nuclear changes in necrotic hl-60 cells

Journal of Cellular Biochemistry Supplement 36:19±31 (2001)

Nuclear Changes in Necrotic HL-60 Cells

Roberta Bortul,1 Marina Zweyer,1 Anna Maria Billi,2 Giovanna Tabellini,1 Robert L. Ochs,3

Renato Bareggi,1 Lucio Cocco,2 and Alberto M. Martelli1,2*1Dipartimento di Morfologia Umana Normale, UniversitaÁ di Trieste, 34138 Trieste, Italy2Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell'Apparato Locomotore, FacoltaÁ diFarmacia, UniversitaÁ di Bologna, 40126 Bologna, Italy3Precision Therapeutics, Pittsburgh, PA 15213, Pennsylvania

Abstract Cell death in eukaryotes can occur by either apoptosis or necrosis. Apoptosis is characterized by well-de®ned nuclear changes which are thought to be the consequence of both proteolysis and DNA fragmentation. On theother hand, the nuclear modi®cations that occur during necrosis are largely less known. Here, we have investigatedwhether or not nuclear modi®cations occur during ethanol-induced necrotic cell death of HL-60 cells. By means ofimmuno¯uorescence staining, we demonstrate that the patterns given by antibodies directed against some nuclearproteins (lamin B1, NuMA, topoisomerase IIa, SC-35, B23/nucleophosmin) changed in necrotic cells. The changes inthe spatial distribution of NuMA strongly resembled those described to occur during apoptosis. On the contrary, the¯uorescent pattern characteristic for other nuclear proteins (C23/nucleolin, UBF, ®brillarin, RNA polymerase I) did notchange during necrosis. By immunoblotting analysis, we observed that some nuclear proteins (SAF-A, SATB1, NuMA)were cleaved during necrosis, and in the case of SATB1, the apoptotic signature fragment of 70 kDa was also present tothe same extent in necrotic samples. Caspase inhibitors did not prevent proteolytic cleavage of the aforementionedpolypeptides during necrosis, while they were effective if apoptosis was induced. In contrast, lamin B1 andtopoisomerase IIa were uncleaved in necrotic cells, whereas they were proteolyzed during apoptosis. Transmissionelectron microscopy analysis revealed that slight morphological changes were present in the nuclear matrix fractionprepared from necrotic cells. However, these modi®cations (mainly consisting of a rarefaction of the inner ®brogranularnetwork) were not as striking as those we have previously described in apoptotic HL-60 cells. Taken together, our resultsindicate that during necrosis marked biochemical and morphological changes do occur at the nuclear level. Thesealterations are quite distinct from those known to take place during apoptosis. Our results identify additionalbiochemical and morphological criteria that could be used to discriminate between the two types of cell death. J. Cell.Biochem. Suppl. 36:19±31, 2001. ß 2001 Wiley-Liss, Inc.

Key words: necrosis; apoptosis; proteolysis; caspases; nuclear proteins; nuclear matrix

Two morphologically distinct pathophysiolo-gical types of cell death which occur in euka-ryotes are apoptosis and necrosis. Whileapoptosis is genetically controlled and is de®ned

by nuclear and cytoplasmic shrinkage, mem-brane blebbing, chromatin margination, andDNA fragmentation, necrosis is characterizedby nuclear pyknosis, cytoplasmic swelling, anda progressive loss of membrane integrity[Wyllie et al., 1980; Bellamy et al., 1995].During apoptosis the cell is broken down inmultiple spherical bodies that retain membraneintegrity and are phagocytosed by neighboringcells without eliciting signi®cant in¯ammatorydamage [Bellamy et al., 1995]. In necrosis,cellular fragmentation takes place with a sub-sequent release of lysosomal content in theextracellular space and the occurrence ofin¯ammation [Wyllie et al., 1980].

Recent ®ndings have highlighted the fact thatthe differences between necrosis and apoptosis

ß 2001 Wiley-Liss, Inc.This article published online in Wiley InterScience, March 13, 2001.

Grant sponsor: Italian ``MURST Co®nanziamento 1999'' toA. M. Martelli and L. Cocco; Grant sponsor: AssociazioneItaliana per la Ricerca sul Cancro (AIRC) to L. Cocco; Grantsponsor: Funds For Selected Research Topics of BolognaUniversity and the Italian CNR Finalized Project ``Bio-technology'' to L. Cocco; Grant sponsor: Italian Ministry forHealth ``Ricerca Finalizzata'' to A.M. Martelli.

*Correspondence to: Dr. Alberto M. Martelli, Dipartimentodi Morfologia Umana Normale, UniversitaÁ di Trieste, viaManzoni 16,34138 Trieste, Italy. E-mail: [email protected]

Received 3 October 2000; Accepted 30 November 2000

might be less numerous than initially believed.Indeed, some of the biochemical changes whichtake place during apoptosis have been reportedto occur also during necrosis. For example, theinner mitochondrial membrane may becomepermeable to ions and small molecules duringboth necrosis and apoptosis. Moreover, activa-tion of cysteine proteases and cleavage ofpoly(ADP-ribose) polymerase has been docu-mented to occur during both modes of cell death[Shah et al., 1996; Dynlacht et al., 1999].

A central biochemical mechanism that leadsto apoptotic cell death is the activation of afamily of cysteinyl-aspartate-speci®c proteasesreferred to as caspases [Patel et al., 1996;Thornberry and Lazebnik, 1998; Utz andAnderson, 2000]. Most of the proteins cleavedby caspases have important functions in thecell, so that their cleavage during early stages ofapoptosis may be expected to silence basiccellular processes and to lead to disintegrationof cellular architecture. However, evidence sug-gests that protease activation is also importantfor cell necrosis and in some cases caspaseactivation has been reported to occur duringthis type of cell death [e.g. Shimizu et al., 1996;Faraco et al., 1999].

