Primary avian tendon (PAT) cells in a permissive cell culture environment will produce 48% of their total protein synthesis as procollagen. PAT cells malignantly transformed by Rous sarcoma virus will produce about 1% procollagen. This drop in procollagen synthesis is accompanied by an approximately 30-fold change in the level of procollagen mRNA. The major aim of this project over the next year is to elucidate the control steps that are altered after transformation that cause PAT cells to radically alter their commitment to procollagen production. We will use a temperature-sensitive mutant in the src gene (LA 24) in order to study the kinetics of the transformation process as they relate to collagen expression. Specifically, PAT cells infected with LA 24 will be shifted from the nonpermissive temperature (41~C) to the permissive temperature (35.5~C), and changes in three critical steps in the collagen pathway will be analyzed over the next 24 hrs: levels of procollagen mRNA, rates of procollagen translation, and ability to secrete procollagen from the cell. Kinetically, we will discriminate the early rate-controlling steps that are directly sensitive to transformation from others that are secondarily controlled. In this way, we plan to gain further insight into the mechanisms that regulate the differentiated state of PAT cells and insight into how these steps can be manipulated by malignant transformation. (B)
