P-glycoprotein (P-gp) and multidrug resistance-related protein 1 (MRP1) are known to extrude a wide range of drugs and xenobiotics from cells. We have previously shown that both P-gp and MRP1 mRNA are expressed by the human placental trophoblast and the JAr choriocarcinoma cell line, but that only MRP1 mRNA is expressed by the BeWo choriocarcinoma cell line in this laboratory (Atkinson et al. 2000). In the present study we used this difference in expression to begin to dissect the relative contribution of P-gp and MRP1 to drug efflux by trophoblast. We measured the accumulation by JAr and BeWo cells of [3H] vinblastine, a substrate for P-gp and MRP1, in the presence and absence of the inhibitor cyclosporin A (cycA). Both time course of [3H] vinblastine accumulation and the dose-response relationship for cycA were studied.
Cells were cultured in 50:50 Ham F12:DMEM with 10 % fetal calf serum. They were seeded into p35 dishes and cultured for 2-3 days prior to drug accumulation assay. Accumulation was measured by addition of buffer containing 0.2 µCi ml-1 [3H] vinblastine (17.7 µM) over a period of 5-120 min in the presence or absence of 20 µM cycA at 37 °C. At the end of the incubation period the cells were lysed in 0.3 M NaOH and the resultant lysate assayed for radioactivity and protein. For dose-response studies accumulation at 15 min was measured in exactly the same way as before, in the presence of a range of cycA concentrations between 0 and 500 µM.
[3H] Vinblastine accumulation by JAr and BeWo cells increased towards a steady state at 120 min (Fig. 1). CycA significantly increased accumulation of [3H] vinblastine by both cell types (P < 0.05, ANOVA).
There was no significant difference in [3H] vinblastine accumulation between JAr and BeWo cells, in the presence or absence of cycA (Fig. 1). Despite the differences observed in mRNA expression, there was also no significant difference in the IC50 for the effect of cycA on [3H] vinblastine accumulation by JAr and BeWo cells (1.4 and 2.1 µM, respectively, Fig. 2)
The cycA-sensitive accumulation by JAr and BeWo cells of a substrate for P-gp and MRP1 is consistent with a role for both these proteins in extruding drugs from trophoblast. The consequences of functional drug efflux in normal syncytiotrophoblast by P-gp and MRP1 will be important to examine, as the former is specifically located to the maternal-facing plasma membrane and the latter to the fetal-facing plasma membrane (Atkinson et al. 2000).
This work was supported by the MRC.
- Atkinson, D.E., Glazier, J.D., Greenwood, S.L. & Sibley, C.P. (2000). J. Physiol. 528.P, 34P.