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Resistance to 1-ß-D-Arabinofuranosylcytosine in Human T-Lymphoblasts Mediated by Mutations within the Deoxycytidine Kinase Gene¹

Departments of Pharmacology [J. K. O., D. S. S., B. S. M.] and Internal Medicine [B. S. M.], University of Michigan, Ann Arbor, Michigan 48109, and Department of Biochemistry and Molecular Biology, The Oregon Health Sciences University, Portland, Oregon 97201 [B. U.]

We have recently identified a complementary DNA clone which encodes the complete amino acid sequence for 2'-deoxycytidine kinase (dCK), the enzyme required for the initial phosphorylation of several deoxyribonucleosides and their analogues that are widely used as chemotherapeutic and antiviral agents. In order to identify the molecular basis for dCK deficiency in two clonal T-lymphoblast cell lines generated by virtue of their resistance to 1-ß-D-arabinofuranosylcytosine (ara-C-8D) or to 2',3'-dideoxycytidine (ddC50), we have cloned and sequenced their dCK complementary DNAs. The ara-C-8D cell line contained two identifiable mutations: (a) a 115-base pair deletion within the coding region, corresponding to the fifth exon of the gene and presumably resulting from a splice site mutation; and (b) a G to A point mutation that substitutes glutamic acid for glycine within the ATP-binding domain of the protein. Expression of each protein in Escherichia coli demonstrated a complete loss of catalytic activity and, in the case of the deletion, a proteolytic degradation product of the altered protein. The substitution of a negatively charged amino acid within the ATP-binding domain resulted in loss of enzyme activity with all nucleoside triphosphates tested. The ddC50 cell line contained a single identifiable structural gene mutation in all clones sequenced resulting in the substitution of arginine for glutamine at amino acid 156 of the protein. This mutation markedly diminished the catalytic activity of the expressed protein with the three substrates, deoxycytosine, deoxyadenosine, and deoxyguanosine. On the basis of the presence of a single point mutation and a marked reduction in dCK mRNA in this cell line, we postulate that the second allele either is not expressed or is expressed at extremely low levels. We conclude that cellular resistance to the toxicity of 1-ß-D-arabinofuranosylcytosine and dideoxycytidine in these cell lines is mediated by specific mutations within the dCK gene. Further elucidation of structural gene alterations in dCK-deficient cells will facilitate a more detailed understanding of the functional domains of this complex enzyme.

¹ This work was supported by NIH Grants 1 RO1 CA34085 and 5 T32 GMO7767 and American Cancer Society Grant MV-492.

² To whom requests for reprints should be addressed. Present address: CB#7365, 1106 Faculty Laboratory Office Bldg., University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365.

Received 11/25/91. Accepted 2/19/92.

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