Proc. Natl. Acad. Sci. USAVol. 93, pp. 654-658, January 1996Biochemistry

Apoptosis induced in Jurkat cells by several agents is preceded byintracellular acidificationROBERTA A. GOTTLIEB*, JUDY NORDBERGt, EVAN SKOWRONSKIt, AND BERNARD M. BABIOR**Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037; tResearch Service, Department of Veterans AffairsMedical Center, San Diego, CA 92161; and tLa Jolla Institute for Allergy and Immunology, La Jolla, CA 92037

Communicated by Ernest Beutler, The Scripps Research Institute, La Jolla, CA, October 2, 1995

ABSTRACT We have previously shown that in neutro-phils deprived of granulocyte colony-stimulating factor, apo-ptosis is preceded by acidification and that the protectionagainst apoptosis conferred on neutrophils by granulocytecolony-stimulating factor is dependent upon delay of thisacidification. To test the hypothesis that acidification could bea general feature of apoptosis, we examined intracellular pHchanges in another cell line. Jurkat cells, a T-lymphoblastoidline, were induced to undergo apoptosis with anti-Fas IgM,cycloheximide, or exposure to short-wavelength UV light. Wefound that acidification occurred in response to treatmentwith these agents and that acidification preceded DNA frag-mentation. Jurkat cells were also found to possess an acidendonuclease that is active below pH 6.8, compatible with apossible role for this enzyme in chromatin digestion duringapoptosis. Incubation of the cells with the bases imidazole orchloroquine during treatment with anti-Fas antibody or cy-cloheximide or after UV exposure decreased apoptosis asassessed by nuclear morphology and DNA content. The alka-linizing effect of imidazole and chloroquine was shown by thedemonstration that the percentage of cells with an intracel-lular pH below 6.8 after treatment with anti-Fas antibody,cycloheximide, or UV was diminished in the presence of baseas compared with similarly treated cells incubated in theabsence of base. We conclude that acidification is an earlyevent in programmed cell death and may be essential forgenome destruction.

Apoptosis is a tightly regulated physiologic process character-ized in part by nuclear condensation and cell shrinkage withpreservation of an intact plasma membrane, culminating in thedestruction of the nuclear chromatin and the digestion of thegenomic DNA, an irreversible event (1). Although manyagents have been reported to trigger apoptosis, the molecularevents involved in the process are poorly understood. DNAdigestion, however, could be accomplished by an endonucleasesimilar or identical to DNase II that is found in many cell typesand is active below pH 6.8. In earlier work we have shown thatthis endonuclease may be responsible for the genome digestionin neutrophils undergoing apoptosis (2).

For this nuclease to participate in apoptosis, however, thepH of the cell interior has to fall to a value at which thenuclease is active. Acidification has been reported to be aconcomitant of apoptosis in HL-60 cells and CTLL cells (3-6).We have shown (7) that in neutrophils, acidification is an earlyfeature of apoptosis and that the protection against apoptosisconferred upon neutrophils by granulocyte colony-stimulatingfactor (G-CSF) depends upon a delay in acidification mediatedby upregulation of a vacuolar H+-ATPase, an observation thatsuggests a possible causal role for acidification in the apoptosisprogram. In this work we examine the relationship betweenacidification and the process of apoptosis in Jurkat cells.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

METHODS

Cell Culture. Jurkat cells were the gift of D. Green (La JollaInstitute for Allergy and Immunology). They were maintainedin RPMI 1640 medium with 5% (vol/vol) fetal calf serum, 2mM L-glutamine, penicillin (100 units/ml), and streptomycin(100 jvg/ml) in 5% C02/95% humidified air at 37°C at adensity of 106 cells per ml. Where indicated, the cells weretreated for the times noted with anti-Fas IgM (Kamiya Bio-chemical, Thousand Oaks, CA; 10 ng/ml), cycloheximide(Sigma; 50 ,ug/ml), or exposure to UV light, followed bycultivation for 4 hr. Treatment with UV light was accom-plished by suspending 5 x 106 cells in 1 ml of Hanks' balancedsalt solution (HBSS), placing the cell suspension in a 60-mmPetri dish, and irradiating with a UV crosslinker (Fisher) setto deliver 260-nm UV radiation at 100 tLW/cm2 over 2.5 sec.The irradiated cells were then centrifuged and resuspended inculture medium. In experiments involving imidazole or chlo-roquine, the bases were brought to pH 7.4 with HCl and thenadded simultaneously with cycloheximide or anti-Fas antibodyor included in the HBSS and culture medium of the cellstreated by UV irradiation.Flow Cytometric Analysis of Intracellular pH. Cells were

cultured for the indicated times in serum-free RPMI 1640medium, then loaded with 10 ,uM carboxy-SNARF-1-AM(Molecular Probes) for 30 min in HBSS, centrifuged, andresuspended in HBSS containing 20mM Hepes (pH 7.4). Flowcytometry was performed on a Coulter Elite flow cytometer,with excitation at 488 nm and emission ratio analysis at 575 and620 nm as described (8). Ten thousand events were analyzed.Emission ratios were converted to pH values by comparison toratios observed in cells treated with nigericin in high potassiumbuffer at a defined pH (9).Flow Cytometric Analysis of DNA Content. Cells were fixed

in 50% ethanol and stored at 4°C until analysis, when they werestained with propidium iodide (50 ,ug/ml), treated with RNase(10 ,ug/ml), washed in Dulbecco's phosphate-buffered saline(PBS), and subjected to DNA content analysis using MULTI-CYCLE software (Phoenix Flow Systems, San Diego) for theCoulter Elite flow cytometer (10).

Preparation of Nuclear Extracts and Plasmid DNA Diges-tion Assay. Nuclear extracts were prepared from nuclei ob-tained by lysis of Jurkat cells in 0.5% Nonidet P-40/10 mMTris-HCl, pH 7.4/1.5 mM MgCl2; the nuclei were extracted in0.4 M NaCl/20 mM Hepes/1.5 mM MgCl2/25% (vol/vol)glycerol for 30 min on ice. The supernatant obtained aftersedimentation at 17,000 x g for 10 min was stored at -20°Cuntil use. For plasmid digestion assays, 1 ,ul of nuclear extractwas incubated at 37°C for 1 hr with 100 ng of supercoiledplasmid DNA (pCAT2, purified over a CsCl gradient) in 50 ,ulof 30 mM sodium phosphate at the indicated pH (11). Thereaction was terminated by the addition of SDS to 0.1%; thesamples were resolved on a 1% agarose gel containingethidium bromide (0.5 p,g/ml).

RESULTSJurkat is a human T-lymphoblast line that rapidly undergoesapoptosis upon engagement of the Fas antigen by ligand (e.g.,

654

Proc. Natl. Acad. Sci. USA 93 (1996) 655

by treatment with anti-Fas IgM). To determine changes inintracellular pH in response to Fas engagement, we treatedcells with anti-Fas IgM (10 ng/ml) for the indicated length oftime, loading them with the pH-sensitive dye carboxy-SNARF-1-AM during the last 30 min of the incubation, and thenexamining intracellular pH by ratiometric flow cytometry.Within 1 hr after the addition of the anti-Fas antibody, an

acidic subpopulation appeared (Fig. 1). Over the ensuinginterval, the population at neutral pH progressively declined infavor of the acidic subpopulation. Acidification was noted incells of normal size, suggesting it precedes volume loss. Withtime, the cells in the acidic subpopulation tended to becomemore acidic and to decrease in size, in part perhaps due toblebbing; some of what appeared to be very small cells may

CONTROLCell no./DN/ FSC/pH

70.

6.2 7.0FAS 1 hour

Cell no./DNA

I

ic -q1I \S

FSC/pH

6.2 7.0FAS 2 hours

Cell no./DNA Cell no./pH FSC/pH

C

6.2 7.0 6.2 70FAS 3 hours

FSC/pH

6.2 7.06.2 7.0

FIG. 1. Time course of acidification in Jurkat cells treated with Fas

antibody. Jurkat cells were cultured for up to 3 hr in serum-free

medium, with the addition of Fas antibody (FAS, 10 ng/ml) at theindicated time, and then subjected to flow cytometric pH analysis.Immediately after the pH analysis was completed, cells were fixed in50% ethanol, washed, and stained with propidium iodide for cell cycleanalysis. (Left) DNA content of the cells in the population. The

subdiploid fraction appears in region C. (Center) Cell number vs.intracellular pH. (Right) Cell size [measured as forward scatter (FSC)]as a function of pH. Intracellular pH values were calibrated bycomparison with pH standards consisting of nigericin-treated cellsloaded with carboxy-SNARF-1-AM and suspended in a high potas-sium buffer of defined pH (8). The percentages of acidified cells andcells with subdiploid DNA content are tabulated in Table 1. The timecourse was similar in three experiments.

actually have been apoptotic bodies (membrane-bound frag-ments including nuclear material). The clear demarcation oftwo cell populations suggests that acidification occurred rap-idly within individual cells and did not represent a gradualdownward drift.DNA cleavage in the same samples was also determined by

flow cytometry. For this purpose, the cells remaining afterwithdrawing samples for pH determinations were immediatelyfixed in ethanol, labeled with propidium iodide, and subse-quently analyzed by flow cytometry for DNA content (Fig. 1).As with the acidified cells, the percentage of cells withsubdiploid DNA content (defined as apoptotic cells) increasedwith time. At every time examined, however, the subdiploidpercentage was less than the percentage of cells that hadacidified (Table 1). This suggests that acidification precedesDNA fragmentation and also suggests it may be mediated bythe acid endonuclease (see below). Apoptosis in response toanti-Fas antibody was confirmed by microscopic examinationafter staining with acridine orange/ethidium bromide (12) andby in situ nick-end-labeling (13), a technique that identifiescells containing DNA strand breaks (data not shown).Treatment of Jurkat cells with other apoptosis-inducing

agents also led to intracellular acidification. Table 2 shows thatapoptosis in Jurkat cells was induced by cycloheximide and byshort-wavelength UV light as well as by anti-Fas IgM. Flowcytometric pH measurements of the same samples showed thateach of them contained two cell populations, one of normal pHand one that was acidified (Fig. 2 and Table 2). With eachagent, the difference in intracellular pH between the normaland acidified populations was -0.5 pH unit, reflecting a meanpH of the acidified population of 6.7-6.9. On the other hand,rates of acidification varied from agent to agent, as indicatedby the differences in the percentages of cells in the acidifiedpopulations. In every case, however, the percentage of acidi-fied cells (operationally defined as cells with a pH <6.8) wasequal to or greater than the percentage of cells with subdiploidDNA, consistent with the idea that intracellular acidificationmay be causally related to programmed cell death. The thresh-old value of pH 6.8 was chosen because it represented nooverlap (>2 SD) with the range of pH values in normalcontrols and because it corresponded to the threshold foractivity of the acid endonuclease (see below). Furthermore,the percentage of acidified cells was directly correlated withthe percentage of cells subsequently shown to contain subdip-loid quantities of DNA. Fas antibody treatment induced themost rapid acidification and similarly was the most cytotoxic,whereas cycloheximide and UV irradiation produced lessacidification and less apoptosis.

If acidification is essential to the process of genome degra-dation that takes place during apoptosis, then prevention ofacidification should prevent the nuclear changes that occurduring apoptosis. To test this, we treated cells with either of thecell-permeable bases imidazole (pK 6.93) or chloroquine (pKi8.4 and pK2 10.8) concurrent with exposure to the apoptosis-inducing agent. For the purposes of these experiments, apo-ptotic cells were defined as those whose DNA content wassubdiploid. The results with all three apoptosis-inducing agentsand both bases are summarized in Fig. 3. These results show

Table 1. Apoptosis (percentage of cells containing subdiploidquantities of DNA) and intracellular pH in Jurkat cells exposed toanti-Fas antibody (10 ng/ml) as a function of time

Time, Subdiploid cells, Acidified cells,hr % of total % of total

0 3.2 1.81 6.3 13.62 12.4 55.93 28.2 78.9

Results shown are representative of three experiments.

Biochemistry: Gottlieb et al.

656 Biochemistry: Gottlieb et al.

Table 2. Apoptosis and intracellular pH of Jurkat cells exposed to anti-Fas antibody, cycloheximide,or UV light

Subdiploid, Neutral population Acidic populationAgent % of total % of total pH % of total pH

None 2.3 + 0.2 94.4 + 2.6 7.40 + 0.06 3.5 + 2.1 6.76 ± 0.13Fas 11.2 ± 3.0 17.6 + 12.0 7.42 + 0.09 80.8 + 13.2 6.90 ± 0.12CHX 7.5 ±- 2.2 77.7 ± 4.7 7.38 ±+ 0.10 20.9 + 4.8 6.72 + 0.14UV 5.3 ±- 1.4 78.3 + 11.6 7.48 + 0.12 23.6 ± 12.1 6.88 + 0.16

CHX- cvcloheximide. Results rernresent the mean + 1 ISD of fouir exneriments.

that both bases confer protection against the apoptosis-inducing effects of all three of the agents tested.Measurements were also made to confirm that incubation

with a base actually affected the pH of cells exposed to anapoptosis-inducing agent. The results (Table 3) indicate thattreatment with imidazole or chloroquine diminished the per-

CONTROLCell no./DN

Cell no./pH FSC/pH

6.2 7.0 6.2 7.0CHX

FSC/pH

62 7.0UV

Cell no./DNA

BA

I. .

6.2 7.0

0

(a

.0

centage of cells with a pH below 6.8. These results are againconsistent with the idea that intracellular acidification may becausally related to the execution of the cell death program.

Recent studies have linked ceramide production to apopto-sis induced by Fas ligation as well as other agents (14-17).Ceramide is produced by hydrolysis of sphingomyelin by asphingomyelinase, of which there are two types-acidic andneutral. To determine whether base was interfering withactivity of the acidic form, we took advantage of the obser-vation that ceramide production by acidic sphingomyelinaseoccurs within the first 5 min of Fas ligation (18-20). We thencompared the protection conferred by imidazole added con-currently with anti-Fas antibody with that conferred by imi-dazole added 60 min later, a time window when ceramideproduction is largely complete but before significant acidifi-cation has occurred. We found that imidazole was similarlyprotective whether added at time zero or 60 min later (Table4). In separate experiments, we measured intracellular pH incells treated with 100 JIM C2-ceramide and observed acidifi-cation in response to this agent as well (Fig. 4).

If the process of apoptosis requires acidification for DNAfragmentation to occur, then one would expect to find anendonuclease that is active at low pH. Indeed, when weexamined nuclear extracts from Jurkat cells, we found an acidendonuclease that was independent of divalent cations and wasactive at low pH (Fig. 5).§ The acid endonuclease activity

§These cells also contain a calcium- and magnesium-dependent neu-tral endonuclease (data not shown).

MEDIA20 F

15 F

10

5

6.2 7.0FIG. 2. Flow cytometric pH analysis of Jurkat cells induced to undergo

apoptosis. Jurkat cells were incubated with Fas antibody (10 ng/ml) (FAS)or cycloheximide (CHX; 100 ,ug/ml) or exposed to UV light (UV) asdescribed in the text. Two hours later, they were subjected to flowcytometric pH analysis. Immediately after the pH analysis was completed,cells were fixed in 50% ethanol, washed, and stained with propidium iodidefor cell cycle analysis. (Left) DNA content of the cells in the population.The subdiploid fraction appears in region B. (Center) Cell number vs.intracellular pH. (Right) Cell size [measured as forward scatter (FSC)] asa function of pH. The neutral and acidic populations are quantified inTable 2. Mean pH values of replicate samples in this experiment differedby <0.02 pH unit.

*

IMZ

CQ

T

*

T*

CON FAS CHX UV

FIG. 3. Effect of base treatment on DNA fragmentation inducedby Fas, cycloheximide, or UV radiation. Jurkat cells were treated asdescribed in Fig. 1, in the presence or absence of imidazole (IMZ) orchloroquine (CO), then fixed, and analyzed for subdiploid DNAcontent as above. Data represent the mean + SEM of 11 or 12(imidazole) or 6 (chloroquine) experiments. Asterisk denotes statisticalsignificance (P < 0.05 for base vs. control) as determined by a one-tailedWilcoxon rank sum test. CHX, cycloheximide.

