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Am J Physiol Cell Physiol 290: C638-C649, 2006. First published September 14, 2005; doi:10.1152/ajpcell.00364.2005
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Changes in intracellular electrolyte concentrations during apoptosis induced by UV irradiation of human myeloblastic cells

F. Arrebola,1,2 E. Fernández-Segura,2 A. Campos,2 P. V. Crespo,2 J. N. Skepper,3 and A. Warley1

1Electron Microscopy Unit, King’s College London, Department of Ophthalmology, The Rayne Institute, St. Thomas' Hospital, London; 2Department of Histology, School of Medicine, University of Granada, Granada, Spain; and 3Multi-imaging Centre, Department of Anatomy, University of Cambridge, Cambridge, United Kingdom

Submitted 20 July 2005 ; accepted in final form 8 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Decreases in the intracellular concentrations of both K+ and Cl– have been implicated in playing a major role in the progression of apoptosis, but little is known about the temporal relationship between decreases in electrolyte concentration and the key events in apoptosis, and there is no information about how such decreases affect different intracellular compartments. Electron probe X-ray microanalysis was used to determine changes in element concentrations (Na, P, Cl, and K) in nucleus, cytoplasm, and mitochondria in U937 cells undergoing UV-induced apoptosis. In all compartments, the initial stages of apoptosis were characterized by decreases in [K] and [Cl]. The largest decreases in these elements were in the mitochondria and occurred before the release of cytochrome c. Initial decreases in [K] and [Cl] also preceded apoptotic changes in the nucleus. In the later stages of apoptosis, the [K] continued to decrease, whereas that of Cl began to increase toward control levels and was accompanied by an increase in [Na]. In the nucleus, these increases coincided with poly(ADP-ribose) polymerase cleavage, chromatin condensation, and DNA laddering. The cytoplasm was the compartment least affected and the pattern of change of Cl was similar to those in other compartments, but the decrease in [K] was not significant until after active caspase-3 was detected. Our results support the concept that normotonic cell shrinkage occurs early in apoptosis, and demonstrate that changes in the intracellular concentrations of K and Cl precede apoptotic changes in the cell compartments studied.

sodium; potassium; chloride; cell shrinkage

APOPTOSIS plays a key role in the regulation of cell numbers and is important in development, remodeling, and immunological processes. Apoptosis was originally characterized morphologically by changes that take place within the dying cells; these changes include blebbing of cell membrane, cell shrinkage, condensation, and fragmentation of chromatin, and formation of membrane-bound apoptotic bodies. These morphological alterations are accompanied by internucleosomal cleavage of genomic DNA (49) due to the activation of cytoplasmic proteolytic enzymes, the caspases (53).

Several lines of evidence have suggested that alterations in the transmembrane gradients of K+ play a major role in apoptosis (31, 52). Reduction of the K+ electrochemical gradient by increasing extracellular K+ inhibits apoptosis induced by different apoptotic inducers (5, 7, 38). Also, pharmacological blockade of K+ channels by 4-aminopyridine, tetraethylammonium, Ba2+, quinine, or clofilium prevents cell death induced by different stimuli in several cellular systems (19, 41, 42, 47). The efflux of K+ and the concomitant decrease of intracellular K+ activate key events in the apoptosis cascade, such as caspase cleavage (14, 33, 38), cytochrome c-dependent formation of the apoptosome (6), and activation of endonucleases (14).

The demonstration that loss of cell volume, termed apoptotic volume decrease, is coupled to regulated volume decrease facilitation suggests the participation of other monovalent ions, such as Cl–, in promoting apoptotic events leading to cell death (19, 24). In this context, some studies demonstrated an activation of volume-sensitive outwardly rectifying Cl– currents in receptor- and mitochondrion-mediated apoptosis in different cell lines (22, 34, 37). The Cl– channel blockers 4,4-disothiocyanateostilbene-2,2'-disulfonic acid, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid inhibited Cl– current and induced a significant delay or inhibition of some apoptotic events, such as apoptotic volume decrease, cytochrome c release, caspase-3 activation, and DNA fragmentation (19, 29, 30, 47). Taken together, these studies suggested that alterations in the transmembrane gradients and intracellular concentrations of the physiologically important ions, K+ and Cl–, play a pivotal role in the regulation, progression, and execution of apoptosis.

However, most research to date has focused on the role of a single ion in apoptosis and has not provided information on the simultaneous compartmentation of different ions within individual cells. In addition, there is no clear definition of the temporal relationship between changes in total electrolyte composition, mainly K+, Cl–, and Na+ at the cellular level, with key events of apoptosis, such as cytochrome c release, caspase-3 activation, chromatin condensation, and DNA fragmentation. In our opinion, this is fundamentally a methodological problem because the techniques used with fluorescent dyes are not able to resolve this issue.

Electron probe X-ray microanalysis (EPXMA)1 is a quantitative technique based on analysis of the element-specific X-rays generated in a specimen by the electron beam in an electron microscope and is complementary to fluorescent ion-binding dyes (1) but provides additional information. EPXMA allows analysis of more than one element at the same time in individual cells, and, when cryosectioned material is analyzed, it is possible to recognize and analyze the electrolyte concentration in single, specific subcellular compartments, i.e., the nucleus, cytoplasm, and mitochondria (45). However, there are few studies (10, 32, 35) that have used this technique to analyze changes in elemental composition in apoptotic cell death. In addition, EPXMA has not been used to determine the changes in electrolyte concentration in subcellular compartments during apoptosis.

