Elastase Complex during Staurosporine-Induced Apoptosis: Role in the Generation of Nuclear L-DNase II Activity

Experimental Cell Research 254, 99 –109 (2000) doi:10.1006/excr.1999.4737, available online at http://www.idealibrary.com on

Nuclear Translocation of a Leukocyte Elastase Inhibitor/Elastase Complex during Staurosporine-Induced Apoptosis: Role in the Generation of Nuclear L-DNase II Activity Chafke´ Ahmed Belmokhtar,* Alicia Torriglia,† Marie-France Counis,† Yves Courtois,† Alain Jacquemin-Sablon,‡ and Evelyne Se´gal-Bendirdjian* ,1 *U496 INSERM, Institut d’He´matologie, Hoˆpital Saint-Louis, 1 avenue Claude Vellefaux, 75010 Paris, France; †U450 INSERM, De´veloppement, Vieillissement et Pathologie de la Re´tine, U450 INSERM affilie´e CNRS, Association Claude Bernard, 29 rue Wilhem, 75016 Paris, France; and ‡UMR1772 CNRS, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94800 Villejuif, France

membrane potential, cell shrinkage, plasma membrane blebbing, and phosphatidylserine externalization [2–7]. It is generally accepted that several of these morphological and biochemical changes result from specific enzymatic activation (e.g., proteases, DNases). During apoptosis, endonucleases present in the cell cleave the DNA at sites located between nucleosomal units, thus resulting in the characteristic ladder of DNA fragments [8]. Several attempts have been made to characterize the nucleases that are responsible for this fragmentation [9 –21]. For instance, DNase I and Nuc 18 have been reported to be associated with the apoptotic DNA cleavage [13, 17, 18, 21]. Recently, a new Ca 21/Mg 21-dependent endonuclease (NUC70) was purified from human hematopoietic cells [22]. It was shown that NUC70 is a cytoplasmic protein that is translocated to the nucleus early in apoptosis. DNase II has also been proposed to be involved in specific cases of apoptosis [23–26]. Recently, it has been shown that specialized proteases could be involved in DNase activation. A caspase-activated deoxyribonuclease (CAD) 2 and its inhibitor (ICAD) have been identified in the cytoplasmic fraction of mouse lymphoma cells and cloned [27, 28]. The caspase cleavage of ICAD resulted in the release of CAD that was shown to be responsible for the internucleosomal DNA degradation during apoptosis. These results provide one example linking the apoptotic caspases to DNA degradation. Furthermore, they identify a specific pathway initiated by caspase cleavage which terminates in activation of a nuclease responsible for internucleosomal DNA digestion.

Using L1210 murine leukemia cells, we have previously shown that in response to treatment with drugs having different targets, apoptotic cell death occurs through at least two different signaling pathways. Here, we present evidence that nuclear extracts from staurosporine-treated cells elicit DNase II activity that is not detected in nuclear extracts from cisplatintreated cells. This activity correlates with the accumulation of two nuclear proteins (70 and 30 kDa) which are detected by an anti-L-DNase II antibody. Partial purification of this DNase II activity suggests that the 30-kDa protein could be the nuclease responsible for staurosporine-induced DNA fragmentation. The 70-kDa protein is also recognized by an anti-elastase antibody, suggesting that it carries residues belonging to both L-DNase II and elastase. Since previous findings showed that L-DNase II was generated from the leukocyte inhibitor of elastase, we propose that the 70-kDa protein results from an SDS-stable association between these two proteins and is translocated from the cytoplasm to the nucleus during staurosporineinduced apoptosis. © 2000 Academic Press Key Words: apoptosis; endonuclease; L-DNase II; elastase; cisplatin; staurosporine; L1210 leukemia cells.

INTRODUCTION

The process of apoptosis, which normally functions to maintain homeostasis in cell populations, can be initiated by a variety of stimuli, including withdrawal of growth factors, various cytokines, ionizing radiation, and cytotoxic drugs [1]. During this process, cells show characteristic morphological changes, such as chromatin condensation, nuclear fragmentation, internucleosomal cleavage of DNA, decrease in mitochondrial

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Abbreviations used: CAD, caspase-activated deoxyribonuclease; cisplatin, cisP, cis-diamminedichloroplatinum(II); G6PDH, glucose6-phosphate dehydrogenase; ICAD, inhibitor of caspase-activated deoxyribonuclease; LEI, leukocyte elastase inhibitor; L-DNase II, LEI-derived DNase II; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; TRITC, tetramethylrhodamine isothiocyanate.

