Rat liver epithelial (RLE) cells provide a very valuable in vitro model to study both spontaneous and chemically induced hepatocarcinogenesis. Therefore, the main objectives of this project are to characterize, using denaturing gradient gel electrophoresis (DGGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), the early cellular biochemical events as a result of genetic alterations and subsequent changes in protein expression during spontaneous and chemically mediated hepatocarcinogenesis. RLE cells spontaneously transform in culture and the phenotype of the tumors produced in nude mice from these transformants was primarily that of a well-differentiated hepatocellular carcinoma-like tumor. Similarly, treatment of RLE cells in vitro with aflatoxin Bl produced a series of transformants which, upon transplantation into nude mice, resulted in a diverse variety of tumors types, some of which resembled hepato-cellular carcinomas. Initial DGGE work was directed toward optimizing separation procedures. 2D-DGGE was utilized to generate 2D maps of at least 250 individual fragments from HaeIII and HinfI digested normal rat liver genomic DNA. Hybridization to 32P-labelled human minisatellite probes 33.6 and 33.15 demonstrated the feasibility of using highly repetitive DNA sequences as hyperpolymorphic loci probes for fragment detection. Procedures for the cloning of selected DNA fragments directly from 2D-DGGE gels were also established for future DNA functional characterization studies.