The study of intracellular compartments is especially important for mitochondria because they are thought to play a pivotal role in initiating the execution phase. The intermembrane space of mitochondria contains various apoptogenic proteins including cytochrome c, apoptosis-inducing factor, and second mitochondria-derived activator of caspases (Smac/DIABLO), the release of which is a central coordinating step in many apoptotic pathways (21). In particular, the release of cytochrome c facilitates the formation of an apoptosome that results in the activation of effector caspases (caspase-3, -6, and -7) (18). However, the mechanism of cytochrome c release is not yet clearly understood, suggesting that various different mechanisms may be involved (17). Several studies suggest initial swelling of the mitochondria and subsequent mechanical rupture of the outer mitochondrial membrane with release of proteins. In contrast, investigators in other studies (11, 13, 39) have reported permeability transition-related changes in the mitochondrial membrane potential that induce mitochondrial swelling. Evidence that alterations in mitochondrial ion homeostasis might underlie the release of cytochrome c is provided by actions of the bcl-2 family of proteins. The antiapoptotic protein bcl-2 is known to prevent release of cytochrome c from the mitochondria (15). Recently, studies of mitochondria isolated from HL-60 cells undergoing etoposide-induced apoptosis showed that bcl-2 upregulated mitochondrial potassium efflux via the K+/H+ exchanger, an action that would stabilize volume under conditions of K+ uptake, whereas tBid, which is proapoptotic, stimulated K+ uptake into the mitochondria and subsequent cytochrome c release (9). Despite the inside-negative mitochondrial membrane potential representing the major force driving mitochondrial ion accumulation, and volume homeostasis being dependent on tightly regulated ion fluxes (4), there is no information about electrolyte concentrations in mitochondria during apoptosis.

For this reason, we used EPXMA to investigate the alterations in electrolyte concentrations at cellular and subcellular levels, i.e., in sections of U937 cells and in the mitochondrial nucleus and cytoplasm during apoptotic cell death induced by ultraviolet (UV) irradiation. UV irradiation is ideal for the purpose of this study because it induces apoptosis rapidly in essentially the entire cell population (>90%), thus minimizing the variability in the cells studied. In addition, we examined the relationship between the time sequence of electrolyte changes at cellular and subcellular levels with the characteristic events of apoptotic cytochrome c release, caspase-3 activation, chromatin condensation, and DNA fragmentation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents and antibodies. RPMI 1640 medium, fetal bovine serum, bovine serum albumin (BSA), L-glutamine, Hoechst 33342, phosphate-buffered saline (PBS), piperazine-N,N'-bis(2-ethane sulfonic acid) (PIPES), Tris·HCl, EDTA, Triton X-100, RNase, proteinase K, agarose, ethidium bromide, and propidium iodide were obtained from Sigma (Poole, UK). Monoclonal antibody for the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp175 (Cell Signaling Technology, Beverly, MA) was used to detect active caspase-3, and the secondary antibody was a biotinylated anti-rabbit IgG (Master Diagnostic, Granada, Spain). A monoclonal antibody for the large fragment (89 kDa) of human poly(ADP-ribose) polymerase (PARP) produced by caspase cleavage adjacent to Asp214 (Cell Signaling Technology) was used to detect PARP cleavage, and the secondary antibody was a biotinylated anti-rabbit IgG (Master Diagnostic). For the detection of cytochrome c and active caspase-3 using confocal microscopy, a monoclonal antibody that recognized residue 62 of cytochrome c (PharMingen) was used with Alexa 488-labeled goat anti-mouse antibody (Invitrogen, Paisley, UK) as the secondary antibody. Active caspase-3 was labeled with a rabbit polyclonal antibody (Promega, Southampton, UK) and visualized using goat anti-rabbit secondary antibody conjugated to Alexa 568 (Invitrogen).

Cell line and culture conditions. The human monoblastoid cell line U937, obtained from European Collection of Cell Culture (Porton Down, UK), was used throughout this study. U937 cells were grown in antibiotic-free RPMI 1640 medium supplemented with 10% (vol/vol) fetal bovine serum and 2 mM L-glutamine and maintained in a humidified incubator at 37°C in an atmosphere of 5% CO2 in air.

Induction of apoptosis. Apoptosis was induced in U937 cells by exposure to UVB irradiation. Cells (1 x 106 cells/ml) were seeded into 75-cm2 tissue culture flasks and exposed 2 cm from below to a dose of 72 J/cm2 of 302-nm UVB from a transilluminator (BTX 20-M; UVItec, Cambridge, UK) for 10 min at room temperature. Cells were returned to 37°C, and samples were removed at appropriate time periods.

Assessment of apoptosis. The fluorescent dye Hoechst 33342 was used to visualize the morphological features of the nuclei. Cells were collected and fixed in 8% buffered formaldehyde for 30 min at room temperature. After fixation, the cells were washed in PBS (pH 7.4) and incubated for 30 min at 37°C in PBS containing Hoechst 33342 (5 µg/ml). Cells were dropped onto a Neubauer chamber and visualized using an epifluorescence microscope (Leitz Laborlux 12; Leica Microsystems, Milton Keynes, UK), an excitation wavelength of 355–425 nm, and a long-pass filter of 470 for emission. We evaluated the cells on the basis of their nuclear morphology, noting the presence of homogeneous chromatin, condensed chromatin, and fragmented nuclei. Fluorescence images were acquired using a digital camera (model DC100, Leica Microsystems) and processed using Photodeluxe (Adobe Systems, San Jose, CA).

