Phosphorylation of uridine and cytidine nucleoside analogs by two human uridine-cytidine kinases.
ABSTRACT 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 approximately 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 approximately 1.8 and approximately 2.7 kb. The 2.7-kb band was ubiquitously expressed in the investigated tissues. The band of approximately 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 9q34.2-9q34.3 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(4)-acetylcytidine, N(4)-benzoylcytidine, 5-fluorocytidine, 2-thiocytidine, 5-methylcytidine, and N(4)-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.
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ABSTRACT: Activation-induced cytidine deaminase (AID) was originally identified as an inducer of somatic hypermutation (SHM) and class switch recombination (CSR) in immunoglobulin genes. However, AID can also cause mutations in host genes and contribute to cancer progression and drug resistance. In this study, molecular docking showed the interaction of free 5-aza-CdR and Zebularine (Zeb) with AID. However, only 5-aza-CdR-incorporated ssDNA bound to the active site of AID and inhibited AID expression through proteasomal degradation. 5-aza-CdR demonstrated cytotoxicity against AID-positive and -negative hematopoietic cancer cells. In contrast, Zeb exhibited a cytotoxic effect only in AID-negative cells due to its inability to inhibit AID expression. This differential effect might be due to the DNMT1 stabilization induced by AID, thus restricting the ability of Zeb to deplete DNMT1 and induce tumor suppressor genes (TSGs), such as p21, in AID-positive cells. Moreover, the in vivo anticancer effect of 5-aza-CdR but not Zeb in AID-positive hematopoietic cancer cells was demonstrated. The study not only displays the association of AID and DNMT1 and identifies a novel biological function of AID, but also provides novel information regarding the use of DNMT inhibitors to treat AID-positive hematopoietic cancers.Oncotarget 11/2013; · 6.64 Impact Factor
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ABSTRACT: Genome-wide association studies (GWAS) have identified multiple common genetic variants associated with an increased risk of testicular germ cell tumors (TGCT). A previous GWAS study reported a possible TGCT susceptibility locus on chromosome 1q23 in the UCK2 gene but failed to reach genome-wide significance following replication. We interrogated this region by conducting a meta-analysis of two independent GWAS including a total of 940 TGCT cases and 1,559 controls for 122 single nucleotide polymorphisms (SNP) on chromosome 1q23 and followed up the most significant SNPs in an additional 2,202 TGCT cases and 2,386 controls from four case-control studies. We observed genome-wide significant associations for several UCK2 markers, the most significant of which was for rs3790665 (PCombined=6.0x10-9). Additional support is provided from an independent familial study of TGCT where a significant over transmission for rs3790665 with TGCT risk was observed (PFBAT=2.3x10-3). Here, we provide substantial evidence for the association between UCK2 genetic variation and TGCT risk.Human Molecular Genetics 03/2013; · 7.69 Impact Factor
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ABSTRACT: The nucleoside analog azacitidine (AZA) is used in the treatment of myelodysplastic syndromes (MDS), but 30-40% of patients fail to respond or relapse after treatment. Hence, to identify new molecular alterations that allow for identification of patients unlikely to respond to AZA could impact the utility of this therapy. We determined expression levels of genes involved in AZA metabolism: UCK1, UCK2, DCK, hENT1, RRM1, and RRM2 using quantitative PCR in samples from 57 patients with MDS who received AZA. Lower expression of UCK1 was seen in patients without a response to AZA (median 0.2 vs 0.49 for patients with response to AZA, P=0.07). This difference in UCK1 expression was not influenced by aberrant methylation of the UCK1 promoter. In addition, the seven polymorphic loci found in the coding sequence were not associated with UCK1 gene expression nor AZA response. Silencing of UCK1 by siRNA leads to blunted response to AZA in vitro. The univariate analysis revealed that patients expressing lower than median levels of UCK1 had a shorter overall survival (P=0.049). Our results suggest that expression level of UCK1 is correlated with clinical outcome and may influence the clinical response to AZA treatment in patients with MDS.Leukemia accepted article preview online, 6 November 2013; doi:10.1038/leu.2013.330.Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 11/2013; · 10.16 Impact Factor