0 1996 Wiley-Liss, Inc. Cytornetry 25:182-190 (1996)

Apoptosis in Hematopoietic Cells Is Associated With an Extensive Decrease in Cellular Phosphotyrosine Content That Can Be Inhibited by the Tyrosine

Phosphatase Antagonist Pervanadate Fridtjof Lund-Johansen, Tom Frey, Jeffrey A. Ledbetter, and Peter A. Thompson

Becton Dickinson Immunocytometry Systems, San Jose, California (F.L.-J., T.F., P.A.T.); Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington (J.A.L.)

Received for publication January 26, 1996; accepted June 8, 1996

In the present study, we investigated whether apoptosis in hematopoietic cells is associated with changes in cellular phosphotyrosine content. Mu- rine thymocytes and B cells, human leukemia cells, and normal peripheral blood leukocytes were in- duced to undergo apoptosis by treatment with spe- cific stimuli or by incubation in growth factor-de- prived medium. hidtiparameter flow cytometry was used to measure changes in phosphotyrosine con- tent that correlated with the appearance of features of programmed cell death, such as cell shrinkage, DNA fragmentation, and loss of membrane integ- rity. The results show that conditions that induced apoptosis also induced a dramatic decrease in cel- lular phosphotyrosine levels. Tyrosine dephospho- rylation preceded the loss of plasma membrane in-

tegrity and, in most cases, was temporally correlated with the onset of DNA hgmentation. The protein tyrosine phosphatase antagonist pervanadate had a dose-dependent inhibitory effect on both dephos- phorylation and apoptosis in murine thymocytes, which were treated with dexamethasone or with the topoisomerase I1 inhibitor etoposide. The results suggest that extensive tyrosine dephosphorylation is an intrinsic part of the apoptotic process of he- matopoietic cells and may be involved mechanisti- cally in the apoptosis induced by certain stimuli. 0 1996 Wiley-Liss, Inc.

Key terms: Apoptosis, vanadate, thymocytes, dexa- methasone, flow cytometry, tyrosine phosphoryla- tion

Apoptosis is associated with the controlled activation of processes that break down cellular components. Well- studied examples of such processes include internu- cleosomal fragmentation of DNA by endonucleases and activation of selected proteases (for reviews, see 3,5,15,25,30,32). Although certain features of apoptosis seem to indicate a final common pathway for physiologic cell death, the suicidal process is still poorly character- ized. The intracellular signaling pathways that lead to activation of the apoptotic program are also largely un- known. Tyrosine kinases and phosphatases, however, are likely to play important roles, because dysregulated tyrosine kinases induce immortality and resistance to chemotherapy in certain cell types (9,19). Studies show- ing the effects of tyrosine kinase and phosphatase inhib- itors on apoptosis also suggest that these enzymes may be targets for pharmacological manipulation of apoptosis (1,2,8,16,18,20,21,23,24,26,29,33,34). Most of these studies have focused on immediate changes in tyrosine phosphorylation induced by apoptotic stimuli. Thus, very little is known about changes in cellular phosphor- ylation status that occur later in the process, when cells

commit to apoptosis. Although they are important for understanding the effects of drug manipulation, such downstream events are difficult to study. One reason is that the phenomena occur over long time periods in an unsynchronized fashion. Any sample, therefore, may con- tain cells at different stages in the process, thereby com- plicating the analysis of data obtained by bulk analysis of the entire sample. Characterization of pathways impor- tant in the cellular commitment to apoptosis or to exe- cution of an apoptotic program in heterogeneous cell populations requires the development of techniques for single-cell analysis.

In the present study a novel approach was taken to study changes in protein tyrosine phosphorylation dur- ing apoptosis. New flow cytometric methods to study apoptosis and tyrosine phosphorylation at the single-cell

Address reprint requests to Fridtjof Lund-Johansen, DNAX Research Institute for Cellular and Molecular Biology, 901 California Avenue, Palo Alto, CA 94304. E-mail: johansen@dnax.org.

