemical characteristics of partially purified DNA polymerase delta from both Novikoff cells and rat liver, the following properties of the enzyme from Novikoff cells were found to be altered: (i) K(50) values for nucleotide analogs (e.g. butylphenyl-dGTP) were lower, (ii) sensitivity to various antineoplastic drugs (e.g. doxorubicin, topotecan and distamycin) was enhanced, (iii) copying fidelity was decreased when primer templates containing O(6)-methylguanine were used, and (iv) the activity of DNA polymerase delta from Novikoff tumor cells was less stimulated by lactate dehydrogenase than the enzyme from normal cells. The altered biochemical characteristics of DNA polymerase delta from Novikoff cells suggest mutator properties. We conclude that the point mutation detected in the cDNA might be causally related to the observed changes in inhibition characteristics and copying fidelity.
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PMID:A mutation detected in DNA polymerase delta cDNA from Novikoff hepatoma cells correlates with abnormal catalytic properties of the enzyme. 1054 66

Hepatitis B virus (HBV) infection is a worldwide public health problem. In France, 150,000 individuals are infected with the HBV. Although many are asymptomatic carriers, about 30% have chronic hepatitis, a condition associated with a risk of cirrhosis and hepatocellular carcinoma. Antiviral treatments, most notably interferon alpha, probably modify the natural history of hepatitis B, decreasing the risk of hepatocellular carcinoma and increasing survival. Nucleoside analogs, particularly lamivudine, have also demonstrated potent antiviral activity, which should however be weighed against the increasing risk over time of mutation development in the YMDD region of the DNA polymerase reverse transcriptase. Antiviral therapy monitoring should include clinical safety evaluations and periodic laboratory tests including blood cell counts, transaminase activities, and serum DNA levels. The improving results provided by antiviral drugs should not deflect attention away from the importance of large-scale hepatitis B immunization of neonates, which has been shown to decrease the incidence of hepatocellular carcinoma in areas with high levels of hepatitis B endemicity.
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PMID:[Hepatitis B: epidemiology, natural history, biology, treatment monitoring]. 1060 72


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