Characterization of Human UMP/CMP Kinase and Its Phosphorylation of D- and 1 L-Form Deoxycytidine Analogue Monophosphates
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[CANCER RESEARCH 62, 1624–1631, March 15, 2002] Characterization of Human UMP/CMP Kinase and Its Phosphorylation of D- and 1 L-Form Deoxycytidine Analogue Monophosphates Jieh-Yuan Liou, Ginger E. Dutschman, Wing Lam, Zaoli Jiang, and Yung-Chi Cheng2 Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520 ABSTRACT with leukemia, lymphoma, or solid tumors (11). Deoxycytidine ana- logues, such as -D-2Ј,3Ј-dideoxycytidine and L-(Ϫ)-SddC (Lamivu- Pyrimidine nucleoside monophosphate kinase [UMP/CMP kinase dine), have been shown to have anti-HIV and antihuman hepatitis B (UMP/CMPK); EC 2.7.4.14] plays a crucial role in the formation of UDP, virus activities (12–17). L-(Ϫ)-SddC was the first nucleoside analogue CDP, and dCDP, which are required for cellular nucleic acid synthesis. Several cytidine and deoxycytidine analogues are important anticancer with an L configuration to show therapeutic activity and, thus, defined  Ј Ј and antiviral drugs. These drugs require stepwise phosphorylation to their a new category for the design of nucleoside analogues. -L-2 ,3 - triphosphate forms to exert their therapeutic effects. The role of UMP/ dideoxy-5-fluoro-3Ј-thia-cytidine and -L-2Ј,3Ј-dideoxy-2Ј,3Ј-dide- CMPK for the phosphorylation of nucleoside analogues has been indi- hydro-5-fluorocytidine have been shown to be potent antihuman hep- cated. Thus, we cloned the human UMP/CMPK gene, expressed it in atitis B virus agents in vitro and in animal studies (18–22). In studies Escherichia coli, and purified it to homogeneity. Its kinetic properties of other -L-(Ϫ)-2Ј,3Ј-dideoxycytidine analogues, it was observed were determined. UMP and CMP proved to be far better substrates than that L-(Ϫ)-OddC was a potent inhibitor of cell growth. Currently, dCMP. UMP/CMPK used all of the nucleoside triphosphates as phosphate L-(Ϫ)-OddC is the only cytotoxic deoxycytidine analogue to act as a donors, with ATP and dATP being the best donors and CTP being the true chain terminator upon incorporation into DNA (23, 24). Preclin- poorest. Furthermore, UMP/CMPK was able to phosphorylate all of ical and Phase I evaluations demonstrated its effectiveness against the deoxycytidine analogue monophosphates that we tested. The rela- both leukemia and solid tumors (25–29). At present, L-(Ϫ)-OddC is tive efficiency was as follows: arabinofuranosyl-CMP > dCMP > -L- 2,3-dideoxy-3-thia-CMP > Gemcitabine monophosphate > -D-2,3- being evaluated in Phase II clinical studies, and the spectrum of its dideoxy-CMP; -L-2,3-dideoxy-2,3-didehydro-5-fluoro-CMP; -L- antitumor activity is anticipated to be significantly different from 2,3-dideoxy-5-fluoro-3-thia-CMP > -L-2,3-dideoxy-CMP > -L- those of 1--D-arabinofuranosylcytosine and Gemcitabine. These dioxolane-CMP. By comparing the relative Vmax/Km values of D- and agents need to be phosphorylated in a stepwise fashion by human L-form dideoxy-CMP, we showed that this kinase lacked stereoselectivity. kinases to their triphosphate forms to exert their activities. The first Reducing agents, such as DTT, 2-mercaptoethanol, and thioredoxin, were phosphorylation step is carried out by deoxycytidine kinase and able to activate this enzyme, suggesting that its activity may be regulated uridine kinases (30). The pyrimidine nucleoside monophosphates are by redox potential in vivo. UMP/CMPK localized predominantly to the further phosphorylated by pyrimidine nucleoside monophosphate ki- cytoplasm. In addition, 196-amino acid UMP/CMPK was the actual form nase, which is thought to be UMP/CMPK. The third step is carried out of UMP/CMPK, rather than the 228-amino acid form as suggested before. by nucleoside diphosphate kinases or other unidentified kinases. Human and mammalian UMP/CMPs have been partially purified INTRODUCTION by a number of investigators, including our group (31–40). Some characterizations have been performed on these partially purified UMP/CMPK3 catalyzes the phosphoryl transfer from ATP to CMP, enzymes. Recently, a pig UMP/CMPK gene has been cloned and UMP, and dCMP, resulting in the formation of ADP and the corre- expressed in Escherichia coli, and the recombinant protein was puri- sponding nucleoside diphosphates (1). Both de novo and salvage fied to homogeneity (7). However, the enzyme activity and properties pathways produce pyrimidine monophosphate, and these two path- have not been well characterized, especially with regard to the phos- ways converge at the level of UMP/CMPK for the synthesis of phorylation of nucleoside analogue monophosphates. In this study, we pyrimidine diphosphate. Thus, UMP/CMPK plays an important role cloned the cDNA of human UMP/CMPK, expressed it in E. coli, and in pyrimidine synthesis. Protein sequences from different species purified the recombinant protein. The enzyme kinetics and phospho- show that the UMP/CMPK gene is highly conserved, suggesting an