Phosphorylation of Uridine and Cytidine Nucleoside Analogs by Two Human Uridine-Cytidine Kinases

Uridine-cytidine kinases (UCK) have important roles for the phosphorylation of nucleoside analogs that are being investigated for possible use in chemotherapy of cancer. We have cloned the cDNA of two human UCKs. The '30-kDa proteins, named UCK1 and UCK2, were expressed in Escherichia coli and shown to catalyze the phosphorylation of Urd and Cyd. The enzymes did not phosphorylate deoxyribonucleosides or purine ribonucleosides. UCK1 mRNA was detected as two isoforms of '1.8 and '2.7 kb. The 2.7-kb band was ubiquitously expressed in the investigated tissues. The band of '1.8 kb was present in skeletal muscle, heart, liver, and kidney. The two isoforms of UCK2 mRNA of 1.2 and 2.0 kb were only detected in placenta among the investigated tissues. The genes encoding UCK1 and UCK2 were mapped to chromosome 9q34.29q34.3 and 1q22-1q23.2, respectively. We tested 28 cytidine and uridine nucleoside analogs as possible substrates of the enzymes. The enzymes phosphorylated several of the analogs, such as 6-azauridine, 5-fluorouridine, 4-thiouridine, 5-bromouridine, N-acetylcytidine, N-benzoylcytidine, 5-fluorocytidine, 2-thiocytidine, 5-methylcytidine, and N-anisoylcytidine. The cloning and recombinant expression of the two human UCKs will be important for development of novel pyrimidine ribonucleoside analogs and the characterization of their pharmacological activation. Uridine-cytidine kinase (UCK) (EC 2.7.1.48) is a pyrimidine ribonucleoside kinase that catalyzes the phosphorylation of uridine and cytidine to UMP and CMP. The enzyme also catalyzes the phosphorylation of several cytotoxic ribonucleoside analogs that have been investigated for possible use as chemotherapeutic agents for treatment of cancer. The nucleoside analogs are dependent on phosphorylation for their pharmacological activity. Once phosphorylated, the compounds interfere with vital cellular processes such as DNA or RNA synthesis or inhibit enzymes involved in nucleotide synthesis (Cihak and Rada, 1976). The ribonucleoside analogs phosphorylated by UCK include 5-fluorouridine, 5-azacytidine, and cyclopentenyl cytosine/-uracil as well as the recently developed 1-(3-C-ethynyl-b-D-ribo-pentofuranosyl)-cytosine/-uracil (Sköld, 1960; Lee et al., 1974; Vesely, 1985; Kang et al., 1989; Hattori et al., 1996; Tabata et al., 1997; Takatori et al., 1999; Verschuur et al., 2000). In addition to the ribonucleoside analogs, UCK may also be important for the pharmacological activation of uridine and cytidine base analogs, such as the clinically used analog 5-fluorouracil. This compound may be converted to 5-fluorouridine by uridine phosphorylase that subsequently will be dependent on UCK catalyzed phosphorylation (Reichard and Sköld, 1957; Reichard and Sköld, 1958). Most of the nucleoside analogs are dependent on phosphorylation to their triphosphate form for pharmacological activity. After the first phosphorylation catalyzed by UCK, pyrimidine ribonucleoside analogs are further phosphorylated by UMP-CMP kinase (Van Rompay et al., 1999) and nucleoside diphosphate kinases (Parks and Agarwal, 1973). However, the first phosphorylation step catalyzed by UCK is considered rate-limiting and the level of UCK activity may be correlated with the cellular sensitivity to the nucleoside analogs (Reichard and Sköld, 1958; Anderson and Brockman, 1964). Loss of UCK activity is also seen in cells resistant to the nucleoside analogs (Reichard et al., 1959; Vesely et al., 1971; Greenberg et al., 1977). Interestingly, several studies suggest that UCK activity may increase in tumor cells compared with normal tissues (Reichard et al., 1959; Herzfeld and Raper, 1979; Shen et al., 1998). Mammalian UCKs have been purified from several different tissues (Reichard and Sköld, 1957; Krystal and Webb, 1971; Anderson, 1973; Cihak and Rada, 1976; Absil et al., 1980). However, the only mammalian UCK cDNA yet cloned is a cDNA isolated from mouse (Ropp and Traut, 1996). We decided to identify and clone human uridine-cytidine kinases to study these enzymes for the pharmacological activation of nucleoside analogs. Our findings show that a UCK enzyme This work was supported by grants from the Swedish Medical Research Council, the Swedish Cancer Foundation, the Swedish Foundation of Strategic Research and the European Commission. The DNA sequence reported in this paper has been deposited in the GenBank database (accession no. AF236636, AF236637, and AF237290). ABBREVIATIONS: UCK, uridine-cytidine kinase; kb, kilobase pairs(s); bp, base pair(s); BSA, bovine serum albumin. 0026-895X/01/5905-1181–1186$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 5 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 706/900016 Mol Pharmacol 59:1181–1186, 2001 Printed in U.S.A. 1181 at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from family with at least two members exists in humans cells. We have further expressed the two enzymes recombinantly and have investigated their ability to catalyze the phosphorylation of several pyrimidine nucleoside analogs. Experimental Procedures Cloning and Expression of Human UCK1 and UCK2 cDNA. We searched the expressed sequence tag library of the GenBank database at the National Institute for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) with the Basic Local Alignment Search Tool (BLAST) to identify human cDNA clones that encoded protein homologs to the cloned mouse UCK (Ropp and Traut, 1996). The expressed sequence tag cDNA clones were obtained from Research Genetics Inc (Huntsville, AL). The DNA sequences of the plasmids were determined with the ABI Prism 310 Genetic Analyzer (PerkinElmer/Applied Biosystems, Norwalk, CT) and with the automatic laser fluorescent sequencer (Amersham Pharmacia Biotech,