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Metabolism of 1-ß-D-Arabinofuranosylcytosine in Leukemia L1210: Nucleoside and Nucleotide Kinases in Cell-free Extracts¹

Cancer Chemotherapy National Service Center, Chemotherapy, National Cancer Institute, NIH, Bethesda, Maryland 20014

The phosphorylation of 1-ß-D-arabinofuranosylcytosine (ara-C) and other nucleosides and nucleotides was studied in cell-free extracts of leukemia L1210 and a subline (L1210/ara-C) resistant to the drug. The specific activities of ara-C kinase, deoxycytidine kinase, and deoxyguanosine kinase were decreased by 95, 98, and 87%, respectively, in the resistant subline. There was no change in the activities of other nucleoside and nucleotide kinases, with the exception of minor decreases in deoxyadenosine and deoxythymidine kinase activities. The results suggest that the phosphorylation of deoxycytidine and ara-C, and apparently also of deoxyguanosine, was mediated by the same enzyme.

Deoxycytidine phosphorylation in cell extracts of the parent L1210 line was not inhibited by extracts of L1210/ara-C. 5'-Nucleotidase activity, measured with ara-C 5'-monophosphate, was identical in extracts of L1210 and L1210/ara-C. These findings strongly suggest that the reduced phosphorylation of deoxycytidine and ara-C is caused by a decreased concentration of deoxycytidine kinase in the resistant cells, and exclude alternative explanations such as the presence of a specific phosphorylation inhibitor or an increased rate of dephosphorylation of the products.

Deoxycytidine and its 5'-triphosphate were found to be competitive inhibitors (K_i = 0.6 µM) of ara-C phosphorylation (K_m = 25 µM) in cell-free extracts of leukemia L1210. The phosphorylation of ara-C is, therefore, mediated exclusively by deoxycytidine kinase. Deoxyguanosine kinase activity was inhibited 84% by ara-C and 98% by deoxycytidine, but deoxyguanosine did not inhibit phosphorylation of ara-C. These results suggest that the affinity of the substrates for deoxycytidine kinase decreases in the order deoxycytidine, ara-C, and deoxyguanosine, assuming that the phosphorylation of the latter nucleoside was mediated by this enzyme.

¹ This investigation was carried out mainly during the author's tenure (1967 to 1968) as a Visiting Investigator at the Scripps Clinic and Research Foundation, La Jolla, Calif. It was supported in part by USPHS Research Grant CA-6522 from the National Cancer Institute to the Scripps Clinic.

Received 10/23/68. Accepted 7/31/69.