The objective of this project is to study the mechanism of carcinogenesis using quantitative two-dimensional gel electrophoresis (2DG). This technique lets us examine both qualitative and quantitative changes in the synthesis of thousands of cellular.polypeptides as 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, our laboratory has undergone a major change and consists now of four Sun Microsystems computer workstations, networked together in a local area network. Major progress has been made in weaning the system from the use of specialized hardware. The system has been successfully ported to a number of different computers. Statistical tests designed to search for spots that may vary over the course of an experiment have been refined. Once interesting spots are flagged, they may be examined using standard image processing software developed in this laboratory. The ELSIE 4 system is being used in the laboratory to study the effects of different transforming oncogenes on the synthesis of proteins. Rat liver epithelial (RLE) cells have been isolated and single-cell cloned. These cells have normal diploid karyotype and represent a homogeneous, clonal system for the study of transformation. Different retroviruses containing transforming oncogenes, such as v-H-ras, v-raf, and v-myc, have been used to transform these cells. All v-H-ras-transformed clones grow in soft agar and are highly tumorigenic. A number of significant variations in polypeptide synthesis have been noted between the 2DG patterns of transformed and non-transformed cells. Further studies are underway to determine if a common pattern exists for proteins whose synthesis are altered by the different oncogenes. A similar series of experiments, utilizing human fibroblasts, has been undertaken.
