The process of TNF cytotoxic or apoptotic signalling in tumor cells is complex, involving several cellular events (protein phosphorylation, liberation of membrane-derived """"""""second messengers"""""""" and the generation of reactive oxygen species) and appears to be mediated through a specific TNF receptor activated process. Understanding the cellular events which initiate or propagate the cell killing activity of TNF will also be of importance in defining the general mechanism of ligand-induced cell death, the substrates importance to this response and the role of defects in this cascade in resistance to TNF-induced cytotoxicity. The objectives of this proposal are to identify, isolate and characterize membrane- associated phosphoproteins expressed in tumor cells which appear to play a role in TNF induced cytotoxicity. Toward this goal, an in vitro assay was developed to measure the influence of TNF on membrane protein phosphorylation in both TNF sensitive and resistant cells. These studies demonstrated the existence of a TNF-stimulated protein tyrosine phosphatase (T-PTPase) active against the membrane receptor for epidermal growth factor (EGFr). TS-PTPase was inhibited by tyrosine phosphatase inhibitors, may be differentially expressed in TNF resistant or sensitive cells and may be activated by changes in the cellular redox state. The first goal of this proposal is to isolate this TS-PTPase and produce antibodies against this activity to be used in the study of its regulation, expression and cellular distribution. An additional observation of membrane protein phosphorylation revealed the presence of a protein (p43) which was highly phosphorylated in TNF-sensitive cells but absent in resistant clones. Incubation with TNF in vitro inhibited p43 phosphorylation suggesting that its expression or phosphoregulation may play a role in ThF cytotoxic signalling. Isolation of and specific antibody production against p43 is the second goal of this proposal and will be useful in studying the role of this protein in ThF responsiveness. Overall these studies may provide important information about the process of TNF signalling in tumor cells and aid in understanding the general process of cytokine-induced cellular suicide or destruction.
