c-erbB2, which has homology to the EGF receptor, is expressed in many ovarian carcinoma cell lines. Based on mRNA phenotyping studies endogenous production of c-erbB2 is elevated regardless of growth conditions with or without serum and may contribute to the proliferation and clonogenicity of these carcinoma cell lines. We have investigated the contribution of c-erbB2 to the autocrine growth of these cells through blocking experiments using an antisense methodology. An inducible expression vector capable of generating antisense c-erbB-2 mRNA modulated gene expression in a target specific fashion. A 1.5 kB fragment of c-erbB2 cDNA was cloned into an IPTG inducible vector in an antisense and sense orientation. These vectors along with the parent expression vector were stably transfected into two ovarian carcinoma cell lines, NIH:OVCAR-8 and OVCA 429. Several sense , antisense and control clones from these transfectants were isolated, expanded into cell lines and investigated by assaying for standard parameters of malignant cell growth in vitro including anchorage independent growth. We examined whether reduction of endogenous c-erbB2 production suppressed growth rates in soft agar and monolayer culture. Upon induction of these transfectants with IPTG, translation of c-erbB2 mRNA was effectively blocked as determined by immunoprecipitation and Western blotting procedures. RT-PCR was performed on induced and uninduced cells to determine relative mRNA levels of c-erbB2. The results showed that message levels of the gene were unaffected in induced cultures. This supports the hypothesis that diminution of c-erbB2 protein occurs at the transnational and not at the transcriptional level. The soft agar assay of the antisense NIH:OVCAR-8 transfectant under inducing conditions showed that the ability of these cells to form colonies was suppressed. However, control and sense transfectants demonstrated a cloning efficiency comparable to that of the parent cell line. Monolayer growth of both NIH:OVCAR-8 and OVCA 429 antisense transfectants were unaffected by treatment with IPTG over a 5 day growth period. The results of the soft agar assay and monolayer proliferation assay support the conclusion that c-erbB2 is more critical to anchorage independent growth than monolayer growth and that blocking translation of c-erbB2 gene product leads to the suppression of the anchorage independent phenotype. Tumorigenicity studies in athymic mice indicated in vivo growth of tumors following subcutaneous or intraperitoneal injection of either NIH:OVCAR-8 or OVCA 429, and tolerance to IPTG either orally, or after i.p. and s.c. injection. Future experiments will address reduced tumorigenicity following IPTG induction. Gordon, AW, Pegues, JC, Johnson, GR, Kannan, B, Auersperg, N and Stromberg,K. mRNA phenotyping of the major ligands and receptors of the EGF supergene family in human ovarian epithelial cells. Cancer Lett., 89, 63-71 (1995)Pegues, JC and Stromperg, K. Effects of a novel, inducible antisense erbB-2 mRNA on growth of human ovarian carcinoma cell lines in monolayer and soft agar. (In preparation)
