Extracellular calcium is not required for gliotoxin or dexamethasone-induced DNA fragmentation: a reappraisal of the use of EGTA.
The immunomodulating agent gliotoxin and related toxins cause apoptotic cell death in a variety of cell types including macrophages and thymocytes [Waring et al. (1988) J. biol. Chem., 263, 18,493-18,499; Waring et al. (1990) Int. J. Immunopharmac., 12, 445-457]. The mechanism of induction of apoptosis by gliotoxin is not yet known, although it does not require protein synthesis [Waring (1990) J. biol. Chem., 14,476-14,480], unlike dexamethasone-induced apoptosis in thymocytes. Because of the reported requirement for extracellular calcium in apoptosis induced by dexamethasone, we studied the effects of extracellular calcium on gliotoxin-induced apoptosis in macrophages. Initial experiments using calcium-depleted media showed no inhibition of apoptotic DNA fragmentation by gliotoxin. By measuring residual 45Ca2+ remaining in cells pulsed with labelled calcium over the time period required for DNA fragmentation, we could demonstrate some uptake of extracellular calcium into treated cells as assessed by residual, slowly exchanging calcium. When cells were treated with the calcium chelator EGTA at 0.5-2 mM, calcium uptake was abolished but DNA fragmentation was unaffected. EGTA at higher concentrations, up to 8 mM, did inhibit DNA fragmentation without any additional inhibition of calcium uptake. Similar results were found for dexamethasone-treated thymocytes. Thymocytes treated with 8 mM EGTA, however, were not rescued from apoptosis but died by necrosis. These results indicate that extracellular calcium is not essential for apoptosis induced by these agents and that the use of high concentrations of EGTA to establish a requirement for extracellular calcium in apoptosis should be treated with caution.
Waring
P
P
John Curtin School of Medical Research, Australian National University, Canberra City.
Sjaarda
A
A
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002478:Cells, Cultured; D004247:DNA / Q000187:drug effects*; D003907:Dexamethasone / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology*; D004996:Ethidium; D005434:Flow Cytometry; D005912:Gliotoxin / Q000494:pharmacology*; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009336:Necrosis / Q000139:chemically induced; D013950:Thymus Gland / Q000378:metabolism; D013997:Time Factors
D007166:Immunosuppressive Agents; D004533:Egtazic Acid; D005912:Gliotoxin; D003907:Dexamethasone; D004247:DNA; D004996:Ethidium; D002118:Calcium