The main objective of this project is to study the mechansim of carcinogenesis using quantitative two-dimensional gel electrophoresis. This technique allows for the simultaneous separation of total cellular polypeptides on a single polyacrylamide gel and allows us to examine qualitative changes in the protein patterns as well as quantitative changes in individual polypeptides as the cell undergoes malignant transformation. Research is, at present, focused on the following areas: (1) continued development and improvements on the computer system (dubbed ELSIE III) currently used to automatically analyze two-dimensional gels; and (2) application of ELSIE III to analyze both the clonal variation among transformed cells and the temporal changes in cellular protein patterns during steroid driven transformation of mouse fibroblasts transfected with MMTV:v-ras chimeras. In the past year we have improved the spot finding program and developed techniques to find spots which vary, either quantitatively or qualitatively, during the course of an experiment. The organization of data on ELSIE III has been modified so that it is now possible to compare gels generated by different experiments. We have examined the polypeptide patterns of 9 single cell clones derived from a clonal cell line, H-4-II-E. Out of 982 proteins found amond the various subclones, only 5 qualitative changes in the protein patterns were detected. About 20% of the proteins, however, were synthesized at quantitatively different rates. Finally, we have examined the polypeptide pattern of a clone of NIH3T3 cells, 433. These cells contain the v-ras oncogene and can be induced, by dexamethasone, to synthesize the ras-p21 protein and express the transformed phenotype. Efforts are underway to identify the p21 protein product on these gels and, using ELSIE III, to search for both qualitative and quantitative protein patterns that are associated with the transformation phenotype.