A distinctive feature of apoptotic cell deathare the marked changes occurring in thenucleus, which most likely re¯ect proteolysisof several polypeptides and fragmentation ofDNA [Earnshaw, 1995; Martelli et al., 1997].Indeed, several nuclear protein are targets ofactivated proteases and include: topoisomeraseI, topoisomerase IIa, lamins, lamin B receptor,NuMA, SAF-A, UBF, SATB1, heterogeneousribonucleoproteins C1 and C2, U1-70 kDa[Earnshaw, 1995; Casiano et al., 1996; Water-house et al., 1996; Weaver et al., 1996; Goehringet al., 1997; Duband-Goulet et al., 1998; Buen-dia et al., 1999; Gotzmann et al., 2000; seeMartelli et al., 1997 for a comprehensive reviewon the issue]. Moreover, recent ®ndings indicatethat the apoptotic process is also characterizedby a caspase-mediated cleavage of nucleic acidsthrough the activation of nucleolytic activities[Degen et al., 2000]. During apoptosis, modi®ca-tions have been described to occur in the nuclearmatrix, i.e. the proteinaceous structure which isthought to be responsible for maintaining thefunctional and topological organization of theinterphase nucleus [Berezney et al., 1995;Martelli et al., 1996, 1999a, 1999b]. In particu-lar, a disassembly of the nuclear matrix is

thought to be a critical step which leads tonuclear collapse [Weaver et al., 1996; Martelliet al., 1997; Gerner et al., 1998]. On the otherhand, the changes that take place in the necroticnucleus have scarcely been investigated, eventhough proteolysis of some key nuclear proteinshas been reported to also occur during necroticcell death [Casiano et al., 1998]. In particular,Dynlacht et al. [1999] recently suggested thatdegradation of nuclear matrix is a commonelement during both apoptosis and necrosisinduced by irradiation of HL-60 cells. However,they came to such a conclusion exclusively byobserving that lamin B was proteolyticallycleaved during the necrotic process. Therefore,we decided to perform a comprehensive inves-tigation of the nuclear structure during ethanol-induced necrosis of HL-60 cells. Our resultsshow that most of the changes in the distribu-tion of several key nuclear components whichoccur during necrosis are clearly distinct fromthose described during apoptosis and the sameis true for the proteolysis of select nuclearpolypeptides. In addition, we show that slightmodi®cations of the nuclear matrix also occurduring necrosis as demonstrated by ultrastruc-tural analysis, albeit the changes are not asstriking as during apoptosis.

MATERIALS AND METHODS

Source of Antibodies and Caspase Inhibitors

The following antibodies were employed inthis study: monoclonal antibody to topoisome-rase IIa (clone Ki-S1, Roche Molecular Bio-chemicals, Milan, Italy); monoclonal antibody toNuMA N-terminal head domain (TransductionLaboratories, Lexington, KY); monoclonal anti-body to lamin B1 (clone 101-B7, Calbiochem, LaJolla, CA); monoclonal antibodies to splicingfactor SC-35 or b-tubulin (Sigma ChemicalCompany, St. Louis, MO); polyclonal antibodyto protein SATB1 was a kind gift by Dr. TerumiKohwi-Shigematsu, University of California,Berkeley, CA; polyclonal antibody to SAF-Awas a generous gift by Dr. Frank Fackelmayer,University of Konstanz, Germany; monoclonalor polyclonal antibodies to nucleolar proteins(C23/nucleolin; B23/nucleophosmin; RNApolymerase I; UBF; ®brillarin) were as recentlyreported [Martelli et al., 2000]. The followingcell-permeable irreversible caspase inhibitorswere from Calbiochem: Ac-DEVD-CMK (aninhibitor of caspase-3); VEID-CHO (an inhibitor

20 Bortul et al.

of caspase-6); Z-IETD-FMK (an inhibitor ofcaspase-8); Z-LEHD-FMK (an inhibitor of cas-pase-9).

Cell Culture and Induction of Apoptosisor Necrosis

HL-60 human promyelocytic leukemia cellswere cultured in RPMI-1640 medium supple-mented with 10% fetal bovine serum. Forinduction of apoptosis, cells were exposed for 5h to 68 mM etoposide (Sigma). Flow cytometricanalysis revealed that this treatment causedabout 85±90% of cells to undergo apoptosis,whereas in control samples < 1% of cells wereapoptotic [data not shown and Martelli et al.,1999a]. For induction of necrosis, cells wereexposed to 10% ethanol for 5 h [Casiano et al.,1998]. In the experiments performed in thepresence of caspase inhibitors, the chemicalswere added 30 min prior to adding etoposide orethanol. The ®nal concentration of the inhi-bitors was 100 mM, from a 25 mM stock indimethylsulfoxide.

Transmission Electron Microscopy (TEM)Analysis

Cells or nuclear matrices were ®xed with 2.5%glutaraldehyde in 0.1 M phosphate buffer for 35min, dehydrated in increasing concentrations ofacetone, embedded in Araldite, thin sectioned,and observed with a Jeol 100S electron micro-scope.

Immuno¯uorescence Staining

Cells were plated onto 0.1% poly-L-lysine-coated glass slides and adhesion was allowed toproceed for 30 min at 378C. Samples were ®xedin freshly-prepared 4% paraformaldehyde inPBS for 30 min at room temperature. Afterseveral washes with PBS, nonspeci®c binding ofantibodies was blocked by a 30 min incubationat 378C with blocking buffer, i.e. PBS containing5% normal goat serum (NGS) and 4% bovineserum albumin (BSA, Sigma fraction V). Slideswere then incubated for 3 h at 378C with theprimary antibodies diluted in blocking buffer.They were subsequently washed three times inPBS and reacted for 1 h at 378C with theappropriate ¯uorescein isothiocyanate (FITC)-conjugated secondary antibodies (from Sigma),diluted 1:100 in blocking buffer. Samples werethen stained for DNA with 0.1 mg/ml 40-6-diamidino-2-phenylindole (DAPI) in PBS,0.05% Tween 20. Slides were observed and

photographed using a Zeiss Axiophot epi¯uor-escence microscope.