-- - --------- ---

Pi-oc. Natl. Acad. Sci. USA 93 (1996)

11Izvl W7 IVI%liilk. \ I UILZ3 1Wll%W16 1 IllCtll I ULJtJ IJUUI A 1 11W13

Proc. Natl. Acad. Sci. USA 93 (1996) 657

Table 3. Effect of base treatment on the percentage of cells with a pH <6.8 after treatment withanti-Fas antibody, cycloheximide, or UVC

P value for P value forimidazole chloroquine

Agent No base Imidazole vs. no base Chloroquine vs. no baseNone 1.3 ± 0.3 (6) 1.2 ± 0.4 (5) NS 2.7 ± 1.6 (2) NSFas 28.6 + 1.5 (6) 10.4 + 1.7 (5) <0.001 4.0 + 3.3 (2) <0.001CHX 10.9 + 2.5 (5) 3.2 ± 0.8 (5) <0.001 2.6 + 0.8 (2) <0.01UV 8.6 + 2.4 (5) 3.3 + 1.1 (4) <0.005 0.4+ 0.3 (2) <0.01CHX, cycloheximide; NS, not significant. Results represent the mean + 1 SEM of the indicated number

of experiments in parentheses.

isolated from Jurkat cells was active below pH 6.8; thus thelevel of acidification observed in cells induced to undergoapoptosis was sufficient to permit activation of the endonu-clease. The constitutive presence of this endonuclease (inunstimulated Jurkat cells) resembles our findings (2) of asimilar activity in neutrophils.

DISCUSSIONWe found that acidification was an early feature of apoptosisin Jurkat cells treated with anti-Fas antibody, cycloheximide,or short-wavelength UV light. From the presence of twodiscrete cell populations, one acidic and the other neutral, weinferred that the transition from the neutral to the acidicsubpopulation was rapid. Acidification preceded DNA frag-mentation, a feature consistent with the idea that the acidendonuclease participated in DNA cleavage during apoptosis.The nuclear condensation and DNA fragmentation of apo-ptosis induced by all three agents were decreased by concur-rent incubation with cell-permeable bases, suggesting thatacidification was essential for these events of apoptosis tooccur. Imidazole, one of the bases used in these experiments,is also an inhibitor of nitric oxide synthase (21), and nitricoxide has been implicated in some cases of apoptosis (22). Itis unlikely, however, that both bases would have similar activityagainst nitric oxide synthase, since only imidazole is a struc-tural analog of arginine, the substrate from which NO isproduced. In addition, 1 mM (Nw-nitro-L-arginine methylester), an inhibitor of NO synthase, did not protect againstapoptosis induced by Fas ligation, cycloheximide, or UV (data

Table 4. Effect of delaying imidazole addition on protectionagainst apoptosis induced by Fas ligation

% cells with subdiploid DNAcontent

Exp. 1 Exp. 2 Exp. 3

Control

Fas

3.7 5.6 7.74.4 6.8 7.3

8.413.4 24.79.6 29.4

Fas/Imz (t = 0) 4.0 20.14.4 22.2

Fas/Imz (t = 60) 8.0 21.25.4 24.1

11.211.911.19.29.88.68.1

10.47.1

Imidazole (10 mM) was added 1 min before (t = 0) or 60 min (t =60) after anti-Fas IgM (10 ng/ml). Cells were then incubated for a totalof 4 hr, fixed in ethanol, and analyzed for DNA content. Apoptosis isscored as the percentage of cells having a subdiploid DNA content.Results from three separate experiments are presented; the groupswere analyzed by the Wilcoxon rank sum test for one-tailed analysis.P < 0.001 for Fas vs. control; P < 0.05 for Fas vs. Fas/Imz (t = 0); notsignificant for Fas/Imz (t = 0) vs. Fas/Imz (t = 60).

not shown). It is more probable that the protective effect ofimidazole and chloroquine was related to their effect onintracellular pH. This is also suggested by the finding that theirrelative efficacy in protecting against apoptosis paralleled theirpK values.

