Altered thymidylate kinase substrate specificity in mammalian cells selected for resistance to iododeoxyuridine

A clone of Syrian hamster melanoma cells was selected for resistance to high levels of the thymidine (dT) analog 5-iododeoxyuridine (IdU). Unlike cell lines previously isolated as IdU resistant (IdU r), these IdU r lines had normal levels of thymidine kinase (EC 2.7.1.21) activity, grew in HAT medium, and readily incorporated exogenous dT and 5-bromodeoxyuridine (BrdU) into DNA. However, these IdU r cells were found to preferentially exclude IdU from their DNA. Analyses of the soluble nucleotide pools of the IdU r cells showed that they were able to take up and phosphorylate exogenous dT as well as the wild-type cells, and both mutant and wild-type cells accumulated dTTP as the major phosphorylated component. In contrast, while the wild-type cells in the presence of exogenous IdU accumulated significant levels of IdUTP (as well as IdUMP), the IdU r cells accumulated only IdUMP. Thus, the mutant cells appear to have a markedly decreased ability to phosphorylate IdU beyond the monophosphate level. Assays of thymidylate kinase (EC 2.7.4.9) activity in extracts of the IdU r cells indicated a marked preference for dTMP as substrate over IdUMP (in comparison to the wild-type enzyme activity). The cell lines described in this study represent a new phenotype arising from selection for resistance to a halogenated dT analog. The resistance appears to involve a change in the substrate specificity of thymidylate kinase, such that the enzyme in the IdU r cells has an enhanced ability to discriminate between very closely related compounds.