This Program Project competing renewal application has as its long-term objective the delineation of molecular events involved in macrophage activation for tumor cell killing. The research proposed will focus on gene activation and products of genes that collectively regulate the development and suppression of nitric oxide-dependent tumoricidal activity, in some cases through autocrine feedback. Early, intermediate and late gene expression will be studied. The three research projects are tightly integrated, both scientifically and fiscally. Project #1, led by David C. Morrison, will investigate molecular mechanisms of LPS- initiated signalling that lead to the development of activation and the expression of tumoricidal activity. He will have three specific aims: (i) to characterize the specific contributions of various LPS receptor molecules (e.g., p73, CD14, and CD11/18) to signal transduction, gene transcription, and activation for tumor cell killing; (ii) to define the molecular basis for LPS-mediated reprogramming of macrophages for altered function; and (iii) to explore the hypothesis that selective macrophage reprogramming also occurs in vivo in a model tumor system that renders mice hypersusceptible to the lethal effect of LPS. Project #2, directed by Tsuneo Suzuki, will investigate the sequence of cytoplasmic activation events that are initiated by LPS, some of which are dependent on auto/paracrine feedback of macrophage gene products.
Two specific aims will be pursued: (i) to investigate LPS-triggered signal transduction mechanisms that lead to the activation of NF-kappa-B, and (ii) to investigate whether or not macrophages can be activated in vitro and in vivo by tumor target cells that have been stably transfected or transduced with the genes encoding for either IFN-beta, IFN-gamma, monocyte chemotactic peptide (MCP-1, aka JE), or combinations thereof inserted into eukaryotic expression vectors. Project #3, led by Stephen W. Russell, will investigate events in the cell nucleus, specifically how LPS/IFN-mediated expresion of the gene that encodes for inducible nitric oxide synthase (iNOS) is regulated. There will be two specific aims in this project: (i) to identify the components of the network of transcription factors and responsive elements that positively/ negatively regulate mouse iNOS gene expression, and (ii) to identify the mechanism(s) by which IFN-beta affects iNOS production, both positively and negatively. The Support Services Core Component, which will also be led by Dr. Russell, is designed to facilitate, coordinate and foster research that is conducted by each of the three projects. It will meet three specific needs, namely those for (i) program coordination and facilitation, (ii) quality control and standardization of reagents and cells, and (iii) clerical support. Overall, the highly integrated approach that is proposed, coupled with a Core Component that will proactively foster research productivity, is expected to lead to extensive collaborations between the component projects and, therefore, to even greater productivity than has characterized the current performance period (34 full length manuscripts either published or submitted in 2+ years).
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