P126	Expression and function of EphB4 in tumor cells.
K.Emmerich, D.Zopf, for CRBA Experimental Oncology
Schering AG, Berlin, DE.

EphB4 is one of 14 members of the Eph receptor tyrosine kinase family, which is the largest of these kinases. EphB4 is activated by its ligand ephrinB2, which itself is membrane-bound. This results in bidirectional signalling. Knock-out animals of EphB4 or ephrinB2, respectively, are fetal lethal due to vasculogenic defects. Further experiments indicate that EphB4/ephrinB2 mediate the functional interaction between venous and arterial endothelial cells (Gerety et al.). Furthermore, expression of the soluble extracellular domain of EphB4 was shown to inhibit tumor growth of A375 cells in vivo (Martiny-Baron et al.). Thus, EphB4 may play an important role in angiogenesis and tumorangiogenesis. More recently EphB4 expression has been detected also in tumor cells where its role is still unknown. We have initiated studies to investigate the potential role of EphB4 as direct target for antitumor therapy. We have screened human tumor cell lines grown in cell culture for the expression of EphB receptors and the ligand ephrinB2 to assess their expression specificity. Protein lysates and RNA were prepared. mRNA expression was analysed by RT-PCR. EphB4 protein expression was analysed by Western Blotting (WB) of total lysates or by immunoprecipitation (IP) using α-EphB4 antibodies. Functional EphB4 analysis was performed by incubation with soluble ephrinB2-Fc and subsequent IP and WB analysis with α-phosphotyrosine antibody and kinase inhibition in the breast cancer cell line MCF7. Protein expression of ephrinB2 was analysed by FACS using the soluble extracellular domain of EphB4 as tag. EphB4 and mostly all other EphB receptors are expressed in almost all tumor cell lines analysed so far, suggesting a functional role for EphB4 in tumor cells. Interestingly, RT-PCR and FACS analysis reveal cooexpression of the ligand ephrinB2. Thus, similar to EphA4 and Ephrin A5 in neurons (Marquart et al.), forward and reverse signalling may occur also in tumor cells. Treatment of tumor cells in culture with soluble EphrinB2-Fc activates EphB4 suggesting its functional expression and proper location in the cell membrane, similar to ephrinB2 which is also detected on the membrane by FACS. However, no Y-phosphorylated EphB4 was observed in cell culture even at confluency, suggesting that autocrine receptor activation does not occur. Further functional analysis of EphB4 (e.g. siRNA knock-downs) are in progress.

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