Raising intracellular free magnesium ions antagonizes caspase activation and apoptosis in mammalian cells

Abstract

We have found that free magnesium ions block Apaf‐1 apoptosome activation of caspase‐9 in vitro by competition with cytochrome c (Abstract 2214). Here we show that raising intracellular free Mg2+ inhibits the induction of apoptosis and activation of caspases ‐9 and ‐3. HeLa cells were treated with PS‐341, a proteasome inhibitor, for 28 h to induce apoptosis. Alternatively, apoptosis was induced by a 5 h treatment with TNFα plus cycloheximide. In both cases, apoptosis was observed by characteristic changes in cell morphology, caspase activation, detachment from the substratum, and cell death. Induction of apoptosis increased caspase‐3 like activity, which was assayed in cell lysates with a fluorogenic substrate, by 13‐fold following proteasome inhibition or 43‐fold following the TNFα?treatment??? Intracellular free Mg2+ was raised by the addition of 20 to 60 mM MgCl2 to the normal culture medium, which contained 0.8 mM Mg2+. Increasing external Mg2+ had no effect on cell viability. Intracellular free Mg2+, which was measured with Mag‐Fluo‐4AM, was 0.6 mM in the normal culture medium and increased to 1.6, 2.2, and 2.4 mM after a 5 or 24 h incubation at 21, 41, or 61 mM external Mg2+, respectively. Raising intracellular free Mg2+ essentially abolished caspase‐3 activation following proteasome inhibition and decreased caspase‐3 activation by the TNFα?treatment by ~60%. Western analysis confirmed the activation of caspase‐3 and indicated that procaspase‐9 was processed to subunits following the treatments. Importantly raising free Mg2+ decreased the processing of both procaspases to subunits. Raising external Mg2+ also protected 911 retinoblasts from apoptosis. These findings support the idea that raising intracellular free Mg2+ antagonizes apoptosis, apparently by decreasing caspase‐9 activation via the apoptosome. Supported by grant GM60383 from NIH.