Polyacrylamide Gel Electrophoresis andImmunoblotting of Cell Lysates

Cells were sedimented at 1,000g for 10 minand washed twice in phosphate buffered saline,pH 7.4 (PBS) containing the COMPLETEProtease Inhibitor Cocktail (Roche MolecularBiochemicals), according to the manufacturer'sinstructions. Cells were then resuspended at� 107/ml in boiling lysis buffer containing 62.5mM Tris-HCl, pH 6.8, 2% sodium dodecylsulfate(SDS), 10% glycerol, 5% 2-mercaptoethanol,and the protease inhibitor cocktail. Lysateswere brie¯y sonicated to shear DNA and reduceviscosity, boiled for 5 min to solubilize protein,and stored at ÿ 808C until required. Cellharvesting and lysate preparation were con-ducted in the presence of the protease inhibitorcocktail as a precaution to prevent furtherproteolysis. Total protein from � 5� 106 cellswas separated by SDS-polyacrylamide gel elec-trophoresis (SDS-PAGE) and electrophoreti-cally transferred to nitrocellulose sheets usinga semi-dry blotting apparatus (Hoefer/Pharma-cia Biotech, Uppsala, Sweden). Sheets weresaturated for 60 min at 378C in blocking buffer,then incubated overnight at 48C in blockingbuffer containing the primary antibodies. Afterfour washes in PBS containing 0.1% Tween 20,they were incubated for 30 min at roomtemperature with peroxidase-conjugated sec-ondary antibodies (from Sigma), diluted 1:5,000in PBS-Tween 20, and washed as above. Bandswere visualized by the enhanced chemilumines-cence method using Lumi-LightPlus (RocheMolecular Biochemicals).

Preparation of Nuclear Matrix fromHL-60 Cells

Cells were washed once in PBS (without Ca2�

and Mg2�) and resuspended to 1.5� 107/ml in10 mM Tris-HCl, pH 7.4, 2 mM MgCl2, 0.5 mMphenylmethylsulfonyl ¯uoride (PMSF), 1 mg/mleach of aprotinin and leupeptin (TM-2 buffer,temperature� 108C). After 5 min at 08C, TritonX-100 was added to 0.5% (w/v) and cells weresheared by one passage through a 22 gaugeneedle ®tted to a 30 ml plastic syringe. Nucleiwere sedimented at 400g for 6 min, washed oncein TM-2 buffer and resuspended to 2 mg DNA/ml in 10 mM Tris-HCl, pH 7.4, 0.25 M sucrose,5 mM MgCl2 plus protease inhibitors as above

Necrosis and Nuclear Structure 21

(STM-5 buffer). They were incubated for 45 minat 08C in STM-5 buffer containing 2 mM sodiumtetrathionate (NaTT). Nuclei were thendigested for 60 min at 08C with 50 U DNase I/mg DNA (from Sigma). An equal volume of 4 MNaCl in 10 mM Tris-HCl, pH 7.4, 0.2 mM MgCl2,1 mM PMSF (LM buffer) was then added,followed by 8 volumes of 2 M NaCl in LM buffer.Structures were sedimented at 1,500g for 10min, washed once in LM buffer, and employedas the nuclear matrix fraction.

Protein Recovery

Assays Were Performed as Described byBradford [1976].

Two-Dimensional Gel Electrophoresis

Nuclear matrix protein (from 5� 107 cells)was resuspended in 400 ml of lysis buffer[O'Farrell, 1975] and incubated for 3 h at roomtemperature. Insoluble material was removedby centrifugation at 10,000g for 5 min and thesupernatant was layered on the ®rst dimensiongel. Non-equilibrium pH gradient gel electro-phoresis (NEPHGE) was carried out as reportedby O'Farrell et al. [1977] in 9.2 M urea, 2%Nonidet P-40, 4% polyacrylamide, 2% ampho-lytes (Bio-Lyte pH 3-10, Bio-Rad Laboratories,Hercules, CA). First dimension gels (10 cm-longwith a diameter of 3 mm) were run for 16 h at300 V (constant). Second dimension gels were8% SDS-PAGE. Gels were stained with Coo-massie Blue R-250. The pH gradient wascalibrated using carbamylated glyceraldehyde-3-phosphate dehydrogenase standards (Phar-macia Biotech). Gels representative of threeseparate preparations are shown.

RESULTS

TEM Analysis of HL-60 Cells

As presented in Figure 1A, the ultrastruc-tural morphology of control HL-60 cells wastypical. Nuclei were indented, with a prevalenceof euchromatin and one or more evident

Fig. 1. TEM analysis of control, apoptotic, and necrotic HL-60cells. (A): control (untreated) cell. (B): apoptotic (etoposide-incubated) cell; nb: nuclear body; arrow points to chromatinmarginations while arrowhead indicates a nucleolar segrega-tion. (C): ethanol-exposed cell; hc: heterochromatin; asteriskindicates a large cytoplasmic vacuole; arrowhead indicates atransparent area, which is often seen in necrotic nuclei; arrowspoints to the damaged plasma membrane. Scale bar: 1 mm.

22 Bortul et al.

nucleoli. In cells exposed for 5 h to etoposide,the distinctive apoptotic nuclear bodies wereobserved. The plasma membrane was wellconserved even at this late stage (Fig. 1B). Inapproximately 90% of ethanol-treated cells weobserved a heavily damaged and swollen cyto-plasm containing numerous vacuoles of dif-ferent size (Fig. 1C). The plasma membraneshowed several breaks. The nucleus was stillsurrounded by a well preserved envelope.Numerous small and regular clumps of hetero-chromatin were scattered in the nuclear inter-ior and juxtaposed to the nuclear envelope (Fig.1C). It is important to emphasize that innecrotic samples we did not ever detect theultrastructural nuclear changes which arecharacteristic of apoptotic cell death, such aschromatin margination, nucleolar segregation,and the appearance of nuclear inclusionsreferred to as HERDS [Earnshaw, 1995; Zweyeret al., 1995, 1997; Biggiogera et al., 1999;Martelli et al., 2000].

Immuno¯uorescence Staining of NuclearAntigens in Necrotic HL-60 Cells

We next investigated by ¯uorescent im-munostaining whether or not changes in thedistribution of nuclear proteins occurred duringnecrosis. Antibody to lamin B1 decorated thenuclear periphery in normal HL-60 cells, deli-neating a ring (Fig. 2). In necrotic samples,besides the nuclear periphery, the antibody alsostained the nuclear interior (Fig. 2). The mono-clonal antibody recognizing topoisomerase IIarevealed a sort of ®brogranular network innormal cells, and spared nucleoli that wereoutlined by DAPI staining (Fig. 2). In necroticHL-60 cells, the antibody characteristicallystained extremely bright and large (diameter:2±3.5 mm) clumps as well as smaller dots(Fig. 2). Interestingly, DAPI counterstainingrevealed that both the smaller dots and thelarge clumps were located within the nucleoli.In control cells, antibody to NuMA gave astaining pattern distribution similar to topoi-somerase IIa (Fig. 2). In necrotic nuclei wedetected numerous brilliant spots of differentsize, which localized both to the center and theperiphery (Fig. 2). Monoclonal antibody to theessential splicing component SC-35 immunos-tained numerous (20±25 for each nucleus)spots, of different size, located both in the centerand at the periphery of the control nuclei (Fig.2). In necrotic samples, the spots were some-

what smaller and mostly concentrated at thenuclear periphery (Fig. 2).

We next investigated a group of proteins thatwere localized to the nucleolus of control cells.The ¯uorescent immunostaining due to anti-bodies recognizing the majority of these pro-teins (®brillarin, UBF, C23/nucleolin, RNApolymerase I) did not change in necrotic cells(Fig. 2). In necrotic samples we did not observeany positive immunostaining of nucleoli whenwe employed a monoclonal antibody to proteinB23/nucleophosmin which, on the other hand,produced a very bright reaction when used innormal samples (Fig. 2). However, some faintimmunoreactivity was seen at the nuclearperiphery of ethanol-treated cells. In Table Iwe report, for each of the antigens studied, thepercentage of necrotic cells in which we detectedthe immuno¯uorescent pattern illustrated inthe pictures. It is evident that the pattern wehave chosen as ``representative'' was seen in themajority of necrotic cells.

Immunoblotting Analysis of Nuclear Proteinsin HL-60 Cell Lysates and Effect of

Caspase Inhibitors

In preliminary experiments, we examined thepolypeptide composition of control, apoptotic,and necrotic cells by performing SDS-PAGE ofwhole cell extracts. As presented in Figure 3,Coomassie Blue staining of gels revealed nomajor differences between control and apoptoticcells. This was true also for extracts fromnecrotic cells apart from the presence of a veryprominent band at 70 kDa. Overall, we feel thatthis observation may suggest that proteincleavage possibly observed in necrotic cellswas unlikely to be due to massive release ofcompartmentalized proteases during cell lysis,in agreement with others [Casiano et al., 1998].

To determine whether or not also necrosis isassociated with cleavage of key nuclear pro-teins, we examined 8 polypeptides by immuno-blotting analysis of HL-60 whole cell lysatesprepared from control (untreated), etoposide-treated (apoptotic), and ethanol-treated (necro-tic) cells.

In apoptotic cells, lamin B1 was cleaved andwe detected a 28 kDa fragment. Such afragment was not present in necrotic cells, inwhich lamin B1 was largely uncleaved, eventhough we saw a very faint band at approxi-mately 50 kDa (Fig. 4). Topoisomerase IIa wasalso cleaved during apoptosis with a predomi-


Fig. 2. Immuno¯uorescence staining of control (C) and necrotic (N) HL-60 cells. Scale bar: 5 mm.

nant cleavage product of approximately 110kDa. Only traces of this fragment were seen innecrotic cells (Fig. 4). In etoposide-treated cells,two major cleavage products of NuMA proteinwere detected, banding at approximately 200-and 185 kDa. In contrast, in necrotic samplesNuMA was proteolytically degraded to multiplesmaller fragments, in the range of 105/130 kDa(Fig. 4). Apoptotic cleavage of NuMA wascompletely blocked by an inhibitor of caspase-6(VEID-CHO) but not by Ac-DEVD-CMK, whichinhibits caspase-3. On the other hand, VEID-CHO could not prevent the necrotic proteolysisof NuMA. Also Ac-DEVD-CMK, Z-IETD-FMK(an inhibitor of caspase-8), and Z-LEHD-FMK(an inhibitor of caspase-9) were completelyineffective in this sense (data not presented).Consistent with other reports [Goehring et al.,1997; Kipp et al., 2000], SAF-A was cleaved

during apoptosis into the signature 97 kDafragment. This fragment was absent fromlysates of necrotic cells, in which instead wedetected a cleavage product banding at approxi-mately 102 kDa (Fig. 4). Ac-DEVD-CMKreduced the amount of the 97 kDa fragmentseen in extracts from apoptotic cells, whereasVEID-CHO could not. All the caspase inhibitorswe tested did not block the necrotic cleavage ofSAF-A (Fig. 4 and data not presented). Thesignature 70 kDa fragment of SATB1 wasdetected in both apoptotic and necrotic samples.However, in ethanol-treated HL-60 cells, inaddition to the signature fragment, we observeda larger fragment with a Mr of about 84 kDa(Fig. 4). Apoptotic proteolysis of SATB1 wasmarkedly inhibited by VEID-CHO but not byAc-DEVD-CMK. In contrast, VEID-CHO couldnot prevent the proteolytic cleavage of SATB1 innecrotic samples (Fig. 4). Ac-DEVD-CMK, Z-IETD-FMK, and Z-LEHD-FMK were also inef-fective in this sense (data not shown).

