THE J O U R N A L 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

OF BIOLOGICAL CHEMISTRY Vol . 264. No. 26, Issue of September 15, pp. 15261-15267, 1989 Printed in U.S.A.

Internucleosomal DNA Cleavage Precedes Diphtheria Toxin-induced Cytolysis EVIDENCE THAT CELL LYSIS IS NOT A SIMPLE CONSEQUENCE OF TRANSLATION INHIBITION*

(Received for publication, April 21, 1989)

Michael P. Chang$$!l, John BramhallQII , Scott Graves$**, Benjamin Bonavida$ll , and Bernadine J. WisnieskiS 11 $$ From the $Department of Microbiology and the Molecular Biology Institute, the §Department of Microbiology and Immunology, and the IlJonsson Comprehensive Cancer Center, The University of California, Los Angeles, California 90024

Diphtheria toxin (DTx) is an extremely potent inhib- itor of protein synthesis. Cell death has been generally accepted as a straightforward effect of translation in- hibition. Using human U937 cells, we found that DTx intoxication leads to cytolysis; indeed, release of “Cr- and ‘%e-labeled proteins could be detected within 7 h. However, little or no cell lysis was observed over a 20- 50-h period when human U937 cells were exposed to cycloheximide, amino acid-deficient medium, or met- abolic poisons even though protein synthesis was rap- idly inhibited to levels observed with DTx. Likewise, investigations with human K562 cells revealed full resistance to the cytolytic action of DTx over a 50-h period despite a severe reduction in translation activ- ity. These observations establish that inhibition of pro- tein synthesis per se is not sufficient to provoke cell lysis. A characterization of DTx-induced cytolysis re- vealed a long lag period (6-7 h) which could be short- ened considerably by a short exposure to low pH. NH,Cl and metabolic poisons blocked the cytolytic ac- tion of DTx, indicating that endocytic uptake of toxin is required for lytic activity. Surprisingly, DTx also induced extensive internucleosomal degradation of cel- lular DNA, a characteristic feature of apoptosis or programmed cell death. DNA-fragmentation preceded cell lysis and did not occur in DTx-treated K562 cells or in U937 cells that were treated with the other protein synthesis inhibitors. From these observations, we conclude that DTx-mediated cytolysis is not a sim- ple consequence of translation inhibition and that in- ternucleosomal DNA fragmentation is a newly identi- fied and relatively early step in the cytolytic pathway of DTx.

* This work was supported in part by Grants CA-43121 (to J. B.), CA-35791 (to B. B.), and GM-22240 (to B. J. W.) from the National Institutes of Health and by grants from the California Cancer Re- search Committee and the UCLA Academic Senate. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ?l Recipient of United States Public Health Service/National Re-

search Service Award Grant CA-09056. Present address: Dept. of Pharmacy, University of California at San Francisco, 513 Parnassus Ave., San Francisco, CA 94143.

**Recipient of National Cancer Institute Training Grant CA- 09120.

$$ TO whom correspondence should be addressed Dept. of Micro- biology, University of California, Los Angeles, CA 90024.

Diphtheria toxin (DTx)’ is a potent cytotoxin for a wide range of eukaryotic cells. The best-characterized activity of DTx is its ability to inhibit target cell protein synthesis, and it has been estimated that a single molecule of DTx is suffi- cient to kill a susceptible cell (1). DTx is secreted by Cory- nebacterium diphtheriae as a single polypeptide chain (Mr = 60,525); subsequent cleavage generates two subunits that re- main connected by a disulfide bond (2). The A subunit (Mr = 20,500) inhibits protein synthesis by catalyzing the transfer of the ADP-ribose moiety of NAD to elongation factor 2 (EF- 2). The B subunit (Mr = 40,000) is the receptor-binding domain of the toxin and has no known enzymic activity (3).

Although protein synthesis inhibition can be monitored precisely and with complete objectivity, target cell death is much more problematical and can be variously monitored as cessation of cell replication, leakage of cytoplasmic solutes, loss of the ability to exclude certain water-soluble dyes, loss of the ability to take up nutrients, or, in the case of specific effector cells, a decline of function. It is not at all clear that these parameters are equivalent to each other or even mu- tually consistent. In studies of immune cytolysis, cell death is usually operationally defined as loss of the ability to retain cytosolic marker solutes such as those radiolabeled with 51Cr. In a previous report (4) we noted that DTx provokes W r release from target cells and that the time course of killing is similar to that observed with tumor necrosis factor-a (TNF), an agent which does not diminish translation activity (5).

