Caspase Independent/Dependent Regulation of K1, CellShrinkage, and Mitochondrial Membrane Potential duringLymphocyte Apoptosis*

(Received for publication, March 10, 1999, and in revised form, May 1, 1999)

Carl D. Bortner and John A. Cidlowski‡

From the Laboratory of Signal Transduction, National Institute of Environmental Health Sciences,National Institutes of Health, Research Triangle Park, North Carolina 27709

The loss of cell volume is a fundamental feature ofapoptosis. We have previously shown that DNA degra-dation and caspase activity occur only in cells whichhave shrunken as a result of potassium and sodium ef-flux (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski,J. A. (1997) J. Biol. Chem. 272, 32436–32442). Further-more, maintaining a normal intracellular potassiumconcentration represses the cell death process by inhib-iting the activity of apoptotic nucleases and suppressingthe activation of effector caspases (Hughes, F. M., Jr.,Bortner, C. D. Purdy, G. D., and Cidlowski, J. A. (1997)J. Biol. Chem. 272, 30567–30576). We have now investi-gated the relationship between cell shrinkage, ion ef-flux, and changes in the mitochondrial membrane po-tential, in addition to the role of caspases in theseapoptotic events. Treatment of Jurkat cells with a seriesof inducers which act via distinct signal transductionpathways, resulted in all of the cell death characteris-tics including loss of cell viability, cell shrinkage, K1

efflux, altered mitochondrial membrane potential, andDNA fragmentation. Interestingly, only cells whichshrunk had a loss of mitochondrial membrane potentialand the other apoptotic characteristics. Treatment ofJurkat cells with an anti-Fas antibody in the presence ofthe general caspase inhibitor z-VAD, abrogated thesefeatures. In contrast, when Jurkat cells were treatedwith either the calcium ionophore A23187 or thapsigar-gin, z-VAD failed to prevent cell shrinkage, K1 efflux, orchanges in the mitochondrial membrane potential,while effectively inhibiting DNA degradation. Treat-ment of Jurkat cells with various apoptotic agents in thepresence of either the caspase-3 inhibitor DEVD, or thecaspase-8 inhibitor IETD also blocked DNA degradation,but failed to prevent other characteristics of apoptosis.Together these data suggest that the cell shrinkage, K1

efflux, and changes in the mitochondrial membrane po-tential are tightly coupled, but occur independent ofDNA degradation, and can be largely caspase independ-ent depending on the particular signal transductionpathway.

Apoptosis is characterized by a distinct set of morphologicaland biochemical features which differ substantially from thoseobserved during necrosis (1–3). Characteristics of apoptosisinclude cell shrinkage, protein degradation, nuclear condensa-tion and fragmentation, and eventual budding of the cells into

what have been termed apoptotic bodies (2, 3). In apoptosis,diverse external or internal signals trigger the cell death proc-ess, which is then executed by the activation of a central orcommon death pathway. Upon receipt of an apoptotic signal,cells activate numerous signaling pathways whose function isto modulate the cell death process. Modulators of cell deathinclude proteins of the Bcl-2 family, p53, and various kinasesand phosphatases, which condemn the cell to death throughtheir action on intracellular effector molecules (4). Notableeffector molecules include cytochrome c, proteases, and nucle-ases which ultimately cleave protein and DNA substrates, re-spectively. The pathway that leads to cell shrinkage, alter-ations in mitochondrial membrane potential, and DNAdegradation are not yet completely understood.

Recently, the study of mitochondria and changes in the mi-tochondrial membrane potential have become a focus of apo-ptosis regulation. A loss of mitochondrial membrane potentialhas been shown to occur in a variety of apoptotic model systems(5–7). The opening of mitochondrial permeability transition(MPT)1 pores, located on the inner mitochondrial membrane, isthought to underlie the loss of mitochondrial membrane poten-tial (8, 9). Activation of these MPT pores permits the redistri-bution of molecules across the inner mitochondrial membrane,thus disrupting the membrane potential of this organelle (10–12). Induction of the MPT has been suggested to result in therelease of several apoptotic factors, including apoptotic pro-teases (apoptotic-inducing factor; Ref. 13) and cytochrome c(14). Other experimental observations suggest that disruptionof electron transport, oxidative phosphorylation, and adenosinetriphosphate (ATP), as well as the generation of reactive oxy-gen species are thought to play an important roles in apoptosis(15–17). Currently, however, the exact role that the MPT playsduring apoptosis is not apparent and its relationship to otherapoptotic events has not been clearly defined.

Along with changes in the mitochondrial membrane poten-tial, expression of a number of genes has also been reported tobe pivotal in apoptosis. Studies in Caenorhabditis elegans haveshown that the cysteine protease Ced-3 is essential for pro-grammed cell death (18). Sequence similarity between Ced-3and the human interleukin-1b-converting enzyme (19) led tothe identification of a family of interleukin-1b-converting en-zyme-related cysteine proteases that become activated duringapoptosis. These proteases, collectively known as caspases,have been shown to play a critical role in apoptosis (20).Caspases are synthesized as inactive proenzymes, which areactivated following cleavage at specific aspartate residues, to

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

‡ To whom correspondence should be addressed. Tel.: 919-541-1564;Fax: 919-541-1367.

