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)

Investigations of mast cell biology have often used immortalized cultured cells which are continuously proliferating. In vivo, however, only 2% or fewer tissue mast cells are actively dividing. We used aphidicolin, an inhibitor of DNA polymerase to induce a proliferative arrest of murine mast cells characterized by an inhibition of cell division and thymidine incorporation, with accumulation of cells in G1 and early S phase of the cell cycle. Uridine incorporation and cell viability were not significantly impaired. DNA synthesis and cell division both resumed rapidly upon removal of the drug. Morphometric analysis demonstrated that cell size, granule size, and number of granules per cell were all increased in aphidicolin-treated cells. Proliferative arrest also produced a 14-fold increase in cellular histamine content, but did not alter the proteoglycans synthesized by the cell. The level of c-myc mRNA was reduced in aphidicolin-arrested cells, but returned to the level observed in untreated cells within 1 hr of removal of the drug. In contrast, the constitutive steady-state RNA levels of tumour necrosis factor-alpha (TNF-alpha), B2-microglobulin, actin, and the c-Ha-ras and c-fes protooncogenes were not altered. Aphidicolin-induced proliferative arrest did not prevent the induction of TNF-alpha, interleukin-6 (IL-6) and c-fos genes in response to calcium ionophore. Both the magnitude and induction kinetics of these messages were similar in aphidicolin-treated and untreated cells. We conclude that proliferative arrest results in morphological and biochemical changes suggestive of cellular maturation, but inhibition of cell division alone is not sufficient to alter mast cell phenotype. Although optimal c-myc expression appears to require active proliferation, cytokine gene induction can occur in non-dividing cells. These data suggest that the proliferative quiescence of in vivo mast cells should not preclude their involvement in biological events via elaboration of multi-functional cytokines.
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PMID:Aphidicolin-induced proliferative arrest of murine mast cells: morphological and biochemical changes are not accompanied by alterations in cytokine gene induction. 138 41

The results presented here demonstrate that expression of a fos ribozyme limits Fos protein synthesis and enhances sensitivity of A2780DDP cells to antineoplastic agents, including cisplatin. Moreover, the reversal of this resistance is associated with down-regulation of dTMP synthase, DNA polymerase beta, topoisomerase I and hMTII-A, genes previously linked to DNA synthesis and repair. Thus these studies further implicate the role of the c-fos gene in DNA synthesis through modulation of expression of dTMP syntase, DNA polymerase beta and topoisomerase I. Finally, the use of ribozymes to circumvent drug resistance suggests their potential utility as agents to inhibit tumor cell growth.
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PMID:The utility of an anti-fos ribozyme in reversing cisplatin resistance in human carcinomas. 149 17

The c-fos gene product Fos has been implicated in many cellular processes, including signal transduction, DNA synthesis, and resistance to antineoplastic agents. A fos ribozyme (catalytic RNA) was designed to evaluate the effects of suppressing Fos protein synthesis on expression of enzymes involved in DNA synthesis, DNA repair, and drug resistance. DNA encoding the fos ribozyme (fosRb) was cloned into the pMAMneo expression plasmid, and the resultant vector was transfected into A2780DDP cells resistant to the chemotherapeutic agent cisplatin. The parental drug-sensitive A2780S cells were transfected with the pMMV vector containing the c-fos gene. Morphological alterations were accompanied by significant changes in pharmacological sensitivity in both c-fos- and fosRb-transfected cells. pMAMneo fosRb transfectants revealed decreased c-fos gene expression, concomitant with reduced thymidylate (dTMP) synthase, DNA polymerase beta, topoisomerase I, and metallothionein IIA mRNAs. In contrast, c-myc expression was elevated after fos ribozyme action. Insertion of a mutant ribozyme, mainly capable of antisense activity, into A2780DDP cells resulted in smaller reductions in c-fos gene expression and in cisplatin resistance than the active ribozyme. These studies establish a role for c-fos in drug resistance and in mediating DNA synthesis and repair processes by modulating expression of genes such as dTMP synthase, DNA polymerase beta, and topoisomerase I. These studies also suggest the utility of ribozymes in the analysis of cellular gene expression.
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PMID:Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein. 166 Jan 42

