Identification of a novel point mutation in ENT1 that confers resistance to Ara-C in human T cell leukemia CCRF-CEM cells.
The genetic basis for the Ara-C resistance of CCRF-CEM Ara-C/8C leukemia cells was investigated. DNA sequencing revealed that these cells expressed an equilibrative nucleoside transporter 1 (ENT1) with a single missense mutation resulting in glycine to arginine replacement (G24R). To test the importance of this residue, additional G24 mutants were created and examined for [3H]-uridine and [3H]-Ara-C uptake. Both a G24E and G24A mutant showed reduced ENT1-dependent activity. An EGFP-tagged G24R ENT1 displayed plasma membrane localization even though it was unable to bind [3H]-NBMPR, an ENT1-specific inhibitor. These results define G24 as critical amino acid for ENT1 nucleoside uptake and suggest that mutations in TM1 may provide a mechanism for Ara-C resistance in CCRF-CEM Ara-C/8C cells.
Zimmerman
Eric I
EI
Department of Pharmacology, 4009 Genetic Medicine Bldg., 120 Mason Farm Rd. CB# 7365, University of North Carolina @ Chapel Hill, Chapel Hill, NC 27599-7365, United States.
Huang
Min
M
Leisewitz
Andrea V
AV
Wang
Yanhong
Y
Yang
Jing
J
Graves
Lee M
LM
D000595:Amino Acid Sequence; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003561:Cytarabine / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D033721:Equilibrative Nucleoside Transporter 1 / Q000235:genetics* / Q000378:metabolism; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015458:Leukemia, T-Cell / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism
D000964:Antimetabolites, Antineoplastic; D033721:Equilibrative Nucleoside Transporter 1; D012333:RNA, Messenger; C438649:SLC29A1 protein, human; D003561:Cytarabine; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D005998:Glycine