Amino acid residue 24 of the human equilibrative nucleoside transporter 1 (ENT1) is important for transport activity

Abstract

Nucleoside analogs such as cytarabine (Ara‐C) and gemcitabine are commonly used to treat cancer (leukemia and breast cancer among others). It has been previously shown that a correlation between drug resistance and reduced uptake of these agents is due to decreased expression of a major equilibrative nucleoside transporter, ENT1. [3H]‐Uridine uptake and [3H]‐NBMPR binding revealed that the CEM‐AraC/8C cells, a leukemia cell line resistant to Ara‐C, are devoid of functional ENTs. Sequence analysis for ENT1 gene from CEM‐AraC/8C revealed a missense point mutation in the ENT1 gene sequence that confers a glycine to arginine substitution at residue 24. To determine the importance of G24 to ENT1 transport activity G24R, G24E, and G24A ENT1 mutants were expressed in PK15 cells, an ENT‐deficient cell line. Expression of wt ENT1 produced a time‐dependent increase in [3H]‐uridine uptake whereas cells that expressed G24R ENT1 were devoid of [3H]‐uridine uptake. Expression of G24E and G24A ENT1 led to only partial [3H]‐uridine incorporation, compared to wt ENT1. Furthermore, the expression of wt ENT1 in PK15 cells increased nucleoside analog‐induced cell death whereas a reduced effect was observed during the expression of the ENT1 mutants. This study reveals that CEM‐AraC/8C resistance to Ara‐C may be attributed to the G24R mutation in ENT1 and that this single amino acid is important for ENT1 nucleoside transport activity.