The current study was undertaken to address the question of whether the observed cytolytic response to DTx is simply an ipso facto hallmark of protein synthesis inhibition, or whether a TNF-like effect is occurring, characterized by target cell DNA damage independent of translation inhibition. By using a cell line in which DTx provokes translation inhibition but not lysis we were able to demonstrate that the cytolytic activity of DTx is strictly correlated with extensive (prelytic) DNA fragmentation akin to that observed during “pro- grammed cell death” or “apoptosis.” This autolytic pathway has been shown to be induced by a number of unrelated agents, including TNF (6, 7), lymphotoxin (8), cytotoxic T- lymphocytes (9), natural killer cells (lo), ionizing radiation (111, and glucocorticoids (12). Our findings lead us to conclude that the ability of DTx to provoke target cell lysis involves a new killing activity.

The abbreviations used are: DTx, diphtheria toxin; TNF, tumor necrosis factor-a; RPMI, tissue culture medium; BCS, bovine calf serum; EF-2, elongation factor 2; NaN3/2-deoxyglucose, sodium azide in combination with 2-deoxyglucose; NH4Cl, ammonium chloride.

15261

15262 The Cytolytic Pathway of Diphtheria Toxin

EXPERIMENTAL PROCEDURES

Celk"U937, RAJI, YAG-1, and K562 target cells (obtained from ATCC) were all maintained in suspension culture (37 "C, 5% CO,, 95% air) in RPMI-1640 medium supplemented with nonessential amino acids, sodium pyruvate, L-glutamine, sodium bicarbonate, and 10% bovine calf serum (RPMI + 10% BCS); PA-1 was grown in the same medium as an adherent culture. U937, RAJI, K562, and PA-1 are all human cell lines; YAC-1 is murine. K562 cells obtained from ATCC in 1985 were susceptible to DTx-mediated cytolysis in contrast to the K562 cells obtained in January 1989 and used in this study. Cells and/or karyotype data are available upon request.

Reagents-DTx was supplied by List Biological Laboratories (Campbell, CA) as a lyophilized powder; after reconstitution it was stored at a protein concentration of 1 mg/ml at -80 "C. As supplied, the preparation was <5% nicked (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% reducing gels). Moreover, DTx preparations purified from toxin obtained from Con- naught Laboratories gave identical results. [3H]Leucine (126 Ci/ mmol), Na2[51Cr]04 (37 MBq/ml), and [75Se]methionine (2.0 Ci/ mmol) were supplied by Amersham (Amersham, England). Trypan blue was supplied by Sigma. Cycloheximide, NH4Cl, sodium azide, and 2-deoxyglucose were purchased from Fluka (Ronkonkoma, NY).

Cell-labeling Procedures-Chromium-labeled cells were prepared by incubating 3-4 X 10' target cells overnight with Na2151Cr]04 (100 pCi) in 5 ml of RPMI + 10% BCS followed by washing three times in RPMI prior to use. Selenium-labeled cells were prepared by incu- bating 3-4 X IO6 target cells in methionine-free RPMI + 10% BCS supplemented with [75Se]methionine (5 pCi) for 2 h; labeled cells were washed (three times) in RPMI prior to use.

Cytolysis Assays-Release of W r and 75Se from cells loaded with Naz["Cr]O, or [75Se]methionine was monitored by measurement of radioactivity in cell-free supernatants removed from cell samples at appropriate times. Labeled cells were distributed in 96-well microtiter plates at a cell density of 105/ml (200 &well); following a 20-h incubation under test conditions, cells were pelleted by gentle cen- trifugation of the plate, and supernatants (100 pl) were collected by aspiration. Each assay was performed in triplicate. The extent of cytolysis was calculated from the following equation:

% Cytolysis = [(A, - A,)/(AT - A.)] X 100 (1)

where A, and A, represent radioactivities of the test sample and control sample (no toxin present), respectively; AT represents the radioactivity of unpelleted samples, containing cells, and is the total "Cr or 75Se associated with each sample. Acid precipitation of labeled proteins was effected in the following manner: samples of cell-free supernatant were adjusted to 0.5 ml with an aqueous solution of bovine serum albumin (1 mg/ml), then 0.5 ml ice-cold 20% (w/v) aqueous trichloroacetic acid was added, and the mixture was allowed to incubate for 5 min at 4 "C. Following incubation the samples were centrifuged at 18,000 X g for 5 min and, for each sample in succession, 0.5 ml of supernatant was removed and transferred to a separate polypropylene tube. The radioactivity of this aliquot of supernatant (Pa), and the radioactivity of the corresponding pellet tube (P,) were measured.

