Degradation of nuclear matrix and DNA cleavage in ... - CiteSeerX

Journal of Cell Science 109, 45-56 (1996) Printed in Great Britain © The Company of Biologists Limited 1996 JCS7023

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Degradation of nuclear matrix and DNA cleavage in apoptotic thymocytes Valerie M. Weaver1, Christine E. Carson1, P. Roy Walker1, Nathalie Chaly2, Boleslaw Lach3, Yves Raymond4, David L. Brown5 and Marianna Sikorska1,* 1Apoptosis

Research Group, Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON, K1A 0R6, Canada 2Department of Biology, Carleton University, Ottawa, ON, K1S 5B6, Canada 3Department of Laboratory Medicine University of Ottawa and Ottawa Civic Hospital, Ottawa, ON, K1Y 4E9, Canada 4Institut du Cancer de Montreal, Montreal, PQ, H2L 4M1, Canada 5Department of Biology, University of Ottawa, Ottawa, ON, K1N 6N5, Canada *Author for correspondence (e-mail: [email protected])

SUMMARY In dexamethasone-treated thymocyte cultures an increase in nuclear proteolytic activity paralleled chromatin fragmentation and the appearance of small apoptotic cells. The elevation of nuclear proteolytic activity was accompanied by site-specific degradation of nuclear mitotic apparatus protein and lamin B, two essential components of the nuclear matrix. Nuclear mitotic apparatus protein phosphorylation and cleavage into 200 and 48 kDa fragments occured within 30 minutes of dexamethasone treatment. Cleavage of lamin B, which generated a fragment of 46 kDa consistent with the central rod domain of the protein, was also detected after 30 minutes of exposure to the steroid hormone. The level of lamin B phosphorylation did not change as a result of the dexamethasone treatment and the lamina did not solubilize until the later stages of apoptosis.

Initial DNA breaks, detected by the terminal transferasemediated dUTP-biotin nick end labeling assay, occurred throughout the nuclei and solubilization of lamina was not required for this process to commence. The data presented in this paper support a model of apoptotic nuclear destruction brought about by the site-specific proteolysis of key structural proteins. Both the nuclear mitotic apparatus protein and lamin B were specifically targeted by protease(s) at early stages of the cell death pathway, which possibly initiate the cascade of degradative events in apoptosis.

INTRODUCTION

implicated in the death of vulnerable neurons in ischemic gerbil brain (Lee et al., 1991; Roberts-Lewis et al., 1994) and in irradiated and dexamethasone-treated thymocytes (Squier et al., 1994). Cathepsins were shown to increase in pyramidal neurons of the hippocampal CA1 region, again, following transient ischemia in gerbils (Nitatori et al., 1995) and during regression of rat prostate and mammary glands (Guenette et al., 1994). Furthermore, an involvement of the ubiquitinmediated proteolytic pathway has been suggested for teniposide-treated and gamma-irradiated lymphocytes (Roy et al., 1992; Delic et al., 1993). ICE or ICE homologes have been shown to affect the survival of serum-deprived fibroblasts (Wang et al., 1994), chicken dorsal ganglion neurons (Gagliardini et al., 1994), TNF-induced cell death in MCF7 breast carcinoma as well anti-Fas-induced apoptosis in a B cell lymphoma line (Tewari and Dixit, 1995). There are also reports showing induction of cell surface peptidase activity in HeLa cells in response to stress (Brown et al., 1994) and a histone H1-specific proteinase in rat liver in response to gammaradiation (Kutsyi and Gaziev, 1994). The list of nuclear proteins targeted by these proteolytic enzymes during apoptosis includes topoisomerases I and II (Kaufmann, 1989;

MacDonald et al. (1980) have shown that glucocorticoids stimulate protein breakdown during apoptosis in lymphocytes and suggested that protein degradation could be a possible mechanism of steroid-induced cell death. This concept gained credence after it was demonstrated that cytotoxic T lymphocytes-mediated killing, which is induced by serine proteases (granzymes) of the cytoplasmic granules, involves apoptosis (Shi et al., 1992a,b; Shiver et al., 1992; Helgason et al., 1993). The discovery that the Caenorhabditis elegans death gene ced3 encodes a protein homologous to the mammalian cysteine protease interleukin-1β-converting enzyme (ICE) intensified the search for protease-dependent steps in apoptosis (Yaun et at., 1993; Kumar et al., 1994; Wang et al., 1994; Earnshaw, 1995). Thus far, every class of protease has been implicated in cell death. For example, serine proteases were shown to play a role in tumor necrosis factor (TNF)-mediated cytotoxicity in fibroblasts (Suffys et al., 1988; Voelkel-Johnson et al., 1995), and in thymocytes in response to dexamethasone and teniposide (Bruno et al., 1992; Weaver et al., 1993). Calpains have been

