The
nucleoside analog 5-azacytidine is an archetypical
drug for epigenetic
cancer therapy, and its clinical effectiveness has been demonstrated in the treatment of
myelodysplastic syndromes (MDS) and
acute myelogenous leukemia (AML). However,
therapy resistance in patients with MDS/AML remains a challenging issue.
Membrane proteins that are involved in
drug uptake are potential mediators of drug resistance. The responsible
proteins for the transport of
5-azacytidine into MDS/AML cells are unknown. We have now systematically analyzed the expression and activity of various
nucleoside transporters. We identified the human
equilibrative nucleoside transporter 1 (hENT1) as the most abundant
nucleoside transporter in
leukemia cell lines and in AML patient samples. Transport assays using [¹⁴C]
5-azacytidine demonstrated Na⁺-independent uptake of the
drug into the cells, which was inhibited by S-(4-nitrobenzyl)-6-thioinosine (
NBTI), a hENT1 inhibitor. The cellular toxicity of
5-azacytidine and its
DNA demethylating activity were strongly reduced after hENT1 inhibition. In contrast, the cellular activity of the
5-azacytidine derivative 5-azacytidine-5'-elaidate (CP-4200), a
nucleoside transporter-independent
drug, persisted after hENT1 inhibition. A strong dependence of 5-azacytidine-induced DNA demethylation on hENT1 activity was also confirmed by array-based DNA methylation profiling, which uncovered hundreds of loci that became demethylated only when hENT1-mediated transport was active. Our data establish hENT1 as a key transporter for the cellular uptake of
5-azacytidine in
leukemia cells and raise the possibility that hENT1 expression might be a useful
biomarker to predict the efficiency of
5-azacytidine treatments. Furthermore, our data suggest that
CP-4200 may represent a valuable compound for the modulation of transporter-related
5-azacytidine resistances.