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Calcium-independent pathway of tumor necrosis factor-mediated lysis of target cells
1Department of Microbiology and Immunology, UCLA School of Medicine 90024.
Abstract:
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.
Insights
Tumor necrosis factor (TNF) mediates tumor cell lysis independently of calcium ions (Ca2+). This Ca2+-independent pathway involves TNF binding, internalization, and DNA fragmentation, crucial for cell-mediated cytotoxicity.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Cell-mediated cytotoxicity involves complex signaling pathways, with both calcium-dependent and -independent mechanisms previously reported.
- Tumor necrosis factor (TNF) has been implicated in cytotoxic activities of Natural Killer (NK) cells and macrophages.
- The precise role of calcium (Ca2+) in TNF-mediated target cell lysis remains incompletely understood.
Purpose of the Study:
- To investigate the hypothesis that TNF-mediated tumor cell lysis occurs via a Ca2+-independent mechanism.
- To determine if extracellular Ca2+ depletion or intracellular Ca2+ mobilization blockade affects TNF-induced tumor cell lysis.
- To examine key events in TNF-mediated lysis, such as receptor binding and DNA degradation, in the absence of Ca2+.
Main Methods:
- Assays of recombinant TNF (rTNF)-mediated lysis of tumor target cells.
- Extracellular Ca2+ depletion using the calcium chelator EGTA.
- Blocking intracellular Ca2+ mobilization and Ca2+ influx using specific inhibitors and Verapamil.
- Assessment of 125I-TNF binding to target cells and TNF-induced DNA fragmentation in the presence and absence of Ca2+.
Main Results:
- Neither extracellular Ca2+ depletion nor blockade of intracellular Ca2+ mobilization inhibited TNF-mediated tumor cell lysis.
- Blocking Ca2+ influx with Verapamil did not affect TNF-induced lysis.
- TNF receptor binding, internalization, and DNA fragmentation occurred effectively without extracellular or intracellular Ca2+.
Conclusions:
- The mechanism of TNF-mediated tumor cell lysis is independent of both extracellular and intracellular Ca2+.
- Key events leading to target cell lysis, including DNA fragmentation, function effectively in the absence of Ca2+.
- These findings support a Ca2+-independent cytotoxic pathway involving TNF interaction with target cells.