The goal of this revised competing application is unchanged; we remain interested in hyperthermia's effect on macrophage/monocyte cytotoxic interactions with tumor cells. However, the focus of the specific aims have evolved toward understanding the interaction of a key cytotoxic molecule of these effector cells, tumor necrosis factor (TNF), with heated tumor cells. We have shown that over-expression of heat shock protein (HSP)27 in TNF-sensitive L929 cells renders these cells highly resistant to TNF. The mechanism(s) for this protection need to be identified and related to our observation of a sequence dependency in heat's sensitization of TNF's cytotoxicity. We will study heat's augmentation of TNF's cytotoxicity and the basis for HSP-mediated protection against TNF-cytotoxicity and the experiments will illuminate the signaling pathways for TNF cytotoxicity and protection and help identify functions of HSP27.
The specific aims test the following hypothesis: (1) The protection from TNF cytotoxicity afforded by human HSP27 is not unique to murine L929 cells and is observed when HSP27 is overexpressed in other TNF-sensitive cells. To address this hypothesis, the murine cell line WEHI-164 has already been transfected with sense and anti-sense HSP27 cDNAs, and the human cell lines CaOV-3 and MCF7 will be similarly transfected. These cells will be tested for their response to TNF relative to parental cells and for protection from heat-induced TNF sensitization. (2) Phosphorylation of HSP27 at Ser sites is required for protection against TNF cytotoxicity and against heat-induced TNF sensitization. To examine this hypothesis, L929 and WEHI-164 cells transfected with mutant HSP27 cDNAs encoding Ser to Gly and Ser to Ala substitutions have already been obtained and will be tested for protection against TNF and heat compared to already established sense and anti-sense L929 and WEHI-164 HSP27 stable transfectants. (3) The TNF protection afforded by HSP27 lies in inhibiting the sphingomyelin signaling pathway initiated by TNF treatment. Transfections expressing wild type or mutant HSP27 will be compared to parental cells for the effect of TNF and heat on, first, proximal and, later, downstream components of this pathway. (4) The TNF protection afforded by HSP27 lies in modulating a diacylglycerol (DAG)/protein kinase C (PKC) signaling pathway initiated by TNF treatment. To test these hypotheses, transfectants expressing wild type or mutant HSP27 will be compared to parental cells for the effect of TNF and heat on, first, proximal and, later, downstream components of this pathway. (5) Hyperthermic enhancement/deregulation of one or both of the signaling cascades in Aims Three and Four leads to enhancement of TNF cytotoxicity by apoptosis and the inhibition of this (these) cascades) by HSP27 inhibits the downstream apoptotic endpoints. Under this aim, parental and HSP27-transfected cells, as obtained as above, will be studied for the effect(s) of TNF and heat on the expression of genes associated with apoptosis and for alterations in the apoptotic endpoints of morphological changes and DNA fragmentation following heat and TNF treatments.
