Cloning and pharmacological characterization of a novel gene encoding human nucleoside transporter 1 (hNT1) from a human breast cancer cDNA library

Abstract: Aims: We isolated a novel gene encoding human nucleoside transporter 1 (hNT1), from a human breast cancer cDNA library. Main methods: A nondirectional cDNA library was screened by an EST clone (GenBank™/EMBL/DDBJ: BU944345). A Xenopus laevis oocyte expression system was used for functional characterization. Membrane localization in the human breast was determined by immunohistochemistry. Key findings: Isolated hNT1 cDNA consisted of 246 base pairs that encoded an 82-amino acid protein. By RT-PCR analysis, hNT1 mRNA was strongly detected in the breast cancer tissues. When expressed in X. oocytes, hNT1 mediated the high affinity transport of [3H]5-fluorouracil (5-FU) with a K m value of 69.2±24.5 nM in time- and pH-dependent, and Na+-independent manners. A cis-inhibition experiment revealed that hNT1 mediated transport of [3H]5-FU is strongly inhibited by various nucleosides such as pyrimidine, uracil, uridine, guanosine, inosine, thymidine, adenosine, cytidine and purine suggesting that hNT1 may be involved in the trans epithelial transport of these endogenous substrates. Immunohistochemical analysis revealed that the hNT1 protein is localized in the lactiferous duct epithelium. Significance: Our present results indicate that a newly isolated cDNA clone, hNT1, is a key molecule for the breast handling of 5-FU in humans. [Copyright &y& Elsevier]