An analysis of the relationship between altered gene expression and the sequential development of cancer following exposure to carcinogens is fundamental to understanding the molecular basis of neoplasia. In the past, this problem has seemed almost intractable, but the recent development of models which reproducibly result in a series of defined stages leading to liver cancer in the rat as well as rapid advances in molecular biology offer new promise of substantial progress in this area. In order to analyze gene expression at the cellular level, in situ hybridization to cellular mRNAs is planned; application of this technique to rat liver will involve preliminary experiments using albumin and Alpha-fetoprotein cDNA probes. Alterations in the expression of known oncogenes will be studied by in situ and northern hybridization in early foci, early nodules, persistent nodules, primary hepatic carcinomas and metastases. These stages will be induced in male Fisher 344 rats by a single intraperitoneal dose of diethylnitrosamine followed by intragastric instillation of acetylaminofluorene 14, 16, 18 and 20 days later and partial hepatectomy on day 21 (a modified Toronto protocol). Changes in oncogene organization (amplification, rearrangement and methylation) will be studied in DNA derived from pooled early nodules, persistent nodules and carcinomas. cDNA libraries, enriched for stage-specific or newly expressed mRNAs by selective hybridization techniques will be prepared and screened by dot hybridization and in situ hybridization for representatives which are stage specific or carcinoma specific. To assess the functional significance of these genes, a second set of cDNA libraries, enriched for full length representatives, will be made from these stages in a eukaryotic expression vector. These stage specific and carcinoma-specific cDNAs, cloned genomic copies of them, DNA from carcinomas and previously characterized oncogenes will be used to transfect cultures of hepatocytes and cells from early foci and nodules, and neoplastic transformation will be assayed by growth in soft agar and carcinoma formation in animals injected with treated cells. Similar experiments are planned for analysis of the genes responsible for conversion of primary tumors to metastatic foci, although in this case the endpoint will be metastases in the whole animals. Application of all of these techniques to other protocols which produce liver cancer in the rat is planned.
