Role of mitochondrial membrane potential in concanavalin A-induced apoptosis in human fibroblasts.

Lectins induce apoptosis in a wide variety of cell types but the mechanisms of apoptotic induction are unknown. We examined the role of mitochondrial membrane potential (Psi m) in concanavalin A-induced apoptosis in human diploid fibroblasts. Cells were treated with Con A for 0.5, 1, 3, 5, and 24 h. Con A induced a time-dependent increase of the proportion of TUNEL+ ve cells over 24 h. Psi m was examined by staining cells with the mitochondria-specific fluorescent cationic dye JC-1. Comparison of JC-1 fluorescence within mitochondria by flow cytometry showed that after 3 h, Con A reduced Psi m in a subpopulation of apoptotic cells with smaller cell volume and with apoptotic nuclear morphology. In contrast, Psi m was unchanged in a separate population of viable cells with normal volume and normal nuclear morphology. Cyclosporin A protected cells against reduction of Psi m and also against nuclear condensation and morphological apoptosis. Measurement of intracellular calcium ion concentration ([Ca2+]i) by ratio fluorimetry of fura 2-loaded cells showed that Con A did not affect [Ca2+]i in viable cells but induced a progressive depletion of [Ca2+]i with generation of calcium oscillations in apoptotic cells. Assessment of Bcl-2 in Con A-treated cells demonstrated an initially strong increase in Bcl-2 protein and mRNA but the appearance of degraded Bcl-2 protein at 3 and 5 h after treatment, indicating an inadequate protective response to the Con A stimulation. Collectively, these data indicate that lectin-induced apoptosis in fibroblasts is associated with breakdown of Psi m, loss of [Ca2+]i homeostasis, and induced Bcl-2 expression.