Fas antigen-mediated DNA fragmentation and apoptotic morphologic changes are regulated by elevated cytosolic Ca2+ level.

Abstract

More than 80% of cells of the human B cell line FMO, which expresses the Fas Ag, underwent apoptosis within 2 h after addition of an anti-Fas mAb. Rises in cytosolic Ca2+ concentration ([Ca2+]i) and their effects were investigated by imaging individual cells continuously and introducing Ca2+ chelators to the outside and/or inside of the cell. The typical Ca2+ response consisted of 1) an early [Ca2+]i rise (basal [Ca2+]i, 85 to 110 nM; peak, 140 to 200 nM; duration, 15 to 20 min), 2) sustained elevation of [Ca2+]i at 140 to 150 nM, 3) a second Ca2+ rise (200 to 500 nM, 15 to 30 min) between 1.5 and 2 h, and 4) a large [Ca2+]i rise (1.2 to 2.0 microM) between 2 and 4 h after addition of mAb. Responses 1, 3, and 4 were mainly caused by Ca2+ entry, and response 2 involved intracellular Ca2+ release from stores. Apoptosis could be induced by mAb even in Ca(2+)-deprived external medium. Chelating cytosolic Ca2+ revealed that the [Ca2+]i rise is a prerequisite for fragmentation of DNA and chromatin, and is also necessary for fragmentation of cells. The critical [Ca2+]i was 140 to 150 nM and a sustained [Ca2+]i rise was more effective. A [Ca2+]i rise itself (without mAb) was ineffective. About 20% of mAb-treated cells showed chromatin condensation at the periphery of the nucleus (possibly an earlier stage of nuclear change) and a bubble-like cell shape even when [Ca2+]i was held below 100 nM. Thus, Ca2+ is mobilized immediately after Fas stimulation and functions as a key factor causing advanced apoptotic changes. Response 4 was related to secondary necrosis.