Gemcitabine Transport in Xenopus Oocytes Expressing Recombinant PlasmaMembrane Mammalian Nucleoside Transporters
Identifieur interne : 003755 ( Main/Exploration ); précédent : 003754; suivant : 003756Gemcitabine Transport in Xenopus Oocytes Expressing Recombinant PlasmaMembrane Mammalian Nucleoside Transporters
Auteurs : John R. Mackey ; Sylvia Y. M. Yao ; Kyla M. Smith ; Edward Karpinski ; Stephen A. Baldwin ; Carol E. Cass ; James D. Young [Royaume-Uni]Source :
- Journal of the National Cancer Institute [ 0027-8874 ] ; 1999-11-03.
Abstract
BACKGROUND: Gemcitabine, a pyrimidine analogue of deoxycytidine, is an anticancer nucleoside drug that requires functional plasma membrane nucleoside transporter proteins to reach its intracellular targets and cause cytotoxicity. Because of technical difficulties inherent in studying nucleoside transport in human cells, we rigorously defined gemcitabine membrane transportability by producing each of the available human (h) and rat (r) recombinant nucleoside transporters (NTs) individually in Xenopus laevis oocytes. METHODS: Oocytes were microinjected with in vitro-transcribed RNAs derived from complementary DNAs encoding (C = concentrative) rCNT1, rCNT2, hCNT1, hCNT2, (E = equilibrative) rENT1, rENT2, hENT1, and hENT2. Uptake of [3H]gemcitabine and [ 14C] uridine was measured 3 days after microinjection to determine kinetic constants. We also used the two-electrode, voltage-clamp technique to investigate the electrophysiology of hCNT1-mediated gemcitabine transport. RESULTS: Gemcitabine was transported by most of the tested proteins (the exceptions being the purine-selective rCNT2 and hCNT2), with the greatest uptake occurring in oocytes producing recombinant rCNT1 and hCNT1. Influxes of gemcitabine mediated by hCNT1, hENT1, and hENT2 were saturable and conformed to Michaelis-Menten kinetics with apparent Km values of 24, 160, and 740 μM, respectively. Gemcitabine had a limited ability to cross the lipid bilayer of oocyte membranes by simple diffusion. External application of gemcitabine to oocytes producing recombinant hCNT1 induced an inward current, which demonstrated that hCNT1 functions as a Na+/nucleoside co-transport protein and confirmed the transporter's ability to transport gemcitabine. CONCLUSIONS: Mammalian nucleoside transporters vary widely in their affinity and capacity to transport gemcitabine. Variation in the tumor and tissue distribution of plasma membrane nucleoside transporter proteins may contribute to the solid tumor activities and schedule-dependent toxic effects of gemcitabine.
Url:
DOI: 10.1093/jnci/91.21.1876
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">Gemcitabine Transport in Xenopus Oocytes Expressing Recombinant PlasmaMembrane Mammalian Nucleoside Transporters</title><author><name sortKey="Mackey, John R" sort="Mackey, John R" uniqKey="Mackey J" first="John R." last="Mackey">John R. Mackey</name></author><author><name sortKey="Yao, Sylvia Y M" sort="Yao, Sylvia Y M" uniqKey="Yao S" first="Sylvia Y. M." last="Yao">Sylvia Y. M. Yao</name></author><author><name sortKey="Smith, Kyla M" sort="Smith, Kyla M" uniqKey="Smith K" first="Kyla M." last="Smith">Kyla M. Smith</name></author><author><name sortKey="Karpinski, Edward" sort="Karpinski, Edward" uniqKey="Karpinski E" first="Edward" last="Karpinski">Edward Karpinski</name></author><author><name sortKey="Baldwin, Stephen A" sort="Baldwin, Stephen A" uniqKey="Baldwin S" first="Stephen A." last="Baldwin">Stephen A. Baldwin</name></author><author><name sortKey="Cass, Carol E" sort="Cass, Carol E" uniqKey="Cass C" first="Carol E." last="Cass">Carol E. Cass</name></author><author><name sortKey="Young, James D" sort="Young, James D" uniqKey="Young J" first="James D." last="Young">James D. Young</name></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:3A790D9EADEE13256A4EF4CB3F37F466F4470BC3</idno><date when="1999" year="1999">1999</date><idno type="doi">10.1093/jnci/91.21.1876</idno><idno type="url">https://api-v5.istex.fr/document/3A790D9EADEE13256A4EF4CB3F37F466F4470BC3/fulltext/pdf</idno><idno type="wicri:Area/Istex/Corpus">002902</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002902</idno><idno type="wicri:Area/Istex/Curation">002902</idno><idno type="wicri:Area/Istex/Checkpoint">001394</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Checkpoint">001394</idno><idno type="wicri:doubleKey">0027-8874:1999:Mackey J:gemcitabine:transport:in</idno><idno type="wicri:Area/Main/Merge">003D34</idno><idno type="wicri:Area/Main/Curation">003755</idno><idno type="wicri:Area/Main/Exploration">003755</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Gemcitabine Transport in Xenopus Oocytes Expressing Recombinant PlasmaMembrane Mammalian Nucleoside Transporters</title><author><name sortKey="Mackey, John R" sort="Mackey, John R" uniqKey="Mackey J" first="John R." last="Mackey">John R. Mackey</name></author><author><name sortKey="Yao, Sylvia Y M" sort="Yao, Sylvia Y M" uniqKey="Yao S" first="Sylvia Y. M." last="Yao">Sylvia Y. M. Yao</name></author><author><name sortKey="Smith, Kyla M" sort="Smith, Kyla M" uniqKey="Smith K" first="Kyla M." last="Smith">Kyla M. Smith</name></author><author><name