Enhancement of tumor necrosis factor cytotoxicity by lithium chloride is associated with increased inositol phosphate accumulation.

We have previously reported that LiCl increases considerably the cytotoxic activity of TNF towards some transformed cell lines such as L929. Here we show that treatment of these cell lines with the combination of TNF and LiCl leads to the prolonged accumulation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate, whereas treatment with TNF or LiCl alone did not. In contrast, both a LiCl-unresponsive TNF-sensitive cell line and TNF-resistant cell lines did not respond with increased accumulation of inositol phosphates (IPn) upon treatment with the combination of TNF and LiCl. Furthermore, the combination of TNF and LiCl induced a transient increase in cytidine diphosphate-diacylglycerol in L929 cells. Increased IPn and cytidine diphosphate-diacylglycerol accumulation preceded the onset of cell killing by approximately 1 h. TNF-mediated cytotoxicity and TNF-induced IPn accumulation were equally sensitive to inhibition by the phospholipase inhibitor neomycin and to stimulation by the protein kinase inhibitor staurosporine. Characterization of the inositol bisphosphate isomers by HPLC analysis revealed that the TNF + LiCl-induced increase in IPn levels was due to activation of a phospholipase C and not of a phospholipase D. In contrast to TNF, several other cytotoxic agents did not increase IPn production upon application in the presence of LiCl. The TNF + LiCl-induced increase in inositol triphosphate suggests a role for intracellular Ca2+ mobilization in TNF action. Moreover, several agents that lower the intracellular Ca2+ concentration inhibited TNF cytotoxicity. In conclusion, our data provide evidence that TNF cytotoxicity and its enhancement by LiCl are mediated by increased IPn accumulation resulting in Ca2+ mobilization.