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)

In extracts of spleen tissue from two patients with haemotological malignancies an RNA dependent DNA polymerase was found in particles with a density of 1.16, that is at the density of oncorna viruses. After treatment with noniomic detergents the enzyme activity was found in particles with a density of 1.23-1.24, similar to the density of oncorna viral cores. A simultaneous detection test with this core fraction material for 70 S RNA and RNA dependent DNA polymerase was positive for both patients. Electron microscopical inspection of the material with a density of 1.16 revealed immature C-type virus like particles, various stages of maturing particles and a number of particles resembling mature C-type oncorna viruses. In two normal spleens from patients with carcinoma of the colon and oesophagus respectively and in three spleens from patients with no history of malignancy no RNA dependent DNA polymerase was found. Material from one normal spleen was examined in the electron microscope and no virus-like particles were seen.
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PMID:Biochemical and electron microscopical evidence for the presence of oncorna viruses in spleen tissue from two patients with haematological malignancies. 6 13

A hybridoma cell line (5F) secreting monoclonal antibodies directed to alpha DNA polymerase has been developed. Kinetic studies on peripheral blood lymphocytes stimulated with mitogen and human colon cancer cell lines established in vitro were made by the two autoradiographic techniques of Thymidine Labelling Index and Primer-dependent alpha DNA polymerase Labelling Index and the immunoperoxidase assay (PAP) with monoclonal antibody to alpha DNA polymerase. We demonstrated the exclusively intranuclear presence of alpha DNA polymerase in lymphocytes induced to proliferate and actively growing colon cancer cells in contrast with the cytoplasmic distribution of the enzyme in resting stage populations. The feasibility of using monoclonal antibodies to alpha DNA polymerase to determine cell growth fraction was evaluated.
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PMID:Monoclonal antibodies to alpha DNA polymerase as a marker of cell proliferative activity. 332 40

We have established and partially characterized a panel of monoclonal antibodies against alpha-DNA polymerase. One of the hybridomas, clone 5F, has been exploited for cell kinetic studies on three colon cancer cell lines, LOVO, SW 620, and SW 403, which are endowed with different growth patterns and differentiation status. By an immunoperoxidase method, we could demonstrate the specific intranuclear localization of alpha-DNA polymerase during the exponential phase of in vitro growth and contrast it with the diffuse distribution of the enzyme throughout the cytoplasm during the resting state. The percentage of intranuclear staining positive cells, evaluated at successive time points of in vitro growth, changed from 75 to 95% (assayed on Days 3 and 7) to 15 to 25% in confluent and resting populations assayed on Days 12 to 14. In agreement with the assumption that the enzyme moves from nucleus to cytoplasm after entering quiescence, alpha-DNA polymerase was still present in the cytoplasm or in the cytoplasmic perinuclear area of cells in resting phase cultures. Comparisons between traditional kinetic parameters (thymidine labeling index and primer-dependent alpha-DNA polymerase) and proliferative state determined by the monoclonal antibody supported the feasibility of this approach to define the proportion of actively proliferating elements in a tumor cell population. Moreover, parallel flow cytometric analysis performed on Days 5 and 14 of continuous culture showed fluctuations of alpha-DNA polymerase content in relation to exponential and steady-state phases, with a significant increase in the amount of alpha-DNA polymerase in actively proliferating populations and a progressive reduction of the enzyme as the cultures entered the resting stage.
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PMID:Evaluation of growth fractions with monoclonal antibodies to human alpha-DNA polymerase. 381 79

Adaptive reversion of a +1 frameshift mutation in Escherichia coli, which requires homologous recombination functions, is shown here to occur by -1 deletions in regions of small mononucleotide repeats. This pattern makes improbable recombinational mechanisms for adaptive mutation in which blocks of sequences are transferred into the mutating gene, and it supports mechanisms that use DNA polymerase errors. The pattern appears similar to that of mutations found in yeast cells and in hereditary colon cancer cells that are deficient in mismatch repair. These results suggest a recombinational mechanism for adaptive mutation that functions through polymerase errors that persist as a result of a deficiency in post-synthesis mismatch repair.
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PMID:Adaptive mutation by deletions in small mononucleotide repeats. 802 53