TYROSINE DEPHOSPHORYLATION DURING APOPTOSIS 183

level were combined to study directly whether apoptosis in hematopoietic cells is associated with changes in cel- lular phosphotyrosine levels. Murine thymocytes and B cells and human leukemia cells were induced to undergo apoptosis by treatment with specific stimuli or incuba- tion in growth factor-deprived medium. Changes in phos- photyrosine content that correlated with the appearance of features of apoptosis, such as cell shrinkage, DNA frag- mentation, and loss of membrane integrity, were mea- sured directly at the single-cell level. The results show that conditions that induced apoptosis also induced a dra- matic decrease in cellular phosphot yrosine levels. Ty- rosine dephosphorylation preceded the loss of plasma membrane integrity and, in most cases, was correlated tightly to the onset of DNA fragmentation. Inhibition of tyrosine dephosphorylation by pervanadate inhibited apoptosis in murine thymocytes treated with dexameth- asone or with the topoisomerase I1 inhibitor etoposide. These results suggest that extensive t yrosine dephospho- rylation is an intrinsic part of the apoptotic process of hematopoietic cells and may be linked mechanistically to the induction of cell death.

MATERIALS AND METHODS Reagents

Staurosporine (Sigma Chemical Co., St. Louis, MO; 0.5 mM in dimethylformamide) and sodium orthovanadate (NaVO,; Sigma; 1 M in H20) were kept at - 20°C. Saponin (Sigma; cat. no. S2149) and phosphotyrosine (Sigma) were dissolved in phosphate-buffered saline (PBS) and were kept at 4°C. Dexamethasone, etoposide, and cam- pothecin were also from Sigma. Ethidium monoazide (Molecular Probes, Eugene, OR) was kept at 1 mqml in ethanol at -20°C and was diluted 1:500 in PBS immedi- ately before use. Pervanadate was prepared fresh every day by mixing NaVO, with a tenfold molar excess of H202. Residual H,O, was then removed by diluting the mixture 1 : l O in PBS containing 1 mg/d catalase (Sigma; cat. no. ClO). The solution was kept on ice until it was used.

Antibodies PT66-fluorescein isothiocyanate (FITC; mIgG 1 an-

tiphosphotyrosine) was obtained from Sigma, and PY20 (mIgG2b antiphosphotyrosine) was obtained from Trans- duction Laboratories (Lexington, KY). The monoclonal antiphosphotyrosine antibody PY20 was conjugated to FITC and R-Phycoerythrin by Qudrat Nasraty and Barney Abrams at Becton Dickinson Immunocytometry Systems (BDIS). Streptavidin allophycocyanin (SA-APC) was from BDIS (San Jose, CA).

Cells Mouse thymocytes were prepared by mechanical dis-

ruption of a freshly excised thymus. Thymocytes were washed, resuspended in 50 ml of prewarmed (37°C) me- dium with 5 X lop5 M 2-mercaptoethanol, and plated in the wells of a 24-well tissue culture plate at 2 mVwell. Dexamethasone was used at 0.1 pM. Raji cells and HL-60

were kept in RPMI containing 10% fetal bovine serum (FCS), 1 mM glutamine, and 1 mM sodium pyruvate. This medium was supplemented with 5 X lop5 M 2-mercap- toethanol for culture of WEHI-231 cells.

Staining of Intracellular Phosphotyrosine Intracellular phosphot yrosine was detected by a mod-

ification of the method of Far et al. ( lo). Cells suspended at 1-5 X 106/ml in PBS containing 1% PBS-FCS were mixed with an equal volume of PBS containing 2% para- formaldehyde (PFA), 2 mM EDTA, 2 mM NaVO,, and 0.1% saponin. Following 10 min of incubation in a water bath held at 20-22"C, 4 ml PBS-FCS were added, the cells were centrifuged, and the supernatant was discarded. The cells were then stained on ice with fluorochrome- conjugated antiphosphotyrosine monoclonal antibody (mAb) for 30 min. In some experiments, 1 mM soluble phosphotyrosine was added to the cells prior to the anti-PY mAb to evaluate the level of nonspecific staining (see Figs. 1-10>. Immunostained cells were either run immediately or resuspended in 0.5% paraformaldehyde and kept at 4°C until analysis or further treatment for analysis of strand breaks.