Internucleosomal DNA cleavage was analyzed by electrophoresis on agarose gels. Cells were washed twice with ice-cold PBS and digested for 15 min at 4°C with lysis buffer containing 20 mM Tris·HCl (pH 7.5), 2 mM EDTA, and 0.4% Triton X-100. Cell lysates were centrifuged at 13,000 g for 15 min and incubated with 100 µg/ml proteinase K and 60 µg/ml RNase for 3 h at 55°C. DNA was extracted with an equal volume of phenol/choroform/isoamyl alcohol (25:24:1) and precipitated with 0.1 vol of 3 M sodium acetate and 2 vol absolute ethanol overnight at –20°C. The samples were centrifuged at 13,000 g for 15 min, and DNA pellets were dried, dissolved in distilled water, and analyzed by electrophoresis on 1.2% agarose gels. Gels were stained with 1 µg/ml ethidium bromide and visualized in a 302-nm UVB transilluminator BTX 20-M (UVItec). Images were acquired using a Sony digital camera (model CCD-Iris SSC-M370CE).

For transmission electron microscopy, the cells were fixed in 4% glutaraldehyde in 0.1 M PIPES buffer, pH 7.2. The cells were washed, postfixed in 1% osmium tetroxide, dehydrated in a graded series of alcohols, and embedded in Spurr's resin. Thin sections were cut using a ultramicrotome (model UCT; Leica Microsystems), mounted on copper grids stained with lead citrate and uranyl acetate, and viewed using an electron microscope (model CM100; Philips, Eindhoven, The Netherlands).

Caspase-3 cleavage and PARP cleveage. Expression of cleaved caspase-3 and PARP was assayed by immuocytochemistry and carried out using the MLINK streptavidin-biotin-immunoperoxidase kit (Master Diagnostic). Briefly, the cells were washed with PBS, dropped onto poly-L-lysine-coated slides, and fixed with 10% buffered formaldehyde for 25 min at room temperature. The samples were then washed twice with PBS, permeabilized with 0.2% Triton X-100 in PBS for 10 min, and washed twice with PBS. Cells were incubated in blocking buffer for 1 h at room temperature and incubated in primary antibody diluted at 1:100 in blocking buffer overnight at 4°C. Cells were then incubated in biotinylated secondary antibody for 1 h in a dark and humid chamber at room temperature and washed with PBS, followed by incubation in streptavidin-biotin-peroxidase complex for 10 min in a dark, humid chamber at room temperature. The peroxidase reaction was visualized using 0.05% diaminobenzidine and 0.01% hydrogen peroxide. Slides were visualized in a Nikon OptiPhot-2 microscope, images were acquired using a Canon PowerShot S40 digital camera and processed using Adobe Photodeluxe.

Measurement of cytochrome c release. The translocation of cytochrome c from mitochondria was evaluated by immunocytofluorescence. Cells were rinsed once with PBS and fixed for 25 min in 0.2% formaldehyde in 0.15 M PIPES buffer at room temperature and washed in 0.15 M PIPES buffer. After fixation, cells were pelleted and incubated with anti-cytochrome c antibody diluted 1:25 in PBS and anti-caspase-3 diluted 1:100 incubated in 3% BSA for 18 h at 20°C. Excess antibody binding was removed by washing with PBS twice for 5 min. The secondary antibodies diluted 1:100 in PBS and 3% BSA were added and incubated for 1 h at room temperature. Cells were washed with PBS, incubated with PBS containing 500 ng/ml of Hoechst 33342, and washed with PBS. Finally, the cells were mounted onto a microscope slide with the use of FluoroGuard antifade reagent (Bio-Rad Laboratories, Hemel Hempstead, UK), analyzed using Vectorshield antifade reagent (Vector Laboratories, Peterborough, UK) with a Leica TCS-AOBS-SP2 laser scanning microscope using a 405-nm laser line to excite Hoechst 33342, a 488-nm line for Alexa 488, and a 568-nm line for Alexa 568.

Electron probe X-ray microanalysis. For EPXMA, control and UV-irradiated cells were removed from the culture medium. The cells were pelleted in a microcentrifuge at 10,000 g for 60 s. Drops of the thick suspension were transferred onto either aluminum specimen pins or wedges of Millipore filter paper and cryofixed by plunging into liquefied propane using a Reichert KF 80 plunge-freezing apparatus. The frozen droplets were stored in liquid nitrogen until required for sectioning. Cryosections (250 nm thick) were cut at –120°C using a RMC cryoultramicrotome. The cryosections were collected onto a Pioloform-covered 100 hexagonal mesh electron microscope grid, placed in a brass grid carrier, and transferred in liquid nitrogen to the precooled stage of an Emitech K775 freeze drier. The sections were freeze dried overnight using controlled conditions and coated with carbon without breaking the vacuum (46).