Ceramide, which can be generated in either an acidic orneutral compartment, has been implicated as a second mes-senger in death signaling (18, 23). Because acidic sphingomy-elinase is active in an acidic compartment, it was important todetermine whether base protection worked by interfering withthis route of ceramide production. Since ceramide productionis largely complete within the first 5 min after Fas ligation(18-20), addition of base 60 min after Fas antibody would notbe expected to interfere with ceramide generation. Indeed, wefind that imidazole added after a 60-min delay is as protectiveas adding it before Fas ligation. It is also possible that ceramideproduction continues beyond 60 min and that imidazole pro-tects against ongoing ceramide production. However, giventhat ceramide also triggers acidification, it is reasonable toconclude that the bases protect by altering cytoplasmic pH,rather than attenuating ceramide production.

It has been suggested that apoptosis represents an abortedmitosis (24). Alkalinization has long been recognized to be anecessary concomitant of mitosis (25); the observations wereport suggest that in an analogous manner, acidification couldbe essential for apoptosis, because treatment with base de-creased both the characteristic nuclear changes and the DNAfragmentation in cells undergoing apoptosis. Our results mayexplain the findings of Kapus et al. (26) that cells lacking afunctional sodium/hydrogen exchanger and grown in acidmedium have an intracellular pH below 6.6 and do not survive.

Cells regulate intracellular pH through two major pathways:sodium/hydrogen exchange and chloride/bicarbonate ex-change. It has been shown that in hemopoietic cells, steel factorand granulocyte/macrophage colony-stimulating factor bothactivate the sodium/hydrogen exchanger and that this activityis necessary for suppression of apoptosis (6). In addition,activity of the sodium/hydrogen exchanger has been impli-cated in the effect of phorbol ester-mediated protection from

Cell no./pH

CONTROL

XCERAMIDE

6.2 7.4

FIG. 4. Effect of ceramide treatment on intracellular pH. Jurkatcells prepared as described in Fig. I were treated with 20 ,uMC2-ceramide for 2 hr and then analyzed for intracellular pH by flowcytometry. The result shown is representative of three experiments.

Biochemistry: Gottlieb et al.

658 Biochemistry: Gottlieb et al.

CON 6.2 6.4 6.6 6.8 7.0 7.2

OCSC

FIG. 5. pH dependence of endonuclease activity in nuclear extractsfrom Jurkat cells. Nuclear extracts were incubated with supercoiledplasmid DNA in 30 mM potassium phosphate at the indicated pH. Thesamples were resolved on a 1% agarose gel containing ethidiumbromide (0.5 ,ug/ml). Figure is representative of three such experi-ments. Activity is reflected by conversion of plasmid DNA fromsupercoiled (SC) to open circle (OC) form.

lovastatin-induced acidification and apoptosis (5). It is impor-tant to also consider the production of metabolic acid, as forexample through glycolysis, which might overwhelm ordinarypH-homeostatic mechanisms. Studies of tumor necrosis factor-mediated apoptosis indicate that inhibition of mitochondrialelectron transport is an important feature (27). In addition,inhibition of mitochondrial respiration (e.g., with oligomycin),which would be expected to increase glycolysis, induces apo-

ptosis (28). Thus, these findings imply that more than one

pathway that could contribute to intracellular acidificationduring apoptosis.

It is of interest that in addition to the acidic DNase, otherproteins thought to participate in apoptosis also operate at lowpH. These include tissue transglutaminase (29), which gener-ates the protein crosslinks that form in apoptotic cells (30-33);acidic sphingomyelinase (18), which liberates ceramide; andgelsolin (34), a cytoskeletal regulatory protein that is activatedby HI (among other things) and whose ability to depolymerizefilamentous actin (35) may be essential for the cytoskeletalchanges that occur in apoptotic cells. It is conceivable thatevery cell contains a set of proteins-enzymes, structuralproteins, and perhaps transcription factors-that are requiredfor the apoptosis program but that only operate at acidic pHvalues, and that by triggering a fall in intracellular pH, apo-ptosis-inducing agents drive the cell into a new metabolic statein which these proteins are awakened from dormancy to helpexecute the apoptosis program.

We thank T. P. Stossel for his suggestion concerning gelsolin. Thiswork was supported in part by U.S. Public Health Service GrantsAI-24227 and HL-17682. R.A.G. is the recipient of Clinical Investi-gator Award K08 AI-01345 from the National Institutes of Health.

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Proc. Natl. Acad. Sci. USA 93 (1996)