Determination of Cell Membrane Integrity Using Ethidium Monoazide

In some experiments, the photoactivatable nonvital DNA dye ethidium monoazide was used to stain cells with increased membrane permeability prior to fixation and permeabilization. Cells ( 107/ml) were suspended in PBS-FCS containing 2 pg/ml ethidium monoazide. The samples were placed under a fluorescent lamp for 15 min to allow photoaffinity binding of ethidium monoazide to DNA. After two washes in PBS-FCS, the cells were stained with antiphosphotyrosine, as described above. Cells with increased cell membrane permeability were detected as ethidiumb"@" cells (13; see Figs. 1-10).

Detection of DNA Strand Breaks With the In Situ Terminal Deoxynucleotidyl Transferase Assay

DNA strand breaks in single-cells were detected by us- ing a modification of the method by Gorczyca et al. ( 14). Cells were stained with ethidium monoazide and an- tiphosphotyrosine antibodies, as described above, and were fixed for at least 12 h at 4°C in 0.5% paraformalde- hyde to stabilize the binding of the mAb. The cells were then washed once in PBS-FCS. Buffers for terminal deox- ynucleotidyl transferase ( TdT) were prepared by using a kit from Boeringer Mannheim (cat. no. 220 582). To each sample, a buffer (20 @I), which consisted of 0.5 p1 TdT solution (stock 25 U/pl), 1 pI CoCI, solution (stock 25 mM), 4 p,l 5 X TdT buffer, and 14.5 pl H,O, was added. In addition, 0.5 p1 biotin-16-dUTP (stock 50 mM; Boeh- ringer Mannheim, cat. no. 1093 070) was added to each sample before incubation in a water bath that was held at 37°C for 30 min. The cells were then washed twice in PBS-FCS and stained on ice with SA-APC. In all experi- ments, the TdT was omitted from at least one sample to evaluate nonspecific binding of biotin- 16-dUTP and SA-

184 LUNDJOHANSEN ET AL.

APC (see Figs. 1-10). Cells that had specifically incorpo- rated biotin-16-dUTP in the presence of TdT were con- sidered to have DNA strand breaks (14). In some experiments, propidium iodide (10 pg/ml) was added to allow measurement of DNA content in addition to cellu. lar phosphotyrosine levels and the presence of DNA strand breaks.

Flow cytometry. The cells were analyzed by using a modified FACSscan flow cytometer equipped with a red Helium-Neon laser in addition to the standard 488 nm argon laser to allow simultaneous measurement of FITC fluorescence, allophycocyanin fluorescence, and fluores- cence from the DNA stains ethidium monoazide and pro- pidium iodide.

Western blotting. Thymocytes were washed once in Tris-buffered saline, centrifuged, and lysed in a buffer containing 150 mM NaCI, 50 mM Tris, 1% NP40, 0.25% sodium deoxycholate, 2 mM EGTA, 1 mM PMSF, 1 mM sodium orthovanadate, and 20 pg/ml aprotinin. Cell de- bris and nuclei were removed by centrifugation at X 10,OOOg for 10 min. Fifty micrograms of cellular pro- tein from each sample were boiled in sodium dodecyl sulfate (SDS) sample buffer with dithiothreitol, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; 8.5% ), and subsequently electroblotted to PVDF mem- brane. After blocking nonspecific binding sites with a buffer containing 6% bovine serum albumin (BSA), the immunoblots were incubated with polyclonal rabbit an- tiphosphotyrosine (0.25 Fg/ml). The blots were rinsed twice in 10 mM Tris, pH 7.4, and 0.9% NaCl (rinse buffer), once in rinse buffer containing 0.05% NP40, and twice in rinse buffer. The blots were incubated with 0.5 pCi/ml '"I protein A (ICN Radiochemicals, Costa Mesa, CA) in blocking buffer containing 6% BSA. The immuno- blots were washed as described above and were exposed to film (X-AR; Eastman Kodak Co., Rochester, NY) at -70°C with intensifying screen overnight.

