Signalling and anti-proliferative effects mediated by gonadotrophin-releasing hormone receptors after expression in prostate cancer cells using recombinant adenovirus

Gonadotrophin-releasing hormone receptors (GnRH-Rs) are found in cancers of reproductive tissues, including those of the prostate, and gonadotrophin-releasing hormone (GnRH) can inhibit growth of cell lines derived from such cancers. Although pituitary and extra-pituitary GnRH-R transcripts appear identical, their functional characteristics may differ. Most extra-pituitary GnRH-Rs have low affinity for GnRH analogues and may not activate phospholipase C or discriminate between agonists and antagonists in the same way as do pituitary GnRH-Rs. Here we have assessed whether GnRH-Rs expressed exogenously in prostate cancer cells differ functionally from those of gonadotrophs. We found no evidence for endogenous GnRH-Rs in PC3 cells, but after infection with adenovirus expressing the GnRH-R (Ad GnRH-R) at 10 plaque forming units (p.Eu.)/cell or greater, at least 80% of the cells expressed GnRH-Rs. These sites had high affinity (K-d for [(125) I]Buserelin 1(.)1 +/- 0(.)4 nM) and specificity (rank order of potency: Buserelin > GnRH >> chicken (c) GnRH-II), and mediated stimulation of [H-3]inositol phosphate (IP) accumulation. Increasing viral titre from 3 to 300 p.fu./cell increased receptor number (2000 to 275 000 sites/cell respectively) and [3 H]IP responses. GnRH also caused a biphasic increase in the cytoplasmic Ca2+ concentration in Ad GnRH-R-infected cells but not in control cells. Mobilization of Ca2+ from intracellular stores contributed to the spike phase of this response whereas the plateau phase was dependent upon Ca2+ entry across the plasma membrane. This effect on Ca2+ and stimulation of [H-3]IP accumulation were both blocked by the GnRH-R antagonist, Cetrorelix. In addition, GnRH reduced cell number (as measured in MTT activity assays) and DNA synthesis (as measured by [H-3]thymidine incorporation) in Ad GnRH-R-infected cells (but not in control cells). This effect was mimicked by agonist analogues and inhibited by two antagonists. Thus, when exogenous GnRH-Rs are expressed at a density comparable to that in gonadotrophs, they are functionally indistinguishable from the endogenous GnRH-Rs in gonadotrophs. Moreover, expression of high affinity GnRH-Rs can facilitate a direct anti-proliferative effect of GnRH agonists on prostate cancer cells.