Journal of Cell Science, Vol 110, Issue 7 819-828, Copyright © 1997 by Company of Biologists

JOURNAL ARTICLES

Cell death of AKR-2B fibroblasts after serum removal: a process between apoptosis and necrosis

A Simm, G Bertsch, H Frank, U Zimmermann and J Hoppe
Department of Physiological Chemistry II, University of Wurzburg, Germany.

AKR-2B cells disintegrate after serum removal. After a delay of approximately 90 minutes, cell death began and reached after six hours a plateau of 40-50% remaining living cells. We used time-lapse video microscopy to monitor dynamic structural changes and to measure the time span of individual cells to die. The first change was the rapid appearance of membrane blebs. Membrane vesicles were rapidly extruded and reintegrated by the cell. This highly dynamic process of an affected cell stopped after 80+/-20 minutes with its death. Conductivity measurements showed that at that time the membrane was electrically permeable. By using fluorescence double staining with propidium iodide and Hoechst 33258, we show that membrane leakage leading to disintegration is accompanied, and for some cells preceded, by nuclear condensation. The energy state of the intact cells was monitored by measuring the intracellular ATP content which remained high (6 mM) throughout the entire time of investigation. Mitochondrial potential was determined by rhodamine 123 fluorescence in parallel to the measurement of membrane permeability via uptake of propidium iodide and lead to the detection of a cell population that exhibits a high mitochondrial potential and an uptake of propidium iodide indicating a membrane disruption of cells which still have a high energy charge. It is shown by electron microscopy that mitochondria were swollen and damaged in parallel to nuclear condensation. There was no DNA fragmentation as shown by two independent methods. Addition of the ICE-like protease inhibitor tyr-val-ala-asp-chloromethylketone immediately after serum starvation lead to an almost complete survival of the cells up to 6 hours. A pronounced protection was still observed after 24 hours, suggesting an involvement of this type of protease in the onset of cell death after serum removal. Apparently, serum withdrawal activates a succession of initial events that are similar to those defined as 'apoptosis', i.e. nuclear condensation and membrane blebbing. These steps are, however, accompanied or rapidly followed by cell lysis and disruption of mitochondria, both of which are characteristic of necrosis.

This article has been cited by other articles:

				T. Antonelli, M. C. Tomasini, J. Fournier, R. Mazza, S. Tanganelli, S. Pirondi, K. Fuxe, and F. Luca Neurotensin Receptor Involvement in the Rise of Extracellular Glutamate Levels and Apoptotic Nerve Cell Death in Primary Cortical Cultures after Oxygen and Glucose Deprivation Cereb Cortex, August 1, 2008; 18(8): 1748 - 1757. [Abstract] [Full Text] [PDF]

				S. Yasuhara, Y. Zhu, T. Matsui, N. Tipirneni, Y. Yasuhara, M. Kaneki, A. Rosenzweig, and J.A. J. Martyn Comparison of Comet Assay, Electron Microscopy, and Flow Cytometry for Detection of Apoptosis J. Histochem. Cytochem., July 1, 2003; 51(7): 873 - 885. [Abstract] [Full Text] [PDF]

				M. H. Beauchamp, A. M. Marrache, X. Hou, F. Gobeil Jr, S. G. Bernier, P. Lachapelle, D. Abran, C. Quiniou, S. Brault, K. G. Peri, et al. Platelet-Activating Factor in Vasoobliteration of Oxygen-Induced Retinopathy Invest. Ophthalmol. Vis. Sci., October 1, 2002; 43(10): 3327 - 3337. [Abstract] [Full Text] [PDF]

				M. Leicht, W. Briest, A. Holzl, and H.-G. Zimmer Serum depletion induces cell loss of rat cardiac fibroblasts and increased expression of extracellular matrix proteins in surviving cells Cardiovasc Res, December 1, 2001; 52(3): 429 - 437. [Abstract] [Full Text] [PDF]

				M. H. Beauchamp, A. K. Martinez-Bermudez, F. Gobeil Jr., A. M. Marrache, X. Hou, G. Speranza, D. Abran, C. Quiniou, P. Lachapelle, J. Roberts II, et al. Role of thromboxane in retinal microvascular degeneration in oxygen-induced retinopathy J Appl Physiol, June 1, 2001; 90(6): 2279 - 2288. [Abstract] [Full Text] [PDF]

				A. Tsugu, K. Sakai, P. B. Dirks, S. Jung, R. Weksberg, Y.-L. Fei, S. Mondal, S. Ivanchuk, C. Ackerley, P. A. Hamel, et al. Expression of p57KIP2 Potently Blocks the Growth of Human Astrocytomas and Induces Cell Senescence Am. J. Pathol., September 1, 2000; 157(3): 919 - 932. [Abstract] [Full Text] [PDF]

				P. Massari, Y. Ho, and L. M. Wetzler Neisseria meningitidis porin PorB interacts with mitochondria and protects cells from apoptosis PNAS, August 1, 2000; 97(16): 9070 - 9075. [Abstract] [Full Text] [PDF]

				L. Gamet-Payrastre, P. Li, S. Lumeau, G. Cassar, M.-A. Dupont, S. Chevolleau, N. Gasc, J. Tulliez, and F. Tercé Sulforaphane, a Naturally Occurring Isothiocyanate, Induces Cell Cycle Arrest and Apoptosis in HT29 Human Colon Cancer Cells Cancer Res., March 1, 2000; 60(5): 1426 - 1433. [Abstract] [Full Text]

				C. Riou, H. Tonoli, F. Bernier-Valentin, R. Rabilloud, P. Fonlupt, and B. Rousset Susceptibility of Differentiated Thyrocytes in Primary Culture to Undergo Apoptosis after Exposure to Hydrogen Peroxide: Relation with the Level of Expression of Apoptosis Regulatory Proteins, Bcl-2 and Bax Endocrinology, May 1, 1999; 140(5): 1990 - 1997. [Abstract] [Full Text]