Studies of Nucleoside Transporters Using Novel Autofluorescent Nucleoside Probes

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• 作者：Jing Zhang ; Xuejun Sun ; Kyla M. Smith ; Frank Visser ; Pat Carpenter ; Geraldine Barron ; Yunshan Peng ; Morris J. Robins ; Stephen A. Baldwin ; James D. Young ; and Carol E. Cass
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：January 31, 2006
• 年：2006
• 卷：45
• 期：4
• 页码：1087 - 1098
• 全文大小：452K
• 年卷期：v.45,no.4(January 31, 2006)
• ISSN：1520-4995

文摘

To better understand nucleoside transport processes and intracellular fates of nucleosides, wehave developed a pair of fluorescent nucleoside analogues, FuPmR and dFuPmR, that differ only in thesugar moiety (ribofuranosyl versus 2'-deoxy, respectively), for real-time analysis of nucleoside transportinto living cells by confocal microscopy. The binding and transportability of the two compounds wereassessed for five recombinant human nucleoside transporters (hENT1/2, hCNT1/2/3) produced inSaccharomyces cerevisiae and/or oocytes of Xenopus laevis. The ribosyl derivative (FuPmR) was usedto demonstrate proof of principle in live cell imaging studies in 11 cultured human cancer cell lines withdifferent hENT1 activities. The autofluorescence emitted from FuPmR enabled direct visualization of itsmovement from the extracellular medium into the intracellular compartment of live cells, and this processwas blocked by inhibitors of hENT1 (nitrobenzylmercaptopurine ribonucleoside, dipyridamole, and dilazep).Quantitative analysis of fluorescence signals revealed two stages of FuPmR uptake: a fast first stage thatrepresented the initial uptake rate (i.e., transport rate) followed by a slow long-lasting second stage. Theaccumulation of FuPmR and/or its metabolites in nuclei and mitochondria was also visualized by livecell imaging. Measurements of fluorescence intensity increases in nuclei and mitochondria revealed rate-limited processes of permeant translocation across intracellular membranes, demonstrating for the firsttime the intracellular distribution of nucleosides and/or nucleoside metabolites in living cells. The use ofautofluorescent nucleosides in time-lapse confocal microscopy is a novel strategy to quantitatively studymembrane transport of nucleosides and their metabolites that will provide new knowledge of nucleosidebiology.