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.
Kulkarni
G V
GV
Faculty of Dentistry, University of Toronto, Toronto, Ontario, M5S 3E2, Canada.
Lee
W
W
Seth
A
A
McCulloch
C A
CA
D017209:Apoptosis; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003208:Concanavalin A / Q000494:pharmacology; D005347:Fibroblasts; D005786:Gene Expression Regulation / Q000187:drug effects; D005881:Gingiva / Q000166:cytology; D006706:Homeostasis; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology*; D008564:Membrane Potentials; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D008934:Mitogens / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D013997:Time Factors
D008934:Mitogens; D019253:Proto-Oncogene Proteins c-bcl-2; D003208:Concanavalin A; D002118:Calcium