The role of cell cycle redistribution in fluoropyrimidine-mediated radiosensitization remains unresolved. To determine if radiosensitization resulted from the redistribution of cells into a sensitive phase of the cell cycle, we assessed fluorodeoxyuridine (FdUrd)-mediated radiosensitization in flow-sorted mid-S phase HT29 human colon cancer cells. We hypothesized that if FdUrd-mediated radiosensitization were strictly the result of cell cycle redistribution, FdUrd-treated mid-S phase cells would remain as radioresistant as mid-S phase cells cultured in the absence of drug. However, we found that the mid-S phase cells from FdUrd-treated populations were markedly radiosensitized. To assess the role of S phase progression in radiosensitization, we exposed FdUrd-treated cells to aphidicolin, an inhibitor of DNA polymerase alpha, prior to irradiation. We found that aphidicolin blocked the radiosensitizing (and cytotoxic) effects of FdUrd. These results, combined with our previous observations that FdUrd-treated cells at the G1/S boundary are minimally sensitized, appear to disprove the hypothesis that sensitization results strictly from cell cycle distribution. Furthermore, they suggest that a key aspect of both FdUrd-mediated radiosensitization and cytotoxicity is S phase progression on a damaged DNA template.
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PMID:Fluorodeoxyuridine-mediated cytotoxicity and radiosensitization require S phase progression. 880 Jan 98

Interferon (IFN) augments the anabolism of 5-fluorouracil (5FU) to its active metabolite, fluorodeoxyuridylate (FdUMP), which inhibits thymidylate synthase (TS). We sought to determine whether this resulted in greater perturbations of nucleotide pools and if so, whether this was associated with an increase in cell lethality, specifically focussing on the lethal cellular lesion, DNA double strand breaks (dsb). To determine whether combination therapy with 5FU + IFN resulted in greater depletion of thymidine nucleotide pools than 5FU alone, a highly sensitive DNA polymerase assay was used. In two human colon cancer cell lines, treatment with 5FU + IFN resulted in a rapid decrease in levels of dTTP by 95%. The addition of IFN to 5FU resulted in greater depletion of dTTP levels over treatment with 5FU alone by up to fourfold, and markedly augmented the dATP/dTTP ratio. The addition of IFN to 5FU had no effect on 5FU-induced perturbations in dCTP, dGTP or dATP pools at 8 and 12 h. Measurement of DNA dsb demonstrated that treatment of HT-29 cells with 10 microM 5FU for 24 h did not increase DNA dsb versus control. The combination of 5FU + 500 U/ml IFN, however, resulted in an increased number of dsb versus both 5FU and untreated control cells (P < 0.01), equivalent to 0.74 +/- 0.12 Gy. The addition of IFN to 5FU resulted in a selective further depletion of pools of dTTP and an increase in the number of DNA dsb versus 5FU treatment alone.
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PMID:Effect of interferon on 5-fluorouracil-induced perturbations in pools of deoxynucleotide triphosphates and DNA strand breaks. 882 94

Weekly administrations of the potent carcinogen 1,2-dimethylhydrazine (DMH) predominantly induce carcinoma of the colon by nearly 100% after six months' treatment in rats. Polyamines, and especially the key enzyme of polyamine de novo synthesis ornithine decarboxylase (ODC) are well-known to play an important role in cell growth and tumor carcinogenesis. Male Wistar rats were s. c.-injected with a single dose of 20 mg DMH/kg b. wt. and five to eight animals were sacrificed 4, 8, 12, 24, 72, 120, 168, and 240 hours after injection of DMH or the basic solution, respectively. Additionally, seven animals were simultaneously treated with the ODC inhibitor alpha-difluoromethylornithine (DFMO) and sacrificed seven days after a single DMH injection. A single s. c.-dosage of the colon carcinogen DMH resulted in dissimilar activation patterns of polyamine metabolism in the various organs studied: in distal and less pronounced in proximal colonic mucosa ODC and putrescine are significantly increased seven days after application of DMH and DNA polymerase after ten days; in small intestinal mucosa ODC activity is significantly elevated after seven days and especially S-adenosylmethionine decarboxylase activity is significantly and prolonged increased between twelve and 72 hours after DMH injection; while spermidine/spermine N1-acetyltransferase activity is significantly elevated in liver after 168 and 240 hours, no changes compared to controls are found in the pancreas. DFMO treatment completely prevents DMH-induced activation of polyamine de novo synthesis and DNA polymerase in colon and small intestine. These data prove completely different and -interestingly-late appearing activation patterns of DMH on intracellular polyamine metabolism in various organ systems and further elucidate the complex metabolic changes following carcinogen treatment.
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PMID:Dissimilar activation patterns of the carcinogen dimethylhydrazine (DMH) on intracellular polyamine metabolism in various organs. 901 96

