The goal of this component will be to determine how tumor cell death or growth is controlled by the LTbeta receptor (LTbetaR). This recently defined member of the TNF receptor family binds to the lymphotoxin alphabeta complex (LTalphabeta), a membrane-anchored cytokine expressed by cytotoxic T lymphocytes (CTL). The LTbetaR appears to play a dual role in the immune system, in host defense mechanisms against viruses such as Epstein-Barr virus (EBV), and in the development of peripheral lymphoid organs. Receptor associated proteins that bind to the cytoplasmic face of LTbetaR are hypothesized to be part of the active signaling complex. A novel RING/zinc finger protein, termed TRAF3 (formerly LAP-1), binds to the LTbetaR and the EBV protein LMP-1, demonstrating a molecular link between the transforming action of EBV with the signal transduction pathways of the LTbetaR. We will address whether the formation of LTbetaR and TRAF3 complex is critical for inducing tumor cell death. Sensitive and resistant clones of the U937 monocytic leukemia and HT-29 adenocarcinoma, characterized for expression of receptors, TRAFs, and cell death responses, will serve as tissue culture models for LTbetaR signaling. Negative sense, dominant negative mutations and direct competitive antagonists of LTbeta and TRAF3 interactions will be introduced into these cell lines and the responses to ligands or agonist anti-LTbetaR antibodies will be measured. Inhibition of cell death by uncoupling TRAF3 binding to LTbetaR will provide evidence that TRAF3 is a regulator of cell death. These two model cell lines will allow for parallel study of cell death pathways induced via related receptors, Fas and TNFR60. A molecular genetic approach will be used to determine whether known proteins, such as sphingomyelinase, can serve to directly couple LTbetaR to cell death. Alternatively, other unidentified proteins that link LTbetaR-TRAF complex will be identified by the two-hybrid system and tested for their functional roles using dominant negative mutants in the U937 and HT-29 cell lines. Finally, mice deleted for LTbetaR gene will be engineered to determine the relationship between cell death in immune responses and for development of lymph nodes. These studies will provide new understanding of the LTbetaR in host defense against persistent viruses and human cancer.
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