Calcium-independent pathway of tumor necrosis factor-mediated lysis of target cells.

The role of Ca2+ in cell-mediated cytotoxicity has been the subject of many investigations and both Ca2+-dependent and -independent pathways have been reported. TNF was suggested to play a role in NK and macrophage cell-mediated cytotoxicity. We assumed that its role in target cell lysis might take place by a Ca2+-independent mechanism. This hypothesis was investigated in assays of rTNF-mediated lysis of tumor target cells. Extracellular Ca2+ depletion by the calcium chelator EGTA (2 mM and 5 mM) and blocking of intracellular Ca2+ mobilization by 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride did not inhibit TNF-mediated tumor cell lysis. Furthermore, blocking of Ca2+ influx in the presence of the Ca2+ channel blocker Verapamil did not inhibit TNF-mediated tumor cell lysis. Previous reports showed that lysis of sensitive tumor cells by TNF is preceded by binding of TNF to TNF receptors, internalization, and DNA degradation. These events were tested in the absence of Ca2+. Treatment with Ca2+ inhibitors did not affect binding of 125I-TNF to target cells. Also TNF induced the fragmentation of cellular DNA in target cells without extracellular or intracellular Ca2+. These findings demonstrate that the mechanism of TNF-mediated tumor cell lysis does not depend on intracellular or extracellular Ca2+ and that events associated with target cell lysis can also function in the absence of Ca2+. Thus, our findings support the contention of a Ca2+-independent lytic pathway in which secreted or membrane-bound TNF may interact with the target cells and ultimately result in DNA degradation and target cell lysis.