1 To whom reprint requests should be addressed. Fax: 33(1)42 40 95 57. E-mail: [email protected].

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0014-4827/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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Recent work by Torriglia et al. [26] established another possible link between the activation of endonucleases and proteases which are activated during apoptosis. In order to study the contribution of DNase II in the apoptotic process, Torriglia et al. [26] purified and cloned the cDNA encoding DNase II extracted from porcine spleen. They reported that this enzyme derives from an anti-protease of the serpin family, leukocyte elastase inhibitor (LEI), by posttranslational modifications. The anti-protease activity was lost during its conversion to active DNase II. As this DNase II was different from other DNases II already described [29, 32], and it was derived from LEI, it was named L-DNase II (for LEI– derived DNase II). These two reports exemplify the diversity of signaling pathways conducive to cell death. Although diverse apoptotic stimuli initially trigger different signal transduction pathways, they may converge to activate a final common death pathway found in all forms of apoptosis. Alternatively, different parallel pathways may be activated by different stimuli and do not overlap from the initial stimulus to the ultimate death of the cell. Indeed, we have previously shown that at least two different apoptosis pathways could be initiated by two unrelated inducers (cisplatin and staurosporine), each leading to the activation of two distinct endonucleases which differ with respect to both ion dependency and intracellular localization [33, 34]. In our work, we propounded the idea that a cytoplasmic endonuclease activity whose specificity resembled that of DNase II (inhibition by Ca 21/Mg 21; optimal activity at acidic pH) was a functional feature of the staurosporine pathway. In keeping with this hypothesis, we have now gained further functional evidence. First, we identify two proteins of 70 and 30 kDa, respectively, whose accumulation in the nuclei of staurosporine-treated cells coincides with both the induction of apoptosis and the development of nuclear DNase II activity. However, only the 30-kDa protein is likely to be the nuclease responsible for staurosporine-induced DNA fragmentation. Second, the 70-kDa protein has been shown to result from an SDS-stable association between the proteinase (elastase) and its inhibitor (LEI) from which L-DNase II could be generated after appropriate stimuli. These results, which exquisitely corroborate biological and biochemical features of the serpin family, are discussed. MATERIALS AND METHODS Chemicals. Cisplatin was purchased from Lilly Laboratory and staurosporine from Boehringer Mannheim (Meylan, France). Stock solutions (1.67 mM cisplatin in 150 mM NaCl; 2.0 mM staurosporine in DMSO) were stored at 220°C. Electrophoresis grade agarose was from Pharmacia (Biotech, Orsay, France). Proteinase K and DNasefree RNase were obtained from Boehringer Mannheim and the DNA molecular weight marker (123-bp ladder) from Gibco/BRL (CergyPontoise, France). Calf thymus DNA and bovine pancreatic DNase I

were from Sigma, porcine spleen DNase II was from Worthington (Coger, France), and porcine elastase was from ICN Biomedicals Inc. (Coger, France). Polyclonal antibodies against DNase I and elastase were from Rockland (Tebu, France). Polyclonal antibodies against glucose-6-phosphate deshydrogenase were from Sigma. Polyclonal antibodies were raised against L-DNase II purified from porcine spleen DNase II, as previously described [25]. Cell culture and media. L1210 murine leukemia cells (parental L1210/0 and cisplatin-resistant L1210/DDP 10 cells) were generously provided by Dr. A. Eastman (Department of Pharmacology, Dartmouth Medical School, Hanover, NH) [35–36]. The resistant cell line was selected in the presence of stepwise increasing cisplatin concentrations and was thereafter maintained in drug-free medium. Cells were grown in suspension in Dulbecco’s minimal essential medium (DMEM, Gibco/BRL, Cergy-Pontoise, France) supplemented with 15% fetal calf serum (Gibco/BRL), 2 mM glutamine, and antibiotics (streptomycin, 200 mg/ml; penicillin, 200 U/ml). The cells were grown in a humidified 5% CO 2 atmosphere. Incubation with drugs and cytotoxicity evaluation. Exponentially growing cells (approximately 3 3 10 5 cells/ml) were treated with various concentrations of either cisplatin (1 mM for L1210/0 and 50 mM for L1210/DDP 10) or staurosporine (50 nM for both cell lines) for up to 3 days. Cell numbers were determined with a Coulter counter. Drug-induced cytotoxicity was estimated using the MTT colorimetric assay, as previously described [34]. The drug concentrations used in these cell lines were already shown to induce more than 70% cytotoxicity. Immunofluorescence microscopy. Treated and control cells (1 3 10 6 per slide) were rinsed in PBS and fixed for 30 min at 220°C with acetone:ethanol (v:v). After permeabilization by 0.25% PBS–Triton X-100 for 5 min at 4°C, the cells were incubated overnight at 4°C with the anti-L-DNase II antibody (dilution 1:25). The cells were stained for 1 h by a secondary antibody coupled to TRITC (tetramethylrhodamine isothiocyanate; Biosys, Compie`gne, France) and excited with the 576-nm line. The nuclei were stained for 5 min with propidium iodide (1 mg/ml). Images were then collected using an Acas-70 (Meridian Instruments, Okemos, Michigan). To exclude nonspecific labeling that could result from the nonspecific fixation of the second antibody, the labeling was performed by omitting the anti-LDNase II antibody. In the confocal analysis 10 optical slices were scanned at 1- or 2-mm z steps. Morphological analyses of cells were performed under light microscopy after May–Gru¨nwald–Giemsa staining. Analysis of DNA fragmentation. DNA was isolated from 2 3 10 6 cells by a salting out procedure described by Miller et al. [38], modified as previously described [33]. Preparation of cytoplasmic and nuclear extracts. Nuclei were isolated according to the method of Nieto and Lopez-Rivas [39], as previously described [33]. Briefly, nuclei were isolated by resuspension of 4 3 10 6 cells in 1.5 mM MgCl 2 after three washings in PBS (pH 7.4). Cells were then incubated in this solution at 4°C for 15 min and disrupted by 20 strokes of a tight-fitting Dounce homogenizer (Kontes, pestle B) to burst open cells and liberate intact nuclei. The release of cytoplasm-free nuclei was monitored by phase-contrast microscopy after staining of a small aliquot of the lysate with trypan blue. Lysates were then centrifuged (1500 rpm at 4°C) and the supernatants were collected as cytoplasmic fractions. Pellets (nuclei fractions) were then washed three times by centrifugation/resuspension in the lysis buffer and resuspended in 10 mM Tris–HCl (pH 7.4), 200 mM sucrose, and 60 mM NaCl. The isolated nuclei were either used intact as substrates in the cell-free assay or lysed for Western blot analysis. Nuclei protein extracts were prepared by lysing isolated nuclei in 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 20 mM EDTA, and 50 mM Tris–HCl (pH 7.4) for 30 min at 4°C. After centrifugation at 14,000g for 15 min, the supernatant (nuclear extract) was recovered.