Key words: NuMA, Lamin B, Phosphorylation, Site-specific proteolysis

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V. M. Weaver and others

Voelkel-Johnson et al., 1995), poly(ADP-ribose) polymerase (Kaufmann et al., 1993; Lazebnik et al., 1994; Tewari et al., 1995; Nicholson et al., 1995), histone H1 (Kaufmann, 1989; Gaziev and Kutsyi, 1994; Voelkel-Johnson et al., 1995), nuclear lamins (Kaufmann, 1989; Ucker et al., 1992; Lazebnik et al., 1993; Oberhammer et al., 1994; Neamati et al., 1995), the U1 small nuclear ribonucleoprotein (Roy et al., 1991; Casciola-Rosen et al., 1994) and a number of constitutive transcription factors (Wang and Pittman, 1993; de Belle et al., 1994). These proteins represent different aspects of nuclear structure and function and some may be directly linked to chromatin degradation in apoptosis. The destruction of the structural organization of chromatin within the nucleus is a central feature of apoptosis and forms the basis of the most characteristic morphological criteria (displacement and aggregation of chromatin and formation of pycnotic nuclei) used to identify this form of cell death (Kerr et al., 1972). In the interphase nucleus each chromosome is anchored to a protein scaffold composed of a peripheral nuclear lamina and an intranuclear proteinaceus network or matrix. Within this framework each chromosome occupies its own three-dimensional space and establishes contacts with the protein scaffold to generate a complex structural organization that is believed to reflect the function of that chromosome in each particular differentiated cell type (Cremer et al., 1993). Despite its structural complexity the nucleus is rapidly disassembled during mitosis and the nuclear structure reassembles again into a functional state after mitosis has been completed. In contrast, during apoptosis the interphase chromosomes collapse into uniformly electron-dense masses which appears to reach a level of compaction similar to that observed during mitosis, but this represents an irreversible step. It is possible that proteolysis occurs in the nucleus at early stages of apoptosis resulting in the degradation of nuclear components which, in turn, are responsible for nuclear destabilization prior to, or in parallel with, endonuclease activation. The lamins, for example, appear to be degraded in parallel with DNA fragmentation in fibroblasts (Oberhammer et al., 1994). In an effort to delineate the early stages of nuclear disassembly we have analyzed changes in nuclear proteolytic activity and the integrity of nuclear matrix components and compared them to the kinetics of chromatin cleavage in dexamethasone-treated thymocytes. We performed a biochemical and morphological study of the Nuclear Mitotic Apparatus (NuMA) protein, which is localized in the nucleoplasm (Lydersen and Pettijohn, 1980; Price and Pettijohn, 1986; Yang et al., 1992; Compton et al., 1992; Zeng et al., 1994; Cleveland, 1995), as well as, two components of the nuclear envelope structure, lamin B, which is the only lamin protein expressed in thymocytes (Guilly et al., 1987), and PI2 antigen, which was shown to be associated with the nuclear membrane (Chaly et al., 1984, 1989). MATERIALS AND METHODS Preparation and treatment of thymocytes Thymocytes were isolated from 5 week old (150-200 g) male Sprague-Dawley rats (bred in this Institute) as previously described (Whitfield et al., 1968). Cell suspensions (3×107 to 4×107 cells/ml) were prepared in RPMI 1640 tissue culture media (Gibco BRL,

Burlington, ON) containing 5% heat-inactivated fetal bovine serum (Unipath, Nepean, ON) and 20 µg/ml gentamycin sulfate (Sigma Cell Culture, St Louis, MO). The thymocyte primary cultures were maintained in an incubator for 30 minutes at 37°C before addition of 1 µM dexamethasone from a 2 mM stock solution in ethanol (Sigma Chemical Co., St Louis, MO). Protease inhibitors such as 2 mM phenylmethylsulphonyl fluoride (PMSF, 200 mM stock in anhydrous propanol, Sigma Chemical Co., St Louis, MO) and 20 µM transepoxysuccinyl-L-leucylamido-(4-guanidino) butane (E64, 10 mM stock in dimethyl sulfoxide, Sigma Chemical Co., St Louis, MO) were added at the same time as dexamethasone. Sample processing and pulsed field agarose gel electrophoresis (PFGE) In order to avoid DNA shearing and to eliminate any possibility for the release of endonucleases during sample handling, thymocytes were immobilized in low melting point (LMP) agarose plugs before treatment with dexamethasone. Approximately 3×107 cells were suspended in 0.25 ml of medium, containing 5% serum, and mixed with an 0.25 ml aliquot of melted 1.5% LMP agarose (at 37°C) in serum-free medium and allowed to solidify in a 1 ml syringe. Slices of 20 µl (2×106 cells) were cut from the plug and incubated, at 37°C, in 5 ml of medium + serum in the presence or absence of 1 µM dexamethasone, on a rotator, in the incubator for 0.5-4 hours. For DNA extraction, the agarose slices were incubated for 3 hours at 37°C in 300 µl of TEN buffer (10 mM Tris-HCl, pH 9.5, 25 mM EDTA, 1 mM EGTA, 10 mM NaCl) containing 4 µg proteinase K and 1% SDS, subsequently washed in TE buffer (10 mM Tris-HCl, pH 8.0, 1.0 mM EDTA) and immediately loaded on an 0.8% agarose gel prepared in TBE buffer (0.089 M Tris-HCl, 0.089 M boric acid, 25 mM EDTA, pH 8-8.5). The wells were sealed with 1.5% (w/v) LMP agarose and PFGE was carried out using a Q-life Autobase Electrophoresis System (Kingston, ON) as described by Weaver et al. (1993) and Walker et al. (1993). Yeast chromosomes, lambda DNA ladder (New England Biolabs, Beverly, MA), the 123 bp ladder and a HindIII digest of lambda DNA (Gibco BRL Life Technologies, Burlington, ON) were used as size markers. The gels were stained with ethidium bromide, placed on a transilluminator and either photographed onto Polaroid MP4 film or the image captured using an Ultra-Lum video imaging data acquisition system. The digitised images were subsequently used to generate scans of pixel intensity vs distance of migration for each lane. The total amount of fragmented DNA in each sample was calculated by integrating the pixel intensity of each scan after subtracting the zero time control from each timepoint. Analysis of cell morphology and in situ DNA breaks For immunofluorescence microscopy, thymocytes were plated onto poly-L-lysine-coated coverslips (Sigma, St Louis, MO), fixed for 5 minutes in 3% paraformaldehyde (J.B. EM Services Inc., Pointe Claire, Dorval, PQ) and permeabilized for 20 minutes in 0.2% Triton X-100 (Pierce/Chromatographic Specialties Inc., Nepean, ON) as described by Chaly et al. (1984). The cells were incubated for 60 minutes with primary and 45 minutes with secondary antibodies and counterstained for 1 minutes with 1 µg/ml Hoechst 33258 (Sigma, St Louis, MO). All incubations were performed at room temperature. For double staining, antibodies were applied sequentially and a blocking step with 0.15% (w/v) gelatin (Bio-Rad, Mississauga, ON) was added before application of the second primary antibody. The following antibodies were used for immunofluorescence staining: a mouse monoclonal IgG1 anti-lamin B (dilution 1:50, clone 119D5-F1 provided by Y. Raymond, Institut du Cancer de Montreal, Montreal, PQ), a mouse monoclonal IgM anti-PI2 (dilution 1:100; Chaly et al., 1984), a mouse monoclonal IgG1 anti-NuMA (dilution 1:500, A-204, Matritech Inc., Cambridge, MA), fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG heavy- and light-chain specific (dilution 1:300, Sigma, St Louis, MO), FITC-conjugated goat anti-mouse IgM (dilution 1:150, Cappel/Organon Teknika, Scarborough, ON) and

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CY3-conjugated goat anti-mouse IgG (Jackson Labs). The cells were examined using an Olympus Bmax Fluorescence Microscope and Photosystems or a Leica confocal laser scanning microscope. DNA breaks were analyzed by the method of terminal transferase-mediated dUTP-biotin nick end labeling (TUNEL) of Gavrieli et al. (1992). Briefly, cells were fixed, stained for lamin B and then incubated for 1 hour at 37°C with 300 units/ml of terminal deoxynucleotidyl transferase (Gibco BRL, Burlington ON) and 10 µM biotin-16-dUTP (Boehringer Mannheim, Laval PQ) in cocadylate buffer. DNA breaks were visualized with 10 µg/ml of streptavidin-CY3 (Jackson ImmunoResearch/BioCan Scientific, Mississauga, ON). For electron microscopy aliquots of thymocytes were fixed in 1.6% glutaraldehyde in 0.1 M sodium cocadylate buffer, pH 7.2. The samples were routinely processed for Epon-Araldite embedding and ultrathin sections were examined under a Philips 301 electron microscope (MacDowell, 1978).

tially as described by Twining (1984). Fluorescence was determined on a SLM 8000C spectrofluorometer (LMM Aminco Inc.), after calibration, using an excitation wavelength of 365 nm and an emission wavelenth of 525 nm. Parallel samples of diluted trypsin (Sigma Chemical Co., St Louis, MO) 0.1-2.0 ng were assayed as standard. Nuclear proteolytic activity was expressed as trypsin equivalent units per mg protein after subtraction of the fluoresence emitted by an appropriate blank.

Biochemical analysis of nuclear matrix proteins Nuclear matrix proteins (from 5×108 to 8×108 cells/time point) were isolated according to a procedure described by He et al. (1990). The following protease inhibitors were used during the isolation procedure: 1 mM PMSF, 20 µg/ml aprotonin, 10 µg/ml pepstatin, 0.5 mM benzamidine, 2 µg/ml leupeptin, 5 µg/ml E64, 5 µg/ml calpain II inhibitor. For western blotting 25-100 µg of nuclear matrix proteins were separated on 8.5% to 10% SDS-PAGE gels and electrotransferred overnight onto a nitrocellulose membrane (0.2 µm, Schleicher and Scheull/Fisher Scientific, Nepean, ON). The filters were blotted for 1.5 hours, with either anti-NuMA (dilution 1:1,000), anti-lamin B (dilution 1:500) or anti-PI2 (dilution 1:1,000), followed by a 1 hour incubation with alkaline phosphatase-conjugated goat anti-mouse IgG (ProMega/Fisher Scientific, Nepean, ON) or goat anti-mouse IgM (Jackson ImmunoResearch Laboratories, Mississauga, ON) secondary antibodies (dilution 1:10,000). Rainbow molecular mass markers (14.3-200 kDa) were run in parallel for size estimations (Amersham Life Science, Oakville, ON).

RESULTS

In vivo phosphorylation of nuclear matrix proteins Thymocytes were preincubated for 30 minutes with phosphate-free RPMI medium (Gibco BRL Life Technologies, Burlington, ON) and 30 minutes with [32P]orthophosphate (50 µCi/4×107 to 6×107 cells, NEN Dupont Research Products, Mississauga, ON). After incubation with radioactive orthophosphate, 1 µM dexamethasone was added and the cells were harvested at different times thereafter. Cells were washed, pelleted and lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% sodium dodecylsulfate, 1% NP-40, 0.5% deoxycholate, pH 7.4) containing protease and phosphatase inhibitors (1 mM PMSF, 20 µg/ml aprotonin, 10 µg/ml pepstatin, 0.5 mM benzamidine, 2 µg/ml leupeptin, 5 µg/ml E64, 5 µg/ml calpain II inhibitor, 10 mM NaF, 100 µM sodium orthovanadate). Lysates were sonicated to shear DNA and cleared, prior to immunoprecipitation, by ultracentrifugation at 55,000 g for 20 minutes at 4°C. Non-specific binding was minimized by preincubation with rabbit anti-mouse IgG (Sigma Immunochemicals, dilution 1:300), and Protein A-Sepharose (Protein A-Sepharose CL-4B, Pharmacia LKB Biotechnology, Baie d’Urfe, PQ). Immunoprecipitations with primary antibodies were carried out for 12 hours at 4°C followed by 1.5 hours incubation with rabbit antimouse IgG and Protein A-Sepharose beads. The immunoprecipitated proteins were collected and resolved on SDS-PAGE. The gels were dried and autoradiographed on Kodak X-OMAT film for 5 days. Analysis of nuclear proteolytic activity Nuclei were isolated from thymocyte primary cultures as previously described (Weaver et al., 1993). Nuclei from 4×107 to 6×107 thymocytes/time point) were resuspended by sonication in 100 mM TrisHCl, 10 mM CaCl2 buffer, pH 7.8, aliquoted into quadruplicates and assayed for proteolytic activity using a FITC-casein substrate essen-

Determination of cellular volume Cell volume profiles were determined by Coulter counting as previously described (Thomas and Bell, 1981; Walker et al., 1991). Cell suspensions (3×107 to 4×107 cells/ml) were diluted 1:400 with PBS and 2 ml of the diluted cultures was counted in a model ZM Coulter counter attached to a computerized Coulter Channelyzer 256.