% Precipitability = [(P, - P,)/(P, + P8)] X 100 (2)

Uptake of trypan blue was monitored by incubation of cells with a filtered solution of trypan blue (0.2% w/v in phosphate-buffered saline) for 5 min at 20 "C, followed by visual microscopic inspection; blue-stained cells were considered to be dead.

Protein synthesis activity was monitored by incubating lo5 target cells in 200 p1 of leucine-free RPMI supplemented with 4% BCS and 1.0 pCi of [3H]leucine. At specified time intervals samples were diluted 1:1 with ice-cold 20% (w/v) trichloroacetic acid and filtered through glass fiber. The filters were washed first with 10% (w/v) trichloroa- cetic acid and then with phosphate-buffered saline; the amount of radioactivity retained by each filter was determined by standard liquid scintillation spectrometry. Inhibition of protein synthesis activity was calculated as the net decrease in the radioactivity (cpm) of filters from test samples divided by the radioactivity of control samples.

DNA Fragmentation Assay-At time zero, cells were distributed into Falcon tubes at a cell density of 5 X 104/ml (4 ml/tube) either with or without DTx (75 ng/ml). At specified times, target cells (0.6 ml) were lysed in 500 pl of buffer containing 100 mM EDTA, 100 mM NaCI, 10 mM Tris, pH 8.0, and 1% sodium dodecyl sulfate. The high molarity of EDTA is essential for inhibiting endogenous nuclease

activity during cell lysis. The samples were incubated with 5 p1 of proteinase K (Sigma; stock 10 mg/ml) at 37 "C for 30 min, and the DNA was extracted once at room temperature with an equal volume of chloroform/phenol (5050) using vigorous shaking by hand. DNA was precipitated in 50 pl of 3 M sodium acetate and 550 pl of 2- propanol at -80 "C for 10 min followed by centrifugation at 13,000 X g for 10 min. Samples were next washed with 500 pl of ice-cold 80% ethanol, dried by evaporative centrifugation, and resuspended in 100 p1 of TE (10 mM Tris and 1 mM EDTA, pH 8.0) before treatment with 2 p1 of RNase A (Sigma; stock 10 mg/ml) for 10 min at 37 "C. Finally, excess EDTA was removed by spin chromatography (13) through 1.0 ml of P-10 resin (Bio-Rad). Samples were concentrated by evaporative centrifugation prior to electrophoresis for 3 h at 50 V in 1.2% agarose gel. DNA was visualized by ultraviolet fluorescence after staining the gel with ethidium bromide.

Ficoll-Hypaque Fractionation-Cells were distributed in Costar 6- well plates a t a cell density of 5 X 104/ml (4 ml/well) either with or without DTx (75 ng/ml). At specified times, the contents of a single well were fractionated on Ficoll-Hypaque as previously described (14). Separated populations of lysed and non-lysed cells were con- firmed by trypan blue staining, and target cell DNA was extracted and analyzed according to the methods described above.

RESULTS

DTx Is Cytolytic for Nucleated Target Cells-DTx admin- istered to human U937 cells caused inhibition of protein synthesis (intoxication) and release of cytoplasmic contents (cytolysis); Fig. 1 shows that these two effects show the same toxin dose dependence with both 50% inhibition of protein synthesis and 50% cell lysis occurring at a DTx concentration of 7.5 ng/ml. The extent of cell lysis monitored by trypan blue exclusion was very similar to that observed with 51Cr-labeled target cells. A number of different mammalian cell lines were tested in parallel for their ability to be lysed by DTx and for their ability to serve as conventional DTx targets as moni- tored by protein synthesis inhibition. Table I shows that there was an overall correlation between the two activities of DTx; cells that were resistant to DTx intoxication also resisted DTx cytolysis, and uice versa. One dramatic exception to this pattern was observed with human K562 cells. These results are discussed below.

Inhibition of Protein Synthesis Alone Is Not Sufficient to Provoke Cell Lysis-Although the preceding results suggested a close association between the two activities of DTx, lysis, and intoxication, experiments conducted under a variety of

100 -

80 -

60 -

40 -

20 -

0- I 1 I I I 4

0 .75 7 5 75 75c T O X I N (ng/ml)

FIG. 1. Toxin-mediated cytolysis and translation inhibition share similar dose dependencies. "Cr-Labeled U937 cells were treated with various concentrations of DTx for a 19-h period prior to the addition of [3H]leucine for 1 h; incorporation of 3H-label into acid-precipitable material (open triangles) was measured as described under "Experimental Procedures." The extent of 51Cr release from 'lcr-labeled U937 cells (open circles) and the appearance of trypan blue-staining in unlabeled U937 cells (filled circles) were both moni- tored after a 20-h incubation with DTx; all incubations were per- formed at 37 "C.

