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

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UMP/CMPK Is Not the Critical Enzyme in the Metabolism of Pyrimidine Ribonucleotide and Activation of Deoxycytidine Analogs in Human RKO Cells UMP/CMPK Is Not the Critical Enzyme in the Metabolism of Pyrimidine Ribonucleotide and Activation of Deoxycytidine Analogs in Human RKO Cells Rong Hu1, Wing Lam1, Chih-Hung Hsu1,2, Yung-Chi Cheng* 1 Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America, 2 Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan, Republic of China 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. 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. Citation: Hu R, Lam W, Hsu C-H, Cheng Y-C (2011) UMP/CMPK Is Not the Critical Enzyme in the Metabolism of Pyrimidine Ribonucleotide and Activation of Deoxycytidine Analogs in Human RKO Cells. PLoS ONE 6(5): e19490. doi:10.1371/journal.pone.0019490 Editor: Jo¨rg Langowski, German Cancer Research Center, Germany Received October 11, 2010; Accepted April 7, 2011; Published May 3, 2011 Copyright: ß 2011 Hu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by the National Institutes of Health [grant CA-63477 and AI-38204]. Y-C.C. is a fellow of the National Foundation for Cancer Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction for CMP, dCMP, UMP and dUMP are around 5–500 mM, 404– 1600 mM, 50–1600 mM and 1300–5900 mM, respectively; the Kcat The enzymatic activity of UMP/CMP kinase has been con- values for CMP, dCMP, UMP and dUMP are 1.7–248 s21, 2.5– sidered to be essential for a variety of biological processes including 216 s21, 0.6–140 s21 and 0.1–7.2 s21, respectively, as summarized RNA synthesis, DNA replication/repair, membrane phospholipids in Table 1 [2,5,8,10]. The physiological concentrations of CMP, synthesis and nucleotide metabolism in cells. The human UMP/ dCMP, UMP and dUMP are around 34 mM, 0.3 mM, 184 mMand CMP kinase (mRNA: AF087865, EC 2.7.4.14) has been identified 2.2 mM, respectively, in cells [11]. The Michaelis-Menten kinetic based on its enzymatic activity catalyzing the phosphorylation of parameters organized in Table 1, however, exhibit much higher CMP, UMP, dCMP and dUMP to their diphosphate nucleotides Km values of this enzyme than the concentration of its natural using partially purified protein from cellular extract as well as its substrates in cells. Based on its enzymatic characteristics, this recombinant protein [1–9]. The gene of this protein was found to enzyme would be inefficient in cells unless other associated protein(s) be located on chromosome 1. (1p34.1-p33). It was assumed that could improve its efficiency. If the amount of this protein is changed this UMP/CMP kinase is responsible for the synthesis of CDP, or the protein is modified such as up-regulation, down-regulation, UDP and dCDP as well as the diphosphates of deoxycytidine/ phosphorylation, dephosphorlyation, glycosylation and methyla- deoxyuridine analog [2–9]. In the in vitro study, the efficiency of tion, it should affect the metabolism of nucleotides in cells. No this protein could be affected by different conditions of the enzyme detailed study currently has yet addressed the role of this protein on reaction, such as the concentration of DTT, Mg+2 and ATP etc. nucleotide metabolism in cells. These could account for the difference of its properties among Deoxycytidine analogs and 5-Fluorouracil (5-FU) are used in different laboratories. For the recombinant protein, its Km values clinic for the treatment of certain tumors and viral infections. PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e19490 Impact of Intracellular UMP/CMP Kinase Table 1. Kinetic properties of recombinant human UMP/CMPK with ATP as phosphate donor. Substrates Km (mM) Vmax (mmol/mg/min) Kcat (s21) CMP 500a 20b 15c 5,29d 4.2a 350.0b 232.0c 