THE INFLUENCE OF MICROENVIRONMENT ON THE CYTOTOXICITY OF TNF-PROPORTIONAL-TO IN-VITRO

Purpose: Investigations were undertaken to study the influence of oxyg en levels on tumor necrosis factor proportional to (TNF proportional t o) toxicity in vitro. Methods and Materials: The cell line used to ass ess the cytotoxicity of TNF proportional to was the mouse fibroblast l ine L929. The cell line was incubated under conditions of 21%, 10%, 5% and 2% oxygen either before or during exposure to TNF proportional to . All incubations with TNF were for 24 h in the presence of 1 mu g/ml actinomycin D. Cell number was assessed immediately following treatmen t by a colormetric method. Results: By preincubating L929 cells under various oxygen conditions for 24 h prior to incubating with TNF propor tional to, we show that pretreatment does influence TNF proportional t o cytotoxicity since up to 50 times more TNF proportional to is requir ed to elicit the same survival level when L929 cells have been preincu bated for 24 h at oxygen levels relevant to those in solid tumors, tha t is 2% rather than at 21%. A 24 h preincubation under an environment of 5% oxygen is not as effective at inducing resistance to TNF proport ional to as incubation under 2% oxygen. However, this resistance could be significantly enhanced by lengthening the preincubation time. Inde ed cells cultured for five passages under 5% oxygen levels are similar to 50 times more resistant to TNF proportional to than cells cultured under 21% oxygen. The resistance induced by conditions of reduced oxy gen tension could be reversed over a 24 h period if cells were returne d to an environment containing 21% oxygen. Conclusion: The oxygen cont ent of the cellular microenvironment has a profound influence on the c ytotoxic action of TNF.