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)

The patterns of DNA fragmentation were evaluated following a brief exposure (2 h) of the human ileocecal adenocarcinoma cell line, HCT-8, to several specific thymidylate synthase inhibitors, a quinazoline (ZD1694) and benz[cd]indole-containing molecule (AG-331). The magnitude and size of DNA fragmentation induced by the two agents were assessed by alkaline elution for DNA single-strand breaks (ssbs), and by pulsed- and constant-field gel electrophoresis for DNA double-strand breaks (dsbs). Both agents induced dose-dependent DNA dsbs. While AG-331 induced ssbs and dsbs only in nascent DNA, ZD1694 affected both genomic and nascent DNA. The fragments of newly synthesized and genomic DNA, estimated by pulsed-field gel electrophoresis assay, were associated with the bands in the range of 0.05 to 1.1 and 1.1 to 5.7 megabases, respectively. 5-fluoro-2'-deoxyuridine (FdUrd), like ZD1694, produced both mature and nascent DNA fragmentation, whereas only nascent DNA breakage induced by 5-fluorouracil (FUra) was detected, similar to AG-331. The induction of both mature and nascent DNA fragmentation by ZD1694 and FdUrd appears to correlate with the higher, but similar, potency of these agents. Aphidicolin, a DNA polymerase inhibitor, protects from DNA dsbs and cytotoxicity by ZD1694 and AG-331. These observations suggest that replicative DNA synthesis is an important factor in ZD1694- and AG-331-induced DNA fragmentation and, subsequently, cell growth arrest. The results indicate that although the new antimetabolites investigated herein were developed and extensively evaluated as specific and potent thymidylate synthase inhibitors, DNA damage appears to be an important additional determinant of drug effect.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Contrasting patterns of DNA fragmentation induced by thymidylate synthase inhibitors, ZD1694 and AG-331. 757 30

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

Although a number of transfection experiments have suggested potential targets for the action of the E2F1 transcription factor, as is the case for many transcriptional regulatory proteins, the actual targets in their normal chromosomal environment have not been demonstrated. We have made use of a recombinant adenovirus containing the E2F1 cDNA to infect quiescent cells and then measure the activation of endogenous cellular genes as a consequence of E2F1 production. We find that many of the genes encoding S-phase-acting proteins previously suspected to be E2F targets, including DNA polymerase alpha, thymidylate synthase, proliferating cell nuclear antigen, and ribonucleotide reductase, are indeed induced by E2F1. Several other candidates, including the dihydrofolate reductase and thymidine kinase genes, were only minimally induced by E2F1. In addition to the S-phase genes, we also find that several genes believed to play regulatory roles in cell cycle progression, such as the cdc2, cyclin A, and B-myb genes, are also induced by E2F1. Moreover, the cyclin E gene is strongly induced by E2F1, thus defining an autoregulatory circuit since cyclin E-dependent kinase activity can stimulate E2F1 transcription, likely through the phosphorylation and inactivation of Rb and Rb family members. Finally, we also demonstrate that a G1 arrest brought about by gamma irradiation is overcome by the overexpression of E2F1 and that this coincides with the enhanced activation of key target genes, including the cyclin A and cyclin E genes.
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PMID:Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. 762 16

Twenty-one independent thymidylate synthase deficient (td) mutants were isolated after proflavin mutagenesis of T4D0 phage. A strikingly high proportion of these mutations (17 of 21; 80%) mapped in a small 122 nucleotide (nt) region which spans the 5' splice site of this intron-containing gene. This region comprises only 14% of the total td exon sequence. RNA sequence analysis of these mutants identified a series of frameshift insertion/deletion mutations and indicated a hotspot for proflavin-induced mutations in the 3' end of exon I of the td gene. The mutant sequences at the hotspot site fully support a previously proposed mutagenic mechanism for proflavin-induced mutations in which frameshifts are produced as a consequence of exonuclease or DNA polymerase activity at the 3' ends of nicks in the DNA produced by perturbation of the T4-encoded type II topoisomerase activity by the acridine. Sixteen of the seventeen DNA mutations in the hotspot region can be explained by the model as a consequence of enzymatic processing of nicks at two phosphodiester bonds staggered by 4 base pairs (bp) and located on opposite strands of the DNA. Thus, these mutants exhibit precisely the symmetry expected of topoisomerase-mediated mutagenesis. The DNA sequences of the td hotspot mutants, when considered with the sequences of proflavin-induced mutants in the T4 rIIB and lysozyme genes, confirm the view that proflavin-induced mutations in diverse bacteriophage T4 DNA sequences are all produced by the topoisomerase-dependent mechanisms and do not support the view that classical misalignments in DNA repeats are hotspots for proflavin-induced mutagenesis in T4.
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PMID:A proflavin-induced frameshift hotspot in the thymidylate synthase gene of bacteriophage T4. 768 30

