FUNCTIONAL EXPRESSION OF THE NITROBENZYLTHIOINOSINE-SENSITIVE NUCLEOSIDE TRANSPORTER OF HUMAN CHORIOCARCINOMA (BEWO) CELLS IN ISOLATED OOCYTES OF XENOPUS-LAEVIS

Cultured human choriocarcinoma (BeWo) cells have previously been shown to exhibit, in comparison with other cultured cell types, elevated ni trobenzylthioinosine (NBMPR)-sensitive transport activity and large nu mbers (> 10(7)/cell) of high-affinity NBMPR-binding sites [Boumah, Hog ue and Cass (1992) Biochem. J. 288, 987-996]. The present study invest igates 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 urid ine transport activity was assayed by comparing rates of uptake (22 de grees C) of 100 mu M [H-3]uridine by RNA-injected oocytes with uptake by water-injected or uninjected oocytes. A 4-fold stimulation of uridi ne uptake (2.0 versus 0.5 pmol/90 min per oocyte) was seen when oocyte s were injected with 50 ng of BeWo poly(A)(+) RNA, and this stimulatio n was abolished when the RNA-injected oocytes were assayed in the pres ence of 10 mu M NBMPR. The expressed uridine transport activity in ooc ytes was highly sensitive to NBMPR, with a 50% reduction seen at 1.1 n M NBMPR (IC50 value). The IC50 value for NBMPR inhibition of uptake of 100 mu M [H-3]uridine by intact BeWo cells was 1.4 nM. Inward fluxes of [H-3]uridine in the RNA-injected oocytes were greatly reduced in th e presence of high concentrations (2 mM) of non-radioactive nucleoside s (adenosine, thymidine, inosine) that are known permeants of NBMPR-se nsitive nucleoside transport processes. These results establish that t he abundance of NBMPR-sensitive nucleoside transporter mRNA in poly(A) (+) RNA preparations from BeWo cells is sufficient to achieve producti on of functionally active transporter protein in Xenopus oocytes and t hat, when expressed in Xenopus oocytes, the transporters exhibit NBMPR sensitivity and permeant selectively similar to that of the native tr ansporters.