Cationic gradient reversal and cytoskeleton-independent volume regulatory pathways define an early stage of apoptosis

Abstract

Cell shrinkage, or apoptotic volume decrease (AVD), is a ubiquitous characteristic of programmed cell death that is independent of the death stimulus and occurs in all examples of apoptosis. Here we distinguished two specific stages of AVD based on cell size and a unique early reversal of intracellular ions that occurs in response to activation of both intrinsic and extrinsic cell death signal pathways. The primary stage of AVD is characterized by an early exchange of the normal intracellular ion distribution for sodium from 12 to 113.6 mm and potassium from 139.5 to 30 mm. This early ionic reversal is associated with a 20-40% decrease in cell volume, externalization of phosphatidylserine, loss of mitochondrial membrane potential, and caspase activation and activity along with nuclear condensation that occurs independent of actin cytoskeleton disruption. Disruption of the actin cytoskeleton, however, prevents a secondary stage of AVD in apoptotic cells, characterized by a loss of both potassium and sodium that results in an 80-85% loss in cell volume, DNA degradation, and apoptotic body formation. Together these studies demonstrate that AVD occurs in two distinct stages with the earliest stage reflecting a cellular cationic gradient reversal.

FIGURE 1. Treatment of Jurkat cells with CB prevents apoptotic body formation and show disruption of the actin cytoskeleton during Fas ligand- and UV-induced apoptosis

A, Jurkat cells treated with either 50 ng/ml FasL or 60 mJ/cm² UV in the presence or absence of 5 μm CB for 4 h examined for morphological changes using differential interference contrast microscopy and Hoechst 33342. In the absence of CB, FasL- and UV-treated Jurkat cells showed the classical apoptotic morphology of shrunken cells, nuclear condensation, membrane blebbing, and apoptotic body formation. In contrast, the presence of CB resulted in only the occurrence of shrunken cells and nuclear condensation with the absence of membrane blebbing and apoptotic body formation. B, Jurkat cells treated as above were examined by confocal microscopy in the presence of Alexa Fluor 488 phalloidin (F-actin, green), Alexa Fluor 594 deoxyribonuclease I (G-actin, red), and Hoechst 333421 (blue). In the presence of CB only, Jurkat cells showed a reduced staining for F-actin along with punctate actin aggregates characteristic of microfilament disruption. Induction of apoptosis resulted in the virtual loss of F-actin staining in cells with condensed nuclei.

FIGURE 2. Treatment of Jurkat cells with CB unmasks two distinct stages of AVD during FasL- and UV-induced apoptosis

Jurkat cells treated with 50 ng/ml Fas ligand or 60 mJ/cm² UV in the presence or absence of 5 μm CB for 4 h were examined for changes in cell size by flow cytometry. A, Jurkat cells were initially analyzed on a FSC (cell size) versus SSC (cell granularity) three-dimensional plot. A decrease in FSC indicates a loss in cell size. The red circle denotes the location of a shrunken population in apoptotic-stimulated cells in these three-dimensional plots that is markedly absent in the presence of CB. B, FSC histograms of control (Con) and apoptotic-stimulated Jurkat cells in the presence and absence of CB. The white lines on the plots denote the various stages of cell shrinkage. The bar graphs show the percentage of cells that comprise the various stages of AVD as determined from the FSC histograms and are the average of at least three individual experiments (#, p < 0.05). C, Jurkat cells analyzed over time on a FSC versus SSC three-dimensional plot for the occurrence of stages of AVD. The presence of the secondary stage of AVD occurs as early as 2 h post-treatment and continues to increase over the course of the study. D, Ramos cells and HL-60 cells after a 4-h treatment with 120 or 20 mJ/cm² UV, respectively, in the presence and absence of CB, analyzed for the occurrence of stages of AVD. Similar to the Jurkat cells, CB prevents the secondary stage of AVD.