The splicing component SC-35 did not showany sign of cleavage during either apoptosis ornecrosis (Fig. 4). However, in samples fromapoptotic cells we observed a slight reduction inthe intensity of the immunoreactivity. Thus, itmight be that some proteolysis had indeedoccurred, but the monoclonal antibody we usedcould not recognize the cleaved fragment(s).The nucleolar protein C23/nucleolin was lar-gely uncleaved during apoptosis, even thoughwe detected an extremely faint band of 97 kDaafter a 5 h treatment with etoposide. Nocleavage at all was detectable in necrotic HL-60 cells. Protein B23 was also uncleaved both inapoptotic and necrotic HL-60 cells (Fig. 4). Thisis in agreement with Casiano et al. [1998]. Adown-regulation of B23/nucleophosmin hadbeen demonstrated to occur in apoptotic JurkatT lymphoblasts [Patterson et al., 1995].

In Table II we summarize the results of boththe immuno¯uorescence staining and theimmunoblotting analysis.

TEM Analysis of HL-60 Cell Nuclear Matrix

As shown in Figure 5A, the nuclear matrixprepared from untreated HL-60 cells exhibiteda characteristic tripartite structure, with anouter lamina, an inner ®brogranular network,and nucleolar remnants. The tripartite organi-zation was somewhat maintained also in necro-tic nuclear matrices, albeit the inner ®bro-granular network was more rari®ed (Fig. 5B).

TABLE I. Percent of Necrotic CellsShowing Typical Changes (as described in

the text) in the Spatial Distribution Patternof Nuclear Polypeptides

Protein

Percent of cells showing changesin the immuno¯uorescence

pattern

Lamin B1 71.6�8.3Topoisomerase IIa 79.5�9.9NuMA 68.7�8.8SC-35 74.5�10.4B23 89.1�9.5

Fig. 3. SDS-PAGE of proteins from whole cell extracts. 80 mg ofprotein was separated on a 10% gel. The position of molecularweight markers is indicated. C: control cells; A: apoptotic cells;N: necrotic cells.


Fig. 4. Immunoblots showing cleavage of some nuclearproteins during apoptosis or necrosis. The position of molecularweight markers is indicated. C: control cells; A: apoptotic cells;N: necrotic cells. Cells (1� 107) were lysed in 1 ml ofelectrophoresis sample buffer, brie¯y sonicated, and boiled.

Protein was separated by SDS-PAGE and transferred tonitrocellulose sheets that were then probed with monoclonalor polyclonal antibodies to various nuclear polypeptides.Bands were visualized by the enhanced chemiluminescencemethod.

26 Bortul et al.

The interchromatin granules were not clus-tered, as observed in control matrices, butinstead they were homogeneously distributed.The nucleolar remnants of necrotic matriceswere sharply separated from the ®brogranularnetwork. Furthermore, ultrastructural analy-sis revealed that preparations of necroticnuclear matrices were always contaminatedby residual elements of the cytoskeleton (seealso later). We could never avoid such acontamination, even if we employed a higher(up to 1%) concentration of detergent (Triton X-100 or Nonidet P-40) in the TM-2 buffer duringthe preparation of nuclei (data not presented).

Protein Composition of NecroticNuclear Matrix

We next investigated the protein compositionof necrotic HL-60 cell nuclear matrix by meansof two-dimensional gel electrophoresis. As pre-sented in Figure 6, it was possible to appreciateboth quantitative and qualitative differencesbetween normal and necrotic samples (compareA with B). As a whole, the necrotic nuclearmatrix fraction retained more protein (about50%) than that prepared from normal cells.However, it is likely that most of the differenceswe detected were due to contamination pro-blems. To prove that the matrix from ethanol-treated cells was contaminated by componentsof the cytoskeleton, we performed immunoblot-ting with an antibody to b-tubulin. As shown inFigure 6 (panel C) b-tubulin was absent frompreparations of control nuclear matrix (lane 1).However, when the nuclear matrix wasobtained from necrotic cells a strong reactionfor b-tubulin (Mr 55 kDa) was observed (lane 2).

DISCUSSION

In this paper, we have reported the results ofan investigation designed to analyze thechanges that possibly occur in the nucleusduring necrosis and to compare them to themodi®cations taking place in the same organelleduring apoptosis. As far as changes in thelocalization of nuclear components are con-cerned, those regarding topoisomerase IIa areof particular interest, because we neverobserved similar modi®cations in apoptoticcells, where the immunoreactivity for topoi-somerase IIa was markedly lower than incontrol cells (R. Bortul, unpublished experi-ments). Given also that the changes in thespatial distribution of topoisomerase IIa havebeen detected with other necrosis-inducingagents (HgCl2, H2O2, heat) and in other celllines (Jurkat T lymphoblasts) (R. Bortul,unpublished experiments), we think thatimmunostaining with an antibody to topoisome-rase IIa might be a useful way to distinguishbetween apoptotic and necrotic cells.

The changes in the spatial distribution ofother nuclear proteins during necrosis were notas striking or were similar to those occurringduring apoptosis. We think that the modi®ca-tions we have seen in the lamin B1 ¯uorescencepattern are conceivably due to changes in theoverall organization of the necrotic nucleus thatunmask intranuclear lamin B1 and render itaccessible to the antibody.