LEI/ELASTASE COMPLEX IN STAUROSPORINE-INDUCED APOPTOSIS Western blot analysis. Protein concentration in cytoplasmic and nuclear extracts was determined using the bicinchoninic acid protein assay reagent. For L-DNase II and elastase detection, samples (100 mg/lane) were fractionated by 12% SDS–PAGE in a Tris– glycine running buffer and blotted on polyvinylidene fluoride membranes (Boehringer Mannheim). The loading homogeneity and transfer efficiency were checked by staining the membrane with Ponceau red and the gel with Coomassie blue. The membranes were preblocked for 2 h at room temperature in PBS containing 5% nonfat milk powder. Afterward, the blots were incubated overnight at 4°C with anti-L-DNase II (dilution 1/660) or anti-elastase (40 ng/ml) antibodies diluted in PBS containing 0.5% nonfat milk powder, followed by a 1-h incubation with horseradish peroxidase-conjugated goat antirabbit IgG (0.025 mg/ml, Calbiochem). Detection was performed using the chemiluminescence procedure (ECL, Amersham, les Ulis, France), according to the manufacturer’s recommendations. To control the specificity of the anti-L-DNase II antibody, we substituted an equivalent dilution of rabbit preimmune serum for the antisera. The purity of the nuclear extracts was checked by hybridization of the blots with a polyclonal antibody which recognized a cytoplasmic enzyme, glucose-6-phosphate dehydrogenase (dilution 1/1000). DNase II activity assays. Two assays were performed for the determination of DNase II activity. In the first one, DNase II activity was determined in an in vitro cell-free system where intact L1210/ DDP 10 nuclei were incubated with the cytoplasmic or nuclear extracts at 37°C, under acidic conditions, as previously described [33]. DNA was then extracted and electrophoresed, as described above. In the second one, DNase II activity was determined in a gel plate assay, according to Yasuda et al. [40] with modifications. Each gel plate was prepared as follows: 30 ml of 1% (w/v) agarose in reaction buffer (Tris–HCl, 40 mM, pH 5.5) had been liquefied and maintained at 55°C. Forty microliters of freshly diluted calf thymus DNA (10 mg/ml) and 4 ml of ethidium bromide (10 mg/ml) were added and the resulting mixture was immediately poured onto a petri dish. After solidification at room temperature, cylindrical sample wells were punched in the gel. Samples of 20 to 40 mg were applied to the wells and the gel plate was incubated at 37°C in a moist chamber. After incubation for 24 or 48 h, DNase II action was vizualized by the appearance of a dark circle zone resulting from the degradation of the DNA, visible under ultraviolet light, on a fluorescent background of dye bound to unhydrolyzed DNA, as the enzyme diffused from the well into the agarose gel containing DNA and ethidium bromide. Ammonium sulfate precipitation. Cytoplasmic extracts were diluted to 2 mg/ml in 10 mM Tris–HCl, pH 7.5, and subjected to a stepwise ammonium sulfate fractionation from 20 to 100% saturation. After desalting, each fraction was diluted in 40 mM Tris–HCl, pH 7.5, and the protein concentration determined. The total amount of precipitated protein was then evaluated in each fraction. The individual fractions were analyzed for both DNAse II activity, using the gel plate assay (20 mg), and the detection of proteins recognized by the anti-L-DNase II antibody on immunoblots (15 mg), using the anti L-DNase II antibody as previously described.