Timecourse of DNA fragmentation To relate changes in other nuclear components to DNA fragmentation it is essential to have an accurate measure of the kinetics of the latter process, particularly in thymocytes, which undergo apoptosis rapidly in response to dexamethasone. To achieve this, cells were immobilized in agarose plugs prior to dexamethasone treatment. Following incubations, the embedded cells were rapidly deproteinized and loaded onto gels, thereby eliminating any physical or subsequent enzymatic damage to, or loss of, DNA. PFGE conditions, that permit fragments from 100 bp - 1 Mbp to be resolved on the same gel, have also been developed so that the rates of production of high molecular mass fragments (>50 kb) can be compared with the rate of appearance of the small oligomers that constitute the DNA ladder (Weaver et al., 1993; Walker et al., 1993). This avoids having to run 2 gels or using 2 techniques of disparate sensitivity (Neamati et al., 1995) to try to compare the kinetics of high molecular mass and oligonucleosomal DNA degradation. As shown in Fig. 1A (lane 0) all of the DNA in the freshly isolated thymocyte cultures was undegraded and remained in the wells, confirming that the sample processing conditions did not introduce artifactual DNA breaks. DNA degradation could be detected as soon as 30 minutes after addition of dexamethasone and the fragmentation proceeded rapidly during the next 4 hours, producing a peak of fragments of approximately 50 kb along with progressively smaller fragments of oligomers and monomers of nucleosomal DNA (Fig. 1A and C). Similar patterns of fragmentation were also observed at 6 and 12 hours (data not shown). Analysis of the total amount of fragmented DNA showed that it increased linearly between 30 minutes and 4 hours (Fig. 1B). None of the cells were Trypan Blue positive by 4 hours, but by 12 hours of exposure to 1 µM dexamethasone, approximately 50-60% of cells had lost viability and became Trypan Blue positive (data not shown). Although a recognisable ‘DNA ladder’ could not be seen before 2 hours (Figs 1A and C) it was not possible to conclude that high molecular mass DNA fragmentation preceded internucleosomal DNA fragmentation to any significant degree. Quite clearly, an insufficient amount of DNA had entered the gel at either 0.5 or 1 hour for small fragments to be seen (assuming that the same relative amounts of large and small fragments were released as seen at 4 hours). It is noteworthy that even


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Fig. 1. Kinetics of dexamethasone-induced DNA cleavage. (A) PFGE of cellular DNA from agarose-embedded thymocytes treated with 1 µM dexamethasone for 0, 0.5, 1, 2 and 4 hours (lanes 0-4, respectively). DNA size standards were yeast chromosomes (lane a), lambda DNA ladder (lane b), HindIII digest of lambda DNA (lane c) and 123 bp ladder (lane d). Position of the 50 kb DNA fragments is indicated on the left. (B) Increase in fragmented DNA (DNA entering the gel) with respect to time after the initiation of dexamethasone treatment. The values represent the integrals of the scans of the individual lanes from C on an arbitary scale. (C) Scans of pixel intensity of lanes 0-4 in A.

at 4 hours, when sufficient DNA has entered the gel for small oligomers to be detectable, they constitute only a small fraction of the total fragmented DNA.

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Activation of nuclear proteolytic activity We have shown previously (Weaver et al., 1993), that internucleosomal DNA fragmentation in thymocytes, induced by either dexamethasone or teniposide (VM26), is prevented by coincubation of thymocyte cultures with serine-specific protease inhibitors. This suggested that proteases play a significant role in nuclear destruction during apoptosis and raised the question as to what extent nuclear proteolytic activity was altered in apoptotic thymocytes. We addressed this issue by measuring ‘total’ proteolytic activity in nuclei isolated from primary cultures of thymocytes incubated with 1 µM dexamethasone for up to 6 hours using FITC-casein as a broadspecificity substrate for nuclear proteases (Fig. 2, open circles). In parallel, the samples were analyzed by Coulter counting to evaluate changes in cellular volume indicative of apoptosis (Fig. 2, filled circles). A burst of the proteolytic activity was reproducibly detected in isolated nuclei during the first hour of dexamethasone treatment and prior to any significant elevation in the number of small apoptotic cells. This activity continued to increase with prolonged dexamethasone exposure and it paralleled both the rate of DNA fragmentation (Fig. 1B) and the rate of appearance of small apoptotic cells (Fig. 2).

Morphological changes in nuclear matrix components: NuMA, lamin B and PI2 To evaluate the changes in nuclear morphology that accompanied chromatin cleavage, thymocytes were immunostained with antibodies specific for distinct compartments of the nuclear matrix, i.e. with anti-NuMA for the nucleoplasm, with anti-lamin B for the nuclear lamina and with anti-PI2 antibodies for the nuclear envelope (Fig. 3). Chromatin morphology was monitored by counterstaining with the DNAspecific fluorochome, Hoechst 33258. The micrographs in Fig. 3 are samples of cells treated with dexamethasone for 2 hours and were selected to show the range of staining patterns observed in control and treated samples. Typically, control thymocytes had roughly spherical nuclei with a slightly crenelated nuclear edge, a broad and irregular band of chromatin juxtaposed the nuclear periphery and a large mass of condensed chromatin usually occupied the center of the nucleus (Fig. 3A′C′). Following exposure to dexamethasone, nuclei with an altered pattern of DNA staining became increasingly frequent (Fig. 3A′-C′, arrowheads). They usually contained only one large mass of uniformly stained, untextured DNA, and were much smaller than adjacent intact nuclei. Cells with such nuclear morphology were interpreted as apoptotic (Kerr et al., 1972). In intact thymocytes, anti-NuMA staining was uniformly distributed in the nucleoplasm, with condensed chromatin masses not stained (Fig. 3A). In apoptotic nuclei, however, anti-NuMA staining was reduced to a single small bright patch closely apposed to, but distinct from, the DNA mass (Fig. 3A). The shape of the patches indicated that they occupied nucleoplasmic regions between the DNA mass and the nuclear envelope. Confocal microscopy of double-stained (NuMA and PI2) cells confirmed the distribution of NuMA in intact cells (Fig. 4A) and clearly demonstrated that the remaining patch of

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Fig. 2. Correlation between nuclear proteolytic activities and rate of apoptosis in dexamethasone-treated thymocytes. The proteolytic activity was assayed using FITC-casein substrate (open circles) and is expressed in trypsin equivalent units per mg of nuclear proteins. Each point represents mean ± s.e.m. of three separate thymocyte experiments with quadruplicate incubations for each isolation. The rate of thymic cell death was assessed from the appearance of small apoptotic cells measured in a Coulter counter (filled circles) within 0 to 6 hours following treatment with 1 µM dexamethasone.

