This proposal will specifically focus on the early biochemical events which occur following TNF treatment of human tumor cell lines displaying either sensitivity or resistance to the antiproliferative effects of this factor. Preliminary results from our laboratory studies suggested that the receptor for epidermal growth factor (EGF-R) is targeted in TNF- sensitive tumor cells. TNF-treatment modulates EGF-R tyrosine kinase activity in sensitive but not resistant human tumor cells. The modulation of EGF-R kinase by TNF was similar but distinct from that of EGF acting on its own receptor and does not appear to be due to a direct binding of TNF to the EGF receptor. In addition, enzymes involved in cellular growth regulation such as ornithine decarboxylase are also affected with TNF treatment. In order to investigate the effect of TNF on EGR-R we propose to 1) fully characterize the affect of TNF on EGF-R in TNF-sensitive cells by measuring receptor phosphorylation and assess its intrinsic tyrosine kinase activity using and EGF-R specific immunoprecipitation assay. 125I-EGF will be used to gauge the effect of TNF on EGF cellular binding and turnover of the receptor. 2) investigate the relationship between EGF receptor number and affinity in tumor cells and their sensitivity to TNF antiproliferative effects by analyzing 125I-EGF binding by Scatchard analysis in sensitive and resistant tumor cells. Additionally a role for autocrine growth factors (e.g. TgF-) secreted from TNF resistant cells will be assessed by measuring their presence in the supernants of these tumor cells, 3) to investigate the divergence of TNF and EGF action on TNF-target cells by comparison of their effects on EGF-r modulated events which occur in nuclear, cytoplasmic or membranous components of tumor cells, 4) complete the characterization of TNF effects on extremely TNF- sensitive or completely TNF-resistant clonally- derived cervical carcinoma ME-180 cells with regard to the biochemical activities mentioned above (1-3); 5) partially purify and characterize a TNF-activated component from TNF-sensitive cells capable of modulating EGF-R kinase activity in reconstitution experiments of cellular components. An understanding of the mechanism of action of tumor necrosis factor (TNF) in mediating its cytotoxic or cytostatic effect on tumor cells might enhance its potential as an antineoplastic agent in treating cancer in humans.
