Studies of nucleoside transporters using novel autofluorescent nucleoside probes.
To better understand nucleoside transport processes and intracellular fates of nucleosides, we have developed a pair of fluorescent nucleoside analogues, FuPmR and dFuPmR, that differ only in the sugar moiety (ribofuranosyl versus 2'-deoxy, respectively), for real-time analysis of nucleoside transport into living cells by confocal microscopy. The binding and transportability of the two compounds were assessed for five recombinant human nucleoside transporters (hENT1/2, hCNT1/2/3) produced in Saccharomyces cerevisiae and/or oocytes of Xenopus laevis. The ribosyl derivative (FuPmR) was used to demonstrate proof of principle in live cell imaging studies in 11 cultured human cancer cell lines with different hENT1 activities. The autofluorescence emitted from FuPmR enabled direct visualization of its movement from the extracellular medium into the intracellular compartment of live cells, and this process was 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 that represented the initial uptake rate (i.e., transport rate) followed by a slow long-lasting second stage. The accumulation of FuPmR and/or its metabolites in nuclei and mitochondria was also visualized by live cell imaging. Measurements of fluorescence intensity increases in nuclei and mitochondria revealed rate-limited processes of permeant translocation across intracellular membranes, demonstrating for the first time the intracellular distribution of nucleosides and/or nucleoside metabolites in living cells. The use of autofluorescent nucleosides in time-lapse confocal microscopy is a novel strategy to quantitatively study membrane transport of nucleosides and their metabolites that will provide new knowledge of nucleoside biology.
Zhang
Jing
J
Membrane Protein Research Group, Department of Oncology, University of Alberta, and Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada.
Sun
Xuejun
X
Smith
Kyla M
KM
Visser
Frank
F
Carpenter
Pat
P
Barron
Geraldine
G
Peng
Yunshan
Y
Robins
Morris J
MJ
Baldwin
Stephen A
SA
Young
James D
JD
Cass
Carol E
CE
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D033721:Equilibrative Nucleoside Transporter 1 / Q000378:metabolism; D033722:Equilibrative-Nucleoside Transporter 2 / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D033703:Nucleoside Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D038761:Photobleaching; D011741:Pyrimidine Nucleosides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism
C509413:3-(2-deoxypentofuranosyl)furo(2,3-d)pyrimidin-2(3H)-one; C509412:3-ribofuranosylfuro(2,3-d)pyrimidin-2(3H)-one; D033721:Equilibrative Nucleoside Transporter 1; D033722:Equilibrative-Nucleoside Transporter 2; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; D033703:Nucleoside Transport Proteins; D011741:Pyrimidine Nucleosides; D011994:Recombinant Proteins; C065171:cif nucleoside transporter; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine