Biol. Cell (2010) 102, 25–35 (Printed in Great Britain) doi:10.1042/BC20090077 Research article

Plasma membrane and nuclearenvelope integrity during theblebbing stage of apoptosis: atime-lapse studyRicardo Andrade*, Lorena Crisol†, Roberto Prado*, Marıa Dolores Boyano†, Jon Arluzea*† and Juan Arechaga*†1

*Biomedical Analytical and High Resolution Microscopy Facility, University of the Basque Country, E-48940 Leioa, Vizcaya, Spain, and

†Laboratory of Stem Cells, Development and Cancer, Department of Cell Biology and Histology, Faculty of Medicine and Dentistry,

University of the Basque Country, E-48940 Leioa, Vizcaya, Spain

Background information. The execution phase of apoptosis is characterized by extensive blebbing of the plasmamembrane, which usually results in secondary lysis in vitro. To analyse the permeability of cellular membranes duringthis process, we induced apoptosis in human melanoma A375 cells that had been transfected with fluorescentlytagged proteins which were targeted to different subcellular locations.

Results. The dual treatment of resveratrol and butyrate produced a synergistic induction of apoptosis by blockingdifferent phases of the cell cycle. Changes in the plasma membrane, nuclear envelope and nucleoli were mon-itored by time-lapse confocal microscopy. Fluorescently labelled proteins were not mis-localized from their originallocations in any of the cells undergoing blebbing for several hours. Thus the maintenance of karyophilic and nuc-leolar proteins within the nucleus during the blebbing stage and the accessibility of vital selective chromatin dyesconfirmed a functional preservation of the nuclear compartment until the final necrotic blister. The translocation ofphosphatidylserine to the outer leaflet of the plasma membrane was not detected during the blebbing period.

Conclusion. These results show that the functional integrity of the nuclear envelope and plasma membrane maybe conserved until the end of the execution phase of apoptosis.

IntroductionIn spite of the vast amount of data, the dynamics of theexecution phase of apoptosis in living cells has onlybegun to be studied during the last few years, whichis due, in part, to the standardization of microscopytechniques. Thus the first morphological evidence ofapoptosis begins with retraction of the cell, whichloses its adherence, while its cytoplasm and plasma

1To whom correspondence should be addressed (emailjuan.arechaga@ehu.es).Key words: apoptosis, butyrate, membrane blebbing, nuclear envelope,resveratrol, time-lapse microscopy.Abbreviations used: AM, acetoxymethyl ester; DAPI,4′,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle’s medium;FBS, fetal bovine serum; GFP, green fluorescent protein; GFP–Np,GFP–nucleoplasmin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; PBS, phosphate-buffered saline; PI, propidium-iodide; PS, phosphatidylserine.

membrane start to convolve, showing extensive andrapid blebbing. This zeotic blebbing can be observedfor several hours, and entails a vast reorganization ofthe organelles and the cell cytoskeleton. At the nuc-lear level, chromatin becomes highly condensed andthe nucleus is usually fragmented in small micronuc-lei. Finally, the cell is divided into several fragmentsthat are called apoptotic bodies, which will be phago-cytosed by neighbouring cells or will degenerate viasecondary lysis, leading to the loss of the cell contentto the surrounding media.

The molecular machinery behind the execution ofapoptosis is based on cytoskeletal forces (Mills et al.,1999). Initially, the actin cytoskeleton (Atenciaet al., 1997), together with myosin II and ROCKI(Rho kinase I) (Mills et al., 1998; Coleman and Olson,2002), are responsible for plasma membrane blebbing

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and nuclear disintegration (Coleman et al., 2001;Croft et al., 2005). At a later stage, reassembly ofmicrotubules contributes in the relocation of chro-matin and cellular fragmentation (Moss et al., 2006).

