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

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.