title: Human concentrative nucleoside transporter 1-mediated uptake of 5-azacytidine enhances DNA demethylation.
abstract: The DNA methyltransferase inhibitors 5-azacytidine (5-azaCyd) and 5-aza-2'-deoxycytidine have found increasing use for the treatment of myeloid leukemias and solid tumors. Both nucleoside analogues must be transported into cells and phosphorylated before they can be incorporated into DNA and inactivate DNA methyltransferases. The members of the human equilibrative and concentrative nucleoside transporter families mediate transport of natural nucleosides and some nucleoside analogues into cells. However, the molecular identity of the transport proteins responsible for mediating the uptake of 5-azanucleosides has remained unknown. To this end, we have generated a stably transfected Madin-Darby canine kidney strain II cell line expressing recombinant hCNT1. An antiserum directed against hCNT1 specifically detected the protein in the apical membrane of hCNT1-expressing Madin-Darby canine kidney cells. Using [14C]5-azaCyd, we show here that hCNT1 mediated the Na+-dependent uptake of this drug with a Km value of 63 micromol/L. Na+-dependent transport of radiolabeled cytidine, uridine, and 5-fluoro-5'-deoxyuridine further showed the functionality of the transporter. hCNT1-expressing cells were significantly more sensitive to 5-azaCyd, and drug-dependent covalent trapping of DNA methyltransferase 1 was substantially more pronounced. Importantly, these results correlated with a significant sensitization of hCNT1-expressing cells toward the demethylating effects of 5-azaCyd and 5-aza-2'-deoxycytidine. In conclusion, our study identifies 5-azaCyd as a novel substrate for hCNT1 and provides direct evidence that hCNT1 is involved in the DNA-demethylating effects of this drug.
authors.affiliation: Division of Tumor Biochemistry, German Cancer Research Center, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. m.rius@dkfz.de
authors.forename: Maria
authors.initials: M
authors.lastname: Rius
authors.forename: Carlo
authors.initials: C
authors.lastname: Stresemann
authors.forename: Daniela
authors.initials: D
authors.lastname: Keller
authors.forename: Manuela
authors.initials: M
authors.lastname: Brom
authors.forename: Esther
authors.initials: E
authors.lastname: Schirrmacher
authors.forename: Dietrich
authors.initials: D
authors.lastname: Keppler
authors.forename: Frank
authors.initials: F
authors.lastname: Lyko
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation; D004285:Dogs; D005786:Gene Expression Regulation; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C065171:cif nucleoside transporter; D004247:DNA; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D001374:Azacitidine