The overall objective of the proposed research is to elucidate LPS- mediated regulatory events during LPS-triggered tumoricidal activation of macrophages. The rationale for these studies is that despite extensive efforts by numerous laboratories, the mechanisms of macrophage activation remain unclear. Based on our initial results, we propose to examine the hypothesis that the sequence of events during LPS-triggered tumoricidal activation begins with the activation of several different protein kinases. This leads to the activation of transcription factors which then translocate to the nucleus and activate various cytokine genes and iNOS gene. Several cytokines, such as IFN-beta, produced by LPS- activated macrophages participate in further development of the macrophage activation process in an auto/paracrine fashion. This hypothesis will be examined under the following specific aims: 1) to investigate LPS-triggered signal transduction mechanisms which lead to the activation of NF-kappa-B; and 2) to investigate the question whether or not macrophages can be activated, in vitro and in vivo, by IFN-beta, IFN-gamma and/or monocyte chemotactic peptide-1 (MCP-1, also known as JE) secreted from various tumor cells which are stably transfected or transduced with IFN-beta, IFN-gamma or MCP-1 genes inserted in eukaryotic expression vectors. Our approaches to Specific Aim #1 are to investigate: 1) the types and properties of I-kappa-B proteins associated with NF-kappa-B proteins in J774 cells; 2) the roles of protein kinases and proteases potentially involved in LPS-triggered phosphorylation and degradation of I-kappa-B proteins; 3) LPS-mediated transcriptional and post-transcriptional regulation of kappa-B biosynthesis; 4) potential role of various endogenous cytokines in LPS-triggered NF-kappa-B activation; 5) the effects of the H-89 and/or H-7 mediated inhibition of LPS-triggered NF-kappa-B and AP-1 on cytokine gene activation. Our approaches to Specific Aim #2 are: 1) to produce tumor cells stably- transfected or transduced with mouse genes for IFN-beta, IFN-gamma, and/or MCP-1; 2) to test whether or not IFN-beta and/or IFN-gamma secreted from gene modified tumor cells will activate J774 cells in vitro; 3) to examine whether or not MCP-1 secreted from tumor cells attracts monocytes/macrophages into tumor site, which can be activated for tumoricidal activity by IFN-beta and/or IFN-gamma secreted from gene- modified tumor cells. The significance of the proposed research is that completion of these specific aims is expected to provide us with better understanding of mechanisms of LPS-triggered tumoricidal activation of macrophages, which may be directed to cancer immunotherapy.
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