T h e Cytolytic Pathway of Diphtheria Toxin 15263

TABLE I A comparison of the cytolytic and protein synthesis inhibition

actiuities of DTx Target cells were preloaded with 51Cr overnight, washed (three

times), and distributed in 96-well microtiter plates (lo4 cells/well). DTx was added to cells for 20 h at 37 "C prior to measuring "Cr- release. In a parallel experiment, cells were incubated with DTx (75 ng/ml) for 19 h at 37 "C prior to measuring the amount of 3H-label incorporated into acid-precipitable material during a 1-h incubation in the presence of 3H-leucine. Details of these methods are described under "ExDerimental Procedures."

Cytolysis Protein synthesis inhibition

19 nglrnl" 38 nglrnl 75 nglml 75 ngjrnl U937 44.8 f 7.8 50.0 f 2.3 76.0 f 4.1 74.5 2 1.5 PA-1 34.1 -t 1.3 37.5 f 3.2 51.8 f 1.4 82.2 2 0.7 RAJI 1.2 c 0.1 0.1 f 0.5 2.6 f 1.1 2.5 2 0.1 YAC-1 2.6 f 0.4 5.0 f 0.9 5.6 f 2.5 ND

~

Concentration of DTx used in assay.

TABLE I1 Inhibition of protein synthesis is not correlated with cell lysis

Experimental conditions were as described for Fig. 1. Cytolysis was monitored using U937 cells preloaded with "Cr; protein synthesis was monitored in parallel using nonloaded target cells. Final concen- trations of reagents used were 2.5 mM, 5 mM, 250 ng/ml, and 75 ng/ ml for NaN3, 2deoxyglucose, cycloheximide, and DTx, respectively.

Treatment Translation inhibition CytOlYSiS 92 %"

Deficient mediumb 97.5 f 0.2 14.2 f 0.1 NaN3/2-deoxyglucose 95.1 f 0.1 5.0 f 1.4 Cycloheximide 87.5 f 1.6 14.8 f 1.5 Dinhtheria toxin 84.7 f 0.9 77.5 f 5.1

- ~~ ~

Net release of 51Cr measured after a 20-h incubation at 37 "C. RPMI medium deficient in leucine, lysine, methionine, and glu-

tamine.

conditions demonstrated that cell lysis was not an automatic consequence of inhibition of protein synthesis activity. Table I1 shows that exposure of U937 cells to cycloheximide (250 ng/ml) at a concentration that caused greater than 85% decrease in protein synthesis activity (measured by the incor- poration of [3H]leucine into acid-precipitable material) caused less than 10% release of trapped 5'Cr during the 20-h incu- bation period. Cytolysis was observed, however, when DTx was added to these cycloheximide-treated cells, suggesting that cycloheximide treatment did not decrease the intrinsic ability of target cells to be lysed (data not shown). Similarly, when U937 cells were maintained in medium deficient in several essential amino acids (leucine, lysine, methionine, and glutamine), protein synthesis was inhibited by greater than 97% whereas only 14% cytolysis was recorded (Table 11). Even when cells were poisoned metabolically with levels of sodium azide/2-deoxyglucose that led to 95% protein synthe- sis inhibition, only 5% cytolysis was observed. In sharp con- trast, Table I1 shows that DTx (75 ng/ml) caused 85% de- crease in protein synthesis and 78% cytolysis during the same standard 20-h incubation period. These results raised the possibility that the cytolytic activity of DTx may not be a direct consequence of inhibition of protein synthesis.

Kinetics of Cell Lysis and Translation Inhibition-Parallel experiments were performed to analyze the kinetics of DTx- mediated cytolysis and intoxication. Fig. 2, A and B shows that DTx-mediated cytolysis occurred much later than inhi- bition of protein synthesis, with half-maximal points at 10 and 3.5 h, respectively. A long lag period (6 h) preceded the sudden onset of marker release whereas inhibition of trans- lation was apparent even at the earliest time points, demon- strating that intoxication began almost immediately after

exposure of the cells to DTx. Protein synthesis was also rapidly inhibited by cycloheximide (1 pg/ml) or NaNd2- deoxyglucose (2.5/5 mM) and the kinetics of inhibition closely resembled that seen in DTx-treated cells (Fig. 2B). However, despite reducing translation activity at the same rate and to the same extent as toxin-exposed cells, the metabolic poisons displayed no lytic activity for 20-30 h, and cycloheximide- treatment led to no appreciable lysis over a 50-h time period (Fig. 2A) . Similar results were obtained when cytolysis was monitored as release of large (>2 kDa) acid-precipitable 75Se- labeled cell proteins (data not shown).

The lag period preceding cytolysis could be shortened by 3- 5 h by maintaining acidic conditions during the initial expo- sure of target cells to DTx (pH 5.3; 15 min). Moreover, acid- pulsed cells showed enhanced susceptibility to DTx-triggered lysis throughout a 20-h assay (data not shown).

Cell Lysis Is Not a Simple Consequence of Protein Synthesis Inhibition-We have observed that the K562 cell line is fully resistant to the cytolytic action of DTx. The basis for this resistance is not explained by a block in the intoxication process, since protein synthesis is severely inhibited by DTx.

z 20

0 0 10 20 30 4 0 50

TIME (nr)

6

o

L b= ""- - " - "

/

0 5 10 1 5

TIME (hr) 20

FIG. 2. Kinetics of target cell lysis (A) or protein synthesis inhibition (B). A, 51Cr-labeled U937 cells were distributed in Falcon tubes (1 X lo6 cells/tube in 20 ml of medium) and exposed at time zero to DTx (300 ng/ml; filled circles), NaN3/2-deoxyglucose (NaN3/ 2-DOG) (2.5/5 mM; filled squares) or cycloheximide (1 pg/ml; open circles). 'lCr-Labeled K562 cells were also examined with an identical concentration of DTx (open squares). Percent cell lysis was deter- mined at specified intervals over a 50-h period as described under "Experimental Procedures." Data represent mean and standard error

inhibition in U937 cells exposed to DTx (filled circles), NaN3/2- of triplicate determinations. B, time course of protein synthesis

deoxyglucose (filled squares) or cycloheximide (open circles). DTx- treated K562 cells (open squares) were monitored in parallel. Reagents were added at concentrations specified in panel A , and translation activity was determined as described under "Experimental Proce- dures."

15264 The Cytolytic Pathway of Diphtheria Toxin

Even at extremely high concentrations of toxin, where trans- lation activity was completely inhibited (>99%), no cytolysis was observed over a 20-h period (Fig. 3). To test whether resistance to lysis could be attributed to a slower rate of intoxication, we monitored the kinetics of DTx-induced pro- tein synthesis inhibition in both K562 and U937 cells. Fig. 2B shows that translation inhibition occurred significantly later in K562 cells as compared with U937 cells, with half- maximal points at 8 and 3.5 h, respectively. However, contin- uous exposure of K562 cells to a high dose of DTx (300 ng/ ml) for 50 h failed to elicit a cytolytic response (Fig. 2 A ) despite the severe inhibition of translation activity observed throughout this period (data not shown). Of considerable interest is the fact that DTx-lysis-resistant K562 cells were found to be cross-resistant to the cytolytic action of TNF, a cytokine with no effect on protein synthesis activity (5). This cell line, however, can be readily lysed by natural killer cells (15) and antibody and complement (16). Our findings with K562 target cells establish a striking lack in correlation be- tween the two activities of DTx; cells that were fully sensitive to DTx intoxication completely resisted cytolysis (Fig. 3) in distinct contrast to the pattern observed with U937 target cells (Fig. 1). What clearly emerges is that protein synthesis inhibition alone is not sufficient to trigger cell lysis. Hence the cytotoxic pathway of DTx appears to include a new but as yet undefined lysis triggering activity.

Internalization of Cytolytically Active DTx Molecules Is Re- quired for Killing Activity-The conventional route of entry to the cytoplasm for DTx, namely, receptor-mediated endo- cytosis followed by escape from the endosomal compartment, shows an absolute requirement for acidic endosomes (17-20) and can be inhibited by depletion of cellular ATP levels (21). Fig. 4 shows that DTx-induced cytolysis was only observed under conditions that allowed for exposure of the toxin to a low pH environment. In the presence of 10 mM NH4C1, a potent inhibitor of endosomal acidification (22), U937 target cells were completely resistant to the lytic effects of DTx, and this resistance was readily overcome when the pH of the external medium was reduced to 5.3. Fig. 5 shows that DTx- induced cytolysis was also sharply inhibited by the metabolic poisons NaNs/2-deoxyglucose; pretreatment of 51Cr-labeled U937 cells with a combination of the two energy poisons resulted in >75% inhibition of DTx-mediated cytolysis. This finding strengthens earlier proposals that cellular ATP is needed in order to effect DTx delivery to the cytosol (21,231.

I ' I

I 0 .75 7.5 75 750 7500

TOXIN (ng / rn l )

FIG. 3. DTx inhibits translation activity in K562 cells but does not induce lysis. DTx-mediated cytolysis and translation inhibition were monitored in K562 cells after a 20-h incubation. Methods were identical to Fig. 1.

2ol 0- I 8 I I

40 60 80 100

TOXIN (og/rnl)

t D T x ApH pH7.4 ?NH,CI

ASSAY I I I I o 5 m i n Z O m i n " 20 h

TIME

FIG. 4. Inhibition of DTx-mediated cytolysis by lysosomo- tropic agents can be overcome by exposure to low pH. "Cr- Loaded U937 cells were exposed to DTx at pH 7.4 either in the presence (filled triangles) or the absence (open triangles) of 10 mM NH4C1. After 5 min at 37 "C, the pH of the medium was lowered to 5.3 in some of the samples, either with (filled circles) or without (open circles) NH4Cl. After 15 min, all samples were washed with fresh medium and incubated at 37 "C in RPMI + 2.5% BCS, pH 7.4, either with or without NH4C1 for 20 h.

40 cn 3 3 20 w 0

0 A 0 C D

FIG. 5. DTx-mediated cytolysis of U937 cells is energy de- pendent. 51Cr-Labeled U937 cells were incubated in RPMI + 10% BCS containing 2.5 mM NaN3 in combination with 5 mM 2-deoxyglu- cose ( B and D) or in medium alone (A and C) for 30 min prior to the addition of DTx to 40 ng/ml (A and B ) or 75 ng/ml (C and D). Incubation was continued for 20 h at 37 "C, and the extent of "Cr release from target cells was measured.

Taken together, these two observations strongly suggest that the cytolytic activity of DTx, much like its translation inhi- bition activity, is normally dependent on internalization of toxin molecules and that cell-surface-bound toxin itself is not sufficient to signal the cytolytic response.

DNA Fragmentation Is a Specific Consequence of DTx Ex- posure-When the integrity of cellular DNA was monitored 18 h after DTx treatment, significant levels of DNA degra- dation were observed. Fig. 6 shows the fragmentation profile has a laddered appearance with the minimum size band being -200 base pairs. Cells treated with cycloheximide (Fig. 6) and other agents/conditions that led to inhibition of protein syn- thesis failed to display significant levels of DNA degradation over the same 18-h time period. When we examined the integrity of cellular DNA from K562 cells (resistant to DTx- mediated lysis), no detectable DNA fragmentation was ob-

The Cytolytic Pathway of Diphtheria Toxin 15265

1 2 3 4

692 - 521 - 284 - 257 - 226 /-

I t

‘“ 40-

1

A - _J

W 0

20 -

FIG. 6. DTx-induced DNA fragmentation. Human U937 cells were treated with (lane 2) or without DTx (lane I) , or incubated with cycloheximide (lune 3) or NaN3/2-DOG (lane 4 ) for 18 h at pH 7.4, 37 “C. Toxin was added a t 75 ng/ml (200 pl/well), cycloheximide a t 250 ng/ml, and NaN3/2-deoxyglucose a t 2.5/5 mM. Molecular weight markers are indicated in base pairs.

2 6 10 14 18 TIME (hours)

FIG. 8. Kinetics of DTx-mediated cytolysis. 51Cr-Labeled U937 cells were exposed to DTx (75 ng/ml) for 18 h a t 37 “C under conditions identical to those employed in Fig. 7. At hourly intervals, aliquots were removed and assayed for “Cr release. Spontaneous release from toxin-free samples was measured at hourly intervals and subtracted as reported under “Experimental Procedures.”

FIG. 7. Time course of DTx-mediated DNA fragmentation. The first three lanes contain the following molecular weight markers: PstI-digested X-phage DNA; Alu-digested pUC8 DNA; HueIII-di- gested pUC8 DNA. Lanes 1-18 represent DNA extracted from DTx- treated cells (75 ng/ml toxin) after 1-18 h of incubation as described under “Experimental Procedures.” The sample shown in lune C is from untreated cells.