All these morphological and biochemical changesdepend on the rupture of key structural andregulatory proteins, which is carried out by a familyof cysteine proteases, the caspases (Earnshaw et al.,1999). At the nuclear level, the profound changesobserved in the apoptotic nucleus rely on the action ofspecific caspases, which have to cross the nuclear en-velope to exert its nuclear activities. Additionally, thenuclear envelope and the nucleocytoplasmic trans-port machinery are molecular targets for the actionof caspases, regulating an ordered disassembly ofthe nucleus during cell death. Proteins from the innernuclear membrane, such as lamins or several nuclearpore complex components, are the most well knownproteins to be degraded in apoptosis. The subcellularlocalization of soluble factors that participate innucleocytoplasmic transport, such as importins or theGTPase Ran, is altered during apoptosis (Ferrando-May et al., 2001), which may help to link specificapoptotic signals between the nucleus and the cyto-plasm. Two main apoptotic pathways have beendescribed: the extrinsic route, which is activated byligands that bind to the plasma membrane, and theintrinsic pathway, which acts at the mitochondriallevel to release factors that promote the activationof caspases. However, the link between the intrinsicand extrinsic routes in the transduction of deathsignals from the cytoplasm to the nucleus and back isnow only starting to be understood (Ferrando-May,2005). The large number of agents able to inducecell death, and the many intracellular routes, makethis issue one of the most challenging tasks in thefield of cell biology.

Among the many inducers of apoptosis, diet-related agents are being thoroughly investigated forthe treatment of several types of cancer. Resveratrol,a natural polyphenol present in red wine, is a prom-ising drug which is under clinical trials (Aggar-wal et al., 2004; Signorelli and Ghidoni, 2005).The histone deacetylase inhibitor butyrate is a fibre-intake-related by-product which has potential in-terest for the treatment of cancer (Marks et al., 2001;Monneret, 2007; VanOosten et al, 2007). In thepresent study, we have observed a synergistic action ofresveratrol and butyrate in the induction of apoptosis

of human melanoma A375 cells. The execution phaseof apoptosis was followed in living cells, using time-lapse confocal microscopy, by expressing fluorescentlytagged proteins that were targeted to different sub-cellular regions. We observed that both a nuclear anda nucleolar protein remain in the nuclear compart-ment during the blebbing stage. Our results showthat the functional integrity of the nuclear envelopeand the plasma membrane may be conserved untilthe end of the execution phase of apoptosis.

ResultsResveratrol and butyrate induce morphologicaldifferentiation and apoptosisIn order to evaluate the changes in permeability ofthe main cellular membranes during apoptosis, wetransfected fluorescently labelled proteins, whichwere targeted to different cell compartments, intohuman melanoma A375 cells. Thus GFP (greenfluorescent protein), which is distributed throughoutthe cell (Figure 1), would allow us to monitor thechanges in plasma membrane permeability, and thenuclear localization of the karyophilic protein GFP–Np (GFP–nucleoplasmin) (Figure 1) would indicatethe changes in nuclear envelope permeability duringapoptosis. Finally, the dynamics of nucleolar organiz-ation was followed by the expression of DsRed2–Nuc,a red fluorescent protein containing three nuclearlocalization sequences which shows a high level ofaccumulation in nucleoli in A375 cells (Figure 1).Subsequently, apoptosis was induced using a com-bination of resveratrol and butyrate, two well knowndrugs that both induce cell death. We observeda significant effect on cellular elongation in cellsexposed to 30 μM resveratrol plus 2 mM butyrate forup to 48 h, together with intense plasma membraneblebbing in detaching cells, which is a morphologicalhallmark of apoptosis. Both cell elongation and theapoptotic morphology were confirmed by scanningelectron microscopy analysis (Figure 1).