RESULTS

We have previously shown that a L1210/DDP 10 cell line, resistant to cisplatin-induced apoptosis, retained at least one pathway for apoptosis that could be activated by staurosporine treatment. It was suggested that this activation was associated with the presence of a cytoplasmic endonuclease activity that resembles DNase II in terms of cation requirements and pH profile [33, 34]. In the present work, the possible involvement of a DNase-II-like enzyme in staurosporine-induced apoptosis was analyzed by using a polyclonal

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antibody recently developed against porcine spleen L-DNase II [25, 26]. Constitutive Cytoplasmic DNase II Activity, Responsible for DNA Fragmentation, Is Translocated in the Nuclei of Staurosporine-Treated Cells Taking advantage of the absence of endonuclease activity in L1210/DDP 10 nuclei [33], we further analyzed, in a reconstituted cell-free system, the acidic DNase activity previously detected in the L1210/DDP 10 cytoplasm. First, we wondered whether the antibodies against L-DNase II [26] were able to block the enzymatic activity of this acidic DNase. For this purpose, L1210/ DDP 10 isolated nuclei were incubated in the absence (Fig. 1A, lanes 1 and 2) or the presence (lanes 3 to 8) of L1210/DDP 10 cytoplasmic fraction, under conditions already known to activate DNase II and induce DNA fragmentation (i.e., acidic medium) [see ref. 31]. As previously reported [33], no DNA fragmentation was detected in isolated L1210/DDP 10 nuclei in either the absence (lane 1) or the presence (lane 2) of 5 mM CaCl 2/10 mM MgCl 2. However, oligonucleosomal-sized DNA fragments were produced after incubation of these nuclei with L1210/DDP 10 cytoplasmic fraction in the absence of any cation (lane 3). This fragmentation was completely inhibited when cations were added (lane 4). Furthermore, this DNase activity was also inhibited when the anti-L-DNase II antibody was added to the incubation medium (lane 5). In contrast, DNase I antibody could not inhibit DNA degradation generated in this reconstituted cell-free assay (lane 7). The corresponding nuclear extracts prepared from the same untreated cells were not able to induce any DNA cleavage under the same conditions (lanes 9 and 11). These results demonstrate that a DNase II enzyme, absent in cell nuclei, but constitutively expressed in the cytoplasmic compartment of the same cell, is functionally active for the internucleosomal DNA cleavage observed in L1210/DDP 10 nuclei. Second, we wondered whether this cytoplasmic DNase II activity could be translocated to its nuclear site of action to induce DNA cleavage upon triggering of apoptosis. Since we have previously shown that staurosporine was able to induce DNA fragmentation characteristic of apoptosis in L1210/DDP 10 cells, while cisplatin failed to do this in the same cell line [33], we expected to detect this activity in the nuclei of staurosporine-treated L1210/DDP 10 cells and not in the nuclei of cisplatin-treated cells. Therefore, nuclear extracts, prepared from L1210/DDP 10 cells treated for 72 h with toxic doses of either cisplatin or staurosporine, were tested for DNase II activity in the same cell-free assay (lanes 10, 12, and 13). Nuclear extracts from staurosporine-treated cells were able to induce

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FIG. 1. Constitutive cytoplasmic DNase II activity is translocated in the nuclei of staurosporine-treated cells. (A) Analysis of DNase II activity in a cell-free assay. Nuclei (4 3 10 6) isolated from L1210/DDP 10 cells were incubated for 2 h at 37°C, in the absence (lanes 1 and 2) or in the presence of either the cytoplasmic fraction (200 mg) prepared from nontreated cells (lanes 3 to 8) or nuclear extracts (200 mg) prepared from L1210/DDP 10 cells treated or not (lanes 9 and 11) with either 50 nM staurosporine (lanes 12 and 13) or 50 mM cisplatin (lane 10). The incubations were performed, as indicated, in either the absence (lanes 1 to 4 and 9 to 12) or the presence of anti-L-DNase II (dilution 1/20) (lanes 5, 6, 13) or anti-DNase I (dilution 1/20) antibodies (lanes 7 and 8). Each set of incubations was carried out at pH 5.0 in the absence (lanes 1, 3, 5, 7, and 9 to 13) or in the presence (lanes 2, 4, 6, 8) of 5 mM CaCl 2 plus 10 mM MgCl 2. DNA was then extracted and the fragments were separated on a 2% agarose gel and stained by ethidium bromide. A 123-bp ladder marker (m) was included as molecular size marker. (B) Western blot analysis of cytoplasmic (Cyto) and nuclear fractions (Nuc) from nontreated (nt) and drug-treated L1210/DDP 10 cells using the anti-L-DNase II antibody. Cells were treated with either cisplatin (cisP, 1 mM, lanes 2 and 5) or staurosporine (STP, 50 nM, lanes 3 and 6) for 72 h. After drug treatment, cytoplasmic (lanes 1 to 3) and nuclear (lanes 4 to 6) extracts were prepared as described under Materials and Methods and analyzed by Western blot using the L-DNase II antibody. Simultaneous electrophoresis of porcine DNase II is shown. The purity of the nuclear fraction was checked using an antibody that recognized a cytoplasmic enzyme, glucose-6-phosphate dehydrogenase (G6PDH) (see the bottom panel).

