The expression of the EGF/erb family of ligands and receptors was examined in ovarian carcinoma cell lines and the several histologic stages of human ovarian carcinogenesis. Using differential RT-PCR techniques, two carcinoma cell lines (even when grown in serum-free medium) concurrently and simultaneously expressed high levels of mRNA for Transforming Growth Factor-Alpha (TGF-a), Epidermal Growth Factor Receptor ( EGFR), and erbB-2 receptor, which is consistent with an autocrine involvement of these three gene products in in vitro ovarian carcinoma cell growth. Human primary and metastatic ovarian cystadenocarcinomas, ovarian carcinomas of low malignant potential (borderline tumors), benign ovarian cystadenomas and normal ovaries were then compared for immunoperoxidase detection of EGF, TGF-a, amphiregulin (AR), and EGFR and c-erbB-2. This in vivo matrix analysis indicated a specific pattern of ligand detection with a particular ovarian histologic category in that AR immunostaining was detected almost exclusively in borderline tumors, and secondly, that TGF-a was least frequently immunopositive in borderline tumors. Thirdly, TGF-a immunoreactivity in the absence of coexpression of cripto or EGF appeared to be associated only with adenocarcinomas of high grade and stage. For example, all six stage I or II adenocarcinomas were immunoreactive for at least two of three of cripto, EGF, or TGF-a; in contrast, six of seven of the stage III carcinomas were immunopositive for only TGF-a. The apparent preferential association of AR immunoreactivity in ovarian carcinomas of low malignant potential and TGF-a in advanced carcinomas was then evaluated at the tumor tissue mRNA level using various RT-PCR methods. Differential display PCR, using GAPDH as the housekeeping reference gene, proved to be problematic and only semi-quantitative. Currently, a competition-based, quantitative RT-PCR method is being developed. Long oligomers (80mers) containing unique primer sequences and amplican base pair sizes specific for each of 5 ligands and 4 receptors were synthesized, joined by 7-cycle PCR, and the resulting template was amplified by 30-cycle PCR. Finally, this multigene RNA template is transfected into bacteria for cloning to obtain sufficient amounts of RNA to use as an internal reference standard for RT-PCR assays. In the interim, we are using Dr. Tarnuzzer's multigene specific template for matrix metalloproteinases (MMPs) to profile mRNA expression in OVCAR-8 cells. Only MMP-1 mRNA is constituitively expressed, but along with MMP-9 mRNA, MMP-1 is upregulated by TPA and IL-1, and marginally by TGF-a. mRNA from TIMPs 1 and 2 were very highly expressed even without induction. Collectively, these MMP/TIMP results are consistent with the cancer biology of ovarian carcinoma in that these tumors do not spread by metastasis but usually by cystic rupture into the peritoneal cavity to result in a malignant ascites.