FIGURE 3. Externalization of phosphatidylserine, loss of mitochondrial membrane potential, caspase activity, and PARP cleavage but not DNA degradation occurs in FasL- or UV-treated Jurkat cells in the presence of CB

Jurkat cells treated with 50 ng/ml Fas ligand or 60 mJ/cm² UV in the presence or absence of 5 μm CB for 4 h were examined for various apoptotic characteristics. A, externalization of phosphatidylserine using annexin-V-fluorescein isothiocyanate and flow cytometry. An increase in the number of cells that had an increased annexin-V-fluorescein isothiocyanate fluorescence before the loss of membrane integrity (lower right-hand quadrant) occurred independent of CB. The dot plots represent a single experiment, and percents on the graph are the average from three independent experiments. B, changes in the mitochondrial membrane potential were examined by flow cytometry using JC-1. Cells that have a depolarized mitochondrial membrane have an increased monomeric fluorescence while having a simultaneous decreased aggregate fluorescence. FasL- or UV-treated Jurkat cell in the presence of CB showed a similar loss of mitochondrial membrane potential compared with analogous samples in the absence of CB. The contour plots represent a single experiment, and the percents on the graph are the average number of depolarized cells from three independent experiments. C, caspase-3-like activity measured using a fluorescent caspase substrate. An increase in the number of cells that are caspase-positive occurred independent of CD. The histograms represent a single experiment, and percents on the graph are the average of three independent experiments. D, Western blot analysis for PARP cleavage. Protein extracts from FasL- or UV-treated Jurkat cells in the presence or absence of CB showed a similar cleavage of PARP resulting in the occurrence of a 24-kDa protein band. E, examination of DNA content by flow cytometry. The occurrence of degraded DNA is shown below the G₁ DNA peak (white line). The histograms represent a single experiment, and the percents on the graph are the average number of cells with subdiploid DNA from three independent experiments (*, p < 0.001).

FIGURE 4. Stages of AVD are reflected in the response of CoroNa Green-AM (Na⁺) and PBFI-AM (K⁺) to measure changes in intracellular sodium and potassium in apoptotic Jurkat cells

Jurkat cells treated with 50 ng/ml FasL or 60 mJ/cm² UV in the presence or absence of 5 μm CB for 4 h were examined for changes in intracellular ions by flow cytometry. A, analysis of sodium using CoroNa Green-AM (Na⁺) showed an increase in intracellular sodium in apoptotic-stimulated cells regardless of the presence or absence of CB. Interestingly, this increase in intracellular sodium upon apoptotic stimulation was enhanced in the presence of CB. The histograms represent a single experiment, and the percents on the graph are the average number of three independent experiments (*, p < 0.001). B, analysis of potassium using PBFI-AM (K⁺) showed an efflux of intracellular potassium in the absence of CB. In contrast, cells treated with CB had a marked inhibition of intracellular potassium efflux upon induction of apoptosis (*, p < 0.001). C, simultaneous analysis of potassium and sodium using PBFI-AM (K⁺) and CoroNa Green-AM (Na⁺), respectively. Treated Jurkat cells that had an increase in intracellular sodium and a concurrent decrease in intracellular potassium regardless of the presence or absence of CB. The presence of CB prevented the continual loss of intracellular ions.

FIGURE 5. Determination of mean cell volume and percentage of change in mean cell volume by the simultaneous examination of intracellular potassium and externalization of phosphatidylserine in apoptotic-stimulated Jurkat cells in the presence and absence of CB

A, Jurkat cells treated with 25 ng/ml Fas ligand or 30 mJ/cm² UV in the presence or absence of 5 μm CB for 4 h were examined for changes in intracellular potassium using PBFI-AM and externalization of phosphatidylserine using biotinylated annexin-V and streptavidin Q dots by NPE cell analysis. In the absence of CB, three distinct populations of cells are observed corresponding to the normal cells (red), the primary stage of AVD (green), and the secondary stage of AVD (blue). In the presence of CB, only the normal (red) and primary stage of AVD (green) cells were observed. The dot plots represent a single experiment of three independent experiments. B, distinct populations of cells were analyzed for changes in their MCV (μm³) using a calibrated scale for the electronic cell size. For each experimental condition, the high potassium/low annexin population is shown in red, the high potassium/high annexin populations is shown in green, and the population of cells with a major loss of intracellular potassium is shown in blue. A clear decrease in MCV was observed based on both the externalization of the phosphatidylserine and the loss of intracellular potassium for each population of cells. The bars represent the average number of three independent experiments (*, p < 0.001; **, p < 0.01; #, p < 0.05). C, percentage of change in MCV for the various stages of AVD in the presence and absence of CB. The control untreated Jurkat cells were used as the standard to which all other populations of cells were compared.