1 The abbreviations used are: MPT, mitochondrial permability tran-sition; PBFI, potassium-binding benzofuran isophthalate; PI, pro-pidium iodide; MMP, mitochondrial membrane potential; PMP, plasmamembrane potential.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 31, Issue of July 30, pp. 21953–21962, 1999Printed in U.S.A.

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become mature active enzymes. Interestingly, many of thepro-caspase cleavage sites are also caspase recognition sites(21), raising the possibility that some caspases sequentiallyactivate others, thus establishing a hierarchy of caspase acti-vation (22–24). This cascade-like activation of activator andeffector caspases permits the amplification of the proteolyticcleavage process during apoptosis only if the normal intracel-lular K1 levels are reduced (25, 26). Currently the precisemechanism by which these proteases contribute to the classicalapoptotic characteristics are still being elucidated.

A fundamental characteristic of apoptosis that occurs in allmodel systems of programmed cell death is the loss of cellvolume. We have previously shown that the loss of cell volumecorrelates with the population of cells which have degradedDNA (25, 27). Additionally, we have determined that cellshrinkage is associated with a dramatic decrease in intracellu-lar ion concentration, particularly potassium, and that theionic environment within cells can affect both the activationand activity of apoptotic enzymes (25, 26). We have now eval-uated the relationship between cell shrinkage and loss of themitochondrial membrane potential during apoptosis and exam-ined the role of caspases in the regulation of cell volume loss,the ion efflux, and alterations in the mitochondrial membranepotential. We show that when the death response is activatedthrough different apoptotic signaling pathways, cell shrinkage,K1 efflux, and the loss of mitochondrial membrane potentialare tightly linked, but all three can occur independently of bothcaspase activation and DNA degradation.

MATERIALS AND METHODS

Cell Culture and Reagents—Jurkat cells (human lymphoma) werecultured in RPMI 1640 media containing 10% heat-inactivated fetal calfserum, 4 mM glutamine, 31 mg/liter penicillin, and 50 mg/liter strepto-mycin at 37 °C, 7% CO2 atmosphere. Jurkat cells (4 3 105 cells/ml) wereinitially treated with 10 ng/ml anti-human Fas IgM (Kamiya Biomed-ical) for 4, 8, and 12 h. In latter studies, Jurkat cells were treated witheither 10 ng/ml anti-human Fas IgM, 1 mM A23187 (Calbiochem), or 7.5mM thapsigargin (Sigma) for 24 h to achieve a similar amount of loss ofmembrane integrity, in the presence or absence of 25 mM of the variouscaspase inhibitors (z-VAD-fmk, DEVD-fmk, and IETD-fmk; KamiyaBiomedical) unless indicated otherwise. Cell viability was determinedby trypan blue exclusion.

Determination of Cell Size by Flow Cytometry—Cell size and changesin the light scattering properties of the cells were determined by flowcytometry as described previously using a Becton Dickinson FACSortequipped with CellQuest software (27). For each sample, 10,000 cellswere examined by exciting the cells with a 488 nm argon laser anddetermining their position on a forward scatter versus side scatter dotplot. Light scattered in the forward direction is roughly proportional tocell size, while light scattered at a 90o angle (side scatter) is propor-tional to cell density (28). Therefore, as a cell shrinks or loses cellvolume, a decrease in the amount of forward scattered light is observed,along with a slight change in side scattered light. A gate based on theproperties of the control cells was set on each forward scatter versusside scatter dot plot to separate the normal and apoptotic populations ofcells, and remained constant throughout the analysis. The percent ofapoptotic cells was determined by statistical analysis of the dot plotsusing CellQuest software. The data was converted to three-dimensionalplots for presentation.

Determination of Intracellular Potassium by Flow Cytometry—Intra-cellular potassium concentrations were determined as described previ-ously using a Becton Dickinson FACSVantage (25). Briefly, Jurkat cellstreated in the presence or absence of various apoptotic agents andcaspase inhibitors were loaded with the potassium-sensitive fluorescentdye potassium-binding benzofuran isophthalate (PBFI-AM; MolecularProbes) to a final dye concentration of 5 mM for 1 h at 37 °C, 7% CO2

atmosphere prior to examination. Immediately prior to flow cytometricanalysis, propidium iodide (PI, Sigma) was added to each sample to afinal concentration of 10 mg/ml. Ten thousand cells were analyzed bysequential excitation of the cells containing PBFI-AM and PI at 340–350 and 488 nm, respectively. Gates were set on a PBFI (K1) versus PIdot plot to individually examine cells which had a reduced potassiumconcentration. Cells which were PI positive, indicating a loss of mem-

brane integrity, were excluded from ion analysis. The data were con-verted to three-dimensional plots using CellQuest software forpresentation.

Measurement of Mitochondrial Membrane Potential—Changes in themitochondrial membrane potential were measured by flow cytometryusing JC-1 (Molecular Probes). Thirty minutes prior to cytometric anal-ysis, JC-1 was added to 1-ml of cells to a final concentration of 10 mM

and incubated at 37 °C, 7% CO2 atmosphere. At the designated time,10,000 cells were examined for each sample on a FL-1 (530 nm) versusFL-2 (585 nm) dot plot on a Becton Dickinson FACSort. JC-1 has dualemission depending on the state of the mitochondrial membrane poten-tial. JC-1 forms aggregates in cells with a high FL-2 fluorescenceindicating a normal mitochondrial membrane potential. Loss of themitochondrial membrane potential results in a reduction in FL-2 fluo-rescence with a concurrent gain in FL-1 fluorescence as the dye shiftsfrom an aggregate to monomeric state. Therefore, retention of the dye inthe cell can be monitored through the increase in FL-1 fluorescence. Thedata were converted to density plots using CellQuest software forpresentation.

DNA Analysis by Flow Cytometry—The DNA content for each samplewas determined as described previously (27). Briefly, 5 ml of cells werepelleted from the culture medium and fixed by the slow addition of cold70% ethanol to a volume of approximately 1.5-ml. The volume of eachsample was adjusted to 5-ml with cold 70% ethanol, and the cells werestored at 4 °C overnight. For flow analysis, the fixed cells were pelleted,washed once in 1 3 phosphate-buffered saline and stained in 1 ml of 20mg/ml of PI, 1 mg/ml RNase in 1 3 phosphate-buffered saline for 20 min.Seven thousand five hundred cells were examined by flow cytometry bygating on an area versus width dot plot to exclude cell debris and cellaggregates. The percent of degraded DNA was determined by the num-ber of cells with subdiploid DNA divided by the total number of cellsexamined under each experimental condition.

RESULTS

The Loss of Intracellular Potassium Is Associated with theShrunken Population of Apoptotic Cells—Previous studies fromour laboratory have shown that DNA degradation and loss ofintracellular potassium (K1) occurs only in the shrunken ap-optotic cells (25, 27). We have now kinetically analyzed therelationship between K1 efflux and the loss of cell volumeduring apoptosis in Jurkat cells treated with anti-Fas. Asshown in Fig. 1A, forward scatter versus side scatter three-dimensional plots showed an increase in the number of cellswhich lose cell volume, which occurred in a time-dependentmanner. Cells which have a decrease in cell volume have areduced ability to scatter light in the forward direction. Fur-thermore, these apoptotic cells also showed a slight increase intheir ability to scatter light at a 90° angle, indicating a concur-rent increase in cellular density. We next analyzed these cellsfor the loss of intracellular K1 at the single cell level by flowcytometry utilizing the fluorescent potassium indicator dyePBFI-AM and propidium iodide to eliminate cells which losstheir membrane integrity. The loss of intracellular potassium,as determined by a decrease in PBFI (K1) fluorescence in theviable shrunken population of anti-Fas-treated Jurkat cells,was also observed in a time-dependent manner with similarkinetics (Fig. 1A). To explore the relationship between cellshrinkage and the decrease in K1 concentration, we evaluatedthe K1 concentration in the population of shrunken and non-shrunken cells obtained from the kinetic study shown in Fig.1A. Fig. 1B shows that cells which fail to shrink show noreduction in intracellular K1 concentration. In contrast, onlyshrunken cells had a decrease in intracellular K1 concentra-tion, indicating that decreased K1 concentration and cellshrinkage are tightly coupled events in apoptosis.

The Loss of Mitochondrial Membrane Potential Is Associatedwith the Shrunken Population of Apoptotic Cells—Since theloss of cell volume is a fundamental characteristic of apoptosis,we next determined if other features of apoptosis were re-stricted to the shrunken population of cells. Recently, severallaboratories have suggested that nuclear features of apoptosis

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are preceded by alterations in mitochondrial structure andtransmembrane potential (5, 8, 29–34). The fluorescent probeJC-1 has been shown to be most specific for measuring changesin the mitochondrial membrane potential (MMP) (35), and wasused for our analysis of the MMP. JC-1 forms either J-aggre-gates or monomers, depending on the state of the mitochondrialmembrane potential, with the emissions of the two dye formsdetectable by flow cytometry at 585 or 530 nm, respectively(35). The high mitochondrial membrane potential of normalcells loaded with JC-1 allows for the formation of J-aggregates,detected by a peak in fluorescence at 585 nm. As the mitochon-drial membrane potential is loss, these aggregates dissipateinto monomers, which are detected as a shift in fluorescencefrom 585 to 530 nm. We initially examined JC-1 along withDiOC6 (Molecular Probes) for their ability to detect acutechanges in both the MMP and plasma membrane potential(PMP). As shown in Fig. 2, JC-1 responds only to an acuteuncoupling of the MMP using CCCP (Sigma). In contrast,DiOC6 responds to both acute changes in the MMP and PMP.These data suggest that JC-1 measures only changes in the

MMP whereas DiOC6 measures both MMP and PMP. Thisobservation cautions against the use of DiOC6 to specificallyexamine mitochondrial events.

Fig. 3A shows the time-dependent loss of the MMP in anti-Fas-treated Jurkat cells, as the population of cells shifts to alower JC-1 aggregate state and a concurrent higher JC-1 mon-omer state. To determine the relationship between the loss ofcell volume and changes in the MMP, we compared the re-sponse of the mitochondrial dye JC-1 to changes in cell sizewhich occur during apoptosis by flow cytometry. Examinationof each individual JC-1 state (J-aggregate and monomeric)versus cell size in control and anti-Fas-treated Jurkat cellsshowed that only the shrunken population of cells had a de-crease in JC-1 aggregates and an increase in JC-1 monomers(Fig. 3A). The increase in JC-1 monomeric fluorescence withthis dye suggests that the loss of JC-1 aggregate fluorescence isnot due to an overall loss of this dye from the cell. Similarresults were observed in A23187 and thapsigargin-treated Ju-rkat cells (Fig. 3B). Therefore, the loss of cell volume and MMPin apoptotic cells appears to be tightly coupled and is independ-

FIG. 1. The occurrence of cell volume loss, and K1 efflux in anti-Fas-treated Jurkat cells. Jurkat cells were treated with 10 ng/mlanti-Fas antibody for 4, 8, and 12 h. A, flow cytometry was used to assess the light scattering properties of the cells and the loss of intracellularK1. Ten thousand cells were examined on a FACSort flow cytometer for the ability of cells to scatter light in the forward direction (forward scatter),which indicates cell size, and at a 90° angle (side scatter), which indicates cell density. Cells which have a decrease in forward scatter light havea decrease in cell size. Cells which have an increase in side scatter light have an increase in cell density. For intracellular K1 analysis, 2 ml of a2.5 mM PBFI-AM stock (5 mM final) was added to 1 ml of cells for each sample 1 h prior to the time of examination. Incubation was then continuedat 37 °C, 7% CO2. Immediately prior to cytometric examination, PI was added to a final concentration of 10 mg/ml. Samples were analyzed on aFACSVantage flow cytometer examining 10,000 cells per sample on a PBFI (K1) versus PI fluorescent dot plot to eliminate the PI positive (dead)cells. Viable cells were then examined on a forward scatter versus PBFI (K1) three-dimensional plot. B, each viable population of cells (normal andshrunken) were analyzed by flow cytometry on a forward scatter versus PBFI (K1) fluorescence contour plot. Only the apoptotic or shrunkenpopulation of cells had a decrease in potassium concentration. All data shown represents one of at least three independent experiments.

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ent of the mechanism used to signal cell death.Diverse Caspase Inhibitors Protect against DNA Degradation

in Jurkat Cells Treated with Numerous Apoptotic Agents—Wenext examined the role that caspase activity plays in the gen-eration of several apoptotic features by using various caspaseinhibitors which are known to block both activator and effectorcaspases. We examined cells after 24 h of apoptotic treatmentto ensure complete activation of the cell death program. SinceDNA degradation has been a persistent characteristic of apop-tosis and is thought to occur downstream of caspase activationduring cell death, we utilized this apoptotic end point to eval-uate the effectiveness of our caspase inhibitors. Jurkat cellstreated with an anti-Fas antibody, A23187, or thapsigarginshowed all of the classical characteristics of apoptosis, includ-ing DNA degradation, as determined by an increase in thenumber of cells with a subdiploid peak of DNA by flow cytom-etry (Fig. 4). z-VAD, a cell-permeable, broad spectrum inhibitorof caspase activity, has previously been shown to prevent ap-optotic death in a number of apoptotic model systems by inhib-iting both early and late proteases (reviewed in Ref. 8). WhenJurkat cells were treated with various apoptotic agents for24 h, the presence of 25 mM z-VAD completely inhibited DNAdegradation (Fig. 4, top). Similar results were observed wheneither 25 mM DEVD (a caspase-3 inhibitor) or IETD (acaspase-8 inhibitor) were used (Fig. 4, middle and bottom),indicating that the concentration of caspase inhibitors used inthese experiments were effective in preventing a late apoptoticevent.

Loss of Cell Viability Occurs in the Presence of CaspaseInhibition in a Signal-specific Manner—Having determinedthe efficacy of the caspase inhibitors in this model system, wenext examined the effects of these various caspase inhibitors ontheir ability to prevent the loss of cell viability. In the absence

of caspase inhibition, a significant increase in trypan bluepositive cells was observed following a 24-h treatment of Jurkatcells with anti-Fas, A23187, or thapsigargin (Fig. 5). z-VADwas effective in preventing the loss of cell viability in theanti-Fas-treated cells (Fig. 5, top). However, z-VAD was onlymarginally effective in preventing the loss of cell viability incells treated with A23187, and was decidedly ineffective in cellstreated with thapsigargin (Fig. 5, top). Interestingly, the loss ofcell viability occurred in the absence of DNA degradation (seeFig. 4), suggesting that membrane permeability changes, asassessed by trypan blue exclusion, can occur independent ofDNA degradation and in a caspase-independent manner. Sim-ilarly, DEVD and IETD significantly blocked the loss of cellviability in the anti-Fas-treated cells. However, these inhibi-tors were ineffective where apoptosis was induced using anon-Fas signaling pathway (Fig. 5, middle and bottom). Thusalthough all caspase inhibition was effective in preventingDNA degradation under each apoptotic condition, the caspaseinhibitors employed only blocked the loss of cell viability in-duced via Fas-receptor signaling. Higher concentration of thecaspase inhibitors (100 mM) were similarly ineffective in pre-venting the loss of cell viability except when anti-Fas antibod-ies were the death signal (data not shown). These data showthat the characteristics of apoptosis can be separated fromcaspases activity and cell death appears to be linked to activa-tion of the death signaling process.

Cell Shrinkage Occurs in a Signal-specific Manner in thePresence of Various Caspase Inhibitors—We have previouslyshown that only the shrunken population of apoptotic cellscontain degraded DNA (25, 27). Since caspase inhibition pre-vented DNA degradation in response to numerous apoptoticstimuli, we wished to determine if cell shrinkage was caspasedependent. We thus examined the loss of cell volume in Jurkat

FIG. 2. JC-1 responds only tochanges in the mitochondrial mem-brane potential. Jurkat cells were ex-posed to RPMI 1640 media containingvarious concentrations of KCl or carbonylcyanide p-chlorophenylhydrazone (CCCP)for 10 min in the presence of 10 mM JC-1or 150 nM DiOC6, were analyzed forchanges in their fluorescent profile byflow cytometry. Both JC-1 and DiOC6 re-sponded to acute uncoupling of the MMPusing CCCP, however, only DiOC6 re-sponded to acute changes in the PMPwith increasing concentrations of KCl.Histograms represent one of two inde-pendent experiments.

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cells treated with various apoptotic agents in the presence orabsence of caspase inhibitors. Jurkat cells activated to undergoapoptosis with anti-Fas, A23187, or thapsigargin in the ab-sence of caspase inhibition showed a significant reduction incell size (Fig. 6, top row). In the presence of 25 mM z-VAD,anti-Fas-treated Jurkat cells did not show a change in cell size,

suggesting the dependence of caspase activity for cell shrink-age under this apoptotic condition, consistent with the data oncell viability. In contrast, z-VAD only marginally prevented thealterations in cell size in Jurkat cells treated with eitherA23187 or thapsigargin (Fig. 6, second row). Jurkat cellstreated with anti-Fas in the presence of either 25 mM DEVD or

FIG. 3. Changes in the mitochon-drial membrane potential occur onlyin the shrunken population of cells.Jurkat cells treated with: A, an anti-Fasantibody as described in the legend to Fig.1, or B, 2 mM A23187 or 10 mM thapsigar-gin (Thaps) for 10 h, were examined forchanges in their mitochondrial membranepotential using JC-1. Ten thousand cellswere examined per sample on a JC-1 ag-gregate versus JC-1 monomer contourplot using a FACSort flow cytometer. JC-1forms aggregates in cells with a high mito-chondrial membrane potential, which is de-tected at 585 nm. The formation of mono-mers, indicative of decreased mitochondrialmembrane potential, is detected as a loss ofaggregate fluorescence, and an increase inJC-1 monomers detected at 530 nm. Eachindividual mitochondrial potential state wasalso examined versus forward scattered light(cell size). All data shown represents one ofat least three independent experiments.

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IETD also had a significant effect on preventing the changes incell size, such that cell shrinkage was reduced by 50 and 70%,respectively (Fig. 6, second column). Both DEVD and IETD atconcentrations which effectively inhibited DNA degradationwere completely ineffective in preventing cell shrinkage wheneither A23187 or thapsigargin was used to induce apoptosis(Fig. 6, third and fourth columns). These results suggest cellshrinkage can occur independently of caspase activation andactivity except when anti-Fas is used to activate apoptosis.

