The role of epidernal growth factor and parathyroid hormone related peptide (1-34) in three choriocariocarcinoma cell lines as a model for implantation of human trophoblast

Bradford, Kathleen Ann Bradford (2002) The role of epidernal growth factor and parathyroid hormone related peptide (1-34) in three choriocariocarcinoma cell lines as a model for implantation of human trophoblast. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

An in vitro model was established using three choriocarcinoma cell lines of human trophoblastic origin, JAr, Jeg-3 and BeWo to determine the effects of EGF upon proliferation. The JAr cell line responded significantly to EGF at a range of doses (0.1ng/ml-20ng/ml) with a maximal increase in proliferation of 90% at 5ng/ml. Similarly, on addition of 5ng/ml BeWo growth was significantly increased by 20% but the Jeg-3 cell line did not show any proliferative response to EGF under my culture conditions. Receptor binding studies showed that following a 2h incubation at pH 7.6 and 4°C ¹²⁵I-EGF bound to all three cell lines with two affinities - a high affinity binding site and a low affinity binding site. In the JAr cell line, the dissociation constants for the high and low binding sites were 3.0±0.72x10¹⁰M^-1 and 5.5±1.4x10⁹M^-1, respectively and 19,874±799 and 37,318±1,435 receptors per cell. BeWo and Jeg- 3 cell lines displayed similar affinities - for BeWo ¹²⁵I-EGF bound with Kds of 2.2±1.65x10⁹M^-1(3,601±572 receptors/cell) and 17.0±x10⁹M^-1 (8,091±484 receptors/cell) for the high and low affinity sites respectively. Similarly, in Jeg-3 two dissociation constants of 2.7±1.43x10⁹M^-1 and 18.2±1.19⁹M^-1 with 5,994±1,321 and 11,752±1,904 receptors per cell, for both high and low binding sites. There are then differences between the effects of EGF upon each cell line, as seen in the proliferation studies, and the ligand binding studies suggest that this could be due to varying receptor number or incongruity in EGF/EGFR association. Parathyroid Hormone related Peptide is another growth factor found in the human placenta and is purported to regulate EGFR expression. I investigated the effect of PTHrP(1-34) upon the JAr cell line and discovered that PTHrP(1-34) up-regulates the expression of the EGFR. Conversely, PTH (1-34) down-regulated EGFR expression. Competitive ligand-binding studies between PTH(1-34) and PTHrP (1-34) in SaOS-2 Cells suggested the peptides were binding to the same receptor whereas in JAr cell lines PTH(1-34) did not displace PTHrP(1-34) suggesting that a novel PTHrP receptor exists in the JAr choriocarcinoma cell line. Clearly there is strong evidence supporting a role for these peptides in the control of trophoblast proliferation and/or differentiation in vitro but whether this is mimicked in vivo remains unknown.