RESULTS Single-Cell Measurement of Phosphotyrosine Content

To measure phosphotyrosine content on a single-cell level, a flow cytometric technique was developed using a mAb that is specific for phosphotyrosine, PT-66, conju- gated directly to FITC (aPY-FITC). Several experiments demonstrate that this assay provides a valid measure of intracellular phosphotyrosine content (Fig. 1 ). Staining of permeabilized cells with aPY-FITC yielded significantly higher cellular fluorescence than that seen with 1) an FITC-conjugated isotype control mAb (Fig. 1B,H), 2) staining of permeabilized cells with aPY-FITC blocked by preincubation with excess phosphotyrosine (Fig. 1B,D), or 3) aPY-FITC staining of nonpermeabilized cells (Fig. 1A,B). Incubation of murine thymocytes with the potent kinase inhibitor staurosporine (27) resulted in a reduc- tion of fluorescence intensity of permeabilized cells stained with aPY-FITC to near background levels (Fig. lB,D,F) but was without effect on aPY-FITC staining of nonpermeabilized cells (Fig. lA,lE). This is in agreement

b 25 3 0 u I4 4 w u

UNFIXED THYMOCYTES

FIXED AND PERMEABILIZED

THYMOCYTES

stauro- sporine

0 -1 F : +500nM]

sporine

0

PT-66 FITC

IgGl FITC

FIG. 1. Staining of murine thymocytes with the antiphosphotyrosine antibody PT-66 (A-F) and isotype control (G,H). Left: Staining of un. fixed cells. Right Cells that were stained after fixation and permeabili. zation, as described in Materials and Methods. The dashed lines indicate the peak channel for the F'Y staining of unfixed and permeabilized cells, respectively. C and D show the staining in the presence of 1 mM soluble phosphotyrosine. E and F show cells that were preincubated for 30 min at 37°C with 500 nh4 staurosporine prior to the staining. The results are representative of four experiments.

with the results of Garcia et al., suggesting that there is an active turnover of phosphotyrosine in resting T cells ( 12). A separate mAb that is specific for phosphotyrosine, PY20, conjugated directly to FITC or phycoerythrin, yielded results similar to those demonstrated with PT66- FITC (data not shown).

Corticosteroid-Induced Apoptosis in Thymocytes is Associated With a Dramatic Decrease in Cellular

Phosphotyrosine Content To investigate whether induction of apoptosis leads to

changes in cellular phosphotyrosine levels, corticoste- roid-induced apoptosis in murine thymocytes was chosen as an initial model system (3 1 ). Freshly isolated thymo- cytes were cultured in the presence or in the absence of the corticosteroid dexamethasone and were assayed for cellular phosphotyrosine content at selected time points. Dexamethasone-treated cultures demonstrated a time-de- pendent accumulation of cells with dramatically lowered cellular phosphotyrosine content (Fig. 2B,C, and data not shown). The relative paucity of cellular events between the discrete phosphotyrosine'ow and the phosphotyro-

TYROSINE DEPHOSPHORYLATION DURING APOPTOSIS 185

PT-66 FITC

FIG. 2. Effect of dexamethasone on thymocyte PY levels. Murine thy- mocytes were incubated for 5 h in medium only (A), for 2.5 h in medium followed by 2.5 h in medium containing 0.1 KM dexamethasone (B), or for 5 h in medium containing 0.1 KM dexamethasone (C). The cells were then permeabilized and stained with anti-F'Y, as described in Materials and Methods. The dashed line indicates the peak channel of the PY staining of cells that were incubated with medium only.

FIG.

sineh'@' cell populations shown in Figure 2B,C argues for rapid transit of cells from the phosphotyrosinehi* to the phosphotyrosine'ow state. Parallel Western blot analysis showed a decrease in the staining of all bands with an- tiphosphotyrosine antibody in dexamethasone-treated cells and confirmed that the results did not represent an interference of dexamethasone with the assay system (Fig. 3A,B). Coomassie staining of the blots showed iden- tical staining of proteins from cells that were incubated in medium or with dexamethasone, suggesting that the de- crease in phosphotyrosine content in dexamethasone- treated cells was not due to general proteolysis (Fig. 4).