K1 Efflux Can Occur Independently of Caspase Activity—Theloss of intracellular ions, particularly potassium, has been pro-posed to be responsible for cell shrinkage during apoptosis (26,27, 36–39). Additionally, the inhibition of intracellular K1 losswas shown to prevent the loss of cell volume in several modelsystems, and in response to numerous apoptotic stimuli (26, 27,36–39). Therefore, we wanted to determine if the viable,shrunken population of cells observed in the presence ofcaspase inhibition also had a decrease in intracellular potas-sium. As shown in Fig. 7 (top row), when Jurkat cells weretreated with anti-Fas, A23187, or thapsigargin, only the viable,shrunken population of cells had a decrease in intracellularpotassium. Similar to the results observed for cell size, thepresence of 25 mM z-VAD completely prevented the loss of

intracellular potassium in anti-Fas-treated Jurkat cells, alongwith the loss of cell size, but was only marginally effective wheneither A23187 or thapsigargin was used as the apoptotic stim-ulus (Fig. 7, second row). Both DEVD and IETD providedsignificant protection from potassium loss upon anti-Fas treat-ment, but failed to inhibit the loss of potassium in A23187- orthapsigargin-treated cells (Fig. 7, third and fourth rows). Thuscaspase inhibition prevents the loss of ions and cell shrinkagewhen the Fas receptor pathway is used to stimulate apoptosis,but not when apoptosis is activated by alternative signalingpathways.

Changes in the Mitochondrial Membrane Potential Accom-pany the Loss of Cell Viability and Cell Size—To determine theeffect of caspase inhibition on other characteristics of apoptosisin response to various apoptotic stimuli, changes in the MMPwere examined using the mitochondrial specific dye JC-1.Treatment of Jurkat cells with anti-Fas, A23187, or thapsigar-gin in the absence of caspase inhibitors resulted in an increasein the percent of cells which showed a loss of MMP (Table I).The presence of z-VAD prevented only the change in MMP inanti-Fas-treated Jurkat cells. Both DEVD and IETD showedsome protection from a loss of MMP in the anti-Fas-treatedcells, however, these caspase inhibitors were ineffective wheneither A23187 or thapsigargin was used to stimulate apoptosis.

FIG. 4. Percent of cells with degraded DNA for Jurkat cellstreated with anti-Fas, A23187, or thapsigargin in the presenceor absence of various caspase inhibitors. Jurkat cells were treatedwith 10 ng/ml anti-Fas antibody, 1 mM A23187, or 7.5 mM thapsigargin(Thaps) in the presence or absence of 25 mM z-VAD-fmk (top), DEVD-fmk (middle), or IETD-fmk (bottom) for 24 h. The percent of cells withdegraded DNA was determined using a FACSort flow cytometer exam-ining 7,500 PI-stained fixed cells, and determining the number of cellswhich had a subdiploid peak of DNA compared with the entire popula-tion of cells. Data is the average of at least three independent experi-ments 6 S.E.

FIG. 5. Percent of dead cells for Jurkat cells treated with anti-Fas, A23187, or thapsigargin (Thaps) in the presence or absenceof various caspase inhibitors. Jurkat cells were treated with anti-Fas, A23187, or thapsigargin, in the presence or absence of z-VAD-fmk(top), DEVD-fmk (middle), or IETD-fmk (bottom) as described in thelegend to Fig. 3. The percent of dead cells was accessed by trypan blueexclusion. Cells which stained blue were considered positive, indicatinga loss of membrane integrity. Data is the average of at least threeindependent experiments 6 S.E.

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The effect of caspase inhibition on MMP is consistent withother data in this study, being protective mainly in anti-Fas-treated cells, but ineffective in preventing the change in mem-brane potential in cells treated with either A23187 orthapsigargin.

DISCUSSION

Apoptosis is defined by a distinct set of morphological andbiochemical characteristics, but it is not clear how these vari-ous apoptotic characteristics relate to each other during celldeath. Additionally, it is unclear what role caspases play in thegeneration of each apoptotic feature. Previously, we haveshown that cells which lost intracellular potassium and de-graded their DNA were largely coincident with cell shrinkage(25–27). We now show that the loss of MMP is also associatedwith the shrunken apoptotic cells. Using JC-1, a dye reliable fordetecting changes in the mitochondrial membrane potential(35), we showed that only cells which have a decrease in cellsize had a loss of MMP. Additionally, we showed that severalcharacteristics of apoptosis, including the loss of cell volume,K1 efflux, and the loss of MMP can be largely caspase inde-pendent depending on the agent used to initiate the cell deathresponse.

Recent studies have suggested that early alterations of mi-tochondrial function may be important for apoptosis (40). Inaddition to alterations in mitochondrial function, the release

from mitochondria of various proapoptotic factors such as cy-tochrome c (14) and apoptosis-inducing factor (13) have beenreported to be critical for apoptosis. The loss of the MMP isthought to release cytochrome c from the outer mitochondrialcompartment, thus potentiating cell death (41). However,Kluck et al. (42) suggest that release of cytochrome c is notaccompanied by mitochondrial depolarization, an event whichfollows the onset of the mitochondrial permeability transition.Additionally, it has been suggested that release of cytochromec, and not the loss of MMP is the required step for initiation ofthe cell death program (43), implying that these events may becell type or inducer specific features of apoptosis.