513,620.0d 1.7a 140.0b 92.0c 205,248.0d dCMP 1600 1000 513 404,1388 602 213.5 65.0 288,540..0 2.5 85.4 14.0 115,216.0 UMP 1600 50 67 N.D 1.6 350.0 218.0 N.D 0.6 140.0 87.8 N.D dUMP 5900 1300 N.D N.D 0.1 17.9 N.D N.D 0.1 7.2 N.D N.D The average values were summarized from the data reported by aVan Rompay et al, 1999; bAlexandre et al, 2007; cLiou et al, 2002; dHsu et al, 2004 in the different enzyme assay condition. doi:10.1371/journal.pone.0019490.t001 Those deoxycytidine analogs have to be phosphorylated stepwise were purchased from Moravek Biochemicals. The double- to their triphosphate forms before they can be incorporated into stranded DNA oligonucleotide encoding the shRNA of UMP/ cellular or viral DNA and/or RNA for anticancer and antiviral CMP kinase with the following sequences were used: 59- effects. UMP/CMP kinase activity should be pivotal for the (+)CACCCGGGGAAATGGATCAGACAATGGCGAACCAT- cytotoxic effect of cytidine analogs in target cells because it is TGTCTGATCCATTTCCC, 59-(2)AAAAGGGAAATGGAT- responsible for the phosphorylation of these cytidine analog CAGACAATGGTTCGCCATTGTCTGATCCATTTCCC (In- monophosphates or 5-FU monophosphate to their diphosphate vitrogen). RKO (human colorectal carcinoma) cells was a gift from metabolites in anticancer and antiviral therapy [2,5,7,9]. Gemci- Dr. Edward Chu’s lab (Department of Internal Medicine, Yale tabine (dFdC) and troxacitabine (L-OddC) are anticancer agents University). that are currently being used or under clinical trials for the treatment of cancer [12]. Although the configurations of dFdC Establishment of a stable cell line overexpressing UMP/ and L-OddC are different, the first two steps in their metabolism CMP kinase appear to be the same. Both dFdC and L-OddC are phosphor- The inducible Tet-On UMP/CMP kinase cell line was ylated by the cytoplasmic deoxycytidine kinase to their respective established by using Tet-On Gene Expression System (Invitro- monophosphate metabolites, and the so-called UMP/CMP kinase gen). The procedures described in the user manual of the Tet-On in vitro can phosphorylate dFdC and L-OddC monophosphates to Gene Expression System were followed. RKO cells were their diphosphate metabolites. Their in vitro Km values are 450– transfected using the regulator plasmid, pcDNA6/TR, which 581 mM with Vmax 3.6–31 mmol/mg/min and 1037 mM with expressed tetracycline repressor (TR) protein. After 48 hr, the Vmax 0.63 mmol/mg/min, respectively [2,5]. There are many cells were grown in medium with 0.5 mg/mL blasticidin for enzymes involved in 5-FU metabolism, in which this UMP/CMP selecting cells with TR expression. The selected cells with TR kinase was thought to play an important role in the activation of were further transfected using the pcDNA5/TO plasmids with 5-FU to 5FUTP/5FdUTP and its incorporation into RNA and the coding sequence (mRNA: AF087865) of human UMP/CMP DNA [12]. kinase, the Tet-responsive element is between 59- BamH I and 39- Although the UMP/CMP kinase is suggested to be the enzyme Xho I sites of pcDNA5/TO vector (Invitrogen). To isolate cells responsible for the phosphorylation of (d)CMP and (d)UMP as well as with co-transfected constructs, selection medium with both some cytidine analog monophosphates, there is little information in blasticidin and hygromycin at 0.5 mg/mL was added after cells to support the current dogma. The current knowledge indicated 48 hr transfection. The selected cells were used for the study. To that the KEGG (Kyoto Encyclopedia of Genes and Genomes) induce the expression of UMP/CMP kinase, the cells grown in metabolic pathway database has represented a network of interacting 5% serum-containing medium to 60% confluence were exposed molecules as well as compensatory and regulatory pathways during to various concentrations of doxycycline for 72 hr.
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