Breakdown of the plasma membrane, which is demon- strable by using nonvital dye staining, is a late event in apoptosis (5). A trivial explanation for our results could be that the reduction in cellular phosphotyrosine content follows membrane permeabilization and protein leakage. To examine this directly, use was made of ethidium monoazide (EMA), a cell-impermeable nucleic acid dye that can be cross linked covalently to its targets upon photoactivation of the azide group. This staining is stable when cells are permeabilized to allow intracellular stain-

MW kDa

Staining a Western t

A B

ts from dexamethasone-tre thymo- cytes with anti-PY polyclonal antibody. Murine thymocytes were incu- bated for 5 h in medium only (A) or for 5 h in medium containing 0.1 pM dexamethasone (B). Western blotting and antiphosphotyrosine staining were performed as described in Materials and Methods.

ing (13) . Simultaneous evaluation of EMA staining and cellular phosphotyrosine content revealed that the de- crease in cellular phosphotyrosine content precedes apoptosis-induced breakdown of the plasma membrane (Fig. 5A,B).

Endonuclease-mediated cleavage of cellular DNA is a common observation in apoptosis, although the extent of DNA fragmentation varies across model systems (15 ,31) , and it is thought to represent a common endpoint in the execution of apoptotic programs. In situ end labeling of cleaved DNA with labeled nucleotides, mediated by TdT, and with detection of labeled cells by flow cytometry is a sensitive assay for DNA strand breaks (14) . To define more precisely the relationship between the accumula- tion of DNA strand breaks and the observed decrease in cellular phosphotyrosine content, the two parameters were measured simultaneously in the thymocyte system.

186 LUNDJOHANSEN ET AL.

FIG. 4. Staining of Western blots from dexamethasone-treated thymo- cyfes with Coomassie blue. Murine thymocytes were incubated for 5 h in medium only (control) or for 5 h in medium containing 0.1 phi dexa- methasone (DEX). Western blotting and anti-Coomassie blue staining was performed as described in Materials and Methods.

The data from these experiments revealed that DNA strand breakage and decreased cellular phosphotyrosine content were effectively coincident (Fig. 6A,B) in thymo- cytes. In other model systems, however, tyrosine dephos- phorylation seemed to precede DNA fragmentation (see below). Thus, apoptosis, defined by the demonstration of DNA strand breaks induced in murine thymocytes by cor- ticosteroid treatment, is associated with a dramatic de- cline in cellular phosphotyrosine content.

Dramatic Reductions in Cellular Phosphotyrosine Content Are Commonly Observed

During Apoptosis To examine whether the decrease in phosphotyrosine

content during apoptosis is peculiar to the thymocyte model or reflects a general characteristic of cells under- going apoptosis, we measured cellular phosphotyrosine content and DNA fragmentation in three additional models of apoptosis in hematopoietic cells (Fig. 7). These in- cluded campothecin treatment of HL-60 cells (Fig. 7A,B; 14), surface Ig engagement of WEHI-231 cells with anti-

mouse IgM (Fig. 7C,D; 6), and serum starvation of Raji cells (Fig. 7E,F; 17). In all cases, induction of apoptosis was associated with the appearance of a phosphotyrosine'ow cell population (Fig. 7). Intriguingly, upon apoptosis in- duction in all of these models, there was a population of phosphotyrosinelow cells with no discernible DNA strand breaks. This was most clearly seen in the Raji model sys- tem (Fig. 7E,F). These phosphotyrosinelow cells without DNA strand breaks had the lowered forward-angle light- scatter characteristics of apoptotic cells (22; data not shown). The data in Figure 7 suggest that dramatic re- ductions in cellular phosphotyrosine content are a com- mon feature of apoptosis and either temporally precede, or are mechanistically independent of, endonuclease- mediated DNA cleavage.