served in the presence of toxin (data not shown). We next determined the onset of DNA damage by examining the DNA profile of U937 target cells a t hourly intervals after DTx treatment. Fig. 7 shows that DNA fragmentation begins at -6 h. Parallel experiments were performed to determine the kinetics of DTx-mediated lysis under conditions identical to those employed in Fig. 7. Thus, Fig. 8, a time course of 51Cr release from human U937 cells, shows that cell lysis began -8-9 h after addition of toxin and reached a maximum a t 16 h. Essentially identical results were obtained when cell death was assayed by trypan blue staining (data not shown). In order to prove that the DNA fragmentation observed a t early time points did not arise from a small fraction of cells that may have undergone cytolysis, DTx-treated cells were sepa- rated into intact and leaky populations a t hourly intervals, and the intact (trypan-blue excluding) cell population was examined for DNA damage. Fig. 9 shows that the onset time of DNA fragmentation in cells harvested by this procedure closely matched the onset time observed in Fig. 7, supporting the conclusion that DNA breakdown has occurred in non- lysed cells and is not the consequence of cell death. These results establish that DNA fragmentation is an “early” event in DTx-mediated cytolysis, preceding lysis by 2-3 h. Hence,

S C 4 5 6 7 8 9 1 0

FIG. 9. Kinetics of DTX-triggered DNA fragmentation in nonlysed targ. cells. U937 cells exposed to DTx (75 ng/ml) were separated on F , Yypaque a t hourly intervals into lysed and non- lysed populat’ ~ n e s 4-20 represent DNA extracted from non- lysed cells r* ’ . : 0 h of incubation with DTx. Lanes S and C represent m ’ . . . weight markers (HaeIII-digested pUC8 DNA) and untreatt .! ’ espectively.

we conclud,. ’ DNA fragmentation represents a newly identified m in the cytolytic pathway of DTx.

DISCUSSION

A very large body of research experience supports the concept that the cytotoxic activity of DTx results from the capacity of the toxin to deliver an enzymically active subunit (A) into the target cell cytoplasm and then to effect inhibition of ribosomal activity by ADP-ribosylation of EF-2 (24). It has been generally assumed that this scenario fully explains the killing mechanism of DTx.

The data presented in this report allow two surprising conclusions to be drawn. First, DTx-triggered cytolysis is not a simple or straightforward consequence of translation inhi- bition. Second, the loss of cellular integrity is preceded by internucleosomal DNA fragmentation, a discovery that raises the possibility that DTx destroys target cells by a novel action. As shown in Table I, DTx triggers the lysis of a variety of target cells. This lysis, measured by the release of ”Cr or ”Se- labeled peptides, is not particularly rapid (half-time of several hours). It has been well documented (17-20, 25, 26) that in

15266 The Cytolytic Pathway of Diphtheria Toxin

order for DTx to inhibit translation activity in susceptible cells, the toxin must be exposed to a low pH environment, presumably supplied by the endosomal compartment. Our results show that DTx-mediated cytolysis is also dependent on acid conditions since NH&l had an inhibitory effect on the lytic activity of DTx, and this inhibition could be over- come by briefly lowering the external pH (Fig. 4).

In direct contrast to results obtained with DTx-treated cells, rapid and complete inhibition of target cell protein synthesis by treatment with cycloheximide, metabolic poison- ing with NaNa/2-deoxyglucose, or incubation in media defi- cient in essential amino acids failed to evoke comparable levels of cytolysis (Table 11). Clearly, cells whose protein synthesis has been irreversibly arrested will ultimately lyse, but our observations establish that the onset time of lysis under these conditions is considerably longer than 6-7 h. Indeed, little lysis is seen before 20-50 h (Fig. 2 4 ) . The data we have obtained with the K562 cell line further demonstrates severe inhibition of translation activity without the induction of cell lysis (Fig. 2, A and B ) . Although the reason is unclear as to why the K562 cell line is resistant to the lytic action of DTx, the explanation may be the failure to initiate DNA breakdown or the presence of a highly efficient DNA repair system.

Of considerable significance is our finding that DTx pro- voked extensive DNA fragmentation in target cells and that this process was initiated a t least 2-3 h before the loss of membrane integrity. The fragmentation of host DNA is a well-characterized biochemical feature of programmed cell death (27). According to this model, cell death inextricably commences when DNA is degraded to oligonucleosome-size fragments. This type of DNA fragmentation has been shown to precede cytolysis in various cell systems, including gluco- corticoid-mediated killing of thymocytes (28) and cytotoxic T-lymphocyte (CTL) killing of targets (9). Many investigators have demonstrated that DNA fragmentation is not merely the result of cell death since killing of target cells by heating, freeze-thawing, or lysis with antibody and complement does not induce DNA degradation (8, 9). Extensive membrane blebbing is another morphological marker for programmed cell death (27) that we consistently observed in DTx-treated target cells (data not shown).

