title: Resistance to 1-beta-D-arabinofuranosylcytosine in human T-lymphoblasts mediated by mutations within the deoxycytidine kinase gene.
abstract: 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-beta-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-beta-D-arabinofuranosylcytosine and dideoxycytidine in these cell lines is mediated by specific mutations within the dCK gene. Further elucidation of structural genes alterations in dCK-deficient cells will facilitate a more detailed understanding of the functional domains of this complex enzyme.
authors.affiliation: Department of Pharmacology, University of Michigan, Ann Arbor 48109.
authors.forename: J K
authors.initials: JK
authors.lastname: Owens
authors.forename: D S
authors.initials: DS
authors.lastname: Shewach
authors.forename: B
authors.initials: B
authors.lastname: Ullman
authors.forename: B S
authors.initials: BS
authors.lastname: Mitchell
mesh_terms: D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D003561:Cytarabine / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D003842:Deoxycytidine Kinase / Q000172:deficiency / Q000235:genetics*; D004351:Drug Resistance; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics*; D013601:T-Lymphocytes
chemical_list: D012333:RNA, Messenger; D003561:Cytarabine; D004247:DNA; D003842:Deoxycytidine Kinase