The sections were analyzed using a Zeiss EM10 electron microscope fitted with an Oxford Instruments EDS detector. Cells for analysis were selected according to morphological criteria; cells that were electron lucent or showed large areas of ice crystal damage (characteristics of necrosis) were avoided. A selected area of the nucleus, cytoplasm, or mitochondrion or an area including all cellular compartments was analyzed in point mode for 60-s live time at 80-kV accelerating voltage and 1-nA beam current in scanning transmission electron microscopy mode at ambient temperature. Spectra were collected and processed using PGT eXcalibur software. Quantification was achieved using the continuum normalization procedure of Hall as described previously (44) with reference to standards composed of gelatin containing known amounts of inorganic salts (43). The quantities of cells analyzed were as follows: for control, 72; for 15 min, 76; for 30 min, 65; for 60 min, 62; for 90 min, 70; and for 120 min, 63. Cells from four independent experiments were analyzed.

Statistical analysis. Data are expressed as means ± SE and were compared using Kruskal-Wallis one-way ANOVA on ranks. When significant differences were found, they were compared with control values using Dunn's method. P values <0.05 were considered statistically significant. Statistical analysis was performed using SigmaStat statistical software (Systat Software, London, UK).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ultrastructural analysis using transmission electron microscopy showed control cells with a rounded nucleus with a thin rim of electron-dense heterochromatin underlying the nuclear membrane. The more electron-lucent euchromatin was evenly dispersed throughout the nucleus. The mitochondria appeared normal and were dispersed throughout the cytoplasm, with no preferential location (Fig. 1A). After 60 and 90 min, UV irradiation-treated cells showed chromatin condensed at the periphery of the nucleus (Fig. 1B) and the appearance of fragmented nuclei (Fig. 1C). After 120 min of UV irradiation, U937 cells showed all of the structural markers of apoptosis described before; in addition, some cells showing signs of secondary necrosis began to appear (Fig. 1D). The compartments that we analyzed (nucleus, mitochondria, and cytoplasm) were clearly visible in the sections of untreated U937 cells (Fig. 1E), and morphological features of apoptosis, such as the presence of condensed chromatin, could be observed in cryosections of apoptotic U937 cells (Fig. 1F).



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  		Fig. 1. Effect of ultraviolet (UV) irradiation on the ultrastructure of U937 cells. Apoptosis was induced by UV irradiation as described in MATERIALS AND METHODS. Samples taken at various time points were either processed for electron microscopy (A–D) or cryofixed and cryosectioned for microanalysis (E and F). Untreated cells showed normal morphology (A). Margination of chromatin became apparent after 60 min (B), and fragmentation of the nuclei began to appear at 90 min after irradiation (C). After 120 min, a small percentage of cells showed secondary necrosis (D) characterized by electron-lucent cytoplasm. In cryosections of untreated cells (E), the normal morphological features, cytoplasm, nuclei (N), and mitochondria (M), were clearly apparent, and in cryosections of UV light-irradiated cells (F), margination of the chromatin was clearly observed (arrow denotes marginated chromatin). Bar, 5 µm.

 
Changes in elemental content expressed as the sum of all compartments in the section area. Element concentrations obtained from the global analysis of whole areas of the cell sections are shown in Fig. 2. There were no significant differences in the concentrations of either magnesium or sulfur throughout the time course studied. Concentrations of Na initially remained level but began to increase at 90 min; this increase became significant 120 min after the induction of apoptosis. The major changes were observed in the elements P, Cl, and K. There was an initial decrease in the concentration of P compared with control that became significant by 30 min after exposure to UV irradiation, and [P] remained level with no further decrease. There was an initial significant decrease in Cl 15 min after exposure to UV light, a time point before structural changes associated with apoptosis were detected. The concentration of this element remained low until the 60-min time point, after which the concentration of this element increased, so that by the end of the experiment it was slightly above the initial value; this increase and the concomitant increase in [Na] occurred after nuclei containing condensed chromatin were detected. [K] fell steadily throughout the experimental period. The decrease in concentration of this element became significantly lower than that in control cells 30 min after the onset of apoptosis, before many of the significant features of apoptosis were detected.



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  		Fig. 2. Concentrations (mmol/kg dry wt) of elements in whole sections of U937 cells at different time points after the onset of apoptosis induced by UV irradiation. Cells were prepared for electron probe X-ray microanalysis (EPXMA) as described in MATERIALS AND METHODS, and for analysis the area scanned was enlarged to cover the whole section of the cell but with the scanned area remaining within the borders of the cell. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control values.

 
Changes in mitochondria of U937 cells after UV-induced apoptosis. The greatest changes in element concentrations occurred more rapidly in the mitochondria than in other compartments (Fig. 3A). [Na] remained stable until 60 min after UV treatment, although there was a nonsignificant decrease in the concentration of this element at 15 min (from 123 to 96 mmol/kg). After this time point, [Na] began to rise from 96 to 265 mmol/kg and was significant only at 120 min (P < 0.01). In the mitochondria, the concentrations of Mg and S did not change significantly throughout the apoptotic period (data not shown). [P] decreased progressively after the onset of apoptosis (from 588 to 472 mmol/kg) and became significant 30 min after UV irradiation (P < 0.05). It reached its maximum drop and significance (P < 0.001) at 60 min. After this time point, [P] recovered slightly without reaching the concentration found in control mitochondria (499 vs. 588 mmol/kg). Of all of the compartments, the mitochondria showed the most rapid and most significant decrease in [Cl]; after 15 min, there was a significant decrease (P < 0.001) that represented just over one-half of the total concentration (70 vs. 146 mmol/kg) of the element in this organelle. Although the low concentration of Cl was maintained throughout the start of the apoptotic process, it began to increase slightly (86 mmol/kg at 30 min, P < 0.001; 94 mmol/kg at 60 min, P < 0.01), and by 90 and 120 min after UV irradiation, [Cl] was similar to that in control cells.



