UMP/CMPK Is Not the Critical Enzyme in the Metabolism of Pyrimidine Ribonucleotide and Activation of Deoxycytidine Analogs in Human RKO Cells

Background Human UMP/CMP kinase was identified based on its enzymatic activity in vitro. The role of this protein is considered critical for the maintenance of pyrimidine nucleotide pool profile and for the metabolism of pyrimidine analogs in cells, based on the in vitro study of partially purified enzyme and recombinant protein. However, no detailed study has yet addressed the role of this protein in nucleotide metabolism in cells. Methodology/Principal Findings Two stable cell lines in which UMP/CMP kinase (mRNA: AF087865, EC 2.7.4.14) can be either up-regulated or down-regulated were developed using Tet-On Gene Expression Systems. The amount and enzymatic activity of UMP/CMP kinase extracted from these two cell lines can be induced up by 500% or down by 95–98%. The ribonucleotides of endogenous pyrimidine as well as the metabolism of exogenous natural pyrimidine nucleosides and their analogs were not susceptible to the altered amount of UMP/CMP kinase in these two stable RKO cell lines. The level of incorporation of pyrimidine nucleoside analogs, such as gemcitabine (dFdC) and troxacitabine (L-OddC), into cellular DNA and their potency in inhibiting cell growth were not significantly altered by up-regulation or down-regulation of UMP/CMP kinase expression in cells. Conclusions/Significance The UMP/CMP kinase (EC 2.7.4.14) expressed in RKO cells is not critical for the phosphorylation of (d)CMP and the maintenance of natural nucleotide pools. It also does not play an important role in the activation of dFdC and L-OddC. The increase by 500% or decrease by 95–98% in the levels of UMP/CMP kinase do not affect steady state levels of dFdC and L-OddC in RKO cells. Overall, the activity and possible mechanisms of recombinant UMP/CMP kinase expressed in the in vitro system can not be extended to that of UMP/CMP kinase expressed in a cell system or an in vivo system.

[1]  I. Burney Cancer Chemotherapy and Biotherapy : Principles and Practice , 2011 .

[2]  Yung-chi Cheng,et al.  Modulation of human UMP/CMP kinase affects activation and cellular sensitivity of deoxycytidine analogs. , 2010, Biochemical pharmacology.

[3]  N. Kemeny,et al.  Decreased levels of UMP kinase as a mechanism of fluoropyrimidine resistance , 2009, Molecular Cancer Therapeutics.

[4]  D. Banerjee,et al.  Epigenetic reversal of acquired resistance to 5-fluorouracil treatment , 2009, Molecular Cancer Therapeutics.

[5]  Nigel Jenkins,et al.  Post-translational Modifications of Recombinant Proteins: Significance for Biopharmaceuticals , 2008, Molecular biotechnology.

[6]  S. Pochet,et al.  Enantioselectivity of human AMP, dTMP and UMP-CMP kinases , 2007, Nucleic acids research.

[7]  D. Geerts,et al.  Comparison of RNAi efficiency mediated by tetracycline-responsive H1 and U6 promoter variants in mammalian cell lines , 2007, Nucleic acids research.

[8]  Chih-Hung Hsu,et al.  Comparison of the Phosphorylation of 4′-Ethynyl 2′,3′-Dihydro-3′-Deoxythymidine with That of Other Anti-Human Immunodeficiency Virus Thymidine Analogs , 2007, Antimicrobial Agents and Chemotherapy.

[9]  C. Leung,et al.  Apurinic/Apyrimidinic Endonuclease-1 Protein Level Is Associated with the Cytotoxicity of l-Configuration Deoxycytidine Analogs (Troxacitabine and β-l-2′,3′-Dideoxy-2′,3′-didehydro-5-fluorocytidine) but Not d-Configuration Deoxycytidine Analogs (Gemcitabine and β-d-Arabinofuranosylcytosine) , 2006, Molecular Pharmacology.

[10]  B. Degrève,et al.  Behavior of Thymidylate Kinase toward Monophosphate Metabolites and Its Role in the Metabolism of 1-(2′-Deoxy-2′-Fluoro-β-l-Arabinofuranosyl)-5-Methyluracil (Clevudine) and 2′,3′-Didehydro-2′,3′-Dideoxythymidine in Cells , 2005, Antimicrobial Agents and Chemotherapy.

[11]  Chih-Hung Hsu,et al.  Phosphorylation of Cytidine, Deoxycytidine, and Their Analog Monophosphates by Human UMP/CMP Kinase Is Differentially Regulated by ATP and Magnesium , 2005, Molecular Pharmacology.

[12]  M. Konrad,et al.  Substrate-induced Conformational Changes in Human UMP/CMP Kinase* , 2004, Journal of Biological Chemistry.

[13]  M. Baba,et al.  Novel 4′-Substituted Stavudine Analog with Improved Anti-Human Immunodeficiency Virus Activity and Decreased Cytotoxicity , 2004, Antimicrobial Agents and Chemotherapy.

[14]  T. Traut,et al.  Physiological concentrations of purines and pyrimidines , 1994, Molecular and Cellular Biochemistry.

[15]  Elizabeth A. Gullen,et al.  Novel Role of 3-Phosphoglycerate Kinase, a Glycolytic Enzyme, in the Activation of L-Nucleoside Analogs, a New Class of Anticancer and Antiviral Agents* , 2003, Journal of Biological Chemistry.

[16]  H. Munier-Lehmann,et al.  Reaction of human UMP-CMP kinase with natural and analog substrates. , 2003, European journal of biochemistry.

[17]  P. O'Connor,et al.  RPR‐115135, a farnesyltransferase inhibitor, increases 5‐FU‐ cytotoxicity in ten human colon cancer cell lines: Role of p53 , 2002, International journal of cancer.

[18]  W. Lam,et al.  Characterization of human UMP/CMP kinase and its phosphorylation of D- and L-form deoxycytidine analogue monophosphates. , 2002, Cancer research.

[19]  H. Castro-Faria-Neto,et al.  Characterization of human UMP-CMP kinase enzymatic activity and 5' untranslated region. , 2001, Life Science.

[20]  Dan L. Longo,et al.  Cancer Chemotherapy and Biotherapy: Principles and Practice , 2001 .

[21]  T. Lawrence,et al.  The role of p53 in gemcitabine-mediated cytotoxicity and radiosensitization , 2000, Cancer Chemotherapy and Pharmacology.

[22]  A. Karlsson,et al.  Phosphorylation of deoxycytidine analog monophosphates by UMP-CMP kinase: molecular characterization of the human enzyme. , 1999, Molecular pharmacology.

[23]  M. Kanehisa,et al.  Reconstruction of amino acid biosynthesis pathways from the complete genome sequence. , 1998, Genome research.

[24]  B. Chabner,et al.  Pyrimidine nucleoside monophosphate kinase from human leukemic blast cells. , 1978, Cancer research.

[25]  S. Fujii,et al.  A new deoxyuridine-5'-triphosphatase in Yoshida sarcoma cells involved in deoxyuridine 5'-triphosphate metabolism. , 1977, Cancer research.

[26]  S. Chen,et al.  Human erythrocyte pyrimidine nucleoside monophosphate kinase. Partial purification and properties of two allelic gene products. , 1976, Journal of Biological Chemistry.