Mitochondrial proliferation and paradoxical membrane depolarization during terminal differentiation and apoptosis in a human colon carcinoma cell line.
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 (DeltaPsim) as determined by the DeltaPsim-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.
Mancini
M
M
Department of Surgery, University of Washington, Seattle, Washington 98195, USA.
Anderson
B O
BO
Caldwell
E
E
Sedghinasab
M
M
Paty
P B
PB
Hockenbery
D M
DM
D000230:Adenocarcinoma; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D016227:Benzoquinones; D002453:Cell Cycle; D002454:Cell Differentiation; D002455:Cell Division; D002470:Cell Survival; D002474:Cell-Free System; D003106:Colon / Q000166:cytology* / Q000648:ultrastructure; D003110:Colonic Neoplasms; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D007425:Intracellular Membranes / Q000502:physiology; D047029:Lactams, Macrocyclic; D008099:Liver / Q000378:metabolism; D008564:Membrane Potentials; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D010084:Oxidation-Reduction; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011809:Quinones / Q000494:pharmacology; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D017828:Rifabutin / Q000031:analogs & derivatives; D014407:Tumor Cells, Cultured
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D047029:Lactams, Macrocyclic; D011809:Quinones; D017382:Reactive Oxygen Species; D017828:Rifabutin; C020754:herbimycin; D011065:Poly(ADP-ribose) Polymerases; D011505:Protein-Tyrosine Kinases