Identification of a novel point mutation in ENT1 that confers resistance to Ara-C in human T cell leukemia CCRF-CEM cells

Abstract

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.

Figure 1. Comparison of [³H]-uridine uptake, [³H]-NBMPR binding, and mRNA expression between CCRF-CEM cells and Ara-C/8C cells

A) 3x10⁵ CCRF-CEM or Ara-C/8C cells (CEM-AraC-8C) cells were incubated with [³H]-uridine in sodium-free transport buffer for 5 min in the presence or absence of 1.0 μM NBMPR, a selective inhibitor of hENT1, as described in Materials and Methods. Data points represent sample mean +/− SD in duplicate (N=4; *p<0.01 different from –NBMPR, #p<0.01 different from parental cell line). B) [³H]-NBMPR binding was performed as described in Materials and Methods. Data points represent mean +/− SD from samples run in triplicate (N=2; *p<0.01). C) RT-PCR analysis of hENT1 mRNA expression was determined as described in Materials and Methods. Representative gel comparing mRNA levels in the parental CCRF-CEM cell line and CCRF-CEM-AraC-8C cell line.

Figure 2. Substitution of G24 in hENT1 disrupts nucleoside and Ara-C transport activity

A) Cross-species comparison of amino acids 13-33 of ENT1 and ENT2. Arrow denotes G24. B) Wt hENT1 and G24A, G24E, and G24R hENT1 mutants were expressed separately in PK15 cells (6×10⁴) and [³H]-uridine uptake was performed for 20, 40, and 60 minutes as described in Materials and Methods. Average transport by wt hENT1 was approx. 4.19 pmol/min/mg protein. Data points represent mean +/− SD of samples run in quadruplicate (N=1; *p<0.01 different from control). C) Wt hENT1 and G24A, G24E, and G24R hENT1 mutants were expressed separately in PK15 cells (6×10⁴) and [³H]-Ara-C uptake was performed for 2 hours in the presence or absence of 1μM NBMPR as described in Materials and Methods. Average transport by wt hENT1 was approx. 2.08 pmol/min/mg protein. Data points represent mean +/− SD of samples run in quadruplicate (N=1; *p<0.01 different from control, #p<0.01 different from –NBMPR).

Figure 3. G24R hENT1 mutant lacks [³H]-NBMPR binding even though plasma membrane expression is maintained

A) Wt and G24R hENT1 expression was observed using confocal microscopy. Image fields are representative of expression. Arrows denote approximate plasma membrane location. B) Wt hENT1 and G24R hENT1 were transfected separately into PK15 cells. Forty-eight hours post-transfection cells were lysed, the membranes were isolated and [³H]-NBMPR binding to the isolated membrane was determined as described in Materials and Methods. Data points represent the mean +/− SD (N=3; *p<0.01 different from control).