Fas-induced B cell apoptosis requires an increase in free cytosolic magnesium as an early event

Ligation of the Fas molecule expressed on the surface of a cell initiates m ultiple signaling pathways that result in the apoptotic death of that cell. We have examined Mg2+ mobilization as well as Ca2+ mobilization in B cells undergoing Fas-initiated apoptosis, Our results indicate that cytosolic le vels of free (non-complexed) Mg2+ ([M2+](i)) and Ca2+ ([Ca2+](i)) increase in cells undergoing apoptosis, Furthermore, the percentages of cells mobili zing Mg2+, fragmenting DNA, or externalizing phosphatidylserine (PS) increa se in parallel as the concentration of anti-Fas monoclonal antibody is rais ed. Kinetic analysis suggests that Mg2+ mobilization is an early event in a poptosis, clearly preceding DNA fragmentation and probably occurring prior to externalization of PS as well. The source of Mg2+ that produces the incr eases in [Mg2+](i) is intracellular and most likely is the mitochondria. Ex tended pretreatment of B cells with carbonyl cyanide m-chlorophenylhydrazon e, an inhibitor of mitochondrial oxidative phosphorylation, produces propor tional decreases in the percentage of cells mobilizing Mg2+, fragmenting DN A, and externalizing PS in response to anti-Fas monoclonal antibody treatme nt. These observations are consistent with the hypothesis that elevated [Mg 2+](i) is required for apoptosis. Furthermore, we propose that the increase s in [Mg2+](i) function not only as cofactors for Mg2+-dependent endonuclea ses, but also to facilitate the release of cytochrome c from the mitochondr ia, which drives many of the post-mitochondrial, caspase-mediated events in apoptotic cells.