The long term goal of this project is the identification of molecular and genetic events associated with the development and progression of human colon cancer. Systems will be developed in which the role of such events in the maintenance of the transformed phenotype can be assessed with the eventual aim of defining new targets for therapeutic manipulations. The specific goal of this proposal is the understanding of the role and regulation of the src-related tyrosine protein kinases in this disease. The activity of the c-src gene product is elevated in colon carcinoma; the lck gene is ectopically expressed in colon carcinoma cell lines derived from metastases. The mechanisms underlying tumor cell expression of lck will be sought, with the aim of identifying a primary transforming event. Activation has already been shown to be transcriptional, from a specific promoter. Regulatory sequences will be cloned, mapped, sequenced, and analyzed in in vitro and intracellular transcription systems. Induction of transcription will be determined to be by cis or trans mechanisms. Regulatory regions will be analyzed by deletion and CAT analysis experiments. Evidence for proteins that bind to these regions and are found only in expresser cells will be sought by gel retardation and DNase footprinting experiments. Purification and cloning of such proteins will lead to an understanding of the events leading to lck expression in metastatic cells. The functional role of the src-related kinases will be studied with transfection and antisense manipulation of their expression. Transfectants will be assayed for invasiveness and metastatic potential, as well as expression of growth factors, protooncogenes, and structural proteins. A system in which colonic carcinoma cells can be terminally differentiated will also be assessed in this way. The in vivo significance of these findings will be studied in tissue samples representing different stages and classes of normal, premalignant, and malignant colon tissue, which will be assayed for lck and other protooncogene expression by in situ hybridization and immunofluorescence.
