Functional Studies Of The Human Uridine Cytidine Kinase Gene

Uridine-cytidine kinase (UCK) (EC 2.7.1.48) is a pyrimidine ribonucleoside kinase that catalyzes thephosphorylation of uridine and cytidine to UMP and CMP. UCK is cloned by homogenous screeningstrategy and submitted to Genebank with accession No. AF125106. The 30kDa proteins were expressed ineukaryotic cells and shown to catalyze the phosphorylation of uridine and cytidine. With confocalmicroscopy, UCK was detected to distribute in the cytoplasma. Northern blot analysis of human UCK was carried out on Multiple Tissue membranes with mRNAsamples from 16 adult human tissues to confirm its natural existence and its tissue distribution. It was foundto be 2kb in length and abundant in liver, testis, heart, middle in brain, skeletal muscle, kidney, pancreas,colon, but little in peripheral blood lymphocyte and ovary. The expressive changes of the UCK on theirtranscriptional levels in liver cancer were detected. It was found to be commonly up-regulated in liver cancertissues. In addition, the expressional levels of UCK in different developmental stage in rat were alsoperformed. The result showed that UCK was highly expressed in fetus and gradually decreases after birth.Tofurther demonstrate the relation between UCK and hepatoma, UCK was expressed in E. coli. and purified bychromatography. The purified proteins immunized rabbits and polyclonal antibody was obtained. With theimmunohistochemistry method, UCK was found obviously highly expressed in the hepatoma tissues than inthe normal liver tissues. It figures out some clues of the relationship between UCK and cell proliferation andprovides some useful indications that over expression of UCK might be a marker of liver cancer. UCK is able to catalyze phosphorylation of its physiological substrate uridine and cytidine, and it alsoplays important roles for the phosphorylation of nucleoside analogs that are being investigated for possibleuse in chemotherapy of cancer, such as 6-azauridine, 5-fluorouridine and so on. Once phosphorylated, thecompounds interfere with vital cellular processes such as DNA or RNA synthesis or inhibit enzymesinvolved in nucleotide synthesis. 6-azauridine was sensitive to the UCK transfected 293 cell, and thesensitivity was in correlation with the plasmid concentration. In addition, 6-azauridine is more sensitive toHepG2 than L02, suggesting the high activity of uridine kinase is helpful for the transformation ofchemotherapeutic analogue, 6-azauridine, to cytotoxic products. The cloning of human UCK gene mayinitiate further investigation of the UCK protein's role in tumor chemotherapy, and is important fordevelopment of novel pyrimidine ribonucleoside analogs and the characterization of their pharmacologicalactivation. TCP10L is a novel gene we cloned recently, and has been deposited to GenBank. In order to study thefunction of the novel gene, we firstly analysed its protein structure and a typical leucine zipper motif wasfound. Since the leucine zipper is indispensable to the biological functions of many transcription factors, we 4复旦大学博士学位论文speculated that the TCP10L might possess transcription regulating activity. We firstly examined thetranscriptional function of the TCP10L protein by transfecting recombinant pM-TCP10L into HEK293,SK-HEP-1, and CHO cells. The subsequent analysis based on the dual luciferase assay system showed thatTCP10L significantly inhibited the expression of reporter gene. Compared with that of the negative control,the luciferase activity were down-regulated in HEK293 and SK-HEP-1, CHO cells by about 2.6, 9.8, and 5.5folds respectively. When the second leucine of zipper was mutated, such transcriptional inhibition disappears.It is suggested that TCP10L encodes a protein bearing transcription inhibition activity. Then, we fusedTCP10L to EGFP and transfected the recombinant TCP10L-EGFP into mammalian cells to explore thecel...