Effects of mutational loss of adenosine kinase and deoxycytidine kinase on deoxyATP accumulation and deoxyadenosine toxicity in cultured CEM human T-lymphoblastoid cells

M. S. Hershfield, J. E. Fetter, W. C. Small, A. S. Bagnara, S. R. Williams, B. Ullman, D. W. Martin, D. B. Wasson, D. A. Carson

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Abstract

Accumulation of deoxyadenosine nucleotides derived from deoxyadenosine may contribute to the lymphocyte-specific toxicity associated with heritable deficiency of adenosine deaminase. To determine which enzymes mediate phosphorylation of deoxyadenosine in intact T-lymphocytes, which are particularly sensitive to the growth inhibitory effects of deoxyadenosine, we have isolated from the CEM human T-lymphoblastoid cell line mutants that are deficient in adenosine kinase, deoxycytidine kinase, or both activities. Loss of deoxycytidine kinase alone abolished the ability to phosphorylate deoxyguanosine and caused a defect in capacity to accumulate deoxyadenosine nucleotides at low concentrations (<20 μM) of deoxyadenosine. Loss of adenosine kinase primarily diminished deoxyadenosine nucleotide accumulation at concentrations of exogenous deoxyadenosine of >20 μM. Deoxycytidine kinase deficiency alone decreased growth sensitivity to deoxyadenosine by ~3-fold, while loss of adenosine kinase had little effect on this sensitivity. Loss of both activities completely eliminated deoxyadenosine phosphorylation and decreased deoxyadenosine toxicity by ~100-fold. A mutant with complete deficiency of adenosine kinase, and with a deoxycytidine kinase that has altered Michaelis constants for both deoxycytidine and deoxyadenosine, was also incapable of phosphorylating deoxyadenosine, and was as resistant. These observations imply that in CEM T-lymphoblasts the toxicity of deoxyadenosine at concentrations below ~100 μM is mediated by deoxyadenosine nucleotides and that both deoxycytidine kinase and adenosine kinase participate in this phosphorylation. Levels of deoxyadenosine phosphorylating activities were compared in extracts of CEM and its kinase-deficient mutants and in extracts of the analogous WI-L2 human B-lymphoblast lines. The differences found were not sufficient to account for the much lower rates of deoxyadenosine nucleotide accumulation by the latter, suggesting the operation of mechanisms that prevent accumulation of these nucleotides in B-cells.

Original languageEnglish (US)
Pages (from-to)6380-6386
Number of pages7
JournalJournal of Biological Chemistry
Volume257
Issue number11
StatePublished - Dec 1 1982

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Hershfield, M. S., Fetter, J. E., Small, W. C., Bagnara, A. S., Williams, S. R., Ullman, B., Martin, D. W., Wasson, D. B., & Carson, D. A. (1982). Effects of mutational loss of adenosine kinase and deoxycytidine kinase on deoxyATP accumulation and deoxyadenosine toxicity in cultured CEM human T-lymphoblastoid cells. Journal of Biological Chemistry, 257(11), 6380-6386.