DNA fragmentation in the intact nuclei (lane 12). This fragmentation was inhibited by anti-L-DNAse II antibody (lane 13). Cations also inhibited DNA cleavage (data not shown). In contrast, L1210/DDP 10 nuclei incubated with nuclear extracts prepared from cisplatintreated cells failed to display significant DNA cleavage (lane 10). Therefore, this assay demontrates that the cytoplasmic L-DNase II activity responsible for DNA fragmentation in L1210/DDP 10 cells is specifically translocated in the nuclei of staurosporine-treated cells. Immunoblot Analyses of L-DNase II Proteins As it neutralized the activity of acidic DNase, the anti-L-DNase II antibody was used to identify DNaseII-like proteins in both cytoplasmic and nuclear extracts prepared from control or cisplatin- or staurosporine-treated L1210/DDP 10 cells. Cellular extracts, prepared as those used for the enzymatic assays (Fig. 1A), were analyzed by Western blot (Fig. 1B).

Specific controls indicated that the anti-L-DNase II antibody recognized porcine spleen L-DNase II, a protein of 32 kDa (Fig. 1B). No cross-reactivity with DNase I was observed (data not shown). A 30-kDa protein was detected in the cytoplasm of nontreated cells (lane 1). This protein has the expected size for murine DNase II, which presents an apparent molecular weight lower than that of porcine spleen DNase II, used as control. This protein was not detected in the nuclear extracts prepared from control cells (lane 4). The L-DNase II antibody reacted also strongly with a protein of 70 kDa present in the cytoplasmic extracts from control L1210/DDP 10 cells (lane 1). A very low level of this protein was detected in the nuclei of nontreated cells (lane 4). The 30- and 70-kDa bands were specific since no signal was obtained with the preimmune serum used at the same dilution (data not shown). The levels of the cytoplasmic 70- and 30-kDa proteins were not subjected to a marked modification by

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FIG. 2. Immunolocalization of L-DNase II in L1210/DDP 10 treated and nontreated cells. (A) Immunofluorescence analyses. After treatments, cells were incubated with polyclonal antibody against L-DNase II (Top). The antibody was then localized using a secondary antibody coupled to TRITC (green). Nuclei were counterstained with propidium iodide (5 mg/ml) (red). Colocalization was vizualized in yellow. (a) Nontreated cells; (b) cells treated for 72 h with 50 mM cisplatin; (c) cells treated for 72 h with 50 nM staurosporine. The bottom panel shows light microscopy pictures of control and staurosporine- and ciplatin-treated L1210/DDP 10 cells after May–Grunwald–Giemsa staining. (B) Confocal analysis of a staurosporine-treated cell. In this analysis, nuclei were not counterstained. The pixel intensities are presented on a color scale from blue (the least fluorescent) to red (the most fluorescent). Twenty slices are presented.

either cisplatin or staurosporine treatments (Fig. 1B, lanes 2 and 3, respectively). In contrast, the patterns of expression of these two proteins in the nuclear compartment were typically correlated to the susceptibility of the cells to the apoptotic inducer (staurosporine versus cisplatin). Indeed, the level of the 70-kDa protein was drastically increased in the nuclei of the cells after treatment with apoptotic doses of staurosporine (lane 6). A simultaneous nuclear accumulation of the 30-kDa protein was also observed. Clearly, cisplatin treatment was able to induce the nuclear accumulation of neither

the 70- nor the 30-kDa protein (lane 5). Appropriate controls (microscopy, immunolocalization of the cytosolic marker, G6PDH) were performed to exclude a contamination of the nuclear fraction by cytosolic proteins that could have occurred in the course of nuclei preparation (see the 36-kDa G6PDH on the lower immunoblot, Fig. 1B). In addition, the nuclear accumulation of both the 70- and the 30-kDa proteins was detected only in staurosporine- and not in cisplatin-treated cells, even though this latter drug was able to induce apoptosis. Therefore, this accumulation could not be the

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FIG. 3. Kinetic analysis of DNA fragmentation (A) and accumulation (B) of both the 70- and 30-kDa proteins after treatment of L1210 cell lines with either cisplatin (cisP) or staurosporine (STP). (A) Cells were incubated with equitoxic doses of either cisplatin (L1210/0, 1 mM; L1210/DDP 10, 50 mM) or staurosporine (50 nM for both cell lines) for 0, 24, 48, and 72 h. The DNAs were then extracted and separated on a 2% agarose gel. (B) Nuclear and cytoplasmic extracts were prepared from L1210/0 and L1210/DDP 10 cells treated with cisplatin or staurosporine as in A and analyzed by Western blot using the L-DNase II antibody. The positions of the 70- and 30-kDa proteins were identified by an arrow. Simultaneous electrophoresis of porcine DNase II is shown.