Thiocoraline, a new anticancer agent derived from the marine actinomycete Micromonospora marina, was found to induce profound perturbations of the cell cycle. On both LoVo and SW620 human colon cancer cell lines, thiocoraline caused an arrest in G1 phase of the cell cycle and a decrease in the rate of S phase progression towards G2/M phases, as assessed by using bromodeoxyuridine/DNA biparametric flow cytometric analysis. Thiocoraline does not inhibit DNA-topoisomerase II enzymes in vitro, nor does it induce DNA breakage in cells exposed to effective drug concentrations. The cell cycle effects observed after exposure to thiocoraline appear related to the inhibition of DNA replication. By using a primer extension assay it was found that thiocoraline inhibited DNA elongation by DNA polymerase alpha at concentrations that inhibited cell cycle progression and clonogenicity. These studies indicate that the new anticancer drug thiocoraline probably acts by inhibiting DNA polymerase alpha activity.
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PMID:Mode of action of thiocoraline, a natural marine compound with anti-tumour activity. 1036 4

We examined cDNAs of the catalytic subunit of DNA polymerase alpha (185 kDa), the 70 kDa subunit of replication protein A (single-stranded DNA-binding protein) and the 140 kDa subunit of replication factor C for mutations. Surgical specimens from 12 patients with sporadic colon cancer and normal mucosae from the same patients were investigated. In addition, we analyzed 3 human colon cancer cell lines that exhibited defects in mismatch repair (DLD-1, HCT116, SW48) and 3 colon cancer cell lines without such a defect (HT29, SW480 and SW620). For detection of mutations, we used reverse transcription of mRNA, amplification of cDNAs by PCR, analysis of single-strand conformation polymorphism and DNA sequencing. Eleven colon cancers and 6 colon cancer cell lines were analyzed for DNA polymerase alpha. Only 2 silent point mutations were detected, in 1 colon carcinoma and in cell line HCT116. Two sequence alterations of the 70 kDa subunit of replication factor A were identified in 15 specimens (9 colon carcinomas and 6 cell lines). Colon carcinomas from 2 patients (CC5MA and CC25HN) exhibited an ACA-->GCA transition in codon 351, which caused a Thr-->Ala exchange. In carcinomas CC5MA and CC8MA, a TCC-->TCT (Ser-->Ser) transition in codon 352 was observed. The deviations in codons 351 and 352 occurred in both cancer tissues and normal mucosae, suggesting a genetic polymorphism. No mutation was found in the 140 kDa subunit of replication factor C from 16 specimens (10 tumors and 6 cell lines). Point mutations were identified in the p53 tumor-suppressor gene in 4 of the 6 colon cancer cell lines and 3 of the 8 carcinoma specimens. We did not find tumor-associated DNA sequence alterations that resulted in amino acid changes in the DNA replication genes analyzed. We infer that the scarcity of mutations found is due to stringent selection, eliminating functionally impaired replication proteins.
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PMID:Mutation analysis of replicative genes encoding the large subunits of DNA polymerase alpha and replication factors A and C in human sporadic colorectal cancers. 1076 Aug 17

Using reverse transcriptase-polymerase chain reaction (RT-PCR), we have recently described a bona fide deletion within the coding sequence of the large subunit of ribonucleotide reductase (R1) mRNA in colon cancer. Consecutive studies have raised questions about the nature of this phenomenon, because the corresponding genomic alteration at the DNA level or an aberrant protein could not be detected. Thus we considered an in vitro artifact during RT-PCR as a possible explanation for this observation. In contrast to reverse transcriptase, Taq DNA polymerase or C. therm DNA polymerase did not generate the aberrant product, suggesting the demand for the template switching activity intrinsic to retroviral reverse transcriptases. In fact, virtually the same deletion was observed in RT-PCR experiments when in vitro transcribed R1 mRNA was used. Considering structural prerequisites for template switching within R1 mRNA, we show that two direct repeats adjacent to a strong stem-loop secondary structure flank the deleted region of 1851 base pairs. Because several mRNAs encoding proteins of clinical and diagnostic importance fulfill these criteria, template switching enhances the potential risk of observing artifacts when interpreting results from RT-PCR studies. As shown in the present example, this may involve the artificial generation and the misinterpretation of PCR fragments amplified from targets relevant to tumor biology or cancer pharmacology. As a possible solution, one-step PCR with C. therm polymerase should be considered. This polymerase eliminates the artificial generation of aberrant mRNA signals observed during cDNA synthesis.
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PMID:Reverse transcriptase template switching during reverse transcriptase-polymerase chain reaction: artificial generation of deletions in ribonucleotide reductase mRNA. 1138 63


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