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  		Fig. 3. Element concentrations and cytochrome c release in mitochondria of U937 cells undergoing UV irradiation-induced apoptosis. A: concentrations (mmol/kg dry wt) of different elements in the mitochondria of U937 cells at different time points after the onset of apoptosis induced by UV irradiation. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control values. B: cytochrome c release and active caspase-3. Sample records from confocal laser-scanning microscopy of U937 cells undergoing UV irradiation-induced apoptosis. Cytochrome c staining (green) shows a punctate form in control cells. The staining was slightly decreased at 30 min after UV exposure and was lost in the later stages of the apoptotic process (90 and 120 min). Staining for active caspase-3 was rarely present in control cells or at 30 min but became apparent in the majority of cells by 60 min. Staining with Hoechst 33342 showed that the chromatin had a dispersed distribution in control cells and at 15 min, but by 90 min it had begun to condense on the nuclear margin and by 120-min fragmentation of chromatin was detected. Three separate experiments were undertaken.

 
The concentration of K decreased steadily by 30 min after the start of UV irradiation. The decrease was significant compared with control cells (412 vs. 531 mmol/kg); after this time point, [K] remained steady and then began to decrease again after 90 min, reaching a final concentration of 362 mmol/kg at the final time point of 120 min.

The release of cytochrome c from the mitochondria is considered to play a pivotal role in the apoptotic process. We therefore determined the time course of cytochrome c release to determine its relationship with the changes in elemental concentration. Cells were also stained with an anti-active caspase-3 and with Hoechst 33342 to enable the correlation between cytochrome c release, activation of caspase-3, and nuclear degradation. The time course for the release of cytochrome c from mitochondria of U937 cells after UV irradiation-induced apoptosis is shown in Fig. 3B. Control cells showed a rounded nucleus with a homogeneous dispersion of chromatin as shown by Hoechst 33342 staining. Staining for cytochrome c showed a punctate distribution throughout the cytoplasm due to its retention within the mitochondria. The same pattern of staining was observed 15 min after treatment with UV light, a time point at which significant changes in Cl had already occurred. At 30 min after UV irradiation, there was a decrease in the punctate green staining for cytochrome c in ~60% of the cells, but the nuclei remained rounded in form and showed a homogeneous distribution of chromatin. At the 60-min time point, the punctate green staining disappeared completely from the majority of cells, indicating that release of cytochrome c from the mitochondria was complete. In addition, at this time point, active caspase-3 was detected in the cytoplasm, and Hoechst 33342 staining showed the appearance of marginated chromatin staining in the majority of the nuclei. Fragmented chromatin was apparent in the nucleus after 120 min (Fig. 3B).

Nuclear changes in UV light-induced apoptosis in U937 cells. Element concentrations in the nucleus are shown in Fig. 4A. [Na] remained stable until 60 min after UV treatment; after this time, the concentration of this element began to rise from the basal level, from 99 to 288 mmol/kg. The concentration of this element was significantly higher than that in control cells at 90 and 120 min after irradiation. The concentrations of Mg and S did not change significantly throughout the apoptotic period (data not shown). [P] was reduced after the onset of apoptosis, but this reduction was slight and showed statistical significance only at the 30-min time point (P < 0.05), when it fell from 548 to 489 mmol/kg. [Cl] fell immediately; within 15 min, it had decreased from 173 to 99 mmol/kg, and it remained significantly lower than control values at 30 and 60 min after UV treatment (P < 0.001). After this time point, [Cl] increased again in parallel with the increase in [Na], and by 120 min after irradiation, the concentration (211 mmol/kg) was higher than control values.



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  		Fig. 4. Changes in the nuclei of U937 cells undergoing UV irradiation-induced apoptosis. A: concentrations (mmol/kg dry wt) of different elements in the nuclei of U937 cells at different time points. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control values. B: changes in nuclear morphology observed at the light microscopic with level using fluorescence after staining with Hoechst 33342. Control cells showed a homogeneous distribution of chromatin within the nucleus, whereas cells that had been incubated for 90 min showed condensation of chromatin at the nuclear borders. C: time course of changes in the number of cells showing changes in nuclear morphology. These results are the means of 5 different experiments. D: DNA fragmentation induced by UV irradiation as determined by agarose gel electrophoresis. DNA laddering began to become apparent at 60 min after exposure to UV light and was more prominent at 90 and 120 min after UV light exposure. E: expression of poly(ADP-ribose) polymerase (PARP) in U937 cells after UV irradiation. The percentage of PARP-positive cells increased between 30 and 60 min after UV irradiation. This sharp increase preceded that of cells showing chromatin condensation (compare with Fig 4C). The results are the means from 3 separate experiments.

 
[K] decreased steadily throughout the progression of apoptosis (590–422 mmol/kg), with a plateau in the concentration observed between 30 and 60 min after UV irradiation. The decrease in the concentration of this element became statistically significant (P < 0.001) at 30 min after UV irradiation. Analysis of the compartments of the nucleus, the condensed chromatin, and the noncondensed chromatin showed that these compartments had a composition similar to that of the nucleus (data not shown).

