Live fluorescence and transmission-through-dye microscopic study of actinomycin D-induced apoptosis and apoptotic volume
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ORIGINAL PAPER
Live fluorescence and transmission-through-dye microscopic study of actinomycin D-induced apoptosis and apoptotic volume decrease Neda R. Kasim • Katerˇina Kuzˇelova´ Alesˇ Holoubek • Michael A. Model
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Published online: 17 January 2013 Ó Springer Science+Business Media New York 2013
Abstract The effect of actinomycin D on HeLa cells was studied by live fluorescence and transmission-through-dye microscopy—a recently developed technique that permits volume measurements in live cells. In particular, it is well suited for the observation and quantification of the apoptotic volume decrease (AVD), which is widely viewed as an essential feature of apoptosis. The main results from our study are as follows. (1) Apoptosis caused in HeLa cells by actinomycin D proceeds in two morphologically distinct stages: the early stage is characterized by extensive blebbing, and the late stage by a more compact shape. The loss of mitochondrial membrane potential occurs at about the same time as blebbing, and chromatin condensation follows 30–90 min later. Caspase-3 and 7 become activated during the late stage. (2) Because blebbing occurs before activation of caspase-3, it has to be initiated by a different mechanism. Although blebbing is one of the earliest observable changes, it can be selectively inhibited without affecting other apoptotic reactions. (3) The majority of cells experience a temporary volume increase after the appearance of blebs. Eventually, AVD takes over and the cells shrink by approximately 40 % of their initial volume; the volume loss becomes noticeable at the end of the blebbing phase and continues through the late stage. Electronic supplementary material The online version of this article (doi:10.1007/s10495-013-0804-z) contains supplementary material, which is available to authorized users. N. R. Kasim M. A. Model (&) Department of Biological Sciences, Kent State University, Kent, OH, USA e-mail: [email protected] K. Kuzˇelova´ A. Holoubek Department of Cellular Biochemistry, Institute of Hematology and Blood Transfusion, Prague, Czech Republic
Sometimes, at the end of long incubations, shrinkage gives way to swelling, possibly indicating secondary necrosis. (4) Both early and late apoptosis are accompanied by intracellular accumulation of Na?, while low-sodium medium prevents apoptosis. Except for a partial protective effect of quinine, all of the tested blockers of Na?, K? and Clchannels failed to prevent apoptosis or AVD. Keywords Apoptotic volume decrease Actinomycin D Transmission-through-dye microscopy Intracellular potassium Intracellular sodium Blebbing Caspase-7 Caspase-3
Introduction Apoptosis was initially recognized as a special type of cell death due to characteristically altered cell morphology [1]. Even today, despite a large array of available biochemical assays, morphological changes remain among the most reliable apoptotic markers [2, 3]. Such easily recognizable signs of apoptosis include blebbing and shrinkage. Blebbing is a common apoptotic (though not exclu
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