The autocrine, paracrine, or systemic growth factors responsible for fetal lung cell growth are not completely defined. The progression-type insulin-like growth factors and epidermal growth factor, or transforming growth factor-alpha acting through the epidermal growth factor receptor, appear to act on the developing lung epithelium. The competence factors that facilitate the actions of progression factors during lung growth are unknown. Fetal rat lung cells in vitro synthesize a platelet-derived growth factor (PDGF)-like polypeptide, which we have hypothesized may play a paracrine role in normal lung development. Slot blot and Northern blot analyses of fetal rat lung mRNA have been used to determine if there is a relationship between expression of message for PDGF-A or PDGF-B chains, or their cognate receptors, and periods of maximal growth during late fetal rat lung development. Whole lung mRNA was extracted on 18, 19, 20, 21, and 22 days of gestation (term = 22 days). The peak of DNA synthesis, as assessed by expression of message for DNA polymerase alpha, histone 3, and the proto-oncogenes c-fos and c-myc, which are stimulated by binding of growth factors including PDGF, occurred during the canalicular stage of lung development on days 19 and 20 of gestation. Expression of message for PDGF-A and PDGF-B chains was low during the pseudoglandular stage on day 18, peaked during the canalicular stage on days 19 and 20, then fell again during the saccular stage at days 21 and 22 of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Platelet-derived growth factor and growth-related genes in rat lung. I. Developmental expression. 191 Aug 22

Peritoneal cells were derived from a patient (PK) with adenocarcinoma of the colon during the course of cisplatin/5-fluorouracil (5-FUra) treatment. Resistance to cisplatin and 5-FUra, characterized by a lack of response to chemotherapy and continued growth of the tumor, was concomitantly associated with a 2-4-fold increase in DNA copy number for dTMP synthase and dihydrofolate reductase. There was a corresponding amplification in DNA copy number of the c-myc (2X), H-ras (4X), and c-fos (15X) oncogenes. Cytogenetic studies revealed an iso (13q) chromosome, but failed to show any double minutes or homogeneously staining regions. In addition, drug-resistant tumor cells from PK and another patient (HG) displayed enhanced expression of dTMP synthase, c-fos and DNA polymerase beta when compared to normal colon tissue and the HCT8 human colon carcinoma cell line. These results suggest that elevated oncogene DNA and gene expression may be involved in the development of cisplatin resistance.
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PMID:Differential oncogene amplification in tumor cells from a patient treated with cisplatin and 5-fluorouracil. 214 97

Biochemical differences were demonstrated between two cell lines derived from a human colon carcinoma (HCT8), one sensitive (HCT8S), and one 4.3-fold resistant to cisplatin (HCT8DDP). The cisplatin-resistant cell line overexpressed five enzymes (dihydrofolate reductase, thymidine 5'-monophosphate synthase, thymidine kinase, and DNA polymerase alpha and beta) believed to be important for DNA replicative and repair synthesis. In addition, the c-fos and c-H-ras oncogenes were also overexpressed in the HCT8DDP cells. This apparent overexpression was not associated with increases in gene copy number, it was related, however, to increased mRNA content. Expression of these key enzymes may be a significant factor in the development of clinical resistance to cisplatin. Further, these specific changes in cellular metabolism associated with cisplatin resistance may be exploited by the use of nucleoside analogues.
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PMID:Overexpression of DNA replication and repair enzymes in cisplatin-resistant human colon carcinoma HCT8 cells and circumvention by azidothymidine. 253 92

1. The structural gene for cholinephosphate cytidylyltransferase (CCT) was isolated from a Saccharomyces cerevisiae genomic library by means of complementation in a mutant of the yeast defective in the enzyme. The cloned DNA restored both the growth and cholinephosphate cytidylyltransferase activity of the mutant. Whereas the enzyme of the mutant was thermolabile, the enzyme produced by the transformant was indistinguishable in heat stability from that produced by the wild type. 2. Strains carrying a multicopy recombinant plasmid overproduced cholinephosphate cytidylyltransferase. The overproduction of the enzyme brought about an increase in the synthesis of CDPcholine in the transformant, but there was no increase in the overall rate of phosphatidylcholine synthesis. 3. The cloned DNA was subcloned into a 2.5-kb DNA fragment. The nucleotide sequence which contained CCT was determined by the dideoxy chain-termination method. The sequence contained an open reading frame capable of encoding a protein of 424 amino acid residues with a calculated relative molecular mass of 49,379.31. Northern blot analysis showed that this DNA segment is transcribed in yeast cells and the length of the transcript is consistent with the putative translation product. 4. Hydropathy analysis according to Kyte and Doolittle indicated that the primary translation product contains extended hydrophilic stretches in its N- and C-terminal regions. 5. The primary translation product contains a region showing local sequence homology with nucleotidyl-transfer enzymes such as DNA polymerase (Escherichia coli), CDPdiacylglycerol pyrophosphatase (E. coli), 3-deoxy-manno-octulosonate cytidylyltransferase (E. coli) and DNA ligase (T4 phage), suggesting that these five enzymes are evolutionarily related. Statistically significant sequence homology was also noted between the human c-fos gene product and the enzyme.
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PMID:Molecular cloning and characterization of the gene encoding cholinephosphate cytidylyltransferase in Saccharomyces cerevisiae. 282 47