To gain knowledge regarding the action of res-veratrol and butyrate in the cell system used, westudied the effect of both drugs in cell viability, cellproliferation and the cell cycle. We observed thatthe dual treatment of resveratrol and butyrate pro-duced a synergistic induction of apoptosis by block-ing different phases of the cell cycle (see Supple-mentary Figure S1 at http://www. biolcell.org/boc/102/boc1020025add.htm). Thus resveratrol blocked

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Figure 1 Expression of fluorescent proteins located to different subcellular compartments and morphological effectsof resveratrol and butyrate in A375 human melanoma cellsUpper panels: overlay of Nomarski and fluorescent micrographs of A375 cells expressing GFP, GFP–Np or DsRed2–Nuc. The re-

combinant protein GFP is localized throughout the cell, whereas the karyophilic protein GFP–Np localizes exclusively to the nuc-

leus, but it is absent from nucleoli. The karyophilic protein DsRed2–Nuc accumulates highly in the nucleoli of A375 cells.

Lower panels: induction of apoptosis by resveratrol and butyrate (rsv+but) in human melanoma A375 cells. Cultured

cells were treated with resveratrol, butyrate or a combination of both drugs. Scanning electron microscopy of A375 cell cultures

exposed to resveratrol and butyrate for 24 h shows acute elongation and extensive blebbing of the plasma membrane, which

are characteristic of apoptotic cells. Scale bar, 10 μm; except for lower right-hand panel, 2 μm. See Supplementary Movie 1 at

http:// www.biolcell.org/boc/102/boc1020025add.htm.

cells in S-phase, whereas butyrate held the cells inG0/G1 after 24 h of treatment. The effect of eachdrug on cell cycle was reversible, as removal of thedrug after 24 h treatment restored the progress ofthe majority of the cells back into the cell cycle.However, we observed that the dual treatment withboth drugs induced a significant increase in the rateof apoptosis, which reached 38% of the total popula-tion by 24 h, as measured by the sub-diploid countin cell-cycle analysis performed by flow cytometry.

Blebbing of apoptotic A375 cells is followed bycell rounding and exposure of PS(phosphatidylserine)The morphological changes during the apoptotic pro-cess were monitored by time-lapse confocal micro-

scopy in living A375 cells. After 20 h of combinedtreatment with resveratrol and butyrate, cell cultureswere observed under the microscope for several hours.As observed in other experimental systems (Rudolfand Cervinka, 2005), there was an enormous vari-ability in the duration of the blebbing stage of ap-optosis of A375 cells. Thus apoptosis was initiatedby rapid cell shrinkage followed by intense blebbingactivity, an active process that can last for severalhours. Afterwards, most of the apoptotic cells usuallystopped blebbing, rounded up and exploded rapidlyin a final necrotic burst, loosing their cellular con-tent during the blebbing period (see Supplement-ary Movie 1 and Supplementary Table S1 at http://www.biolcell.org/boc/102/boc1020025add.htm). Itis important to mention that the start of blebbing

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Figure 2 Annexin V and PI staining are absent during the execution phase of apoptosis in A375 cellsInduction of apoptosis was performed with resveratrol plus butyrate (Rsv+But) or with Fas ligand for 24 h. Monitoring of the

presence of fluorescently labelled annexin V in the plasma membrane or PI nuclear staining were followed in living cells by

time-lapse confocal microscopy. Cells in active blebbing (arrows) for several hours were not positive for any of the fluorescent

dyes, which only labelled cells which had stopped apoptotic movement. See Supplementary Movies 2 (resveratrol plus butyrate)

and 3 (Fas ligand) at http:// www.biolcell.org/boc/102/boc1020025add.htm.

leads to an irreversible death process, as, after ex-amining several thousand cells undergoing blebbing,we did not observe any cell that could return to aprevious non-apoptotic stage. The extreme variabil-ity in the timing of each phase did not permit us toestablish the duration of the blebbing phase or therounded phase prior to cell lysis, which always tookplace in a very fast process that lasted a few seconds.