Lipopolysaccharide (LPS), known as one of the potent activators of macrophages, has inhibitory effects on the proliferation of normal macrophages and macrophage-like cell lines. We report here that LPS dose- and time-dependently suppressed the tritiated thymidine ([3H]TdR) incorporation into the acid-insoluble fraction with a significant inverse correlation to the tumour necrosis factor-alpha (TNF) production in the J774.1 macrophage cell line. Among the three tested enzymes involved in DNA synthesis, only thymidine kinase (TK) activity decreased progressively in parallel with the decline in [3H]TdR incorporation, reaching 97% inhibition within 12 hr of LPS treatment, while changes in the activities of other two enzymes, DNA polymerase alpha and thymidylate synthase (TS), were less significant. On the other hand, LPS inhibited the cell proliferation only incompletely, as judged by 62% inhibition of cell growth at 36 hr. Even in the experiments done in a TdR-free medium, cell growth was inhibited by LPS to the same extent, suggesting that TK was not directly involved in the proliferation of J774 cells. LPS also inhibited the conversion of TdR to thymidine monophosphate (TMP) in murine peritoneal exudate macrophages (PEM). Thus LPS-induced suppression of TdR salvage related to TNF production is common in both normal and neoplastic macrophages, and therefore may be of potential importance in the process of macrophage activation.
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PMID:Effect of lipopolysaccharide on thymidine salvage as related to macrophage activation. 775 Oct 1

N-Substituted indan-1.3-diones have proven to be potent cytotoxic agents effective against the growth of single cell leukemia tumors and cell lines derived from solid tumors. A number of the derivatives were active against growth of solid tumors e.g. colon, lung bronchogenic and osteosarcoma for which few effective agents are available to inhibit their growth. These agents inhibited DNA and RNA synthesis of L1210 cells. The de novo purine synthetic pathway was inhibited at PRPP amido transferase and IMP dehydrogenase. The pyrimidine synthetic pathway was inhibited at aspartate transcarbamylase. Other sites which demonstrate minor inhibition were DNA polymerase alpha, r- and t-RNA polymerase, ribonucleoside reductase, dihydrofolate reductase, nucleoside kinases and thymidylate synthetase. In addition d(NTP) pool levels were reduced by the drugs. L1210 DNA strand scission was evident after exposure to drugs for 24 hr. at 100 microM.
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PMID:Cytotoxicity and mode of action of substituted indan-1, 3-diones in murine and human tissue cultured cells. 784 49

We have reported that noncytotoxic concentrations of 3'-azido-3'-deoxythymidine (AZT) increase the cytotoxicity of ICI D1694, a folate-based thymidylate synthase (TS) inhibitor, with increasing AZT incorporation into DNA. We postulated that the inhibition of TS by ICI D1694 would decrease 5'-deoxythymidine triphosphate (dTTP) pools, which compete with AZT triphosphate (AZT-TP) as a substrate for DNA polymerase. Furthermore, the inhibition of TS would increase the activity of both thymidine kinase (TK) and thymidylate kinase (TdK). Each of these consequences of TS inhibition would favor more incorporation of AZT into DNA. Thus, we reasoned that other TS inhibitors should also result in increased AZT incorporation into DNA and, perhaps, in increased cytotoxicity. N6-[4-(Morpholinosulfonyl)benzyl]-N6-methyl-2,6-diaminobenz[ cd]indole glucuronate (AG-331) differs from ICI D1694 in that it is a de novo designed lipophilic TS inhibitor, it does not require a specific carrier for cellular uptake, and it does not undergo intracellular polyglutamation. This potent TS inhibitor causes minimal cytotoxicity in MGH-U1 human bladder cancer cells. A 24-h exposure to 5 microM AG-331 causes almost complete TS inhibition but only 35% cell kill. The combination of AZT and AG-331 in MGH-U1 cells resulted in an enhanced antitumor effect relative to that of each agent alone; 50 microM AZT, noncytotoxic alone, increased the cell kill of induced by AG-331 from 35% to 50%. Biochemical studies of this combination revealed that simultaneous treatment with 5 microM AG-331 plus 1.8 microM [3H]-AZT produced as much as a 68% +/- 7% increase in AZT incorporation into DNA. This observation was associated with an increase in DNA single-strand breaks, measured as comet tail moment, of up to 6.6-fold. These studies support our original premise that TS inhibition favors increased incorporation of AZT into DNA and that the combination causes more cell kill than either drug alone in MGH-U1 cells.
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PMID:Cytotoxic and biochemical implications of combining AZT and AG-331. 785 Sep 19

