Chemotherapeutic nuclosides such as deoxyguanosine, guanine arabinoside (araG) and cytosine arabinoside (araC) require activation by deoxycytidine kinase for anti-tumor activity. Studies of this enzyme to date have yield conflicting results regarding its relative activity in leukemic cells, substrate affinity and nucleotide regulation. The investigations proposed here will attempt to resolve these controversies through a comprehensive analysis of deoxycytidine kinase activity in both a highly purified enzyme preparation and intact cells. The human cell lines that will be employed in these studies are leukemic T lymphoblasts, B lymphoblasts and mature T cells which display prominent differences in sensitivity to deoxyguanosine, araG and araC, apparently through differential abilities to accumulate the corresponding cytotoxic 5'-triphosphates. Since intracellular nucleotide accumulation is the net result of nucleoside phosphorylation and nucleotide degradation, the studies described here will examine both anabolic and catabolic pathways in the lymphoid cell lines. Initially, deoxycytidine kinase will be purified from each cell line using a procedure that results in a more highly purified preparation of this enzyme than that previously reported. Relative enzyme activity, Km and Vmax values will be measured for deoxycytidine kinase from each cell source. Since deoxycytidine kinase is allosterically regulated, the effects of deoxy- and ribonucleotides on the purified enzyme activity will be studied using the normal cellular concentrations of these nucleotides. For the studies intact cells, the intracellular dCTP and dGTP pool levels will be either elevated by administering the corresponding deoxyribonucleoside, or depleted by incubating the cells with phosphonoacetyl-L-aspartate and mycophenolic acid, which are inhibitors of pyrimidine and purine biosynthesis, respectively. After alteration of the desired nucleotide pool, cellular phosphorylation of the substrates deoxycytidine, deoxyguanosine, araG and araC will be analyzed by the extent of radioactive precursor incorporation into the corresponding nucleoside 5'-triphosphate pool. These studies will also determine the effects of alteration of nucleotide pools on the intracellular half-lives of araGTP and araCTP. The proposed investigations will elucidate mechanisms by which human leukemic cells can be manipulated to increase the intracellular activation of araG and araC, thus increasing their therapeutic potential.
|Chottiner, E G; Shewach, D S; Datta, N S et al. (1991) Cloning and expression of human deoxycytidine kinase cDNA. Proc Natl Acad Sci U S A 88:1531-5|