result of a contamination by proteins released from fragile lysosomes during the apoptotic process. These results show that the detection of nuclear DNase II activity in staurosporine-treated cells (Fig. 1A, lane 12) correlates with the nuclear translocation of two proteins of 30 and 70 kDa, respectively, recognized by the anti-L-DNase II antibody (Fig. 1B, lane 6). Immunolocalization of L-DNase II in L1210 Staurosporine-Treated Cells In situ immunofluorescence analyses were performed on whole cells to further demonstrate the trans-

location of L-DNase II in nuclei of cells triggered to death by staurosporine. Figure 2A (top) shows the immunolocalization of L-DNase II protein in control (a) and cisplatin (b)- and staurosporine-treated (c) L1210/ DDP 10 cells, respectively. In control cells (Fig. 2A, a), the immunocytochemical analyses showed a diffuse cytoplasmic immunoreactivity (green signal). Counterstaining with propidium iodide, a nuclear staining (red signal), revealed a very low colocalization of the two signals (yellow staining where the green and red signals overlap). After staurosporine treatment, an increase in yellow staining was observed (Fig. 2A, c).

LEI/ELASTASE COMPLEX IN STAUROSPORINE-INDUCED APOPTOSIS

This suggested a redistribution of the proteins recognized by the anti-L-DNase II antibody to the nucleus. Such a redistribution was not observed after cisplatin treatment (Fig. 2A, b). When the anti-L-DNase II antibody was omitted, no labeling was observed, excluding nonspecific labeling with the FITC-conjugated antibody (data not shown). In Fig. 2A, the bottom panel shows the morphology of the cells after May–Gru¨nwald– giemsa staining. The characteristic features of apoptosis in staurosporine-treated L1210/DDP 10 cells were observed (condensation of the chromatin associated with nuclear fragmentation). These features were absent in cisplatin-treated L1210/DDP 10 cells. A confocal analysis of a staurosporine-treated cell was performed in order to confirm the nuclear distribution of the protein recognized by the anti-L-DNase II antibody (Fig. 2B). Importantly, these in situ experiments are in agreement with those obtained by enzymatic assays (Fig. 1A) and immunoblot analyses (Fig. 1B). They confirm that staurosporine induces the accumulation of L-DNase II protein in the nucleus of the treated cells. Consistently with the biological response of these cells to cisplatin, this latter drug fails to induce this accumulation. Time Course of DNA Fragmentation and Accumulation of Nuclear 70- and 30-kDa Proteins As previously shown [33], only staurosporine was able to induce internucleosomal DNA degradation in both L1210/0 and L1210/DDP 10 cells. This degradation could be detected after 48 h of exposure to this drug. Conversely, cisplatin could not induce any DNA fragmentation in L1210/DDP 10 cells, while it was detected in L1210/0 cells, after 48 h of drug exposure. We took advantage of these distinctive features to perform time course analyses, in order to relate DNA fragmentation occurring during apoptosis to the accumulation of the 70- and 30-kDa proteins. Cells were treated with either cisplatin or staurosporine for 24, 48, and 72 h. After treatment, oligonucleosomal DNA degradation was evaluated by agarose gel electrophoresis (Fig. 3A). In the same experiment, nuclear and cytoplasmic extracts were prepared and analyzed by immunoblot with the anti-L-DNase II antibody (Fig. 3B). Figure 3B shows that the accumulation of both the 70- and the 30-kDa proteins in the nuclear extracts of both cell lines exposed to staurosporine began at 48 h and progressed at 72 h. As previously noticed, no marked modification in the level of either the 70- or the 30-kDa protein was observed in the cytoplasm of treated cells. Therefore, the nuclear accumulation of proteins recognized by the anti-L-DNase II antibody correlated with the DNA fragmentation kinetics observed during staurosporineinduced apoptosis (Fig. 3A). Moreover, this experiment

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FIG. 4. Partial purification of L-DNAse II activity in L1210/0 cells. Cytoplasmic extract (C.E.) prepared from L1210/0 cells was fractionated by ammonium sulfate saturation at 20 to 100%. In each fraction, the amount of precipitated proteins was evaluated. Equal amounts of protein (15 mg) were loaded on 12% SDS–PAGE gel and analyzed by immunoblot (A) using the L-DNase II antibody, as described under Materials and Methods. The same fractions (20 mg) were analyzed for DNase II activity in a fluorescent substrate gel assay (B) under conditions known to activate DNase II activity but not DNase I (40 mM Tris–HCL, pH 5.0, without cations), as described under Materials and Methods.