Dose-Dependent Inhibition of Apoptosis by a Protein Tyrosine Phosphatase Inhibitor

To evaluate whether tyrosine dephosphorylation was functionally linked to apoptosis, thymocytes were incu- bated with 0.1 FM dexamethasone or 20 FM etoposide in the presence of varying concentrations of the protein tyrosine phosphatase inhibitor pervanadate (28). The data Erom these experiments demonstrate dose-depen- dent inhibition of apoptosis, defined by the demonstra- tion of DNA strand breaks (Fig. 8), decreased staining with the nuclear dye LDS-751 ( 1 I), or lowered forward- angle scatter (data not shown). In both model systems, the concentration of pervanadate (30-60 FM) that yielded the greatest inhibition of apoptosis, thus defined (30-60 pM), was also the lowest concentration that pre- vented the decrease in cellular phosphotyrosine content (Figs. 8B,D, 9, 10). These data suggest that apoptosis in- duced in thymocytes by dexamethasone or etoposide re- quires protein tyrosine dephosphorylation either for the commitment of cells to undergo apoptosis or for the ex- ecution of apoptotic programs leading to DNA strand breakage. Higher concentrations of pervanadate resulted in DNA strand breakage in untreated control thymocytes (Fig. 8G). Under these conditions, dramatic decreases in cellular phosphotyrosine content were no longer evident (Figs. 8G, 9, 10). This suggests that dephosphorylation is not required for action of the enzymes responsible for DNA fragmentation.

DISCUSSION The results from the present study show that apoptosis

of hematopoietic cells is associated with a dramatic de- crease in cellular phosphotyrosine content. The majority of proteins that were phosphorylated in resting cells, as determined by Western blotting, were affected, and the decrease was not due to a general breakdown of cellular proteins. Multiparameter flow cytometric analysis showed that the loss of intracellular phosphotyrosine occurs in cells with intact plasma membranes, thereby eliminating artifacts from protein leakage or soluble phosphatases. Simultaneous measurement of phosphotyrosine levels and DNA strand breaks provided direct evidence that the de- phosphorylation occurs in cells that are apoptotic. Fur-

TYROSINE DEPHOSPHORY LATION DURING APOPTOSIS 187

Medium Dex. 5.0h

ETHIDIUM MONO-AZIDE FLUORESCENCE

RG. 5. Combined determination of cell membrane integrity and cel- lular phosphotyrosine content. Thymocytes were incubated for 5 h in medium only (A) or with medium containing 0.1 pM dexamethasone (B). The cells were then stained with ethidium monoazide, permeabi- lized, and stained with antiphosphotyrosine, as described in Materials and Methods. Cells that were considered to have increased ethidium monoazide staining and, thus, membrane defects are to the right of the dashed lines. The results are representative of four experiments.

u k 2

rr Medium 0

-1A icells with

- Dex. 5.0h

c)

m 0 4

N 0

0 d

0

El 1 lo3 to4

BIOTIN d-UTP + SA-APC FIG. 6. A-D: Combined determination of DNA strand breaks and cel-

lular phosphotyrosine content. Thymocytes were incubated for 5 h in medium with (B,D) or without (A,C) 0.1 pM dexamethasone. Combined staining of phosphotyrosine and DNA strand breaks w a s performed as described in Materials and Methods. A and B show cells that were stained in the presence of terminal deoxynucleotidyl transferase. C and D show the background staining in samples where the enzyme was omitted from the reaction buffer. Cells with higher staining in the presence of terminal transferase are to the right of the dashed lines and are considered to have DNA strand breaks. The events were gated on ethidium monoazided" cells (see Fig. 4). The results are representative of four experiments.

thermore, the process is likely to have rapid kinetics, because cells at any time point were found to be distrib- uted in two discrete populations with high and low phos- photyrosine levels. Because the decrease in phosphoty-

CONTROL INDUCED APOPTOSIS

0 - M 0

N 0 - - 0

0 0

4

1

BIOTIN d-UTP + SA-APC

FIG. 7. Correlated measurement of cellular PY levels and the extent of DNA strand breaks in various cell types undergoing apoptosis. Com- bined staining for measurement of cellular PY levels and DNA strand breaks was performed as described in Materials and Methods. A,B: HL-60 cells incubated for 4 h in the absence (A) or presence (B) of 0.1 p@nl campothecin. CP: WEHI-231 cells incubated for 24 h in the absence (C) or presence of 10 pP/d goat-antimouse IgM (D). E,F: Raji cells cultured for 48 h in medium with 10% (E) or 1% fetal bovine serum (F). The results are representative of four experiments.

rosine staining was inhibited by pervanadate, the results above suggest that there is rapid dephosphorylation of proteins by intracellular tyrosine phosphatases during apoptosis of hematopoietic cells.