Degradation of nuclear matrix in apoptosis NuMA positive staining seen in apoptotic cells was contained within the nuclear envelope (Fig. 4B) and was not released into the cytoplasm. Control thymocytes exhibited a continuous rim of peripheral nuclear staining when labeled with anti-lamin B (Figs 3B and 4C). The staining was considerably fainter or barely detectable in apoptotic nuclei, indicating possible solubilization of the lamina and/or loss of the protein. Labeling with anti-PI2 in intact thymocytes was indistinguishable from anti-lamin B staining, though brighter (Figs 3C and 4B). Unlike lamin B, however, PI2 was only slightly fainter and it was largely retained in the apoptotic nuclei. In some apoptotic nuclei, segments of the rim staining were brighter. These segments appeared to be in regions of the nuclear periphery that were not in contact with the collapsed DNA. Relationship between nuclear lamina and DNA fragmentation There are already several reports in the literature suggesting degradation of nuclear lamins in apoptotic cells, including those of dexamethasone-treated thymocytes (Kaufmann, 1989; Ucker et al. 1992; Lazebnik et al., 1993; Oberhammer et al., 1994; Neamati et al., 1995). These are based upon studies of the behavior of the whole cell population, which do not necessarily reflect true kinetic or temporal changes, since at any given timepoint (e.g. 2 hours or 4 hours after dexamethasone treatment) cells are at all stages of apoptosis (Fig. 3). Therefore, a timecourse of changes with reference to the point of addition of dexamethasone reflects events that are changing temporally within some but not all of the cells. For example, the changes in Coulter volume (Fig. 2) reflect the accumulation of cells that have suddenly undergone a rapid volume change, not the timecourse of volume change of the whole population. To correlate disassembly of the nuclear lamina with chromatin cleavage in the same cell we have used confocal microscopy to examine cells double labeled for lamin B and TUNEL. As shown in Fig. 4D, the nucleus of a cell selected at an early stage of DNA fragmentation has a brightly stained lamina. The labeling pattern with anti-lamin B in these TUNEL positive cells was not different from that seen in TUNEL negative cells (Fig. 4C). Moreover, there was no obvious concentration of DNA breaks associated with the nuclear periphery or any other defined area. Indeed, the breaks were evident throughout the nucleus, indicating that chromatin collapse into small dense masses had not yet occurred. However, late-stage apoptotic nuclei with collapsed DNA were typically lamin B negative (Fig. 3B), in agreement with the previous studies described above. Biochemical analysis of NuMA Nuclear matrix fractions were isolated and western blot analysis was performed with an anti-NuMA antibody, which was generated against the 96 kDa C-terminal fragment of the recombinant protein (Fig. 5). The antibody specifically recognized a 240 kDa single band of the native NuMa protein in matrix fractions prepared from freshly isolated and untreated thymocytes (Fig. 5A, lane 1). However, as early as 30 minutes after dexamethasone treatment an additional 200 kDa protein band became labeled by the antibody and the level of the lower 200 kDa band continued to increase with prolonged (up to 4 hours) dexamethasone treatment (Fig. 5A, lanes 2-5). Over the same timecourse a number of smaller protein bands became

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evident, particularly one of approximately 48 kDa. None of these lower molecular mass bands could be identified in matrix preparations isolated from untreated cells (lane 1), suggesting that they must have arisen, in response to the dexamethasone treatment, as a result of specific proteolytic cleavage of the native NuMA protein. Since the majority of cells in the primary culture are non-proliferating immature thymocytes that are spontaneously undergoing apoptosis, at a low rate, when placed in culture, NuMA integrity was also analyzed in control untreated cells over the same period of time (Fig. 5B). Under these conditions NuMA degradation was detectable after 2 hours of incubation, at which time the same 200 and 48 kDa fragments also became evident (Fig. 5B, lane 2). This showed that both spontaneous and dexamethasone-induced apoptosis triggered the proteolysis of NuMA protein. As mentioned earlier, serine-specific protease inhibitors alter the thymocyte apoptotic pathway by inhibiting internucleosomal DNA cleavage in response to dexamethasone and teniposide (Weaver et al., 1993). Here we tested the effects of PMSF and E64 on the integrity of NuMA protein (Fig. 5C). Clearly, neither of them prevented NuMA cleavage and the level of 200 kDa fragment observed after 2 hours of co-treatment with either PMSF (lane 1) or E64 (lane 2) was the same as found in the absence of these inhibitors (cf. Fig. 5A lane 4), suggesting that the protease involved has a high degree of specificity. Interestingly, since the antibody could recognize only the Cterminal region of NuMA, the initial cleavage of the protein must have occurred within its N terminus, yielding the 200 kDa band and an undetectable 40kDa fragment. This initial proteolytic step preceded any substantial level of DNA fragmentation. Whereas the 200 kDa NuMA fragment first appeared at 30 minutes of dexamethasone treatment and by 1 hour its level was equal to that of the remaining intact protein, DNA cleavage was evident in only a small fraction of the cells at this time (Fig. 5A, lanes 2-3 and Fig. 1A, lanes 0-1). Similarly, in control thymocytes NuMA degradation was detected after 2 hours in culture, which also preceded internucleosomal DNA cleavage, which does not occur until 4 to 6 hours (Weaver at el., 1993). NuMA is a phosphoprotein (Price and Pettijohn, 1986; Yang et al., 1992). Consistent with its role in mitotic spindle formation its phosphorylation, possibly by p34cdc2 kinase, is believed to be mitosis-specific (Cleveland, 1995; Compton and Luo, 1995). To establish whether any change in NuMA phosphorylation occurred prior to its degradation we radiolabeled the cellular pool of ATP by preincubating freshly isolated thymocytes for 30 minutes with [32P]orthophosphate. Cells were harvested at different times, lysed with RIPA buffer, immunoprecipitated with anti-NuMA and the precipitated proteins were analyzed by autoradiography after separation by SDS-PAGE (Fig. 6A and B). Initially, control precipitations were performed to demonstrate, by western blotting, that this procedure permitted the specific immunoprecipitation of NuMA. As shown in Fig. 6C, a 240 kDa protein band was found in samples treated with two different dilutions of anti-NuMA serum (lanes 1 and 2) but not in a mock precipitation with a non-specific rabbit serum (lane 3). Phosphorylation of NuMA occurred in dexamethasone-treated thymocytes as seen in Fig. 6A. Phosphorylated NuMA and its fragments were detected as early as 30 minutes after dexamethasone addition and their level increased in a time-dependent manner (lanes 2 to 4). NuMA

