Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We demonstrate that l-ATP is recognized by some enzymes that are involved in the synthesis of nucleotides and nucleic acids. l-ATP, as well as its natural d-enantiomer, acts as a phosphate donor in the reaction catalysed by human deoxycytidine kinase, whereas it is not recognized by either enantioselective human thymidine kinase or non-enantioselective herpes virus thymidine kinase. l-ATP strongly inhibits (Ki 80 microM) the synthesis of RNA primers catalysed by DNA primase associated with human DNA polymerase alpha, whereas RNA synthesis catalysed by Escherichia coli RNA polymerase is completely unaffected. Moreover, l-ATP competitively inhibits ATP-dependent T4 DNA ligase (Ki 25 microM), suggesting that it interacts with the ATP-binding site of the enzyme. Kinetic studies demonstrated that l-ATP cannot be used as a cofactor in the ligase-catalysed joining reaction. On the other hand, l-AMP is used by T4 DNA ligase to catalyse the reverse reaction, even though a high level of intermediate circular nicked DNA molecules accumulates. Our results suggest that a lack of enantioselectivity of enzymes is more common than was believed a few years ago, and, given the absence of selective constraints against l-nucleosides in Nature, this may depend on chance more than on evolutionary strategy.
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PMID:L-ATP is recognized by some cellular and viral enzymes: does chance drive enzymic enantioselectivity? 989 5

In this study, human oropharyngeal epidermoid carcinoma KB cells that were resistant to 2,2-difluorodeoxycytidine (dFdCyd) were selected and designated the KB-Gem clone. The KB parental cell line IC50 was 0.3 microM dFdCyd, as compared with the KB-Gem clone IC50 of 32 microM dFdCyd. The KB-Gem clone demonstrated overexpression of ribonucleotide reductase (RR) M2 subunit mRNA (9-fold) and overexpression of M2 protein (2-fold); RR activity was 2.3-fold higher than the KB parental cell line. Both the dATP and dCTP pools of the KB-Gem clone increased 2-fold over the parental cell line, with no change in the dGTP and dTTP pools. Reverse transcriptase-PCR was used to clone the cDNA of deoxycytidine kinase (DCK). Resulting sequences revealed two silent mutations in the KB-Gem clone. The amino acid sequence of the DCK protein and mRNA expression remained unchanged. The KB-Gem clone's DCK enzyme activity was 56% of that of the parental cell line. After the endogenous dNTPs were removed with a G-25 column, no difference was evident between the enzyme activities of the KB-Gem clone and parental cells. Thus, contrary to previous hypotheses, DCK deficiency does not play the primary role in the resistance mechanism of dFdCyd, accepting a secondary role to the overexpression of the target gene, RR, and pool expansion.
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PMID:Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-difluorodeoxycytidine in the human KB cancer cell line. 1048 55

In vitro studies have demonstrated that deoxycytidine kinase (dCK) plays a crucial role in the mechanism of resistance to cytarabine (AraC). The resistant phenotype in vitro is always a result of mutational inactivation of dCK, leading to defects in the metabolic pathways of AraC. Although inactivation of dCK has shown to be one of the major mechanism of resistance to AraC in vitro, limited in vivo data are available. To improve research concerning the involvement of dCK inactivation in patients with acute myeloid leukemia (AML), we have set up a protocol that allows direct assessment of dCK expression and activity in primary human cells. In this protein activity truncation assay (PAT assay), the complete coding region of dCK is amplified by RT-PCR and a T7 RNA polymerase promoter sequence is introduced upstream of the coding region in a nested PCR reaction. After in vitro transcription-translation dCK proteins are analyzed for their molecular weight and phosphorylating capacities. We show that this relatively quick method can be used in purified, primary human leukemic blasts. In addition, inactivation of dCK by point mutations, deletions or genomic rearrangements can easily be detected in AraC-resistant cell lines. This novel assay may contribute to further elucidate the mechanism of AraC resistance in vivo.
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PMID:A novel RT-PCR-based protein activity truncation assay for direct assessment of deoxycytidine kinase in small numbers of purified leukemic cells. 1136 49

