MODULATION OF TNF-MEDIATED CELL-LYSIS IN-VITRO - FURTHER ANALYSIS OF INTRACELLULAR SIGNALING

We have investigated the post-receptor events governing the Tumor Necr osis Factor (TNF)-mediated cytotoxicity in vitro. As calcium has been reported to be an essential mediator in the cell killing processes, we asked whether an early increase in intracellular calcium could be inv olved during TNF-induced cell death. Using the ACAS methodology (adher ent cell analysis and sorting), we could not detect any significant in crease in intracellular calcium following TNF treatment (40 s) within the TNF-sensitive human breast carcinoma MCF7 cell line. In addition, A23187 (0.1-0.4 muM) did neither enhance TNF-mediated MCF7 cell lysis, further confirming that TNF-mediated cell lysis can occur in the abse nce of an early calcium increase. Given the potentiating effect of cAM P-inducing agents, such as forskolin, on TNF-mediated cytotoxicity, we have investigated the relationship between cAMP accumulation and the TNF signaling pathway during cell death. Our results indicate that the potentiating effect of forskolin (50 muM) on TNF-mediated MCF7 cell l ysis did not involve a modulation in the TNF-induced activation of the nuclear factor NF-kB but was associated with an increase in the DNA f ragmenting capacity of TNF as assessed by agarose gel electrophoresis of target cell DNA.