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Mitochondrial Proliferation and Paradoxical Membrane Depolarization during Terminal Differentiation and Apoptosis in a Human Colon Carcinoma Cell Line

Mariangela Mancini, Benjamin O. Anderson, Elizabeth Caldwell, Monireh Sedghinasab, Philip B. Paty and David M. Hockenbery
The Journal of Cell Biology
Vol. 138, No. 2 (Jul. 28, 1997), pp. 449-469
Stable URL: http://www.jstor.org/stable/1618157
Page Count: 21
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Mitochondrial Proliferation and Paradoxical Membrane Depolarization during Terminal Differentiation and Apoptosis in a Human Colon Carcinoma Cell Line
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Abstract

Herbimycin A, a tyrosine kinase inhibitor, induces cellular differentiation and delayed apoptosis in Colo-205 cells, a poorly differentiated human colon carcinoma cell line. Cell cycle analysis in conjunction with end labeling of DNA fragments revealed that G2 arrest preceded apoptotic cell death. Ultrastructural examination of herbimycin-treated cells demonstrated morphologic features of epithelial differentiation, including formation of a microvillar apical membrane and lateral desmosome adhesions. A marked accumulation of mitochondria was also observed. Fluorometric analysis using the mitochondrial probes nonyl-acridine orange and JC-1 confirmed a progressive increase in mitochondrial mass. However these cells also demonstrated a progressive decline in unit mitochondrial transmembrane potential (Δ Ψ m) as determined by the Δ Ψ m-sensitive fluorescent probes rhodamine 123 and JC-1 analyzed for red fluorescence. In concert with these mitochondrial changes, Colo-205 cells treated with herbimycin A produced increased levels of reactive oxygen species as evidenced by oxidation of both dichlorodihydrofluorescein diacetate and dihydroethidium. Cell-free assays for apoptosis using rat-liver nuclei and extracts of Colo-205 cells at 24 h showed that apoptotic activity of Colo-205 lysates requires the early action of mitochondria. Morphological and functional mitochondrial changes were observed at early time points, preceding cleavage of poly (ADP-ribose) polymerase. These results suggest that apoptosis in differentiated Colo-205 cells involves unrestrained mitochondrial proliferation and progressive membrane dysfunction, a novel mechanism in apoptosis.

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