Characterization of the apoptotic effects of human tumor necrosis factor: development of highly rapid and specific bioassay for human tumor necrosis factor and lymphotoxin using human target cells.

Currently available bioassays for most cytokines require several days and therefore must be performed under sterile conditions. In this report we describe a bioassay for tumor necrosis factor (TNF) and lymphotoxin (LT) that is extremely rapid and specific and does not require sterile conditions. Using tritiated thymidine release, we could conveniently monitor degradation of DNA into small fragments following the incubation of human myelogenous leukemia ML-1a cells with TNF. The assay showed that TNF-dependent DNA fragmentation was potentiated by cycloheximide and occurred within 90 min. Treatment of cells to TNF lead to apoptosis as indicated by thymidine release, DNA laddering on agarose gels and morphological alterations. Under these conditions, plasma membrane were not damaged as indicated by lack of chromium release. This effect was linear with TNF concentration. This assay had high throughput, did not require sterile conditions, could be carried out in the absence of serum, and was sensitive only to TNF and LT and not to interferon (IFN)-alpha, IFN-beta, IFN-gamma, transforming growth factor beta, interleukin-4, leukemia inhibitory factor and granulocyte-monocyte colony stimulating factor; all cytokines known to inhibit different cell types. Besides detection of TNF in biological fluids, this assay may prove useful for the identification of novel inhibitors of TNF action.