c-erbB2 which has homology to the EGF receptor is expressed in several ovarian carcinoma cell lines. Based on mRNA phenotyping studies endogenous production of c-erbB2 is elevated regardless of growth conditions 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 two types of antisense methodologies. One approach to the modulation of gene expression is the use of transfected inducible expression vectors that generate RNA capable of modulating eukaryotic gene expression in a target specific manner. c-erbB2 cDNA was cloned into an inducible vector system in an antisense and sense orientation and stably transfected into three ovarian carcinoma cell lines, OVCAR 8:NIH, OVCA 429 and OVCA 433. Several sense and antisense clones from these cell lines were isolated and investigated by assaying for standard parameters of malignant cell growth in vitro including reduced ability to grow in soft agar, loss of the ability to grow in serum reduced conditions and reduced growth rates. A second antisense approach to modulation of gene expression in these cell lines involves the use of short synthetic derivatized oligonucleotides that are complementary to the mRNA sequences of interest. These oligonucleotides can down regulate transcription and/ or translation in these cells. Antisense, sense and missense oligonucleotides (18 bp) were used to investigate antisense mediated changes in cellular activity of c-erbB2 mRNA, DNA and protein expression levels. The reduction of endogenous c-erbB2 production necessary to effect a change on anchorage dependence and individual cell growth rates in vitro was investigated. Current results show that upon induction of stable transfectants with IPTG, there is a decrease in message expression (as assessed by RT-PCR) and a dramatic increase in the amounts of protein in isolated cell lysates. A soft agar assay of the cell line OVCAR 8:NIH revealed the loss of the ability of these cells to grow in soft agar upon induction of the antisense clones. The sense clones, transfected with c-erbB2 cloned in the sense orientation and control clones transfected with the parent inducible vector grew for two to three weeks in soft agar with and without induction by IPTG. These results support the importance of c-erbB2 in the maintenance of a transformed phentoype. GRAMT=Z01BL03003 Our studies are directed towards understanding the mechanism of action and biological role of the newly-discovered human growth factor, amphiregulin. To address these issues, we utilize techniques common to protein biochemistry, cell biology and molecular biology. Recent work has focused on the identification of the cell surface receptor for amphiregulin. We have determined that amphiregulin acts by binding to the extracellular domain of the epidermal growth factor receptor, resulting in autophosphorylation on tyrosine residues, activation of the receptor tyrosine kinase activity and cell division. In addition, we have shown that the interaction of amphiregulin and the epidermal growth factor receptor results in activation of the c-erbB-2 receptor tyrosine kinase, a kinase is believed to be involved in the pathogenesis of a number of human malignancies. Other studies have focused on the structural characterization of the amphiregulin molecule and identification of protein domains critical to the bioactivity of amphiregulin. We have identified and purified multiple heparin-binding, glycosylated biologically active forms of the amphiregulin molecule. The predominant form of amphiregulin which is secreted by human epithelial cells is a 16.5 kDa molecule that contains complex N-linked oligosaccharide which is rich in sialic acid. The 16.5 kDa form of AR also contains disaccharides linked to serine/threonine residues. However, a smaller 9.5 kDa non-glycosylated biologically active form of amphiregulin has been isolated which contains the epidermal growth factor-like domain but possesses a truncated NH2-terminal extension. These results revealed that the NH2-terminal region of the amphiregulin is not critical to the ability of amphiregulin to activate the epidermal growth factor receptor tyrosine kinase. Current studies are directed towards identifying the heparin-binding domain of amphiregulin and understanding the role of cell surface proteoglycan in the mechanism of amphiregulin action. In addition, more recent studies have focused on elucidation of how amphiregulin and epidermal growth factor signal through the epidermal growth factor receptor.