Since DTx-triggered cytolysis is not, in fact, an automatic or straightforward consequence of translation inhibition, it must result either from (i) toxin-induced disruption of the cell boundary membrane(s) or alternatively from (ii) toxin- mediated perturbation of some other vital cell system. Asso- ciation of DTx (or toxin fragments) under acidic conditions with a variety of model membranes results in the formation of lesions (pores/channels) capable of being penetrated by a number of water-soluble solutes and ions (29-32). I t is con- ceivable that the long (>6 h) lag period between the initial exposure of the cell to DTx and the first signs of cytolysis results from the time taken for lesions introduced into specific endosomes to recycle back to the cell surface. However, even when DTx was driven directly into the plasma membrane by an acid pulse, a condition calculated to lead to toxin translo- cation to the cytoplasm within 20 s (33), there was still a time lag of 4-5 h before cytolysis occurred. This is slow in compar- ison with lysis triggered by melittin or Stuphylococcus a-toxin (34); indeed, even the complex sequence of events leading to lysis of nucleated cells by serum complement can be completed within 30 min (35). Another argument against direct mem- brane damage is derived from studies demonstrating that target cell lysis induced by isolated perforin granules (36) or activated complement (37, 38) occurs in the absence of chro-

matin breakdown or any of the morphological changes asso- ciated with programmed cell death. Thus, a mechanism for DTx-mediated lysis based solely on direct pore formation would not account for the critical changes in target cell integrity observed in this report. These facts coupled with our recent findings with CRM197, a mutant form of DTx in which a single amino acid substitution in the A domain renders it inactive as an ADPr-transferase yet fully active in membrane channel assays,’ lead us to favor a mechanism for toxin- induced cytolysis that involves a relatively slow accumulation of membrane damage caused by as yet to be defined intracel- lular events, rather than the direct formation of toxin lesions within the target cell boundary membrane.

The killing mechanism we currently favor is the existence of a second messenger substrate which is tightly coupled to the activation of an endogenous endonuclease. DTx-induced modification (e.g. ADP-ribosylation) of such a substrate could trigger the cell suicide response. In speculation, this pathway might involve the direct activation of a constitutive endonu- clease, the increased transport of critical ions such as Ca2+ into the nucleus, or the mobilization of essential cofactors for endonuclease activity. Of interest is the fact that protein synthesis is clearly not required for DTx-triggered DNA frag- mentation and cell lysis, resembling the cytotoxic properties exhibited by cytotoxic T-lymphocyte effector cells (9). This observation discounts the involvement of a “death gene” product as proposed in models for glucocorticoid- (39) and radiation-induced (11) killing of lymphocytes.

EF-2 is the only protein known to be ADP-ribosylated by physiological concentrations of DTx, but specificity for EF-2 is not absolute and additional proteins are ADP-ribosylated with low efficiency (40). In addition, a broad range of intra- cellular proteins have been identified that are modified by cytosolic and chromatin-bound (41) ADP-ribosyltransferases (EC 2.4.2.30) with a catalytic mechanism similar to DTx. These enzymes are responsible for the synthesis of the mono-, oligo-, and poly-ADP-ribosylated proteins that have been detected in practically every major compartment of the cell and in a variety of eukaryotic tissues (41). Although little is known about the precise identity and biological role of these modified proteins, EF-2 was recently identified as a target substrate (42). Thus, cellular components proven to be ADP- ribosylated by endogenous transferases under physiological conditions may likewise be susceptible to modification and hence regulation by DTx. We propose that a connection exists between substrate modification, induced directly or indirectly by DTx, and the activation of an endonuclease-mediated death program.

The DNA fragmentation profile of cells treated with DTx is identical to that of cells treated with TNF and dexameth- a ~ o n e , ~ molecules which do not inhibit translation activity. The fact that a phage-encoded bacterial product elicits DNA fragmentation of a sort that resembles profiles observed in response to several completely unrelated agents truly suggests the activation of a common cell death pathway. On the basis of data presented in this report, we propose that the conven- tional view of DTx-mediated killing must be expanded to include activation of a cytolytic pathway that displays the characteristic features of programmed cell death.

Acknowledgment-We thank Dr. David A. Campbell for his gen- erous assistance.

* M. P. Chang, B. Kagan, B. J. Wisnieski, and J. Bramhall, sub- mit,ted for publication.

M. P. Chang and B. J. Wisnieski, unpublished results.

T h e Cytolytic Pathway of Diphtheria Toxin 15267

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