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phosphorylation also occurred in control thymocytes (Fig. 6B), but like its degradation, it did not take place until after 2 hours of incubation (lane 3). No phosphorylation of NuMA was detected at time 0, i.e. immediately after 30 minutes of preloading with [32P]orthophosphate (lane 1), suggesting that a protein kinase system involved in NuMA phosphorylation, inactive in quiescent thymocytes, became activated in response to the apoptotic stimuli. Biochemical analysis of lamin B and PI2 In order to compare the kinetics of lamin B degradation with those of NuMA and chromatin we prepared nuclear matrices from dexamethasone-treated cells for up to 12 hours of continuous drug exposure and analyzed them by western blotting with lamin B antibody (Fig. 7). In addition to the 67 kDa band of intact lamin B, the antibody also detected a 46 kDa protein doublet, which appeared within 30 minutes of dexamethasone exposure and its level increased with time (lanes 2 to 9). This 46 kDa band was not detected in matrix preparations isolated directly from intact thymus tissue (Fig. 7B), suggesting that it

Fig. 3. Immunofluorescent staining of nuclear matrix components. Thymocytes treated with 1 µM dexamethasone for 2 hours were immunostained with anti-NuMA (A), anti-lamin B (B) and anti-PI2 (C) sera and counter-stained with Hoechst 33258 dye (A′, B′, C′). Nuclei of apoptotic cells are indicated by arrowheads. Bar, 10 µm.

represented a proteolytic fragment of lamin B generated in response to dexamethasone. The effect of protease inhibitors on lamin B degradation were also examined (Fig. 7C). The data showed that neither PMSF (lane 1) nor E64 (lane 2) prevented the cleavage of lamin B into 46 kDa fragment, pointing out, again, that a highly specific proteolytic system is also involved in the degradation of lamin B. Comparing the kinetics of lamin degradation (Fig. 7) with that of DNA cleavage (Fig. 1) it seemed that the lamin fragments also appeared before detectable levels of chromatin cleavage. However, the confocal images displayed in Fig. 4D clearly showed that breaks in DNA occurred prior to the lamina solubilization. It is well established that the nuclear lamina structure is controlled by phosphorylation and two different protein kinase systems, protein kinase C (Hornbeck et al., 1988; Hocevar et al., 1993), and p34cdc2 kinase (Dessev et al., 1991; Heitlinger et al., 1991; Luscher et al., 1991), have been implicated in this process. In order to assess the phosphorylation of lamin B in dexamethasone-treated thymocytes, we also performed immunoprecipitation of the protein from thymocytes preloaded


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D

Fig. 4. Confocal microscope images of nuclear matrix components and DNA breaks. Thymocytes treated with 1 µM dexamethasone for 2 hours were double stained for NuMA and PI2 (A,B) or lamin B and TUNEL (C,D). Both intact (A,C) and apoptotic (B,D) nuclei are depicted. In A and B, the NuMA (red) and PI2 (green) staining patterns are shown either separately (on the left), or together in the overlay image (on the right). In C and D only the overlay images of lamin B (green) and TUNEL (red) are depicted.

with [32P]orthophosphate prior to dexamethasone treatment as described for Fig. 6. Again, the specificity of this assay was verified by performing western blot analysis of the proteins precipitated from control cell lysates by either anti-lamin B (Fig. 8B, lanes 1 and 2) or non-specific rabbit serum (Fig. 8B, lane 3). A 67 kDa band equivalent to intact lamin B was precipitated only by the anti-lamin B serum. Autoradiography of immunoprecipitated proteins from dexamethasone-treated thymocytes revealed that lamin B was rapidly phosphorylated even in cells not incubated with dexamethasone. The incorporation of 32P into both intact lamin B and the 46 kDa fragments remained at the same level throughout 4 hours of dexamethasone treatment; however, there was a timedependent accumulation of a number of radioactive bands in the 30 kDa range, indicating increasing degradation of lamin B (Fig. 8A). These lower molecular mass fragments were not seen on western blots of nuclear matrix preparations (Fig. 6), but they were immunoprecipitated by the antibody from cell lysates, suggesting that they represented soluble fragments of lamin B that were released from nuclei. A monoclonal antibody specific for the nuclear envelope structure, designated PI2, was developed several years ago and has been used extensively in immunofluorescence and EM studies; however, the constituent(s) of the nuclear envelope recognized by this antibody have not been well characterized (Chaly et al., 1984, 1989). We performed western blot analysis of nuclear matrix preparations obtained from dexamethasonetreated thymocytes with this antibody and were able to show, in agreement with previously reported data (Chaly et al., 1984),

Fig. 5. Degradation of NuMA in thymocytes undergoing dexamethasone-induced apoptosis. Western blots with NuMA antibody carried out on a 8.5% SDS-PAGE gel loaded with 100 µg/lane of nuclear matrix proteins prepared from thymocytes treated with 1 µM dexamethasone for 0, 0.5, 1, 2 and 4 hours (A, lanes 1-5, respectively), from untreated control thymocytes maintained in culture conditions for 1, 2, and 4 hours (B, lanes 1-3, respectively) and from thymocytes treated for 4 hours with both dexamathasone and either 2 mM PMSF or 20 µM E64 (C, lanes 1 and 2, respectively). Size standards are indicated in kDa.

that it recognized two protein species of molecular masses 80 and 140 kDa (Fig. 9). Their identity is unknown at present, but a number of proteins ranging in size from 45 to 210 kDa have been reported in association with the nuclear envelope structure (Snow et al., 1987). Significantly for this study, both proteins were remarkably stable and they were present in matrix preparations obtained from thymocytes treated with dexamethasone for up to 12 hours (Fig. 9, lanes 1-12). There was no detectable level of 32P associated with the immunoprecipitated PI2 antigens (data not shown), suggesting that their phosphorylation did not occur in dexamethasone-treated thymocytes. Electron microscopy Arends et al. (1990) have reported that the nuclear matrix is largely intact in apoptotic thymocytes, which would appear to contradict the degradation of matrix proteins observed here. Therefore, samples were examined by electron microscopy with particular attention to apoptotic cells, which were easily recognized by the characteristic morphology of the nuclei (Fig. 10). As has been previously described (Kerr et al., 1972), the nuclei generally contained a single, large, electron-dense mass of chromatin with a fibrogranular texture within an approximately circular nuclear profile (Fig. 10). The structural integrity of nucleoplasm was destroyed, appearing largely amorphous in texture with only a few identifiable filaments and granules.

