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Live fluorescence and transmission-through-dye microscopic study of actinomycin D-induced apoptosis and apoptotic volume decrease

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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. 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 Cl− channels failed to prevent apoptosis or AVD.

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Notes

  1. There is no doubt that some latent changes were developing in visibly normal cells prior to blebbing and depolarization of mitochondria, and therefore the terms “early” and “late” should be understood as a purely operational definition.

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Acknowledgments

We are grateful to Akwasi Minta (Teflabs) for providing Asante Potassium dyes. Various parts of the work have been discussed with Francisco Javier Alvarez-Leefmans (Wright State University, Dayton) and Aleksey A. Vereninov (Institute of Cytology, St. Petersburg), who also suggested important improvements to the manuscript. We also thank Sarah Khoncarly for critically reading the draft. We acknowledge the contribution of Mariana Pelts who was involved in some of the initial experiments. The work was supported by NIH grant 1R15GM186816, by the Kent State Initiative for Clinical and Translational Research, and by the Ohio Board of Regents.

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  1. Department of Biological Sciences, Kent State University, Kent, OH, USA

    Neda R. Kasim & Michael A. Model

  2. Department of Cellular Biochemistry, Institute of Hematology and Blood Transfusion, Prague, Czech Republic

    Kateřina Kuželová & Aleš Holoubek

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Kasim, N.R., Kuželová, K., Holoubek, A. et al. Live fluorescence and transmission-through-dye microscopic study of actinomycin D-induced apoptosis and apoptotic volume decrease. Apoptosis 18, 521–532 (2013). https://doi.org/10.1007/s10495-013-0804-z

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