c-erbB-2 (HER-2, neu), a member of the epidermal growth factor gene family of ligands and receptors is a protooncogene that encodes a 185 kD transmembrane glycoprotein (p185). This receptor has intrinsic tyrosine kinase activity and has been impicated in the pathogenesis of several adenocarcinomas including ovarian carcinoma. We previously reported the construction of inducible c-erbB-2 antisense cell lines and the reduction of endogenous p185 after addition of IPTG in two ovarian carcinoma cell lines. The biological consequence of the reduction of p185 in the antisense cell line of NIH:OVCAR-8 was a suppression of the anchorage-independent phentoype. Monolayer growth was unaffected by the reduction in protein. Constitutive antisense ovarian carcinoma cell lines were recently constructed to (1) continue the study of the role of c-erbB-2 in the pathogenesis of ovarian cancer and (2) to gain insight into ligand induced activation of erbB receptors in the presence or absence of c-erbB-2. A 1.5 kB fragment of c-erbB-2 cDNA was cloned in a sense and antisense orientation in pZeoSV and transfected into NIH:OVCAR-8, OVCA 429 and SkOV3. EGFR antisense cell lines were also constructed using a 1.8 kB fragment of EGFR c-DNA. Several NIH:OVCAR-8 and OVCA 429 antisense clones were screened for changes in endogenous c-erbB-2 by immunoprecipitation and Western blot analysis. Clones screened to date failed to show a reduction in p185 levels relative to the parent cell ine. However, blots containing the immunoprecipitated lysates of NIH:OVCAR-8 antisense transfectants were stripped and reprobed with 4G10, an antiphosphotyrosine antibody. One of the clones (C17) showed substantial reduction in phosphorylation of c-erbB-2 relative to the sense and control cell lines. This clone was assessed for anchorage independent growth in a soft agar assay. Preliminary results show that C17 grows in soft agar with the same efficiency as the parent, sense and control cell lines. It is not clear whether the reduction in phosphorylation is directly related to antisense expression. Additional clones are being screened. With these recently constructed receptor knockout cell lines, experiments examining ligand induced activation of these receptors, phosphorylation of intracellular substrates and the resulting biological effect on these ovarian carcinoma cell are planned.
