Front-line induction chemotherapy regimens containing cytosine arabinoside (Ara-C) and anthracyclines result in 80% complete remission rate in childhood acute myeloid leukemia (AML) but their cure rate is about 35 – 50%, one of the lowest of all childhood cancers. Understanding the factors that contribute to emergence of chemoresistant leukemic cells is crucial to improving treatment outcome in children with AML. We are interested in studying the role of variation in Ara-C transport and biotransformation pathway genes in the efficacy and toxicity of treatment of childhood AML. To permeate the cell membrane, Ara-C is mainly dependent on human equilibrative nucleoside transporter 1 (hENT1; SLC29A1; gene localized to 6p21.1). Several studies have suggested an important role for altered levels of hENT1 in the chemosensitivity of AML blasts to Ara-C (
Galmarini et al. Leukemia 2001; 15(6):87
; Gati et al. Leuk Lymphoma 1998; 32(1–2):45
). Osato and colleagues identified two single nucleotide polymorphisms (SNPs) in the hENT1 coding sequence that led to missense changes, but their in vitro analysis did not detect differences in the activity of variant alleles in a yeast transfection system (Osato et al. Pharmacogenetics 2003;13(5):297
). To identify variation in hENT1 that might influence its expression, we sequenced 1.6Kb of the proximal 5′-flanking sequence of the gene in 42 unrelated individuals and identified three SNPs at positions C-1345G, G-1050A, and G-706C. TRANSFAC analysis (www.genomatix.de) predicted that two of these (C-1345G & G-706C) would alter consensus transcription factor binding site sequences. We cloned four naturally occurring haplotypes (CGG, CAG, CGC, and GAG) using the TOPO-TA cloning kit, then transfected Cos-1 cells using the Lipofectamine 2000 protocol. Gene expression was assayed using the Promega Dual-Luciferase Reporter Assay System and read on a Molecular Devices HT Analyzer. Luciferase activity was measured at 24 and 48 hours after transfection for six replicates of every condition during three separate transfections. To correct for differences in transfection efficiencies, experimental (Photinus pyralis) luciferase activities were normalized by co-transfection with control (Renilla reniformis) luciferase plasmid. Compared to the wild type CGG haplotype, variant haplotypes CAG, CGC, and GAG drive luciferase expression at approximately 2x (p <0.0001), 1.4x (p <0.001) and 1.2x (p =0.08), respectively. This leads to the hypothesis that individuals carrying CAG or CGC haplotypes (17% of the population) exhibit higher levels of hENT1 expression and are more sensitive to Ara-C exposure. Experiments are underway to quantify gene transcripts in people of known hENT1 haplotypes. We also plan to genotype a large cohort of children with de novo AML for these three SNPs in hENT1 and correlate clinical outcomes in individuals carrying the low- versus the high-expressing haplotypes.
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