Expression of high levels of nitrobenzylthioinosine-sensitive nucleoside transport in cultured human choriocarcinoma (BeWo) cells.
We have examined binding of [3H]nitrobenzylthioinosine (NBMPR) and influx of [3H]thymidine in adherent cultures of human choriocarcinoma (BeWo) cells and, for comparison, cervical-carcinoma (HeLa) cells. Specific association of NBMPR with BeWo cells at 22 degrees C required 1.5 h to reach an equilibrium between free and bound ligand, whereas association with HeLa cells required 20-30 min. Scatchard analysis of NBMPR binding to low-density cultures of BeWo cells revealed a total of 27 x 10(6) sites per cell, consisting of two distinct populations that differed in their affinities for NBMPR. One population bound NBMPR with 'high' affinity (Bmax.1 15.0 pmol/10(6) cells; Kd1 0.6 nM) and the other, larger, population bound NBMPR with 'low' affinity (Bmax.2 29.0 pmol/10(6) cells; Kd2 14.5 nM). By contrast, HeLa cells possessed only 4.1 x 10(5) sites per cell, and these sites all bound NBMPR with the same affinity (Bmax. 0.7 pmol/10(6) cells; Kd 0.5 nM). Interaction of NBMPR with both populations of sites in BeWo cells could be blocked by nitrobenzylthioguanosine (NBTGR), dilazep or dipyridamole. Concentration-effect relationships for dilazep inhibition of binding of 1 nM- and 25 nM-NBMPR to BeWo cells were monophasic, with virtually complete inhibition achieved at 0.1 microM and 1 microM respectively. Plasma-membrane preparations from BeWo cells also had high numbers of NBMPR-binding sites, and u.v. irradiation of site-bound [3H]NBMPR in such preparations labelled polypeptides that migrated in electrophoretograms as a broad band with a peak M(r) of 60,000. The concentration-effect relationship for NBMPR inhibition of thymidine transport by BeWo cells was biphasic, with an IC50 for inhibition of the 'NBMPR-sensitive' component of 1.6 nM and a substantial (15-20%) component of flux that was not inhibited by 10 microM-NBMPR and was thus 'NBMPR-insensitive'. Vmax. values for thymidine transport by BeWo cells were 20-30-fold larger than the corresponding values for transport by HeLa cells. Elimination of the Na+ gradient had no effect on initial rates of thymidine fluxes measured in either the presence or the absence of NBMPR. Our results demonstrate that BeWo cells have an unusually large capacity for NBMPR-sensitive nucleoside transport, apparently resulting from high levels of expression of 'erythrocyte-like' transport elements, identified by their high-affinity interaction with NBMPR. The relationship of the low-affinity binding sites to NBMPR-sensitive transporter elements is uncertain.
Boumah
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Department of Biochemistry, University of Alberta, Edmonton, Canada.
Hogue
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DL
Cass
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CE
D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D002822:Choriocarcinoma / Q000378:metabolism* / Q000503:physiopathology; D004586:Electrophoresis; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000187:drug effects / Q000502:physiology*; D033703:Nucleoside Transport Proteins; D012680:Sensitivity and Specificity; D012964:Sodium / Q000502:physiology; D013868:Thioinosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D013936:Thymidine / Q000493:pharmacokinetics*; D013997:Time Factors; D014316:Tritium; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D014316:Tritium; D013868:Thioinosine; D012964:Sodium; C001789:4-nitrobenzylthioinosine; D013936:Thymidine