When the possible cleavage patterns of eightnuclear components were examined employingwhole cell extracts from necrotic and apoptoticsamples, our results were essentially similar to

TABLE 2. Summary of the Results Obtained by Immuno¯uorescence Staining andImmunoblotting Analysis

ProteinImmuno¯uorescence

changes in necrotic cells

Proteolysis in apoptotic cellsand size of predominant

fragments(s)

Proteolysis in necrotic cellsand size of predominant

fragments(s)

Lamin B1 Yes Yes:28 kDa Very slight: 50 kDaTopoisomerase IIa Yes Yes:110 kDa Very slight: 110 kDaNuMA Yes Yes:200-and 185 kDa Yes: multiple at 130 /105 kDaSC-35 Yes No NoSAF-A n.p. Yes:97 kDa Yes:102 kDaSATB1 n.p. Yes:70 kDa Yes:84-and 70 kDaFibrillarin No n.p. n.p.UBF No n.p. n.p.C23 No Very slight: 97 kDa NoRNA polymerase I No n.p. n.p.B23 Yes No No

n.p., not performed


those reported by Casiano et al. [1998] whodemonstrated that during necrosis severalnuclear proteins are either not cleaved orcleaved in a pattern which differs from thatpeculiar to apoptosis.

The only notable exception we have observedis represented by SATB1, because the apoptoticsignature cleavage fragment of 70 kDa was alsogenerated in necrotic cells in an amount similarto apoptotic cells.

We would like to point out that in an ourprevious report we did not detect apoptoticcleavage of SATB1 protein after treatment with

camptothecin [Martelli et al., 1999a]. However,in the present study, in which we employedetoposide, we observed the characteristic apop-totic signature cleavage pattern of SATB1, asreported recently also by others [Gotzmannet al., 2000]. A possible explanation is thatdifferent apoptotic effectors cause the genera-tion of different cleavage patterns, in agreementwith the recent ®ndings by Dynlacht et al.[2000].

As far as the results of the inhibition ofcaspases are concerned, our data are in linewith the data reported by others [Casiano et al.,1998] who showed that the proteolytic cleavagewhich takes place during necrosis is not blockedby the commonly employed caspase inhibitors.

Fig. 5. TEM analysis of control and necrotic HL-60 cell nuclearmatrix. (A): control nuclear matrix. (B): nuclear matrix obtainedfrom ethanol-exposed cells; the asterisk points to the ®brogra-nular network; arrow indicates a transparent area; arrowheadpoints to a nucleolar remnant sharply separated from the®brogranular network. Scale bar: 1 mm.

Fig. 6. Two-dimensional gel electrophoresis analysis ofprotein recovered in the nuclear matrix prepared from control(panel A) and ethanol-treated (panel B) HL-60 cells. Migration inthe ®rst dimension (NEPHGE) was from left to right. The dashedline indicates pH 7.0. Molecular weight markers are indicatedon the left. Panel C: immunoblot showing the presence of m-tubulin in the nuclear matrix prepared from necrotic cells (lane2). Lane 1: nuclear matrix obtained from control cells. 50 mg ofprotein was blotted to each lane. The observed Mr was 55 kDa.

28 Bortul et al.

Thus, other proteases, that await precise iden-ti®cation, must be activated during necrosis.However, at least in the case of SATB1, thecaspase inhibitor-insensitive proteolytic activ-ity was capable of generating the apoptoticsignature fragment of 70 kDa in necrotic cells.

The fact that apoptotic cleavage of SAF-A wasinhibited by Ac-DEVD-CMK is consistent withthis protein being a target of caspase-3 [Kippet al., 2000]. At variance with Gotzmann et al.[2000], the cleavage of SATB1 in apoptoticsamples was not prevented by treatment of cellswith an inhibitor of caspase-3, but it was indeedlargely reduced by VEID-CHO, which inhibitscaspase-6. However, Gotzmann et al. [2000]have mapped a potential caspase-6 cleavage site(VEMD) in the N-terminal site of SATB1. Thus,it would seem more logical that an inhibitor ofcaspase-6 could prevent proteolysis.

In necrotic cells, three nuclear proteins whichare thought to play an important structural role(i.e. NuMa, SAF-A, and SATB1) were cleaved.Indeed, these proteins (as well as topoisomeraseIIa) bind to speci®c DNA sequences, calledScaffold or Matrix Associated Regions (S/MARs) that would represent 300±1000 bp longstretches of nucleotides, A-T rich (over 70%),conceivably de®ning the base of loops in whichgenomic DNA is organized during the inter-phase. As strong evidence has been presentedthat S/MARs are essential determinants ofchromatin structure, the proteins binding tothese elements can be considered putativestructural components of the nuclear matrix[Strick and Laemmli, 1995].

It may, therefore, appear somewhat surpris-ing that we did not detect dramatic changes inthe nuclear matrix structure of ethanol-treatedcells. This could not be the consequence of thestabilization with NaTT, because our ownprevious results have demonstrated that, ifthe cells are apoptotic, morphological modi®ca-tions of the nuclear matrix were evident eventhough nuclei had been exposed to NaTT priorto DNase I digestion and 2 M NaCl extraction[Martelli et al., 1999b]. A few points should betaken into account for a possible explanation. Asfar as NuMA is concerned, previous investiga-tions, in which a mutated form of NuMA wasoverexpressed, have suggested that it couldplay a major structural role in the architectureof the normal interphase nucleus [Gueth-Hall-onet et al., 1998], even if more recent resultshave questioned such a conclusion [Taimen

et al., 2000], because NuMA can be lost bynon-proliferating cells without dramatic effectson nuclear structure and shape. Moreover, thecleavage pattern of both NuMA and SAF-A innecrotic cells differs from that observed inapoptotic samples, as emphasized above. Thesedifferences might affect in a speci®c manner theoverall organization of the nuclear matrix sothat it did not degrade as much as duringapoptosis. Furthermore, in necrotic samples animportant structural protein such as topoisome-rase IIa [e.g. Adachi et al., 1989] was almostcompletely uncleaved. However, it might bethat in this respect the most critical issue is theabsence of lamin B1 proteolysis in necrotic cells.Evidence indicates that lamin cleavage is veryimportant for the occurrence of the nuclearchanges typical of apoptosis. Indeed, Rao andcoworkers [1996] have shown that overexpres-sion of uncleavable mutant lamin A or B blocksthe appearance of chromatin condensationand nuclear shrinkage typical of apoptotic celldeath, even though the late stages of apoptosiswere morphologically unaltered and formationof apoptotic bodies was evident. Lazebnik et al.[1995] had previously reported that laminproteolysis is a prerequisite also for chromatinpackaging into apoptotic bodies.