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Fig. 6. Phosphorylation of NuMA following dexamethasone treatment. Autoradiographs of 32P-labeled proteins immunoprecipitated with NuMA antibody at a dilution 1:500 from cell lysates obtained at 0.5, 1, 2 and 4 hours after dexamethasone treatment (A, lanes 1-4, respectively) and from untreated control thymocytes maintained in culture for 0, 1, and 2 hours (B, lanes 1-3, respectively). Western blot of samples immunoprecipitated wit two dilutions of NuMA antibody (C, lane 1-1:500, lane 2-1:750), and with a nonspecific rabbit anti-mouse serum (C, lane 3). 14C-radiolabeled molecular size markers (Amersham) are shown in lane M (values indicated in kDa). Proteins were separated on a 8.5% SDS-PAGE gel.

In contrast, the nuclear envelope was well preserved but it was exceptionally smooth, exhibiting few of the crenelations common in intact nuclei. Nuclear pores and both nuclear membranes were clearly visible. Where the envelope was in contact with the nucleoplasm some ribosomes could be identified on its cytoplasmic face. In these conventional electron microscope preparations, it was not possible to determine whether the lamina was also present.

DISCUSSION The proteolytic cleavage of NuMA and lamin B appears to be one of the earliest of the nuclear changes observed in dexamethasone-treated thymocytes. It occurred in a substantial fraction of the cells within 30 minutes of steroid treatment and before a detectable level of DNA fragmentation. The accumulation of protein fragments of definite sizes, i.e. 200 and 48 kDa for NuMA and 46 kDa for lamin B, suggested that the cleavage occurred at specific sites. Moreover, the lack of effect of general protease inhibitors (PMSF and E64), further implied the involvement of a highly specific proteolytic system(s) such as those of the ICE/CED-3 family of proteases. Thus far, only one other protein, poly(ADP-ribose) polymerase, has been shown to be a target of site-specific proteolytic degradation during apoptosis (Kaufmann et al., 1993; Lazebnik et al., 1994; Tewari et al., 1995; Nicholson et al., 1995). Several members of ICE/CED-3 family of cysteine proteases, namely ICEs, Nedd-2/ICH-1 and CPP32, have been identified so far and shown to initiate apoptotic events when transfected into host cells (Yuan et al., 1994; Wang et al., 1994; Kumar et al., 1994; Fernandes-Alnemri et al., 1994; Tewari et al., 1995). They cleave protein substrates at aspartic acid residues and their activity can be blocked by specific tetrapeptides representing the cleavage site of the appropriate substrate (Nicholson et al., 1995). The identity of the protease(s) responsible for NuMA and lamin B cleavage is unknown. It is possible, however, that they are pre-existing nuclear enzymes, since we have shown here and elsewhere (de Belle et al., 1994) that there is a significant increase in nuclear proteolytic activity in response to dexamethasone. Using a broad-specificity substrate, casein, we demonstrated that this increase was detected within the first

hour of the steroid treatment (Fig. 2), coincident with the degradation of both NuMA and lamin B (Figs 5 and 7). Using a zymogram assay with gelatin as a substrate we have shown (de Belle et al., 1994) that this nuclear proteolytic activity is associated with a high molecular mass, SDS-stable, protein complex. At present we are characterizing this proteolytic activity further. NuMA is an abundant nuclear protein (2×105 molecules/cell), and plays an essential role in mitotic spindle formation (Lydersen and Pettijohn, 1980; Price and Pettijohn, 1986; Yang et al., 1992; Compton and Cleveland, 1993; Cleveland, 1995). Its function in the interphase nucleus is less clear, however, but the coiled-coil nature of the protein implies that it could form core filaments of the nucleoskeleton or

Fig. 7. Degradation of lamin B in thymocytes undergoing dexamethasone-induced apoptosis. Western blots with lamin B antibody carried out on a 10% SDS-PAGE gel loaded with 100 µg/lane of nuclear matrix proteins prepared from thymocytes treated with 1 µM dexamethasone for 0, 0.5, 1, 2, 4, 6 and 12 hours (A, lanes 1-7, respectively), nuclear matrix proteins prepared directly from a thymus tissue (B, lane 1-100 µg and lane 2-75 µg of proteins/lane), and from thymocytes treated for 4 hours with both dexamathasone and either 2 mM PMSF or 20 µM E64 (C, lanes 1 and 2, respectively). Mr, molecular mass markers.


Fig. 8. Phosphorylation of lamin B following dexamethasone treatment. (A) Autoradiograph of 32P-labeled proteins immunoprecipitated with lamin B antibody at a dilution 1:50 from cell lysates obtained at 0, 1, 2 and 4 hours after dexamethasone treatment (lanes 1-4, respectively). 14C-radiolabeled molecular size markers (Amersham) was shown in lane M. (B) Western blot of samples immunoprecipitated from control untreated thymocytes with two dilutions of lamin B antibody (1:100 and 1:50, lanes 1 and 2), and with a non-specific rabbit anti-mouse serum (lane 3). Proteins were separated on a 10% SDS-PAGE gel.

intranuclear matrix (Zeng et al., 1994; Cleveland 1995). In control thymocytes, NuMA was distributed throughout the nucleoplasm (Figs 3A and 4A), but did not appear to be in direct contact with DNA at MAR or SARs (matrix or scaffold attachment regions), which are the likely starting points of DNA fragmentation (Walker and Sikorska, 1994; Walker et al., 1995). However, as a result of its degradation the nucleoskeleton structure became destabilized and eventually collapsed. Specific degradation of NuMA also occurs in other cell types (Jurkat, MCF7) exposed to different apoptotic stimuli (anti-Fas, serum deprivation, VM-26), suggesting that NuMA degradation is a common process in apoptosis (unpublished data). Two significant functional features are encoded in the NuMA amino acid sequence. The C-terminal tail of the protein carries the sequence necessary for nuclear import and spindle binding and the N-terminal head is required for the post-mitotic reassembly of nuclei (Compton and Cleveland, 1993). Our data show that it was the N terminus of the protein that was preferentially targeted by the degradative process. The significance

Fig. 9. Identification of PI2-specific antigens in dexamethasonetreated thymocytes. Western blot with PI2 antibody carried out on a 8.5% SDS-PAGE gel loaded with 100 µg/lane of nuclear matrix proteins isolated from thymocytes treated with 1 µM dexamethasone for 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10 and 12 hours (lanes 1-9 and 11-12, respectively). Molecular size markers (Rainbow markers, Amersham) are shown in lane 10.

