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

• Published in: The Journal of cell biology Vol. 138; no. 2; pp. 449 - 469
• Main Authors: Mancini, Mariangela, Anderson, Benjamin O., Caldwell, Elizabeth, Sedghinasab, Monireh, Paty, Philip B., Hockenbery, David M.
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 28.07.1997; The Rockefeller University Press
• Subjects: Adenocarcinoma; Animals; Apoptosis; Apoptosis - physiology; Benzoquinones; Cell Cycle; Cell death; Cell Differentiation; Cell Division; Cell growth; Cell lines; Cell nucleus; Cell Survival; Cell-Free System; Cells; Cellular biology; Cellular differentiation; Colon - cytology; Colon - ultrastructure; Colon cancer; Colonic Neoplasms; Enzyme Inhibitors - pharmacology; Fluorescence; Fluorescent Dyes; Humans; Intracellular Membranes - physiology; Lactams, Macrocyclic; Liver - metabolism; Membrane Potentials; Membranes; Mitochondria; Mitochondria - drug effects; Mitochondria - physiology; Mitochondria - ultrastructure; Oxidation-Reduction; Poly(ADP-ribose) Polymerases - metabolism; Protein-Tyrosine Kinases - antagonists & inhibitors; Quinones - pharmacology; Rats; Reactive Oxygen Species - metabolism; Rifabutin - analogs & derivatives; Tumor Cells, Cultured
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.138.2.449

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.