STRUCTURAL-ANALYSIS OF THE DEOXYCYTIDINE KINASE GENE IN PATIENTS WITHACUTE MYELOID-LEUKEMIA AND RESISTANCE TO CYTOSINE-ARABINOSIDE

Deficiency of deoxycytidine kinase (dCK) activity represents one possi ble cause of resistance to cytosine arabinoside (ara-C). Mutations of the dCK gene have recently been shown to be responsible for dCK defici ency and increased resistance in vitro. In order to define the relevan ce of this mechanism in vivo, we analyzed the dCK gene in 16 adult pat ients with relapsed/refractory acute myeloid leukemia (AML) and clinic al resistance to standard-dose and/or high-dose ara-C. Southern blot a nalysis using genomic DNA from peripheral blood or bone marrow samples containing greater than or equal to 70% leukemic blasts and agarose g el electrophoresis of cDNA obtained by RT-PCR did not reveal gross rea rrangements of the dCK gene. Sequencing of the dCK coding region showe d point mutations in seven patients. Besides two silent mutations (or RFLPs) in codon 42 and 86, base pair mutations resulting in amino acid replacements were found in five patients affecting codon 20, 93, 98, 99, and 154, respectively. dCK cDNA clones from three patients with gr eater than or equal to 50% of sequenced clones revealing the specific base pair alteration were bacterially expressed in E. coil and analyze d for dCK activity. Normal enzyme activity was found in two patients ( codon 20 and 98), and a complete loss of activity in one patient (codo n 99). We conclude that structural alteration of the coding region of the dCK gene represents one possible mechanism for ara-C resistance in vivo, but, considering the frequency of this event, other mechanisms may play a more important role for clinical resistance to ara-C in pat ients with AML.