J Biol Chem, Vol. 274, Issue 11, 7059-7066, March 12, 1999

Fas-induced B Cell Apoptosis Requires an Increase in Free Cytosolic Magnesium as an Early Event

Millie M. Chien, K. Elizabeth Zahradka^§, M. Karen Newell, and John H. Freed^§

From the  Division of Basic Immunology, Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206 and the ^§ Department of Immunology, University of Colorado Health Sciences Center, Denver, Colorado 80262

Ligation of the Fas molecule expressed on the surface of a cell initiates multiple 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 levels of free (non-complexed) Mg²⁺ ([Mg²⁺]_i) and Ca²⁺ ([Ca²⁺]_i) increase in cells undergoing apoptosis. Furthermore, the percentages of cells mobilizing Mg²⁺, fragmenting DNA, or externalizing phosphatidylserine (PS) increase in parallel as the concentration of anti-Fas monoclonal antibody is raised. Kinetic analysis suggests that Mg²⁺ mobilization is an early event in apoptosis, clearly preceding DNA fragmentation and probably occurring prior to externalization of PS as well. The source of Mg²⁺ that produces the increases in [Mg²⁺]_i is intracellular and most likely is the mitochondria. Extended pretreatment of B cells with carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of mitochondrial oxidative phosphorylation, produces proportional decreases in the percentage of cells mobilizing Mg²⁺, fragmenting DNA, and externalizing PS in response to anti-Fas monoclonal antibody treatment. These observations are consistent with the hypothesis that elevated [Mg²⁺]_i is required for apoptosis. Furthermore, we propose that the increases in [Mg²⁺]_i function not only as cofactors for Mg²⁺-dependent endonucleases, but also to facilitate the release of cytochrome c from the mitochondria, which drives many of the post-mitochondrial, caspase-mediated events in apoptotic cells.