Apoptotic changes induced in the nucleus of U937 cells by UV irradiation are shown in Fig. 4, B and C. These results agree with our previous findings (10) and the results from electron microscopy studies. The evaluation of the nuclear morphology stained with Hoechst 33342 showed that control cells had a rounded nucleus with a homogeneous dispersion of chromatin. Clear changes in the morphology of the nucleus with the appearance of marginated condensed chromatin became apparent 60 min after UV irradiation (29%). The number of cells showing marginated chromatin continued to increase until 90 min after treatment (61%). After this time point, the number of cells with condensed chromatin decreased (53%), but there was a steady increase in the number of cells showing fragmented nuclei (38%). At this stage, 91% of cells were apoptotic.

The DNA ladder technique showed that typical internucleosomal DNA cleavage occurred during UV irradiation-induced apoptotic cell death. DNA ladders first became visible 60 min after treatment with UV light; the ladders were more prominent 90 min after treatment and were still present 120 min after UV irradiation (Fig. 4D).

We assayed the appearance of cleaved PARP in the nucleus as a consequence of active caspase-3 activity. While cleaved PARP was not detected in control cells (Fig. 4E), the number of nuclei expressing the cleaved form of this enzyme increased after UV irradiation. Low levels of cleaved PARP were detected between 15 and 30 min after UV treatment (2% and 8%, respectively; see Fig. 4E). There was a significant increase in the number of nuclei positive for cleaved PARP at 60 min after UV irradiation (65%), and this number increased at 90 min after that stained UV irradiation (90%) and was maintained (85%) at 120 min after UV treatment (Fig. 4E).

Our results of analysis of the nucleus thus show that the decreases in Cl and K precede the morphological changes, DNA fragmentation, and PARP cleavage and that these changes in the nuclear compartment occurred in the time period when [Cl] had begun to increase toward control levels and intranuclear [Na] was also rising.

Changes in the cytoplasm of U937 cells after UV light-induced apoptosis. The cytoplasm was the compartment least affected by the induction of apoptosis. The concentrations of the different elements in this compartment at the different time points after the induction of apoptosis are shown in Fig. 5A. There was an initial significant decrease (P < 0.05) in [Na] at 15 min after the induction of apoptosis from 162 to 119 mmol/kg, but after this time point the concentration of this element began to increase (119–398 mmol/kg), with statistical significance reached at 120 min (P < 0.001). Although there were variations in the concentrations of S and P during the course of apoptosis, with a decrease in [P] at 60 min after the onset of apoptosis (from 650 to 550 mmol/kg), these changes were not statistically significant. The concentration of Cl decreased within 15 min after the onset of apoptosis from 200 to 129 mmol/kg, and this decrease was statistically significant (P < 0.001). The low concentrations of Cl were maintained up to 60 min after UV irradiation, but at 90 min, [Cl] began to increase. By 120 min, [Cl] was slightly higher than the concentrations in control cells. [K] fell throughout the experimental period (from 651 to 483 mmol/kg); this fall became statistically significant after 90 min (P < 0.01) and reached its lowest concentration at 120 min (P < 0.05).



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  		Fig. 5. Changes in the cytoplasm of U937 cells undergoing UV irradiation-induced apoptosis. A: concentrations (mmol/kg dry wt) of different elements in the cytoplasm of U937 cells at different time points after the onset of apoptosis induced by UV irradiation. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control values. B: appearance of cells showing active caspase-3 after the onset of UV light-induced apoptosis. The results are the means from 3 separate experiments.

 
A major hallmark in apoptosis is the appearance of active caspase-3 in the cytoplasm. We therefore assayed the appearance of this enzyme. While active caspase-3 was not detected in control cells, the number of cells expressing the active form of this enzyme increased after UV irradiation. Low levels of active caspase-3 were detected between 15 and 30 min after UV treatment (5 and 20%, respectively). There was a significant increase in the number of cells that stained positive for active caspase-3 at 60 min after UV irradiation (73%), and this number increased at 90 min after treatment with UV (99%) and was maintained (96%) at 120 min after UV treatment (Figs. 5B and 3B). Thus our results show that the appearance of active caspase-3 is preceded by a significant decrease in cytoplasmic [Cl] but not in [K].


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have previously used UV treatment to study element changes in whole U937 cells during the later stages of apoptosis (10). The advantage of this model is that apopotosis is induced rapidly in almost the entire cell population, producing a relatively synchronous population of cells for study. Herein we have used EPXMA for the analysis of cryosectioned U937 cells, which allowed simultaneous determination of the elements of interest, sodium, magnesium, phosphorus, chloride, and potassium, and also allowed us to determine how any changes affected different subcellular compartments. In addition, we have determined cytochrome c release, PARP cleavage, degradation of DNA, and activation of caspase-3, so that the results presented herein enable direct correlation between changes in cellular elemental content and well-recognized markers of apoptotic progress.

Global elemental concentrations where the analyzed area includes all cell compartments. The results from the analyses that included all cell compartments are considered first because these exemplify occurrences in the other individual cellular compartments, and can be related most closely to work in which ionic concentrations have been studied in whole cells. The major finding of this study is that decreases in the concentrations of both K and Cl occurred very early in the apoptotic process and that the pattern of the response of these two elements differed. [K] continued to decrease throughout apoptosis, whereas [Cl] increased in the later stages, with this rise occurring after many of the manifestations of the later stages of apoptosis became apparent. Our results are in agreement with other studies that have used EPXMA for the determination of element changes in apoptosis. Decreases in [Cl] of 27% and [K] of 50% have been recorded in whole U937 cells after the induction of apoptosis in U937 cells by UV irradiation or staurosporine (3, 10), but these results were recorded for single time points later in the apoptotic process, when indicators of the execution stage such as condensation of chromatin were already apparent. Our results show that the decreases in [Cl] and [K] occur early in the apoptotic process and precede all of the other manifestations that we studied. Similar findings have been reported for monocytes/macrophages exposed to LDL, where decreases in both [Cl] and [K] were shown to precede DNA degradation estimated by TdT-mediated dUTP nick-end labeling (35).