Finally, the possibility exists that the ethanoltreatment we employed to induce necrosis actedas a mild ®xative also for the nuclear matrix(and not only for the cytoskeletal elements) andthis fact would have prevented marked changesin the nuclear matrix.

In any case, our results, taken together,unequivocally show that marked nuclearchanges occur also during necrotic cells death.The changes are partly similar and partlydifferent form those taking place during apop-tosis. The nuclear matrix is affected by this typeof death (even though only partially) and this issomewhat consistent with the report by Dyn-lacht et al. [1999]. It might be hypothesized thatboth the rarefaction of matrix inner ®brogra-nular network and the homogeneous distribu-tion of interchromatin granules seen duringnecrosis are the consequence of the speci®ccleavage of SAF-A and NuMA. Indeed, both ofthese proteins associate with nuclear ribonu-cleoprotein complexes [Zeng et al., 1994; Kippet al., 2000], that are among the most abundantcomponents of the nuclear matrix [Berezney,1991; Berezney et al., 1995]. A different proteo-lytic cleavage of SAF-A and NuMA, associated


with cleavage of SATB1 and lamin B1, may leadto the more striking changes that are typical ofthe nuclear matrix prepared from apoptoticcells [Martelli et al., 1999a, 1999b].

It may be that the differential immunostain-ing and proteolytic cleavage patterns we haveidenti®ed prove to be useful in the future forallowing a differential diagnosis between necro-tic and apoptotic samples, especially in thecontext of pathological processes in vivo, whichhas never been an easy task if ultrastructuralanalysis could not be employed [Farber, 1994;Columbano, 1995; Majno and Joris, 1995].

ACKNOWLEDGMENT

We thank Giovanna Baldini for helpfultechnical assistance.

REFERENCES

Adachi Y, Kas E, Laemmli UK. 1989. Preferential,cooperative binding of DNA topoisomerase II to scaf-fold-associated regions. EMBO J 13:3997±4006.

Bellamy OC, Malcomson DG, Harrison DJ, Wyllie AH.1995. Cell death in health and disease: the biology andregulation of apoptosis. Semin Cancer Biol 6:3±16.

Berezney R. 1991. The nuclear matrix: a heuristic model forinvestigating genomic organization and function in thecell nucleus. J Cell Biochem 47:109±123.

Berezney R, Mortillaro M J, Ma H, Wei X, Samarabandu J.1995. The nuclear matrix: a structural milieu for genomicfunction. Int Rev Cytol 162A:1±65.

Biggiogera M, Trenatani A, Martin TE, Pellicciari C. 1999.Terminal differentiation of erythroblasts leads toRNP segregation and formation of heterogeneous ectopicRNP-derived structures. Histochem Cell Biol 112:473±477.

Bradford MM. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizingthe principle of protein dye binding. Anal Biochem 72:248±254.

Buendia B, Santa-Maria A, Courvalin JC. 1999. Caspase-dependent proteolysis of integral and peripheral proteinsof nuclear membranes and nuclear pore complex proteinsduring apoptosis. J Cell Sci 112:1743±1753.

Casiano CA, Martin SJ, Green DR, Tang EM. 1996.Selective cleavage of nuclear autoantigens duringCD95(Fas/APO-1)-mediated T cell apoptosis. J ExpMed 184:765±770.

Casiano CA, Ochs RL, Tan EM. 1998. Distinct cleavageproducts of nuclear proteins in apoptosis and necrosisrevealed by autoantibody probes. Cell Death Diff 5:183±190.

Columbano A. 1995. Cell death: current dif®culties indiscriminating apoptosis from necrosis in the context ofpathological processes in vivo. J Cell Biochem 58:181±190.

Degen WGJ, Pruijn GJM, Raats JM, van Venrooij WJ.2000. Caspase-dependent cleavage of nucleic acids. CellDeath Diff 7:616±627.

Duband-Goulet I, Courvalin JC, Buendia B. 1998. LBR, achromatin and lamin binding protein from the innernuclear membrane, is proteolyzed at late stages ofapoptosis. J Cell Sci 111:1441±1451.

Dynlacht JR, Earles M, Henthorn J, Roberts ZV, HowardEW, Seno JD, Sparling D, Story MD. 1999. Degradationof the nuclear matrix is a common element duringradiation-induced apoptosis and necrosis. Radiat Res152:590±603.

Dynlacht JR, Earles M, Henthorn J, Seno JD. 2000.Different patterns of DNA fragmentation and degrada-tion of nuclear matrix proteins during apoptosis inducedby radiation, hyperthermia or etoposide. Radiat Res152:590±603.

Earnshaw WC. 1995. Nuclear changes in apoptosis. CurrOpin Cell Biol 7:337±343.

Farber E. 1994. Programmed cell death: necrosis vs.apoptosis. Modern Pathol 7:605±609.

Faraco PR, Ledgerwood EC, Vandenabeele P, Prons JB,Bradley JR. 1999. Tumour necrosis factor inducesdistinct patterns of caspase activation in WEHI-164 cellsassociated with apoptosis or necrosis depending on cellcycle stage. Biochem Biophys Res Comm 261:385±392.

Gerner C, Seelos C, Sauermann G. 1998. Alteration ofnuclear matrix protein composition during apoptosis inrat embryo cells. Exp Cell Res 238:472±480.

Goehring F, Schwab BL, Nicotera P, Leist M, FackelmayerFO. 1997. The novel SAR-binding domain of scaffoldattachment factor A (SAF-A) is a target in apoptoticnuclear breakdown. EMBO J 16:7361±7371.

