This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. The goal of the proposed research is to establish the structural and functional roles of TBI gene in 1) counteracting TNF cytotoxic function, 2) blocking TGF-beta signal transduction, and 3) regulating cancer development. Overexpression of transforming growth factor-beta1 (TGF- beta1) is associated with the development of benign prostatic hyperplasia to malignant prostate cancer. We have determined that TGF-beta1 induces resistance to the TNF cytotoxic response in murine L929 fibrosarcoma, which is related in part with the TR1 gene expression. Hyaluronidase enhances TNF cytotoxic response in L929 cells by decreasing TGF-beta gene expression and down-regulating a novel intracellular TNF binding protein, TBI. Functionally, an amplified PCR fragment encoding a truncated TBI protein restricts TNF cytotoxicity in L929 cells. Notably, TBI converts TGF-Beta1-mediated growth enhancement of L929 to growth inhibition and blocks the cellular apoptotic event. Two dysfunctional and mutated cDNA fragments have been isolated from L929 cells. TBI gene appears to undergo alternative splicing differently in lung cancer cells than in normal cells. These observations lead to our hypothesis that malignant formation of lung and prostate cancers is associated with mutation of TBI gene, which allows these cells to evade immune attack and promote TGF- beta1-mediated cell growth in vivo. Accordingly, the Specific Aims of the proposed study are: 1) To determine the full length TBI cDNA sequence and to obtain its deduced amino acid sequence for analyzing homologies and structural motifs in existing databases, 2) To determine the structural motifs in conferring TNF resistance and alerting TGF-beta function by expressing selected coding regions and by site-directed mutagenesis, 3) To produce polyclonal antibodies against recombinant TBI protein for studying its intracellular distribution, binding interactions with TNF, and intracellular associated-proteins, 4) To characterize TBI- mediated TNF-resistance, TGF-beta growth regulation, and other metabolic processes in L929 and cancer cells, and 5) To amplify known mutated regions in TBI cDNA by PcR from lung and prostate cancer cells for mutation analysis and sequence determination, and to express mutant proteins in normal fibroblasts for assessing alterations of their physiologic functions. These proposed studies will increase out understanding of how TBI blocks TNF and TGF-beta functions, and provide evidence to test the hypothesis that mutation of this gene links to malignant development of prostate and lung cancers.