Although protein tyrosine dephosphorylation seems to be an intrinsic part of the suicide program in hematopoi- etic cells, the exact role of this step for the induction of cell death remains to be determined. Extensive dephos- phorylation does not seem to be essential for activation of the enzymes responsible for DNA fragmentation, because high concentrations of pervanadate greatly increased cel- lular phosphotyrosine content and also induced DNA fragmentation. The observed dephosphorylation, there fore, may be a consequence rather than a requisite step of the apoptotic process. The differences in the relative ki- netics of tyrosine dephosphorylation and DNA fragmen- tation observed between the models could also indicate

LUNDJOHANSEN ET AL. 188

Fig. 8

v, m i( a

FIG. a

U-

rc 15pM

- 4 I

& 7-77 ! 10

* 30pM

+ DEXAMETHASONE

d 125pM

14

BIOTIN d-UTP + SA-APC

A-H: Effect of nervanadate on dexamethawne-induced de- . ~~

phosphorylation and DNA strand breaks. DNA strand breaks and cellular phosphotyrosine content were measured as described in Materials and Methods and in the legend to Figure 6. Thymocytes were incubated for 5 h in medium containing 0.1 FM dexamethasone (bottom) or not (top).

that the two phenomena are unrelated. On the other hand, DNA fragmentation never preceded dephosphory- lation in the absence of pervanadate. It is possible that the large population of Raji cells with low phosphotyrosine content but without detectable DNA fragmentation in Figure 7 reflects the slow rate of apoptosis in this model. This may allow the individual events to be resolved bet- ter than in the other models. Thus, dephosphorylation may precede DNA fragmentation in all models, although strong support for this interpretation was only obtained by the results from Raji cells. The view that tyrosine dephosphorylation is a necessary step upstream of DNA fragmentation in some systems is supported by the results showing that low concentrations of pervanadate inhibited apoptosis in thymocytes. It is intriguing that pervanadate inhibited apoptosis induced both by dexamethasone and by etoposide, because the two compounds induce apop- tosis by different mechanisms. Etoposide induces apop- tosis by inhibiting topoisomerase I1 and activating a p53- dependent pathway, whereas dexamethasone signals are

A and B show cells that were incubated in the absence of pervanadate. C-H show cells that were incubated in the presence of indicated con- centrations of pervanadate. The results are representative of four exper- iments.

mediated through the glucocorticoid receptor and lead to p53-independent apoptosis (4). It is tempting to spec- ulate that the observed dephosphorylation may reflect a common phosphatase-dependent step downstream in the two pathways. Further investigation of the effect of ty- rosine phosphatase inhibitors in different models of apop- tosis may address this possibility and determine whether these enzymes represent possible pharmacological tar- gets for the manipulation of apoptosis.

Previous studies on the role of tyrosine phosphoryla- tion in apoptosis have reported variable results, depend- ing on the model of apoptosis used. Apoptosis induced by engagement of certain cell surface receptors, such as lym- phocyte antigen receptors and the Fas antigen, is fre- quently inhibited by tyrosine kinase inhibitors or by an- tisense nucleotides to tyrosine kinases (7,8,33). In contrast, tyrosine kinase inhibitors have been shown to prevent certain growth factors from rescuing cells from apoptosis in culture (2,20,26). Tyrosine phosphorylation is among the first events to occur after stimulation via the

TYROSINE DEPHOSPHORYLATION DURING APOPTOSIS 189

80

70

60

50

40

30

20

10

(

I Medium

r O.1pM Dexamethasone

1

0 3.5 7.5 1 15

30 60 125 250

"" I Medium

0 O.1pM T Dexamethasone

- 1 n / 50

40

30

20

I0

0 0 3.5 7.5 15 30 60 125 250

Pervanadate conc pM FIG. 9. Dose-dependent effects of pervanadate on dexamethasone-

induced dephosphorylation and DNA strand breaks. Cells were incu- bated for 5 h in the presence (open bars) or absence (solid bars) of 0.1 pM dexamethasone and the indicated concentrations of pervanadate. DNA strand breaks and cellular phosphotyrosine content were measured as described in Materials and Methods and in the legend to Figure 6. The bar plots show percentage of phosphotyrosine low cells (upper plot) and cells with DNA strand breaks (lower plot). The bars and error bars represent the mean and standard deviation of three experiments, respec- tively.

