Structural analysis of the deoxycytidine kinase gene in patients with acute myeloid leukemia and resistance to cytosine arabinoside.
Deficiency of deoxycytidine kinase (dCK) activity represents one possible cause of resistance to cytosine arabinoside (ara-C). Mutations of the dCK gene have recently been shown to be responsible for dCK deficiency and increased resistance in vitro. In order to define the relevance of this mechanism in vivo, we analyzed the dCK gene in 16 adult patients with relapsed/refractory acute myeloid leukemia (AML) and clinical resistance to standard-dose and/or high-dose ara-C. Southern blot analysis using genomic DNA from peripheral blood or bone marrow samples containing > or = 70% leukemic blasts and agarose gel electrophoresis of cDNA obtained by RT-PCR did not reveal gross rearrangements of the dCK gene. Sequencing of the dCK coding region showed 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 > or = 50% of sequenced clones revealing the specific base pair alteration were bacterially expressed in E. coli and analyzed for dCK activity. Normal enzyme activity was found in two patients (codon 20 and 98), and a complete loss of activity in one patient (codon 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 patients with AML.
Flasshove
M
M
Innere Klinik und Poliklinik (Tumorforschung) Universitätsklinikum Essen, Westdeutsches Tumorzentrum Essen, Germany.
Strumberg
D
D
Ayscue
L
L
Mitchell
B S
BS
Tirier
C
C
Heit
W
W
Seeber
S
S
Schütte
J
J
D000328:Adult; D000368:Aged; D001483:Base Sequence; D015139:Blotting, Southern; D003062:Codon; D003561:Cytarabine / Q000627:therapeutic use*; D004252:DNA Mutational Analysis; D003842:Deoxycytidine Kinase / Q000235:genetics* / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012334:RNA, Neoplasm / Q000032:analysis; D012194:RNA-Directed DNA Polymerase
D003062:Codon; D012334:RNA, Neoplasm; D003561:Cytarabine; D003842:Deoxycytidine Kinase; D012194:RNA-Directed DNA Polymerase