The goal of the proposed research is to increase our understanding of how the TR1 and its-related genes confer resistance to TNF-mediated cytotoxicity in normal and cancerous cells. Transforming growth factor-beta1 (TGF-beta1) promotes metastatic potential in certain cancer cells and increases some cells resistant to the cytotoxic effects of TNF-alpha or TNF-beta. We have determined that a novel gene, TR1,is involved in TGF-beta1-mediated TNF-resistance in murine L929 fibrosarcoma cells (L929S). TGF-beta1 induces TR1 gene expression, and TR1 expression appears to confer TNF-resistance: Transfection of L929S cells with the TR1-vector construct induces resistance to the TNF cytotoxic actions. These findings led to the development of our hypothesis that expression of TR1 gene allows normal and cancerous cells to escape TNF cytotoxicity.
The Specific Aims of the proposed study are designed to establish the role of TR1 and its related genes in the development of TNF-resistance in cancer and normal cells. Accordingly, the Specific Aims of the proposed research are as follows: 1. To determine the entire TR1 cDNA sequence and its deduced amino acid sequence, and to isolate TR1-related genes. This will provide information concerning the nature of TR1 gene family, and may provide clues regarding its mechanisms of action. 2. To produce polyclonal antibodies against recombinant TR1. The antibodies will be utilized to study TGF-beta1-mediated TR1 synthesis, processing and possible activation of intrinsic kinase or others, thus further defining the role of TR1 in antagonizing TNF cytotoxic function. 3. To determine the biological aspect of TR1-transfected cells and to correlate the expression of TR1 gene and protein with TNF-sensitivity in a variety of normal and cancerous cells. TNF-sensitive cells will also be transfected with the TR1 gene and the influence of this transfection on TNF-sensitivity will be determined. 4. To transfect TNF-resistant L929R, monocytic THP-1, and lung cancer - cells, SCLC and NSCLC, with TR1 cDNA designed to continuously produce antisense TR1 mRNA. We expect this treatment to eliminate resistance to the cytotoxic actions of TNF. These studies will increase our understanding of TNF resistance in cancer cells, and are likely to suggest reasonable therapeutic goals in cancer treatment (e.g., suppression of TR1 expression in target cells, thereby facilitating their destruction by host immune cells and cytotoxic effector molecules).