To monitor for the presence of the early apoptoticmarker PS on the outer leaflet of the plasma mem-brane, we added fluorescently labelled annexin Vand PI (propidium iodide) to A375 cells undergoingapoptosis that had been induced by resveratrol plusbutyrate. We observed that annexin V labelling wasabsent during the active blebbing stage (Figure 2,Rsv+But, and see Supplementary Movie 2 at http://www.biolcell.org/boc/102/boc1020025add.htm).Similar results were obtained when apoptosis wasinduced by the addition of Fas ligand to cell cultures(Figure 2, Fas, and see Supplementary Movie 3at http://www.biolcell.org/boc/102/boc1020025add.htm). In some cases, the presence of PS was faintlydetected during the initial cytoplasmic shrinkage,prior to the beginning of blebbing, which was prob-ably due to the detection of PS on the inner plasma

membrane fragments which remained attached tothe culture surface. The presence of annexin V couldonly be detected after the end of the blebbing stage.Finally, entry of PI and annexin V labelling wasobserved when the integrity of the membraneswas lost. We also observed that a minor proportionof cells were annexin-V-positive, but did not exhibitany signs of active apoptosis.

Maintenance of subcellular permeability duringthe execution phase of apoptosisThe localization of the different fluorescently la-belled proteins during the apoptotic process wasmonitored by time-lapse confocal microscopy in liv-ing A375 cells. Our results showed that the conser-vation of the plasma membrane, evaluated by themaintenance of cytoplasmic GFP, was not altereduntil the final necrotic process (Figure 3, GFP,and see Supplementary Movie 4 at http://www.biol-cell.org/boc/102/boc1020025add.htm). This resultwas not specific to the action of resveratrol andbutyrate, as induction of apoptosis with camp-tothecin also maintained the localization of GFP inthe cytoplasm during blebbing (Figure 4, and seeSupplementary Movie 5 at http://www.biolcell.org/

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Figure 3 Permeability of the plasma membrane, the nuclear envelope and the nucleolar organization remainfunctionally stable during the execution phase of apoptosisA375 cells stably expressing fluorescently labelled proteins that localized to different subcellular compartments were treated with

resveratrol plus butyrate and their morphological changes during apoptosis were followed by time-lapse confocal microscopy.

GFP, the karyophilic protein GFP–Np or the nucleolar protein DsRed2–Nuc remained in their subcellular compartments from

the beginning of the execution phase of apoptosis (panels A) and through the blebbing stage (panels B), which usually lasted

several hours. Finally, the cells rounded up, stopped movement and the loss of the fluorescent proteins, together with the rest

of the cellular content, took place in a very fast final necrotic blister (panels C for GFP and GFP–Np, arrows). The apoptotic

cell expressing DsRed–Nuc shown in panel D is at the onset of the final burst, when the internal components are lost (arrow).

At this very initial stage of final lysis, this bubble is still highly fluorescent, and dilutes rapidly in the surrounding medium. See

Supplementary Movies 4 (GFP), 8 (GFP–Np) and 9 (DsRed–Nuc) at http:// www.biolcell.org/boc/102/boc1020025add.htm.

boc/102/boc1020025add.htm). The use of the smallmolecular-mass probe calcein/AM (acetoxymethylester) to ensure that the plasma membrane waspreserved during the blebbing stage of apoptosis pro-duced similar results to those obtained with GFP,both after treatment with camptothecin or withresveratrol plus butyrate (Figure 4, and see Sup-plementary Movies 6 and 7 at http://www.biolcell.org/boc/102/boc1020025add.htm).

