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Functional expression of the nitrobenzylthioinosine-sensitive nucleoside transporter of human choriocarcinoma (BeWo) cells in isolated oocytes of Xenopus laevis.

ABSTRACT: Cultured human choriocarcinoma (BeWo) cells have previously been shown to exhibit, in comparison with other cultured cell types, elevated nitrobenzylthioinosine (NBMPR)-sensitive transport activity and large numbers (> 10(7)/cell) of high-affinity NBMPR-binding sites [Boumah, Hogue and Cass (1992) Biochem. J. 288, 987-996]. The present study investigates whether NBMPR-sensitive nucleoside transport activity could be induced in Xenopus laevis oocytes by microinjection of poly(A)+ RNA isolated from proliferating cultures of BeWo cells. Expression of uridine transport activity was assayed by comparing rates of uptake (22 degrees C) of 100 microM [3H]uridine by RNA-injected oocytes with uptake by water-injected or uninjected oocytes. A 4-fold stimulation of uridine uptake (2.0 versus 0.5 pmol/90 min per oocyte) was seen when oocytes were injected with 50 ng of BeWo poly(A)+ RNA, and this stimulation was abolished when the RNA-injected oocytes were assayed in the presence of 10 microM NBMPR. The expressed uridine transport activity in oocytes was highly sensitive to NBMPR, with a 50% reduction seen at 1.1 nM NBMPR (IC50 value). The IC50 value for NBMPR inhibition of uptake of 100 microM [3H]uridine by intact BeWo cells was 1.4 nM. Inward fluxes of [3H]uridine in the RNA-injected oocytes were greatly reduced in the presence of high concentrations (2 mM) of non-radioactive nucleosides (adenosine, thymidine, inosine) that are known permeants of NBMPR-sensitive nucleoside transport processes. These results establish that the abundance of NBMPR-sensitive nucleoside transporter mRNA in poly(A)+ RNA preparations from BeWo cells is sufficient to achieve production of functionally active transporter protein in Xenopus oocytes and that, when expressed in Xenopus oocytes, the transporters exhibit NBMPR sensitivity and permeant selectively similar to that of the native transporters.

SUBMITTER: Boumah CE

PROVIDER: S-EPMC1138087 | biostudies-other | 1994 May

REPOSITORIES: biostudies-other

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Project description: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.

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Functional expression of the nitrobenzylthioinosine-sensitive nucleoside transporter of human choriocarcinoma (BeWo) cells in isolated oocytes of Xenopus laevis.

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