showed that, even in L1210/0 cells, which were proficient in cisplatin-induced apoptosis, cisplatin treatment was not able to induce the nuclear accumulation of either of these two proteins. Enrichment in DNase II Activity by Stepwise Ammonium Sulfate Precipitation DNA SDS–PAGE gels can be used to identify both the size and the activity of nucleases [39]. This technique involved the restoration of enzymatic activity with a minimum of denaturation and degradation. It was efficient in detecting DNase I activity, but inefficient in detecting L-DNase II activity. This might indicate that L-DNase II activity is highly susceptible to conformational changes and, because it was not possible to properly renature the protein, the nuclease activity was lost. In an attempt to partially purify the proteins responsible for DNase II activity, ammonium sulfate fractionation at 20 to 100% saturation was performed on cytoplasmic extracts prepared from L1210/0 cells. Figure 4A shows that about 88% of the total proteins precipitated in the two first fractions (7.4 mg of a total amount of about 8.5 mg). However, in each fraction, about the same amount of the 70-kDa protein was

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FIG. 5. Western blot analysis of L1210/0 (A) and L1210/DDP 10 (B) cytoplasmic and nuclear extracts using anti-L-DNase II and anti-elastase antibodies. (A) Cytoplasmic extracts were prepared from nontreated L1210/0 cells. Lanes 1 and 2 were loaded with 20 and 100 mg of protein, respectively. Nuclear extracts were prepared from control (C) or staurosporine-treated (STP) L1210 cells. Staurosporine treatment was performed as described in Fig. 2. (B) Cytoplasmic and nuclear extracts prepared from control (C) and cisplatin (cisP)- and staurosporine (STP)-treated cells. The treatments were performed as in Fig. 2. The sizes of the marker proteins are shown at the side of the blots and the sizes of the immunoreactive bands are indicated with an arrow.

detected by the anti-L-DNase II antibody. Since all lanes of the gel (lanes 1 to 5) were equally loaded (15 mg), it can be concluded that, in fact, most of the 70kDa protein precipitated in the two first fractions. In contrast, the detection of the 30-kDa protein began in the fraction corresponding to the 60% ammonium sulfate saturation (lane 3) and increased with the percentage of the saturation, suggesting that this protein was better precipitated at 60 to 100% ammonium sulfate saturation. As shown on the substrate gel assay (Fig. 4B), this progressive enrichment in the 30-kDa protein paralleled the detection of L-DNAse II activity. Therefore, this experiment provides clear evidence that the 70-kDa protein has no DNase II activity. In addition, although these results are merely semiquantitative, they suggest that the 30-kDa protein is responsible for DNase II activity. Characterization of the 70-kDa Protein The 30-kDa protein was likely to be the protein endowed with DNase II activity. However, we wanted to characterize the 70-kDa protein that was also recognized by the anti-L-DNase II antibody and accumulated specifically in the nuclei of staurosporine-treated L1210 cells. In the course of cloning porcine spleen DNase II, Torriglia et al. [26] showed that this DNase II was generated from a 42-kDa serpin, LEI. For this reason,

this DNase II was called L-DNase II. It was previously shown that LEI was able to form, with its target proteinase, elastase, an SDS-stable covalent protein complex [42– 45]. Therefore, the possibility that elastase (molecular mass 24 –29 kDa) might be a component of the 70-kDa protein recognized by the anti-L-DNase II antibody was investigated. Protein extracts were prepared from L1210/0 or L1210/DDP 10 cells treated or not with cisplatin or staurosporine. Western blot analyses using anti-L-DNase II and anti-elastase antibodies are shown in Fig. 5. Control experiments showed that the anti-elastase antibody did not cross-react with purified DNase II (data not shown). However, in cytoplasmic extracts prepared from L1210/0, it recognized specifically the same 70kDa protein band as the anti-L-DNase II antibody (Fig. 5A, lanes 3 and 4 versus lanes 1 and 2). This recognition of elastase was specific since commercial porcine elastase was capable of depleting the anti-elastase antibody of its ability to recognized both elastase and the 70-kDa protein (data not shown). If the same protein were recognized by both antibodies, it would be expected to detect the nuclear accumulation of this protein in staurosporine-treated cells using either L-DNase II or anti-elastase antibodies. Conversely, no 70-kDa protein would be detected in the nuclei of cisplatin-treated cells. Indeed, after treatment of L1210/0 or L1210/DDP 10 cells with toxic doses of staurosporine,

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FIG. 6. Hypothetical role of the LEI/elastase complex in staurosporine-induced apoptosis. The LEI/elastase complex is localized in the cytoplasm of living cells as an inactive complex. Staurosporine treatment induces the nuclear translocation of the LEI/elastase complex. Posttranslational modifications of LEI lead to the appearance of L-DNase II, which is responsable for DNA degradation. The role of elastase remains unknown. Thus, the association between LEI and elastase might be a means for the regulation of nuclease activity.

the nuclear accumulation of the 70-kDa protein was detected using both antibodies (Figs. 5A, lane 8, and 5B, lane 6). As expected, no nuclear accumulation of this protein was detected after treatment with toxic doses of cisplatin (Fig. 5B, lane 5). Thus, the 70-kDa protein, whose nuclear accumulation is specifically induced by staurosporine treatment, is recognized by both anti-L-DNase II and anti-elastase antibodies. DISCUSSION