We have previously shown that a loss of intracellular potas-sium occurs in the shrunken population of apoptotic cells (25,27). We now show that the loss of MMP is also restricted to theshrunken population of cells, suggesting that loss of cell vol-ume, K1 efflux, and loss of the MMP are tightly coupled. Arecent study by Dallaporta et al. (39) supports our originalpublication that the loss of intracellular potassium occurs incells which have lost their MMP; however, they suggest thatthese events precede the cell shrinkage. However, the relation-ship between intracellular potassium and MMP as determineby Dallaporta et al. (39) was based on measurements usingDiOC6 as a fluorescent indicator. We show (Fig. 2), along withstudies from other laboratories (35, 44), that DiOC6 measures

FIG. 6. Flow cytometric analysis of the light scatter properties for Jurkat cells treated with anti-Fas, A23187, or thapsigargin(Thaps) in the presence or absence of various caspase inhibitors. Jurkat cells were treated with anti-Fas, A23187, or thapsigargin, in thepresence or absence of z-VAD-fmk, DEVD-fmk, or IETD-fmk as described in the legend to Fig. 4. Flow cytometry was used to assess changes inthe ability of cells to scatter light in the forward direction (forward scatter), and at a 90° angle (side scatter) as described in the legend to Fig. 1.A representative forward scatter versus side scatter three-dimensional plot containing 10,000 cells is shown for each experimental condition. Gateswere set up based on the control cells to determine the percent of cells which had a decrease in forward scatter light (cell size) and an increase inside scattered light (cell density) compared with the entire population of cells. Percentages shown are the average of at least three independentexperiments 6 S.E.

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both mitochondrial membrane potential and plasma mem-brane potential. Using the specific MMP dye JC-1, which didnot respond to the PMP, we have shown that the loss of MMPoccurs only in apoptotic cells which shrink (see Fig. 3).

Caspases play a central role during apoptosis by cleavingand thereby altering critical cellular substrates which arethought to mediate the dramatic morphological and biochem-

ical changes of apoptosis (21). Shrunken apoptotic cells haveincreased caspase-3-like activity and degraded DNA (25–27).These studies also showed that high extracellular potassiuminhibits caspase-3 activation and DNA degradation, and pre-vents the loss of cell viability and cell shrinkage (25, 27).Caspase-3 is an effector caspase, involved in the later stagesof cell death in many model systems. Thus, caspase-3 wouldbe activated during the common or execution phase of theprocess which results in the classical characteristics apo-ptotic, such as loss of cell volume, DNA degradation, andapoptotic body formation. Interestingly, little is known aboutthe contribution of upstream, or activator caspases to theseapoptotic events. We have used several caspase inhibitors foractivator and/or effector proteases to determine the relation-ship between caspase activity and apoptotic events. Our re-sults show that depending on the particular signal transduc-tion pathway employed to induce cell death, caspaseactivation and activity is not an essential requirement forseveral features of apoptosis.

Diverse stimuli induce programmed cell death through theactivation of different signaling mechanisms which ultimatelylead to the induction of a common apoptotic pathway. The

FIG. 7. Flow cytometric analysis of intracellular potassium for Jurkat cells treated with anti-Fas, A23187, or thapsigargin (Thaps)in the presence or absence of various caspase inhibitors. Jurkat cells were treated with anti-Fas, A23187, or thapsigargin, in the presenceor absence of z-VAD-fmk, DEVD-fmk, or IETD-fmk as described in the legend to Fig. 4 and examined for intracellular K1 as described in the legendto Fig. 1. A gate was set up based on the control sample on a PBFI (K1) versus a PI fluorescent dot plot to eliminate cells which have loss theirmembrane integrity, thus only viable cells were used for further ion analysis. Gates were then set on a PBFI (K1) fluorescence versus forwardscatter (cell size) dot plot of viable cells to determine the percent of cells which had a change in PBFI (K1) fluorescence. A representativethree-dimensional plot is shown for each experimental condition. Percentages shown are the average of at least three independent experiments 6S.E.

TABLE IJC-1 flow cytometric analysis of Jurkat cells treated with anti-Fas,

A23187, or thapsigargin in the presence ofabsence of various caspase inhibitors

The percent of cells with a loss in mitochondrial membrane potentialas detected by JC-1 fluorescence was determined by gating on thepopulation of cells which had a decrease in JC-1 aggregates and asimultaneous increase in JC-1 mononers as demonstrated in Fig.3A. Percentages are the average of at least three independentexperiments 6 S.E.