Gotzmann J, Meissner M, Gerner C. 2000. The fate of thenuclear matrix-associated-region-binding protein SATB1during apoptosis. Cell Death Diff 7:425±438.

Gueth-Hallonet C, Wang J, Harborth J, Weber K, OsbornM. 1998. Induction of a regular lattice by overexpressionof NuMA. Exp Cell Res 243:434±452.

Kipp M, Schwab BL, Przybylski M, Nicotera P, Fack-elmayer FO. 2000. Apoptotic cleavage of scaffold attach-ment factor A (SAF-A) by caspase-3 occurs at anoncanonical cleavage site. J Biol Chem 275:5031±5036.

Lazebnik YA, Takahashi A, Moir RD, Goldman RD, PoirierGG, Kaufmann SH, Earnshaw WC. 1995. Studies of thelamin proteinase reveal multiple parallel biohemicalpathways during apoptotic execution. Proc Natl AcadSci USA92:9042±9046.

Majno G, Joris I. 1995. Apoptosis oncosis and necrosis: anoverview of cell death. Am J Pathol 146:3±15.

Martelli AM, Cocco L, Riederer BM, Neri LM. 1996. Thenuclear matrix: a critical appraisal. Histol Histopathol11:1035±1048.

Martelli AM, Bareggi R, Bortul R, Grill V, Narducci P,Zweyer M. 1997. The nuclear matrix and apoptosis.Histochem Cell Biol 108:1±10.

Martelli AM, Bortul R, Fackelmayer FO, Tazzari PL,Bareggi R, Narducci P, Zweyer M. 1999a. Biochemicaland morphological characterization of the nuclear matrixfrom apoptotic HL-60 cells. J Cell Biochem 72:35±46.

Martelli AM, Bortul R, Bareggi R, Grill V, Narducci P,Zweyer M. 1999b. Biochemical and morphologicalchanges in the nuclear matrix prepared from apoptoticHL-60 cells. Effect of different stabilizing procedures. JCell Biochem 74:99±110.

Martelli AM, Robuffo I, Bortul R, Ochs RL, Luchetti F,Cocco L, Zweyer M, Bareggi R, Falcieri E. 2000. Behavior

30 Bortul et al.

of nucleolar proteins during the course of apoptosis incamptothecin-treated HL-60 cells. J Cell Biochem 78:264±277.

O'Farrell PH. 1975. High-resolution two-dimensional elec-trophoresis of proteins. J Biol Chem 250:4007±4021.

O'Farrell PZ, Goodman HM, O'Farrell PH. 1977. High-resolution two-dimensional electrophoresis of basic aswell acidic proteins. Cell 12:1133±1142.

Patel T, Gores GJ, Kaufmann SH. 1996. The role ofproteases during apoptosis. FASEB J 10:587±597.

Patterson SD, Grossman JS, D'Andrea P, Latter GI. 1995.Reduced numatrin/B23/nucleophosmin in apoptotic Jur-kat T-lymphoblasts. J Biol Chem 270:9429±9436.

Rao L, Perez D, White E. 1996. Lamin proteolysis facilitatesnuclear events during apoptosis. J Cell Biol 135:1441±1455.

Shah GM, Shah RG, Poirier GG. 1996. Different cleavagepatterns for poly(ADP-ribose) polymerase during necro-sis and apoptosis in HL-60 cells. Biochem Biophys ResComm 229:838±844.

Shimizu S, Eguchi Y, Kamiike W, Waguri S, Uchiyama Y,Matsuda H, Tsujimoto Y. 1996. Retardation of chemicalhypoxia-induced necrotic cell death by BCL-2 and ICEinhibitors: possible involvement of common mediators inapoptotic and necrotic signal transductions. Oncogene12:2045±2050.

Strick R, Laemmli UK. 1995. SARs are cis DNA elements ofchromosome dynamics: synthesis of a SAR repressorprotein. Cell 83:1137±1148.

Taimen P, Viljamaa M, Kallajoki M. 2000. Preferentialexpression of NuMA in the nuclei of proliferating cells.Exp Cell Res 256:140±149.

Thornberry NA, Lazebnik Y. 1998. Caspases: enemieswithin. Science 281:1312±1316.

Utz PJ, Anderson P. 2000. Life and death decisions:regulation of apoptosis by proteolysis of signalingmolecules. Cell Death Diff 7:589±602.

Waterhouse N, Kumarz S, Song Q, Strike P, Sparrow L,Dreyfuss G, Alnemri ES, Litwack G, Lavin M, Watters D.1996. Heteronuclar ribonucleoproteins C1 and C2,components of the spliceosome, are speci®c targets ofinterleukin 1b-converting enzyme-like proteases in apop-tosis. J Biol Chem 271:29335±29341.

Weaver VM, Carson CE, Walker PR, Chaly N, Lach B,Raymond Y, Brown DL, Sikorska M. 1996. Degradationof nuclear matrix and DNA cleavage in apoptoticthymocytes. J Cell Sci 109:45±56.

Wyllie AH, Kerr JFR, Currie AR. 1980. Cell death:the signi®cance of apoptosis. Int Rev Cytol 68:251±306.

Zeng C, He D, Berget SM, Brinkley BR. 1994. Nuclear-mitotic apparatus protein: a structural protein betweenthe nucleoskeleton and RNA splicing. Proc Natl Acad SciUSA91:1505±1509.

Zweyer M, Bareggi R, Grill V, Soranzo MR, Marugg RA,Riederer BM, Narducci P, Martelli AM. 1995. Behaviourof nuclear matrix proteins during camptothecin-inducedapoptosis in HL-60 human leukemia cells. Exp Cell Res221:27±40.

Zweyer M, Riederer BM, Ochs RL, Fackelmayer FO,Kohwi-Shigematsu T, Bareggi R, Narducci P, MartelliAM. 1997. Association of nuclear matrix proteins with®brogranular nuclear bodies in cell lines undergoingapoptosis. Exp Cell Res 230:325±336.


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