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To clarify the mechanisms of anti-tumor activity of human recombinant tumor necrosis factor alpha (rTNF alpha), an established cell line KU-2, derived from a patient with human renal cell carcinoma (RCC), was treated with rTNF alpha alone or in combination with anti-cancer agents: actinomycin-D (ACD), vinblastine sulfate (VLB), nimustine hydrochloride (ACNU), and methotrexate (MTX). Cytotoxic assay by crystal violet dye exclusion test showed that 21.0 +/- 4.0% and 34.8 +/- 4.7% of the cells were killed by 72 hours incubation with 100 ng/ml of rTNF alpha, alone and 1 ng/ml of ACD alone, respectively. Synergistic cytotoxicity of 75.0 +/- 0.3% was observed at 72 hours when 100 ng/ml of rTNF alpha and 1 ng/ml of ACD were added simultaneously. Furthermore, additive cytotoxicity of 48.5 +/- 1.1% was observed by 0.1 ng/ml of VLB and 100 ng/ml of rTNF alpha. However, when KU-2 was treated in conjunction with both 100 ng/ml of rTNF alpha and 3 micrograms/ml of ACNU or 2.5 ng/ml of MTX, no significant increase in cytotoxicity was demonstrated. When KU-2 was pretreated with 1 ng/ml of ACD for 24 hours, followed by adding 100 ng/ml of rTNF alpha, a synergistic cytotoxicity by ACD and rTNF alpha was observed at 24 hours. On the other hand, when KU-2 was pretreated with 100 ng/ml of rTNF alpha for 24 hours, followed by adding 1 ng/ml of ACD, no significant increase in cytotoxicity was demonstrated. In clonogenic assay studies, the colony forming efficiency (CE) of the control cultures was 31.8 +/- 8.1%. A 92.3 +/- 1.8% reduction in CE was observed when 100 ng/ml of rTNF alpha was added to the cultures. No significant synergistic or additive effects were demonstrated between rTNF alpha and chemotherapeutic agents in clonogenic assay studies. The effects of rTNF alpha on exponentially growing KU-2 cells were analyzed by studying the distribution of cells in the cell cycle. No cell cycle specific effect of rTNF alpha was demonstrated, regardless of whether or not chemotherapeutic agents were added. These results indicated that the cytotoxic and cytostatic activities of rTNF alpha may be mediated by separate mechanisms of action. Moreover, it was postulated that rTNF alpha may more significantly affect KU-2 cells having clonogenic potentials. rTNF alpha was concluded to have significant anti-tumor effects on renal cell carcinoma cells based on clonogenic assay studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies on in vitro mechanisms of anti-tumor activity of the tumor necrosis factor .ALPHA. against human renal carcinoma cell line (KU-2).