Control Anti-Fas A23187 Thapsigargin

Control 8.2 6 0.6% 75.0 6 1.8% 86.3 6 7.4% 71.5 6 1.4%z-VAD 7.4 6 0.4% 7.6 6 0.5% 77.7 6 11.6% 59.2 6 3.9%DEVD 7.9 6 0.6% 42.5 6 2.3% 84.9 6 7.6% 70.7 6 9.1%IETD 8.6 6 0.6% 24.9 6 1.5% 86.6 6 8.7% 72.7 6 3.5%

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induction of cell death via Fas cross-linking initially signalsand activates caspase-8 at the cytosolic face of the transmem-brane receptor. In contrast, other apoptotic stimuli, such as thecalcium ionophore A23187 or the Ca21-ATPase inhibitor thap-sigargin, directly activate intracellular signals to initiate thecell death process. In this study, all caspase inhibitors exam-ined (z-VAD, DEVD, and IETD) completely prevented DNAdegradation after 24 h of apoptotic treatment, regardless of theinitial cell death signal, affirming their effectiveness and sug-gesting that DNA degradation is largely caspase dependent.However, our observation that caspase inhibition permittedprogrammed cell death as judged by membrane permeability,cell shrinkage, K1 efflux, and changes in the MPP suggest thatcaspase activity may also not be essential for apoptosis.

Activation of Fas receptor transduces an extremely rapidapoptotic signal through the cytoplasmic domain of the recep-tor which permits the binding of several cytoplasmic proteins,including FADD and caspase-8 (FLICE), forming what hasbeen termed the death-inducing signaling complex (45–47).z-VAD has been shown to inhibit the activation of FLICE at thelevel of the death-inducing signaling complex (45), suggestingthat Fas-induced cell death is triggered from the death-induc-ing signaling complex and that the activation of caspase-8 isrequired for all the downstream events. Our results supportthese observations, as z-VAD completely prevented all anti-Fasinduced characteristics of apoptosis, including loss of cell vol-ume and changes in the MMP. In contrast, when Jurkat cellswere treated with either A23187 or thapsigargin in the pres-ence of z-VAD, a loss of cell viability, cell volume, and intracel-lular potassium, concurrent with changes in the MMP weremanifest. Although the severity of these apoptotic featuresupon A23187 or thapsigargin treatment of Jurkat cells in thepresence of z-VAD was not as great as observed in the absenceof caspase inhibition, an alternative or secondary caspase-in-dependent pathways must exist.

Specific protease inhibitors for caspase-3-like (DEVD) andcaspase-8-like (IETD) enzymes were only marginally effectivein preventing the loss of cell viability, cell volume, and potas-sium, along with the change in MMP that occurs in response toanti-Fas. Furthermore, these specific caspase inhibitors wereineffective in preventing several of the aforementioned charac-teristics of apoptosis in A23187- or thapsigargin-treated Jurkatcells, while remaining potent inhibitors of DNA degradation. A4-fold increase in DEVD (or IETD) in combination with variousapoptotic stimuli also did not prevent the occurrence of theseapoptotic features (data not shown). Thus, depending on theparticular signal transduction pathway activated, alternativeapoptotic pathways appear to exist in the cell which can com-pensate for or by-pass caspase-mediated apoptosis. Our resultssuggest that cells exhibit an inherent commitment to cell deathin response to an apoptotic signal, regardless of caspaseinhibition.

The idea of caspase independent pathways for cell deathhave been proposed in other apoptotic model systems (48, 49).The expression of the pro-apoptotic protein Bax in Jurkat cellswas shown to induce cell death even in the presence of thegeneral caspase inhibitor z-VAD (48). Bax localization to themitochondrial membranes, along with the changes in the mi-tochondrial membrane potential observed during apoptosis,implies that dysfunction of this organelle may promote analternative cell death process. Acid-induced apoptosis in tumorcells was shown to depend on the SAP kinase pathway, to bemarkedly enhanced by Bax, and to be caspase independent(49). Caspase-1- or caspase-3-deficient hepatocytes were alsopartially sensitive to Fas (50). Alternatively, these character-istics may depend on an unidentified protease, not inhibited by

current caspase substrates, as has been proposed for Bax-induced cell death (48).

The present study has shown that the loss of mitochondrialmembrane potential and the loss of cell volume are tightlycoupled features of apoptosis. Additionally, we show that cellviability, cell volume, and efflux of intracellular potassium,along with the concomitant change in mitochondrial membranepotential, can occur either independently or dependently ofcaspase activity, depending on the specific signal transductionpathway activated by a particular apoptotic stimulus. Whilecaspase inhibition effectively prevented DNA degradation un-der all apoptotic conditions, the presence of other classical celldeath characteristics suggest that other features of apoptosiscan be caspase independent.

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Carl D. Bortner and John A. CidlowskiMitochondrial Membrane Potential during Lymphocyte Apoptosis

, Cell Shrinkage, and+Caspase Independent/Dependent Regulation of K

doi: 10.1074/jbc.274.31.219531999, 274:21953-21962.J. Biol. Chem. 

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