Early events in the antiproliferative action of tumor necrosis factor are similar to the early events in epidermal growth factor growth stimulation

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Abstract
The process of TNF‐induced cytotoxicity is complex but appears to be mediated through a TNF‐specific cell surface receptor. Recent evidence suggests that TNF action on tumor cells may be antagonized by epidermal growth factor (EGF) and other EGF‐receptor modulatory peptides implicating a role for EGF‐R in the process of TNF‐induced cytotoxicity. In the present report, we investigated the biochemical actions of TNF of several biochemical events known to occur in the process of EGF signal transduction in intact cells. The actions of TNF were compared directly to those of EGF in both TNF‐sensitive and ‐resistant tumor cell lines. In TNF‐sensitive ME‐180 cervical carcinoma cells, TNF (20 ng/ml) stimulated the tyrosine protein kinase activity of the EGF‐receptor (EGF‐R) fivefold when measured by receptor autophosphorylation in an immune complex kinase assay. TNF activation of EGF‐R kinase activity in ME‐180 was measurable 10 min after TNF incubation and enzymatic activity remained elevated 20 min after TNF addition. Activition of the receptor by TNF correlated with increased 32P incorporation into EGF‐R protien when receptor was immunoprecipitated from 32P‐equilibrated cells following a 20 min incubation with TNF. Acid hydrolysis of EGF‐R protein isolated from TNF‐treated ME‐180 cells demonstrates an increase in the phosphotyrosine content of EGF‐R when compared to receptor isolated from untreated cells. The results suggest that TNF increased EGF‐R tyrosine protein kinase activity and the state of EGF‐receptor tryosine phosphorylation in a manner similar to that reported for EGF. However, TNF does not appear to be structurally related to EGF since TNF was unable to directly activate EGF‐R when incubated with extensively washed immunoprecipitates of EGF‐R. In TNF‐resistant T24 bladder carcinoma cells, TNF failed to alter EGF‐R tyrosine protein kinase activity although both EGF and phorbol ester were shown to modulate the enzymatic activity of the receptor in these cells. These results indicate that the ability of TNF to modulate EGF‐R kinase in target cells may correlate with its cytotoxic actions on TNF‐sensitive tumor cells. Other biochemical activities associated with the induction or regluation of cellular growth were examined in TNF‐ or EGF‐treated tumor cells. EGF stimulated a rapid 8–16‐fold increase in the expression of the proto‐oncogene c‐myc when analyzed by dot‐blot analysis of total cellular RNA or Northern blot hybridization of polyadenylated RNA. TNF treatment failed to alter c‐myc expression in ME‐180 cells when analyzed by either technique. The two structurally distinct peptides, TNF and EGF, induced similar patterns of ornithine decarboxylase activity (the rate‐limiting enzyme in polyamine biosynthesis) in ME‐180 cells but differed in their relative magnitude of maximal induction. Similar results were obtained in TNF‐ or EFG‐treated T24 cells, suggesting the effects of TNF on polyamine biosynthesis are not related to its cytotoxic mechanism of action. These results indicate that TNF shares some of the early biochemical actions of EGF in tumor cells and some of these effects may be related to the mechanism of TNF‐induced cytotoxicity.