In order to monitor the integrity of the nuc-lear envelope, we followed the localization of severalfluorescently labelled components during the activephase of apoptosis. We observed that the karyophilicprotein GFP–Np was maintained in the nucleus

throughout the blebbing stage (Figure 3, GFP–Np, and see Supplementary Movie 8 at http://www.biolcell.org/boc/102/boc1020025add.htm). The re-combinant fluorescently labelled protein DsRed2–Nuc, which accumulates in the nucleoli of A375cells, was co-localized to the nucleoli structures dur-ing the blebbing period, and usually collapsed andfused together during the process (Figure 3, DsRed2–Nuc, and see Supplementary Movie 9 at http://www.biolcell.org/boc/102/boc1020025add.htm). Inall cases, release of the fluorescently labelled pro-teins from the cells did not occur until the finalnecrotic burst, together with the rest of the cellularcontent.

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Figure 4 Permeability of the plasma membrane is not altered in camptothecin-induced apoptotic A375 cellsCells expressing GFP or loaded with the vital dye calcein/AM were treated with camptothecin and monitored for several

hours by time-lapse confocal microscopy. Both fluorescent markers remained inside the cells during the blebbing period. See

Supplementary Movies 5 (GFP) and 6 (calcein/AM) at http:// www.biolcell.org/boc/102/boc1020025add.htm.

The localization of nuclear DNA and the entryof vital selective dyes during the execution phase ofapoptosis were monitored by adding the permeantdye Hoechst 33342 or the non-vital dye DAPI(4′,6-diamidino-2-phenylindole) to cell cultures ex-pressing the different fluorescently labelled proteins.In A375 cells, the apoptotic nucleus usually did notfragment during the blebbing stage, but remainedhighly condensed in a single chromatin area, whereasin some cells it split up into several micronuclei (Fig-ure 5, and see Supplementary Movie 10 at http://www.102biolcell.org/boc/102/boc1020025add.htm).The condensed DNA in fragmented micronucleiremained inside the apoptotic cell, and the exit ofDNA was concomitant with the release of GFPafter the final necrotic eruption. Similarly, accessof the non-permeant dye DAPI to DNA was notobserved until the end of the blebbing stage,just prior to cell-cycle block and rapid final lysis(Figure 5, and see Supplementary Movie 11 athttp:// www.biolcell.org/boc/102/boc1020025add.htm).

The morphological and functional changes ob-served during apoptosis are associated with an orderedmolecular degradation, which is governed by the bio-chemical apoptotic machinery. As a consequence, keystructural proteins are specifically fragmented by cas-pases, leading to an ordered apoptotic disassembly.Using immunofluorescence microscopy, we found thepresence of active caspase 3 in all blebbing cells un-dergoing apoptosis (Figure 6), indicating that thiseffector caspase may be implicated in this apoptoticprocess in A375 cells. Further studies will providemore details of the molecular mechanisms underly-

ing apoptosis induced by resveratrol plus butyrate inthis and other cell lines.

DiscussionIn the present study, we have monitored the local-ization of several fluorescent dyes and fluorescentlylabelled proteins in different subcellular compart-ments during the blebbing stage of the executionphase of apoptosis in human melanoma A375 cells.Among the various apoptotic agents tested, we ob-served that the combined treatment of resveratrol andbutyrate induced a significant proportion of cells toundergo apoptosis, together with profound morpho-logical changes that resembled a process of differen-tiation. Apoptotic death could already be observedduring the first 24 h of treatment, whereas the ac-tion of resveratrol or butyrate by themselves was onlyevident after 48 h exposure. Cell-cycle analysis re-veals that the effect of both drugs depended on time,drug concentration and cell type (Heerdt et al., 1997).We have observed that, in human melanoma A375cells, resveratrol at 30 μM caused the arrest of cellsin S-phase, whereas 2 mM butyrate induced a G0/G1

block. This blockage has been shown to be transit-ory in human colonic (Schneider et al., 2000) andleukaemia cancer cells (Park et al., 2001). We con-firmed a similar effect in A375 cells by removingall of the drugs after 24 h of treatment and perform-ing cell-cycle analysis 24 h later. Our results con-firmed that resveratrol or butyrate have a cytostaticeffect, whereas their combination exerts a markedcytotoxic effect in this melanoma cell line. Althoughboth resveratrol and butyrate have been extensivelyused as inductors of apoptosis in tumour cell lines of