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Fig. 10. Electron micrograph of dexamethasone-treated apoptotic thymocyte.

of this is apparent in the study of Compton and Cleveland (1993) in which they have demonstrated that overexpression of an N-terminal truncated, headless NuMA in HeLa cells leads to their post-mitotic micronucleation identical to that seen in the temperature-sensitive mutant hamster tsBN2 cell line (Ohtsubo et al., 1989). NuMA is shown to be absent from these micronuclei and the protein undergoes degradation in the cytoplasm. Coincidentally, many apoptotic cells package fragmented chromatin into apoptotic bodies morphologically similar to micronuclei. The NuMA protein became phosphorylated in response to the dexamethasone treatment, suggesting that the hormone engaged a signalling pathway capable of NuMA modification. This is the first demonstration of its phosphorylation in a nonmitotic cell. It is known that within the NuMA primary structure there are p34cdc2 consensus phosphorylation sites, which are essential for protein function in mitosis. These sites have been mapped to the 45 kDa carboxy-terminal tail of the protein (Compton and Luo, 1995). As mentioned earlier, the NuMA antibody employed in this study that immunoprecipitated the 200 and 48 kDa NuMA fragment were also specific for the C-terminal portion of the molecule, suggesting that the dexamethasone-induced phosphorylation could be mediated by p34cdc2 kinase in a fashion similar to that in mitosis. Phosphorylation could trigger a conformational change within the NuMA protein at an inappropriate phase of the cell cycle, rendering it a target for a proteolytic enzyme. The protein was not completelety degraded, but rather cleaved at specific sites, possibly leading to a loss of interactions with other nuclear proteins. Interestingly, the predicted structural features of NuMA, such as globular head and tail domains and central α-helical rod domain, are similar to those of members of the family of intermediate filament proteins (Yang et al., 1992). This suggest that NuMA can oligomerize through coiled-coil interactions to form a filamentous structures (Zeng et al., 1994). The cleavage of NuMA would be expected to lead to changes in nuclear morphology, causing a destabilization

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and, ultimately, a breakdown and collapse of the nuclear proteinaceous meshwork supporting chromatin organization, as seen in the electromicrograph of Fig. 10. The function of the nuclear lamina is well defined. It has been shown to provide some of the attachment sites for higher order chromatin domains, support the organization of the nuclear envelope structure and maintain a direct link with the cytoplasm through its interaction with intermediate filaments (Georgatos and Blobel, 1987; Gerace, 1988; Paddy et al., 1990; Luderus et al., 1992). This nuclear structure contributes not only to the organization of interphase chromosome architecture, but also the nucleo-cytoplasmic movement of macromolecules. Its alteration might be expected to bring about major changes in chromatin organization as well as a disruption of nuclear-cytoplasmic transport. Such changes are typically seen in apoptotic cells. Earlier studies have suggested that destruction of the nuclear lamina is an early apoptotic event preceding, or triggering, chromatin cleavage and collapse. Here we showed that, in thymocytes, solubilization of the lamina was not required for the initiation of DNA cleavage as shown by confocal microscopy of lamin B/TUNEL double-labeled cells. In cells at early stages of DNA degradation breaks were detected throughout the nucleus, not just at the periphery (Fig. 4B). This is not to say that an apoptotic signal did not modify the lamina in some other ways that altered its interaction with the cytoplasm and/or the nucleus and destabilized it. Indeed, degradation of lamin B could be detected within 30 minutes of dexamethasone exposure and the protein was cleaved at a specific site to generate the 46 kDa fragment that persisted and accumulated with time. The length of this fragment corresponded to the central rod domain region of lamin B, which might have been protected from further degradation due to its coil-coiled dimerization mode, required for the first level of the structural organization of the lamina (Heitlinger et al., 1991). This would imply that the C-terminal end domains, which are normally folded into globular heads essential for further association of the lamin B dimers within the lamina structure, were destroyed as the result of this site specific proteolysis. This would clearly compromise its functional integrity. Nevertheless the lamina structure did not solubilize until late stages of nuclear collapse, suggesting that cleavage was not sufficient to completely dissolve it. In mitotic cells, hyperphosphorylation of lamins triggers their depolymerization (Ottaviano and Gerace, 1985; Gerace, 1988; Heitlinger et al., 1991). We have detected phosphorylation of lamin B (and its fragments) in non-mitotic thymocytes (Fig. 8). However, there was no increase in the level of phosphorylation in response to dexamethasone. This implied that the process of lamin phosphorylation, which was described to occur continuously throughout all interphase periods on the assembled lamina, was uninterrupted in dexamethasonetreated thymocytes (Ottaviano and Gerace 1985). As mentioned earlier, two different protein kinase systems, protein kinase C (Hornbeck et al., 1988; Hocevar et al., 1993) and p34cdc2 (Dessev et al., 1991; Heitlinger et al., 1991; Luscher et al., 1991; Dessev, 1992), have been shown to participate in the lamina phosphorylation. Whereas protein kinace C might be responsible for the modification of lamins during interphase, p34cdc2 is implicated in the triggering of their depolymerization during mitosis. From our phosphorylation data obtained for both NuMA and lamin B, participation of p34cdc2 protein kinase could not be excluded even though its role apoptosis is

still controversial. For example, Shi et al. (1994) have shown in YAC-1 lymphoma cells that an inappropriate activation of the kinase during the cell cycle leads to apoptotic cell death preventable by kinase inactivation. On the other hand, Oberhammer et al. (1994) and Neamati et al. (1995) using serumdeprived HPV-transformed rat cells and dexamethasonetreated thymocytes, respectively, did not find any evidence of apoptosis-specific activation of p34cdc2 kinase. Taken together, the data presented in this manuscript support a model of apoptotic nuclear destruction that is triggered by site-specific proteolysis of key structural proteins. Their modification and cleavage lead to the exposure of highly sensitive sites on chromatin to endonucleolytic attack. These sites probably lie within the unpaired regions of DNA attached to the nuclear matrix, generating high molecular mass fragments (50-300 kb) of DNA (reviewed by Walker et al., 1994, 1995) that are subsequently degraded to much smaller fragments in some, but not all, cells. Both the NuMA and lamin B proteins, because of their essential role in the maintenance of nuclear structure, appear to be critical targets of site-specific proteases that initiate the cascade of degradative events in apoptosis. The authors thank Julie Leblanc and Andrew Vaillant for excellent technical assistance.

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