The long-term goal of this research is to harness the anti-cancer therapeutic poteintial of activated macrophages. The central hypothesis is that activation of intratumoral macrophages for the expression of inducible nitric oxide synthase (iNOS) and, thus, nitric oxide (NO) will have therapeutic efficacy. Accordingly, the overall objective for the proposed period of funding is to understand how expression of the iNOS gene is regulated in mouse macrophages. Application of such understanding to human macrophages could allow transient conversion of these cells to producers of NO, which could have value for cancer therapeutic purposes. Such transient conversion would undoubtedly also have broad general significance, because NO mediates microbial host defense, as well as tumor cell killing. Thus, these studies could be an important first step in harnessing the iNOS system as a therapeutic modality in several different venues.
Two specific aims will be pursued, each in close collaboration with other members of the Program Project. The first specific aim will be to identify the network that regulates expression of the mouse iNOS gene after macrophages are activated for NO production by protocols involving LPS, LPS + IFN-gamma, and TNF-alpha + IFN-gamma. In vivo footprinting will be used to identify directly which candidate responsive elements (two kappa-B, octamer, ISRE, and GAS) in the promoter bind protein(s). Protein-binding to these specific elements will be confirmed by electrophoretic mobility shift assays (in conjunction with antibody that will allow identification of specific proteins, where possible). The functional role of protein-binding responsive elements will be confirmed by either oligonucleotide-directed mutational analyses or through synthetic reconstruction of the iNOS promoter. In the latter approach, the responsive elements of interest will either be deleted or altered, thereby rendering them functionless. Putative inactivating mutations have been identified in Region II of the human promoter. If these prove to be in functional elements of the mouse promoter, studies will be undertaken to determine whether or not the human promoter can be reconstructed to become responsive to LPS/IFN- gamma. The second specific aim will be to determine the mechanism(s) by which IFN-beta affects iNOS production, both positively and negatively. IFN-beta has been shown during the current performance period to augment LPS-induced expression of the iNOS gene by a mechanism that differs from that of IFN-gamma. If the effect is shown to be transcriptionally based in nuclear run-on studies, DNA upstream of the 1.7 kb fragments thus far characterized will be assayed for augmenting activity. Similar studies of the first and second introns will also be made. If the effect is post-transcriptional, evidence for prolongation of the half-life of iNOS mRNA will be sought. As a second phase under this specific aim, the mechanism through which prolonged exposure to IFN-beta suppresses subsequent activation by LPS/IFN-gamma will be sought. Two possible explanations will be investigated: (i) exhaustion of a key transcription factor, and (ii) down-regulation of LPS and/or IFN-gamma receptors needed for the subsequent response.
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