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© The Rockefeller University Press, 0021-9525/1997//449 $5.00
The Journal of Cell Biology, Volume 138, Number 2, , 1997 449-469

Article

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



Mariangela Mancini*,{ddagger}, Benjamin O. Anderson*,{ddagger},||, Elizabeth Caldwell§, Monireh Sedghinasab*,{ddagger}, Philip B. Paty||, and David M. Hockenbery{ddagger}

* Department of Surgery, University of Washington, Seattle, Washington 98195; {ddagger} Division of Clinical Research and Molecular Medicine, and § Electron Microscope Laboratory, Shared Resources, Administrative Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98195; and || Department of Surgery, Memorial Sloan Kettering Cancer Center, New York 10021

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 ({Delta}{Psi}m) as determined by the {Delta}{Psi}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.

Abbreviations used in this paper: {Delta}{Psi}m, mitochondrial membrane potential; DHE, dihydroethidium; H2-DCF-DA, dichlorodihydrofluorescein; NAO, nonyl-acridine orange; PARP, poly (ADP-ribose) polymerase; ROS, reactive oxygen species; TdT, terminal deoxynucleotidyl transferase.

The authors are indebted to Andrew Berger for his excellent technical assistance in flow cytometry experiments and analysis of cell cycle data; to Judy Groombridge for the preparation of electron microscopy specimens; and to Paul Goodman, Paula Salewsky, and Tim Knight for their assistance with computer graphics and image analysis. We are grateful to Michael Wright and Stephen Schwartz for providing the rat liver nuclei preparation for in vitro assays of apoptosis.

D.M. Hockenberry is a Lucille P. Markey scholar, and this work was supported by a grant from the Lucille P. Markey Charitable Trust.

Received for publication 11 July 1996 and in revised form 5 May 1997.

Please address all correspondence to Benjamin O. Anderson, Department of Surgery, University of Washington, Box 356410, Seattle, WA 98195. Tel.: (206) 543-6352; Fax: (206) 543-8136.


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