In this work, we show that after staurosporine treatment, cytoplasmic nuclease is translocated to the nucleus. The nuclear accumulation of this nuclease activity correlates with both chromatin DNA fragmentation and detection of two proteins of 70 and 30 kDa, respectively, both detected by the anti-L-DNase II antibody. Partial purification of L-DNase II activity by selective ammonium sulfate precipitation demonstrates that the 30-kDa protein is likely to be the nuclease responsible for the DNA fragmentation in staurosporine-treated L1210 cells. Indeed, nuclease activity, enriched in the 80 and 100% saturated ammonium sulfate fractions, parallels the presence of the 30-kDa protein. The correlation between the presence of the 30-kDa protein and the expression of DNase II activity is in agreement with previous experiments using the recombinant protein. Indeed, it was shown that this molecular form was responsible for DNase II activity

[26]. However, we cannot rule out some contribution from another protein. Characterization of the 70-kDa protein showed that this protein, recognized by the anti-L-DNase II antibody, was also recognized by the anti-elastase antibody, suggesting that it carries residues belonging to both L-DNase II and elastase. Importantly, previous findings showed that L-DNase II is derived from a serpin, LEI, by an acidic-dependent posttranslational modification or by digestion with elastase [26]. This serpin is able, with its target serine proteinase, to form elastase, an SDS-stable protein complex [42– 45], this association leading to an inhibition of the proteinase. Thus, the immunoreactive 70-kDa protein, which has no DNase activity, would be the result of an SDS-stable association between elastase (24 –29 kDa) and LEI (42 kDa). A hypothetical mechanism for DNase activation is proposed in Fig. 6. Staurosporine treatment would stimulate (directly or indirectly via a decrease in pH) the release of L-DNase II activity after the dissociation of the inhibitory complex formed between LEI (42 kDa) and elastase (29 kDa). This hypothesis is supported by recent results from the literature: (i) intracellular acidification, which was previously shown to be associated with staurosporineinduced apoptosis [46], is a favorable condition for the release and the activation of L-DNase II; (ii) Torriglia et al. [26] showed that LEI (which is devoid of any

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nuclease activity) could, after acidic-dependent posttranslational modifications leading to a shift in its molecular weight (from 42 to 30 kDa), generate active L-DNase II capable of degrading DNA; (iii) these findings are reminiscent of biochemical and functional features of other proteins in the serpin family [42, 43]; (iv) numerous studies have demonstrated that serine proteases are involved in apoptotic cell death [49]: apoptosis of endothelial cells could be promoted by the neutrophil serine protease elastase [50], a serine protease (AP24) purified from apoptotic human monocytic U937 cells, demonstrating elastase-like properties, and able to cause DNA fragmentation in isolated nuclei [51, 52]. It was hypothesized that AP24 may play a key role in transmitting apoptotic signals from cytosol to nucleus, where it directly or indirectly activates endogenous endonucleases resulting in DNA digestion. Together with those of the current literature, the results presented in this paper suggest that an association between LEI and elastase might be involved in the regulation of nuclease activity. Similar kinds of regulatory mechanisms have already been described. Actin specifically inhibits DNase I [53]. Its proteolysis by ICE-like proteases could release the enzyme and generate its activation. Another case is illustrated by CAD, a 40-kDa nuclease, located in the cytoplasm as an inactive form [27]. CAD forms a complex and copurifies with an inhibitor of CAD (ICAD) of 29 –30 kDa that can be cleaved by caspase 3 during apoptosis [28]. However, this is certainly not the only method of regulation of DNA degradation since CAD is not expressed in every tissue. Interestingly, preliminary results showed that in the cellular system used in this study, staurosporine-induced DNA fragmentation was not dependent on caspase activation (Se´gal-Bendirdjian, unpublished results). Conversely, cisplatin, despite its capacity to induce apoptosis and DNA fragmentation in parental L1210/0 cells, could not induce nuclear DNase II activity or activate the process responsible for the translocation of this DNase-II-like nuclease. Therefore, these results have contributed to demonstrate further that a variety of endonucleases, activated by distinct signals and different transduction pathways, are stimulus- and cell-specific. The skillful technical assistance and valuable help of M. Thonier (CNRS, UMR1772) are particularly aknowledged. We also acknowledge the assistance of Z. Mishal (Laboratoire de Cytome´trie, CNRS, UPS 47), who performed all the immunofluorescence analyses. We thank Dr. A. Eastman (Dartmouth) for generously providing the L1210/0 and cisplatin-resistant L1210/DDP 10 cell lines and Dr. M. Lanotte (INSERM U496) for many helpful discussions and comments on the manuscript. We also thank Dr. A. P. Se`ve and Dr. G. Chabot (INSERM U496) for critical reading of the manuscript. This work was supported in part by grants from the Association pour la Recherche contre le Cancer (Villejuif, France) and from the Ligue Nationale contre le Cancer (Paris, France). C. A. Belmokhtar is supported by a Ligue Nationale contre le Cancer fellowship.

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