rylation of both natural nucleoside and nucleoside analogue mono- important role of this kinase in all of these organisms (2–9). Recently, phosphates were studied, with special emphasis on the enzymatic a conditional lethal mutant isolated from Saccharomyces cerevisiae conversion of several clinically important L-form nucleoside analogue was described in which UMP/CMPK was mutated, indicating the monophosphates. The subcellular localization and protein form were essential role of the kinase in this organism (10). However, a detailed determined using a specific antibody. Independently, van Rompay et study of this enzyme has not been performed to date. al. (41) have also cloned the human UMP/CMPK gene and have done Deoxycytidine and cytidine analogues, such as 1--D-arabino- some enzymatic characterization using the recombinant kinase. De- furanosylcytosine, 5-azacytidine, and 2Ј,2Ј-difluorodeoxycytidine tailed comparisons will be presented in the “Discussion” section. (Gemcitabine), were shown to be useful for the treatment of patients MATERIALS AND METHODS Received 10/3/01; accepted 1/16/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with Cloning of the Human UMP/CMPK Gene. Total RNA was extracted 18 U.S.C. Section 1734 solely to indicate this fact. from KB cells (human epidermoid carcinoma). Five g of total RNA were 1 Supported by NIH Grants AI38204 and CA63477. used in the reverse transcription reaction using SuperScriptII (Life Technolo- 2 To whom requests for reprints should be addressed, at Department of Pharmacology, gies, Inc., Rockville, MD) and oligo(dT) primer. The reverse transcription Yale University School of Medicine, P. O. Box 802066, New Haven, CT 06520. Phone: Ј (203) 785-7119; Fax: (203) 785-7129; E-mail: [email protected]. product was subjected to PCR using the primers 5 -GTCAGCTCCCT- 3 The abbreviations used are: UMP/CMPK, UMP/CMP kinase; L-(Ϫ)-SddC (3TC), CAGCGTCCGG-3Ј and 5Ј-TGGTCCACAAATTCCTAAGG-3Ј, which were -L-2Ј,3Ј-dideoxy-3Ј-thiacytidine; L-(Ϫ)-OddC, -L-dioxolane-cytidine; aa, amino acid; designed according to the assembled EST sequences. Amplification was per- Ϫ  Ј Ј Ј  Ј Ј L-( )-SddCMP, -L-2 ,3 -dideoxy-3 -thia-CMP; ddCMP, -D-2 ,3 -dideoxy-CMP; formed in a PTC-100 thermal controller (MJ Research, Watertown, MA). The L-(Ϫ)-FSddCMP, -L-2Ј,3Ј-dideoxy-5-fluoro-3Ј-thia-CMP; HPLC, high-performance liq- uid chromatography; AraCMP, 1--D-arabinofuranosyl-CMP; ORF, open reading frame; amplified 1.2-kb product was cloned into the Eukaryotic TA Cloning Vector GST, glutathione-S-transferase; GFP, green fluorescent protein. (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The 1624 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2002 American Association for Cancer Research. PHOSPHORYLATION OF L-FORM DEOXYCYTIDINE ANALOGUE MONOPHOSPHATES plasmid clones were checked by EcoRI cutting to contain the 1.2-kb fragment. cific binding of antibodies. UMP/CMPK protein was targeted first by rabbit Potential clones were confirmed by sequencing. polyclonal anti-UMP/CMPK antibody (1:200) and subsequently by FITC- Expression, Purification, and Thrombin Cutting of Recombinant UMP/ conjugated antirabbit IgG (1:100: Sigma). In the control experiment, purified CMPK. Two sets of primers were used to amplify the desired fragments for the UMP/CMPK protein was added to compete for binding with anti-UMP/CMPK expression of 228-aa and 196-aa UMP/CMPK forms. The primers were designed antibody in the primary hybridization step. Nuclei were counterstained with to contain an NdeI site at the 5Ј end and a BamHI site at the 3Ј end for cloning propidium iodide (50 ng/ml). Cells were sealed in antifade reagent (Molecular purposes. The primer sets used were as follows: 5Ј-GGGAATTCCATATGCT- Probes, Eugene, OR). Confocal micrographs were scanned by a laser scan GAGCCGCTGCCGCAGC-3Ј and 5Ј-CGCGGATTCGAATTAGCCTCCCTTG- confocal microscope (LSM 510; Zeiss). GTC-3Ј (for 228-aa UMP/CMPK); 5Ј-GGGAATTCCATATGAAGCCGCTG- Cell Culture, Transfection, and Cloning of Stable Transfectants. HeLa GTCGTGTTC-3Ј and 5Ј-CGCGGATTCGAATTAGCCTCCCTTGGTC-3Ј (for S3 cells were cultured in DMEM supplemented with 10% fetal bovine serum 196-aa UMP/CMPK). The amplified fragments were cut with NdeI and BamHI in a humidified incubator at 37°Canda5%CO2 atmosphere. Transfections restriction enzymes and cloned into the pET-28a expression vector (Novagen, using the calcium phosphate method were performed as described previously Madison, WI), which carries an NH2-terminal His-tag/thrombin configuration. (44). The transfectants were selected with 1 mg/ml G418 (Life Technologies, The 196-aa UMP/CMPK fragment was also cloned into vector pCR3.1 (In- Inc.) from day 3 and continued for 3 weeks. The colonies were then picked out vitrogen) under the control of CMV promoter for eukaryotic expression. The and cultured in separate wells in 24-well plates. The activity of each clone was clones containing the desired NdeI-BamHI fragments were selected and analyzed, and positive clones were further cloned by serial dilution.