This report concerns the utility of the reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (QPCR) assay to detect the drug-resistance of related genes. The expression of some drug-resistance genes was compared with the sensitivity and resistance-acquired cancer cell lines to anti-cancer drugs by Northern blot analysis and PCR assay. The resistance cell lines exhibited an enhanced expression of multi-drug resistance (MDR-1), thymidylate synthase (TS), c-fos and DNA polymerase beta genes. Then these genes that expressed mRNA were quantitated using RT-PCR. The expression of the genes was dependent on their sensitivity (IC50) to anti-cancer drugs. Additionally, the QPCR assay has been developed as a rapid method for the expression of drug-resistance genes and applied to the PCR products amplified by the RT-PCR. Thus the QPCR assay for the expression of genes will allow rapid detection of the drug-resistance to chemotherapy in human cancers.
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PMID:Rapid diagnosis of drug-resistant genes by PCR assay. 760 96

The results presented here demonstrate that expression of a fos ribozyme limits Fos protein synthesis and enhances sensitivity of A2780DDP cells to antineoplastic agents, including cisplatin. Moreover, the reversal of this resistance is associated with down-regulation of dTMP synthase, DNA polymerase beta, topoisomerase I and hMT II-A, which are genes previously linked to DNA synthesis and repair. Thus, these studies further implicate the role of the c-fos gene in DNA synthesis through modulation of expression of dTMP synthase, DNA polymerase beta and topoisomerase I. Finally, the use of ribozymes to circumvent drug resistance suggests their potential utility as agents to inhibit tumor cell growth.
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PMID:[Circumventing drug resistance in human tumors by antisense ribozyme]. 810 89

DNA damage results from a wide variety of external agents such as chemicals and radiation. The consequences of exposure to agents that damage DNA have been traditionally studied from the perspective of cell survival and mutagenesis. Mutations are late endpoints of DNA damage. Cells respond to the earlier stages of DNA damage by inducing the expression of several genes, including those specific of the nature of the lesion. These early transcriptional responses are likely to predetermine the later fate of the damaged cell. Genes activated during this early response include those involved in DNA repair, replication, and growth control. We are interested in the transcriptional mechanisms by which cells respond to DNA damaging agents. To facilitate the measurement of gene induction, we used seven different reporter constructs integrated stably into the RKO cell line derived from a human colon carcinoma. These constructs were derived from promoters and/or response elements isolated from genes associated with DNA damage responses in human cells, and were fused to the bacterial reporter gene, choramphenicol acetyl transferase (CAT). The cell lines generated in this manner contain the promoters and/or response elements representing DNA polymerase beta, p53, gadd (growth arrest and DNA damage) 45 and 153, c-fos, TPA response element, and tissue-type plasminogen activator. These recombinant cell lines were assembled in a 96-well microtiter plate permitting their simultaneous exposure to compounds and subsequent CAT protein measurement. This assembly has been designated the CAT-Tox (D) assay. These cell lines were exposed to different classes of DNA damaging agents including those which covalently join bases to form dimers (e.g., UVC irradiation), generate DNA adducts by alkylation (e.g., methylmethane sulfonate [MMS], ethylmethane sulfonate [EMS], N-methyl-N-nitro-N-nitrosoguanine [MNNG], dimethylnitrosamine [DMN]), cross-link DNA (e.g., mitomycin C), and inhibit DNA replication by intercalative (e.g., actinomycin D) and nonintercalative (e.g., hydroxyurea) mechanisms. The transcriptional responses were measured as a function of the accumulation of CAT protein using antibodies against CAT protein in a standard ELISA. Endogenous cellular responses were evaluated for a number of the genes represented in the assay at both the mRNA and protein levels by Northern and Western blot analysis, respectively. These data corroborate the stress-induced responses measured by CAT ELISA in the CAT-Tox (D) assay, demonstrating the usefulness of this assay as a rapid and sensitive method for detection of DNA damaging agents in human cells.
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PMID:Stress responses to DNA damaging agents in the human colon carcinoma cell line, RKO. 895 Mar 45


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