The results from EPXMA are also in line with those from other techniques that have been used to study ionic activity after the onset of apoptosis. Activation of K+ channels and of Cl– channels has been reported after apoptotic stimulation in several different cell types (16, 22, 25, 29, 31, 37, 41, 47). Our results provide evidence that such activation leads to loss of both intracellular potassium and chloride.

Apoptotic cells decrease in volume. The appearance of the shrunken pyknotic cells was one of the characteristics that led to the first descriptions of apoptosis; however, initially, it was considered that this shrinkage was associated with the later stages. Herein we have shown that in U937 cells, the concentrations of both potassium and chloride decreased immediately after UV irradiation, with the loss of these two elements on an equimalar basis being more or less equivalent. Because the initial intracellular [Cl] is low compared with that of potassium, when the results are expressed on a percentage basis, these changes result in a much higher decrease of Cl compared with that of potassium (a loss of 40% Cl compared with 16% for K after 30 min; see Table 1). This pattern of loss of Cl is characteristic of cells undergoing isotonic cell shrinkage (27). Maeno et al. (19) showed that in several different cell types, induction of apoptotic cell death was coupled with facilitation of regulated volume decrease and that volume decrease was an early event in the apoptotic process. The results presented herein support these ideas and also provide direct evidence, without the need to provoke a hypotonic challenge, that an apoptotic stimulus, UV irradiation, induced immediate isotonic shrinkage in U937 cells.


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  		Table 1. Changes in the concentrations of Cl and K after UV irradiation of U937 cells expressed as %decrease from control value

 
Mitochondria. Several ion transporters have been identified in both inner and outer mitochondrial membranes, but little is known about their roles in either intact cells or the apoptotic process. Our results from the analyses that included all cell compartments indicate that cell shrinkage is an immediate effect of UV irradiation-induced apoptosis in U937 cells. We also show an immediate and rapid decrease in Cl in the mitochondria, a decrease of 52% after 15 min compared with the control cells, that is much greater than that which occurs in the other cellular compartments. It is possible that the observed decrease represents the result of UV light-induced activation of ion transporters similar to those activated in the plasma membrane with resulting effects on mitochondrial volume. It has been suggested that efflux of Cl through the inner membrane anion channel acts as a safeguard against excessive mitochondrial swelling (28) and is active under alkaline conditions in the mitochondrial matrix. The decrease in Cl that we describe herein could reflect an attempt to stabilize mitochodrial volume and is compatible with the idea that during apoptosis, loss of volume control by the mitochondria leads to cytochrome c release.

The situation regarding mitochondrial K is not quite so straightforward. K+ channels have been identified on the inner mitochondrial membrane, and opening of these channels would be expected to lead to influx of K+ and swelling of the mitochondria (12). Our results instead show a steady decrease in [K]. Under control conditions, mitochondrial [K] is known to be high (4, 12), so that net K+ transport has little overall effect on the matrix concentration but a greater effect on matrix volume (12). It is therefore perhaps not surprising that an increase in mitochondrial [K] was not detected. In addition, the first time point that we investigated was 15 min after exposure to UV light. Significant disruption of mitochondrial structure and a decrease in mitochondrial membrane potential were shown to occur within 30 s in U937 cells after treatment with dolichyl monophosphate to induce apoptosis (51). It may therefore be necessary to investigate earlier time points for an increase in mitochondral [K] to be detected.

Our results show that cytochrome c loss occurs very rapidly in most of the cells and that this occurs ~30 min after UV irradiation. Before this time point, the pattern of change in the elements Cl and K begins to differ. At 30 min, the [Cl] was greater than it was at the 15-min time point, and the increase in the concentration of this element continued throughout the remainder of the experiment, whereas [K] continued to decrease, although it remained steady between the 30- and 60-min time points. The increase in mitochondrial [Cl] was accompanied by a gradual increase in [Na] in the later stages of apoptosis after release of cytochrome c occurred. These values probably represent equilibration of mitochondrial ion concentrations with the surrounding cytoplasm.

In the mitochondria, there was a loss of P that became significant at 30 min after UV irradiation, after cytochrome c release. This probably reflects a loss of high-energy phosphate from this compartment.

Nucleus. The results presented herein regarding the nuclear compartment show that the changes in intranuclear ion concentrations occur much earlier than the overt manifestations of nuclear damage, PARP cleavage, DNA laddering, and chromatin condensation. The main changes are the decrease in chloride concentration that occurs within 15 min of UV irradiation and the steady loss of potassium throughout the period of apoptosis. A decrease in intracellular [Cl] has been implicated as a cofactor in the activation of DNA fragmentation factor endonuclease that is responsible for internucleosomal fragmentation of DNA (30), and these authors suggested that the decrease in Cl due to shrinkage late in apoptosis favored endonuclease activation. In contrast, we have shown here that the decrease in [Cl] is an early event, with [Cl] returning toward control values in the later stages. Nevertheless, [Cl] remains below the value in control cells in the period between 15 and 60 min after UV irradiation, the period during which PARP cleavage occurs, and thus could provide an intranuclear environment favorable for chromatin degradation.