sortKey="Karpinski, Edward" sort="Karpinski, Edward" uniqKey="Karpinski E" first="Edward" last="Karpinski">Edward Karpinski</name></author><author><name sortKey="Baldwin, Stephen A" sort="Baldwin, Stephen A" uniqKey="Baldwin S" first="Stephen A." last="Baldwin">Stephen A. Baldwin</name></author><author><name sortKey="Cass, Carol E" sort="Cass, Carol E" uniqKey="Cass C" first="Carol E." last="Cass">Carol E. Cass</name></author><author><name sortKey="Young, James D" sort="Young, James D" uniqKey="Young J" first="James D." last="Young">James D. Young</name><affiliation wicri:level="4"><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Affiliations of authors: J. R. Mackey, C. E. Cass,Department of Oncology, University of Alberta, Canada, and Cross CancerInstitute, Edmonton, AB; S. Y. M. Yao, K. M. Smith, E. Karpinski, J. D.Young, Department of Physiology, University of Alberta; S. A. Baldwin,School of Biochemistry and Molecular Biology, University of Leeds</wicri:regionArea><placeName><settlement type="city">Leeds</settlement><region type="country">Angleterre</region><region type="région" nuts="1">Yorkshire-et-Humber</region></placeName><orgName type="university">Université de Leeds</orgName></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of the National Cancer Institute</title><title level="j" type="abbrev">JNCI J Natl Cancer Inst</title><idno type="ISSN">0027-8874</idno><idno type="eISSN">1460-2105</idno><imprint><publisher>Oxford University Press</publisher><date type="published" when="1999-11-03">1999-11-03</date><biblScope unit="volume">91</biblScope><biblScope unit="issue">21</biblScope><biblScope unit="page" from="1876">1876</biblScope><biblScope unit="page" to="1881">1881</biblScope></imprint><idno type="ISSN">0027-8874</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0027-8874</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">BACKGROUND: Gemcitabine, a pyrimidine analogue of deoxycytidine, is an anticancer nucleoside drug that requires functional plasma membrane nucleoside transporter proteins to reach its intracellular targets and cause cytotoxicity. Because of technical difficulties inherent in studying nucleoside transport in human cells, we rigorously defined gemcitabine membrane transportability by producing each of the available human (h) and rat (r) recombinant nucleoside transporters (NTs) individually in Xenopus laevis oocytes. METHODS: Oocytes were microinjected with in vitro-transcribed RNAs derived from complementary DNAs encoding (C = concentrative) rCNT1, rCNT2, hCNT1, hCNT2, (E = equilibrative) rENT1, rENT2, hENT1, and hENT2. Uptake of [3H]gemcitabine and [ 14C] uridine was measured 3 days after microinjection to determine kinetic constants. We also used the two-electrode, voltage-clamp technique to investigate the electrophysiology of hCNT1-mediated gemcitabine transport. RESULTS: Gemcitabine was transported by most of the tested proteins (the exceptions being the purine-selective rCNT2 and hCNT2), with the greatest uptake occurring in oocytes producing recombinant rCNT1 and hCNT1. Influxes of gemcitabine mediated by hCNT1, hENT1, and hENT2 were saturable and conformed to Michaelis-Menten kinetics with apparent Km values of 24, 160, and 740 μM, respectively. Gemcitabine had a limited ability to cross the lipid bilayer of oocyte membranes by simple diffusion. External application of gemcitabine to oocytes producing recombinant hCNT1 induced an inward current, which demonstrated that hCNT1 functions as a Na+/nucleoside co-transport protein and confirmed the transporter's ability to transport gemcitabine. CONCLUSIONS: Mammalian nucleoside transporters vary widely in their affinity and capacity to transport gemcitabine. Variation in the tumor and tissue distribution of plasma membrane nucleoside transporter proteins may contribute to the solid tumor activities and schedule-dependent toxic effects of gemcitabine.</div></front></TEI><affiliations><list><country><li>Royaume-Uni</li></country><region><li>Angleterre</li><li>Yorkshire-et-Humber</li></region><settlement><li>Leeds</li></settlement><orgName><li>Université de Leeds</li></orgName></list><tree><noCountry><name sortKey="Baldwin, Stephen A" sort="Baldwin, Stephen A" uniqKey="Baldwin S" first="Stephen A." last="Baldwin">Stephen A. Baldwin</name><name sortKey="Cass, Carol E" sort="Cass, Carol E" uniqKey="Cass C" first="Carol E." last="Cass">Carol E. Cass</name><name sortKey="Karpinski, Edward" sort="Karpinski, Edward" uniqKey="Karpinski E" first="Edward" last="Karpinski">Edward Karpinski</name><name sortKey="Mackey, John R" sort="Mackey, John R" uniqKey="Mackey J" first="John R." last="Mackey">John R. Mackey</name><name sortKey="Smith, Kyla M" sort="Smith, Kyla M" uniqKey="Smith K" first="Kyla M." last="Smith">Kyla M. Smith</name><name sortKey="Yao, Sylvia Y M" sort="Yao, Sylvia Y M" uniqKey="Yao S" first="Sylvia Y. M." last="Yao">Sylvia Y. M. Yao</name></noCountry><country name="Royaume-Uni"><region name="Angleterre"><name sortKey="Young, James D" sort="Young, James D" uniqKey="Young J" first="James D." last="Young">James D. Young</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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