majority of these receptors, and the effects of inhibitors on apoptosis correlate frequently with the ability to pre- vent rapid tyrosine phosphorylation after engagement of the receptors. It seems likely that the results from those studies reflect interference with signal transduction path-

80 Phosphotyrosine l o w cells

60 -

40 -

20 -

n+

- - 0 Cells with DNA strandbreakr

" 0 15 30 60 Pervanadate conc pM

FIG. 10. Dose-dependent effects of pervanadate on etoposide-in- duced dephosphorylation and DNA strand breaks. Cells were incubated for 5 h in the presence of 20 (rM etoposide. DNA strand breaks and cellular phosphotyrosine content were measured as described in Mate- rials and Methods and in the legend to Figure 6. The solid bars show the mean percentage of phosphotyrosine low cells, and the open bars show the mean percentage of cells with DNA strand breaks. The bars and error bars represent the mean and standard deviation of three experiments, respectively.

ways immediately downstream of the respective recep- tors rather than the apoptotic process, per se. A recent report showed that long-term treatment with the protein tyrosine phosphatase inhibitor phenylarsine oxide could inhibit spontaneous apoptosis in neutrophils and in eosinophils (34). It was suggested that this effect of phe- nylarsine oxide was due to stimulation of tyrosine phos- phorylation in a way similar to that induced by granulo- cyte-macrophage colony-stimulating factor (GM-CSF). The data presented here may indicate that this effect was could be due to interference with tyrosine phosphatases in these model systems. This view is supported by a study showing that phosphotyrosine and Zn2 + effectively in- hibited apoptosis in a cell-free system at concentrations that inhibit tyrosine phosphatases (23). Collectively, the data in these studies suggest that tyrosine phosphatases may be involved in the apoptotic process at a level down- stream immediate of receptor signaling. This should prompt a more detailed investigation of the function of tyrosine phosphatses at different stages in apoptosis.

In conclusion, the present study shows that apoptosis is associated with a dramatic decrease in cellular phospho- tyrosine content. Antiphosphotyrosine staining of perme- abilized cells allows detection of a death-associated event that occurs prior to loss of cell membrane integ- rity. The inhibitory effect of pervanadate on dephospho- rylation suggests that the loss of cellular phosphotyrosine may be mediated by tyrosine phosphatses and that acti-

190 LUNDJOHANSEN ET AL.

vation of one or more phosphatases is an intrinsic part of the suicide program in hematopoietic cells.

ACKNOWLEDGMENTS We thank Jim Bishop and Louis Picker for helpful dis-

cussions and Ken Davis, Barny Abrams, and Qudrat Nas- rati for conjugation of monoclonal antibodies.

LITERATURE CITED 1. Azuma Y, Onishi Y, Sat0 Y, Kizaki H. lnduction of mouse thymoqte

apoptosis by inhibitors of tyrosine kinases is associated with dephos- phorylation of nuclear proteins. Cell Immunol 152:271-278, 1993.

2. Bergamaschi G, Rosti V, Danova M, Ponchio L, Lucotti C, Cazzola M: Inhibitors of tyrosine phosphorylation induce apoptosis in human leukemic cell lines. Leukemia 72012-2018, 1993.

3. Bonner C, Oldenburg N, Cidlowski J: The role of DNA 6ragmentation in apoptosis. Trends Cell Biol 5:21-26, 1994.

4. Clarke AR, Purdie CA, Harrison DJ, Morris RG, Bird CC, Hooper ML, Wyllie AH: Thymocyte apoptosis induced by p53-dependent and independent pathways [see comments]. Nature 362849-852,1993.

5. Cohen JJ: Apoptosis. Immunol Today 14:126-130, 1993. 6. Cuende E. Ales MJ, Ding L, Gonzalez GM, Martinez C, Nunez G:

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