Previously constructed Swiss mouse 3T3 fibroblasts producing polyomavirus large T antigen after addition of dexamethasone were used to study the transcriptional activation by the viral protein of five genes coding for enzymes involved in DNA synthesis and precursor production, namely, dihydrofolate reductase, thymidine kinase, thymidylate synthase, DNA polymerase alpha, and proliferating-cell nuclear antigen. It was found that all these genes, whose expression is stimulated at the G1/S boundary of the cell cycle after growth stimulation by serum addition, are coordinately trans activated when T antigen is induced in cells previously growth arrested by serum withdrawal. Cell lines carrying the information for a mutant form of large T antigen, in which a glutamic acid residue in the binding site for the retinoblastoma protein was changed into aspartic acid, were constructed to test the involvement of an interaction of T antigen with the retinoblastoma protein in this reaction. It was found that the mutated T protein is incapable of stimulating transcription of any one of the genes. The promoter of three of the genes (dihydrofolate reductase, thymidine kinase, and DNA polymerase alpha) unequivocally carries binding sites for transcription factor E2F, suggesting that complexes forming with this growth- and cell cycle-regulating transcription factor are the targets for T antigen. Although there is so far no evidence that thymidylate synthase and proliferating cell nuclear antigen are regulated via E2F, our data indicate that the retinoblastoma protein still is involved in the control of these genes. mRNA for E2F itself increases in amount at the G1/S border in serum-stimulated cells but not during polyomavirus T antigen-induced transcriptional activation of DNA synthesis enzymes in arrested cells.
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PMID:Coordinated trans activation of DNA synthesis- and precursor-producing enzymes by polyomavirus large T antigen through interaction with the retinoblastoma protein. 790 59

A series of selective antiherpetic compounds were found to exert pronounced cytostatic activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) thymidine kinase (TK) gene-transfected mammary carcinoma FM3A cells. Based on their potency and mechanism of cytostatic action, the antiherpetic compounds could be divided into two different classes. The first class encompasses (E)-5-(2-bromovinyl)-2'-deoxyuridine and structurally related analogues thereof [i.e., the cytosine derivative (E)-5-(2-bromovinyl)-2'-deoxycytidine and the 4'-thio derivative (E)-5-(2-bromovinyl)-2'-deoxy-4'-thiouridine]. These compounds are exquisitely cytostatic against FM3A/TK-/HSV-1 TK+ and FM3A/TK-/HSV-2 TK+ cells (50% inhibitory concentrations ranging from 0.047 to 0.001 microM) and inhibit tumor cell proliferation by inhibiting cellular thymidylate synthase. The second class consists of the acyclic guanosine derivatives penciclovir, buciclovir, and ganciclovir. These compounds are also more inhibitory to the HSV-1 TK or HSV-2 TK gene-transfected FM3A cells than to FM3A/0 or FM3A/TK- cells, but at concentrations that are higher than the concentrations at which the (E)-5-(2-bromovinyl)-2'-deoxyuridine derivatives proved to be inhibitory. These acyclic guanosine analogues appear to be targeted at the cellular DNA polymerase. From this study, (E)-5-(2-bromovinyl)-2'-deoxy-4'-thiouridine emerged as a promising candidate compound for the treatment of HSV-1 TK gene-transfected tumors in vivo, due to its metabolic stability (i.e., resistance to hydrolysis by thymidine phosphorylase).
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PMID:Comparative cytostatic activity of different antiherpetic drugs against herpes simplex virus thymidine kinase gene-transfected tumor cells. 802 17

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


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