The objective of this project is to study the mechanism of carcinogenesis using quantitative two-dimensional gel electrophoresis. This technique allows the separation of total cellular polypeptides on a single gel and lets us examine both qualitative and quantitative changes in the pattern of protein synthesis a the cell undergoes neoplastic transformation. Research is focused on (1) continued development of the computer system (dubbed ELSIE 4) used to automatically analyze gels and (2) use of ELSIE 4 to analyze experiments requiring computerized analysis of two-dimensional gels. In the past year we have continued developing software tools to aid the investigator in identifying interesting spots. Statistical tests designed to search for spots that may vary over the course of an experiment have been refined, and new tests, such as heuristic clustering, have been included. The system is now running on a number of mid-sized computers as well as the IBM PS/2, model 60 personal computer. The ELSIE 4 system is being used in a number of projects in the Laboratory, including one designed to study the effects of different transforming oncogene on the synthesis of proteins in rat liver- derived epithelial (RLE) cells. RLE cells have a normal chromosome count (2N=42), the chromosomes appear karyotypically normal except for a duplication in q arm of chromosome #1. Different retroviruses containing transforming oncogenes, such as v-ras, v- raf, and v-myc have been used to infect and transform these cells. The v-ras-transformed cell have been single-cell cloned. All transformed cell clones tested grow in soft agar and are highly tumorigenic. Two-dimensional gels have been run on the normal and transformed cells and a number of significant variations in polypeptide synthesis have been noted.