We established a variant of MIAPaCa-2 human pancreatic cancer cells that is resistant to 2',2'-difluorodeoxycytidine (gemcitabine, dFdCyd), MIAPaCa-2/dFdCyd, and elucidated the biochemical characteristics and mechanism of dFdCyd-resistance in these cells. We also evaluated 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106, RNA polymerase inhibitor), a new anticancer ribonucleoside, for antitumor activity against the resistant cells in vitro and in vivo. MIAPaCa-2/dFdCyd cells were 2541-fold more resistant to dFdCyd than parental MIAPaCa-2 cells, and the major mechanism of the dFdCyd-resistance was found to be a decrease in the intracellular pool of dFdCyd and its active metabolites, which would result in a decrease in incorporation of dFdCyd triphosphate into DNA. This finding was confirmed by the discovery of decreased deoxycytidine kinase activity, increased cytidine deaminase and ribonucleotide reductase activity, and increased 5'-nucleotidase mRNA expression in the MIAPaCa-2/dFdCyd cells. The cytotoxicity of TAS-106 as an antitumor nucleoside analog was similar in both parental and dFdCyd-resistant cells, with IC(50) values of 6.25 and 6.27 nM, respectively, and this finding was supported by similar intracellular uptake and metabolism of TAS-106 in both cell lines. We also evaluated the in vivo antitumor activity of TAS-106 against MIAPaCa-2 and dFdCyd-resistant MIAPaCa-2/dFdCyd tumors implanted into nude mice. The tumor growth inhibition rate of weekly additions of TAS-106 (7 mg/kg, iv) against parental and dFdCyd-resistant tumors was 73% and 76%, respectively, while that of dFdCyd administered twice a week (240 mg/kg, iv) was 84% and 34%, respectively. These results suggest that TAS-106 would contribute to the treatment of patients with advanced pancreatic carcinomas in whom dFdCyd-based chemotherapy has failed.
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PMID:Possible antitumor activity of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106) against an established gemcitabine (dFdCyd)-resistant human pancreatic cancer cell line. 1590 71

The aim of the present study was to estimate the expression of mRNA, specific for thymidine kinase 1 (TK1), deoxycytidine kinase (dCK), and thymidine phosphorylase (dThdPase), i.e. enzymes involved in pyrimidine and purine metabolism in human papillary thyroid carcinoma (PTC) tissue. Additionally, the expression of dCK was estimated, in medullary thyroid carcinoma (MTC). For control, the RNA expression levels for all the enzymes were measured in macroscopically unchanged thyroid tissue. Reverse transcriptase-polymerase chain reaction (RT-PCR) and densitometry were employed for mRNA expression measurements, with the beta-actin gene as a control housekeeping gene. The levels of mRNA expression for TK1, dCK and dThdPase in human PTC, as well as mRNA expression for dCK in MTC, were significantly higher than mRNA expressions for those enzymes found in macroscopically unchanged thyroid tissue. It is concluded that an increased expression of mRNA, specific for TK1, dCK and dThdPase, may be involved in carcinogenic processes in the human thyroid.
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PMID:Increased expression of mRNA specific for thymidine kinase, deoxycytidine kinase or thymidine phosphorylase in human papillary thyroid carcinoma. 1597 30

Beta-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5'-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2'-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a Km of 81 microM and a kcat of 0.007 s-1, but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (Ki) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 microM. Similar results were obtained with 2'-C-methyladenosine triphosphate (Ki=1.5 microM) and 2'-C-methylcytidine triphosphate (Ki=1.6 microM). NS5B with the S282T mutation, which is known to confer resistance to 2'-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.
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PMID:Mechanism of activation of beta-D-2'-deoxy-2'-fluoro-2'-c-methylcytidine and inhibition of hepatitis C virus NS5B RNA polymerase. 1710 74