title: UMP/CMPK is not the critical enzyme in the metabolism of pyrimidine ribonucleotide and activation of deoxycytidine analogs in human RKO cells.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, USA.
authors.forename: Rong
authors.initials: R
authors.lastname: Hu
authors.forename: Wing
authors.initials: W
authors.lastname: Lam
authors.forename: Chih-Hung
authors.initials: CH
authors.lastname: Hsu
authors.forename: Yung-Chi
authors.initials: YC
authors.lastname: Cheng
mesh_terms: D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000378:metabolism*; D015536:Down-Regulation; D004798:Enzymes / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007700:Kinetics; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism*
chemical_list: D004798:Enzymes; D009711:Nucleotides; D011743:Pyrimidines; D011994:Recombinant Proteins; D012265:Ribonucleotides; D003841:Deoxycytidine; C023276:uridine monophosphate kinase; D009703:Nucleoside-Phosphate Kinase; C030986:pyrimidine