It has been suggested that high intranuclear [K] is essential for high-order structure in mammalian chromosomes (36). Herein we show that there is an initial decrease in intranuclear [K] but that the concentration of this ion remains level throughout the period between 30 and 60 min after UV irradiation, when there is a major increase in the number of cells positive for PARP and when the DNA becomes more fragmented as demonstrated by the appearance of laddered DNA. [K] falls again in the later stages at 90 and 120 min after UV irradiation suggesting that DNA is released as chromatin degrades.

In the nucleus, there was a slight decrease in [P] that was significant at the P < 0.05 level 30 min after UV irradiation. In the nucleus, the detected P was most likely a constituent of DNA. Our results suggest that there is no great loss of nuclear material during the course of chromatin condensation.

Cytoplasm. In the cytoplasm, the concentrations of [Cl] and [K] both decrease after UV irradiation. The major decrease in Cl occurs in the first 15 min after the apoptotic stimulus. As in other compartments, after 30 min, [Cl] begins to increase compared with the previous time point while remaining significantly lower than that of control cells, whereas [K] continues to decrease, and, unlike the mitochondria and nucleus, this decrease does not plateau at the 60-min time point but continues at a steady rate. The initial decline in both of these elements is probably a result of the opening of K+ and Cl– channels in response to UV irradiation as previously reported (41). [Na] also begins to increase at 30 min after UV treatment. The changing pattern at 30 min indicates that the mechanisms for transport of these ions changes some time around this time point. The cells were subject to an intense dose of UV irradiation in an attempt to induce apoptosis in a synchronous manner, and it is possible that the increase in [Cl] and parallel increase in [Na] represent the occurrence of necrosis as a late stage of apoptosis in cells in culture. However, we think that this explanation is not likely. Unfixed portions of the cells used for experimentation were routinely treated with Trypan blue to assess viability. The results obtained were similar to those published previously (10). The cells excluded the dye, indicating a lack of permeablization of the plasma membrane as would occur in necrosis. In addition, electron-lucent cells showing necrosis were not analyzed. A similar pattern of change of these elements has been reported in monocyte macrophages after apoptosis induced by oxidized LDL, but the possible mechanisms underlying the changes were not discussed (35). Several studies (20, 23, 50) have shown that ouabain might induce apoptosis in different cell types. In addition, an increase in [Na] in U937 cells 5 h after staurosporine-induced apoptosis was shown to be associated with decreased Na+-K+-ATPase activity (2). A similar mechanism operating after UV light-induced apoptosis would account for the results presented herein.

Studies using cell fractions have shown that high [K] acted to suppress activation of caspases and DNA degradation (14), suggesting that in intact cells, K loss should occur before these events are detected. Here we show that, although the decrease in K in the nucleus precedes the appearance of DNA degradation, the loss of K from the cytoplasm does not reach significant levels until after activation of caspase-3, suggesting that a loss of cytoplasmic K does not control caspase activation in our experimental system. Our results, however, are not in complete disagreement with the idea that a decrease in cytoplasmic [K] is necessary for caspase activation, because in later studies (40), caspase inhibitors were found to be less effective in preventing cell shrinkage and K loss after UV irradiation-mediated induction of apoptosis in Jurkat T lymphocytes compared with Fas receptor-induced apoptosis.

It is perhaps not surprising that in the cytoplasm, the lowered [K] reaches signifcance only at later stages. The significant decreases in the concentration of this element occur earliest in the mitochondria and nucleus, and K that is lost from these compartments must pass through the cytoplasm to exit the cell. In studies of dexamethasone-treated lymphocytes (8), it was shown that the breakdown of mitochondrial membrane potential occurs before loss of the plasma membrane potential. Although the reason for this was not clear, the authors suggested that subtle changes in K+ fluxes might be involved in the induction phase of apoptosis, with major losses of this ion occurring later in the degradation phase. Our results confirm such a pattern of K+ loss. We found that decreases in [K] occur earliest in the mitochondria, but because the mitochondria represent only a small fraction of the cell volume, such changes would almost certainly not be detected when potassium concentration is measured in whole cells.

In summary, our data show that in U937 cells undergoing UV light-induced apoptosis, decreases in intracellular concentrations of Cl and K precede other manifestations of apoptosis and the pattern of electrolyte loss changes during the course of apoptosis. We also have shown that the pattern of electrolyte loss affects intracellular compartments differently. A significant decrease in [Cl] occurs in all compartments. In the mitochondria, this precedes cytochrome c release. In the nucleus, significant decreases in the concentrations of both Cl and K occurred before nuclear fragmentation is detected. The cytoplasm is the compartment that is least affected by loss of potassium. Although there is a steady decrease in the concentration of this element, this decrease does not become significant until late in the execution phase.


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This work was supported by The Wellcome Trust Grant Ref. 0578811 and Spanish Ministry of Health Grant FIS PI02622. F. Arrebola was supported by grants from University of Granada and Spanish Ministry of Education and Science (PF 00 33380884).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Warley, Centre for Ultrastructural Imaging, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK (e-mail: alice.warley{at}kcl.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 EPXMA measures element concentrations and cannot detect oxidation states; for this reason, the oxidation state is not given when referring to microanalytical results. Back


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