Cyclic amp enhances toxicity of tumor necrosis factor alpha (tnf-α) in l929 cells

Mouse L929 cells undergo necrosis in response to TNF-α and are used as a standard assay for biologically active TNF-α. Therefore determining the effect of agents on TNF-α cytotoxicity in this cell line could contribute to understanding the mechanism of cell death and also is important because of consequences in a standard assay. L929 cells were grown to confluence in 96 well plates. Human recombinant TNF-α was added in increasing concentrations (12.5 pg/ml to 400 pg/ml) for 24 hr. and cytotoxic activity was determined by staining cells with crystal violet. The influence of 8-Br-cAMP on TNF-α cytotoxity was also determined Intracellular CAMP was measured by RIA. L929 cells were killed by TNF-α dependent on concentration. Maximum % cytotoxity was 60 ± 0.1% at 400 pg/ml TNF-α. 8-Br-cAMP, alone, produced no cytotoxicity. However, the addition of 0.1, 0.5, or, 1 mM 8-Br-cAMP to the medium significantly shifted the TNF-α cytotoxicity curve upward and to the left in a concentration-dependent manner. Maximum cytotoxicity of TNF was increased to 74 ± 0.1% with 1 mM 8-Br-cAMP (P < 0.05) and 50% cytotoxicity occurred at 25 pg/ml TNF vs. 200 pg/ml in the absence of 8-Br-cAMP. Intracellular CAMP was 8 4 ±0.5 pmol/mg cell protein in control cells and increased to 17.4 ±1.5 (p < 0.05) in cells exposed to 1 mM 8-Br-cAMP Forskolin also potentiated cytotoxicity with concentrations of TNF-α up to 100 pg/ml but did not alter maximum cytotoxicity. These results suggest that elevated Intracellular cAMP potentiates the cytotoxic effect of TNF-α in L929 cells. Such findings suggest caution when using the L929 cell cytotoxicity assay for TNF-α in situations where alterations in cAMP might be encountered.