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Figure 5 Chromatin is not accessible to the non-vital dye DAPI until the end of the execution phase of apoptosisIn experiments similar to those described in Figure 3, the permeant DNA dye Hoechst 33342 or the non-permeant dye DAPI

(pseudo-coloured in red to increase contrast) were added to cells expressing GFP or GFP–Np undergoing apoptosis. The

loss of both GFP and the nuclear content (chromatin stained with Hoechst 33342) took place during the final necrotic lysis

of the cell (arrow). Similarly, in cells expressing the karyophilic protein GFP–Np, the entrance of DAPI could only be detected

immediately before the end of blebbing. See Supplementary Movies 10 (GFP+Hoechst) and 11 (GFP–Np+DAPI) at http://

www.biolcell.org/boc/102/boc1020025add.htm.

different origins (Joe et al., 2002; Mertens-Talcottand Percival, 2005; Daehn et al., 2006; Garvin etal., 2006; Schwab et al., 2006), the combination ofboth of them has not been thoroughly tested yet. Ithas been shown that resveratrol intensifies the dif-ferentiating effect of butyrate in colonic carcinomacells (Wolter and Stein, 2002) and their combin-ation in cancer chemotherapy has been suggested(Blumenstein et al., 2005; Galfi et al., 2005). We be-lieve that the synergistic apoptotic effect may rely onthe different molecular targets of both drugs, which

exert contradictory signals on cell-cycle control. Ourresults suggest that a combined therapy with res-veratrol and butyrate may be an interesting approachto the treatment of malignant melanoma.

Although it is assumed that the integrity of theplasma membrane is maintained until the end of ap-optosis, little is known about the changes in sub-cellular compartments during the active executionof apoptosis. To further investigate this matter, wefollowed three fluorescently labelled proteins thatlocalize to different subcellular regions during the

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Figure 6 Caspase 3 is present in apoptotic A375 cellsinduced by resveratrol and butyrateImmunolocalization of active caspase 3 (red) by confocal

microscopy shows that it is exclusively present during the

blebbing stage of apoptosis, whereas it is absent in adhered

non-apoptotic cells. The Nomarski image (right-hand panel) of

the same field and DNA stain (left-hand panel; Hoechst 33342,

blue) are included to recognize interphase and apoptotic cells.

Figure 7 Model of the preservation of the permeabilitybarrier of the plasma membrane and the nuclearenvelope during the execution phase of apoptosis inA375 cellsThe exit of the fluorescently labelled proteins GFP (A), GFP–Np

(B) or DsRed2–Nuc (C) from their subcellular locations and the

entrance of non-vital DNA dyes was only observed after

the end of the long and variable blebbing phase, taking place

in a fast necrotic blister that derived in cell lysis. Presence of

PS in the outer leaflet of the plasma membrane is only detec-

ted in the final stages of apoptosis.

process of apoptosis using time-lapse confocal mi-croscopy. Surprisingly, during the highly dynamicblebbing stage, which usually lasted for several hours,GFP, the karyophilic protein GFP–Np and the nuc-leolar protein DsRed2–Nuc were not redistributedfrom their proper locations. In all cases, the loss offluorescence from the cell occurred at a very latenecrotic stage, ulterior to apoptotic blebbing, afterthe final rapid cell lysis (Figure 7). Interestingly,

the nuclear envelope of apoptotic A375 cells seemsto function in the maintenance of the karyophilicproteins GFP–Np or DsRed2–Nuc. Several stud-ies have reported an alteration in the nucleocyt-oplasmic transport components during apoptosis(Ferrando-May, 2005). Despite the activation ofcaspases (Faleiro and Lazebnik, 2000), the reloca-tion of karyophilic proteins or changes in nuc-lear envelope permeability (Kihlmark et al., 2001;Mason et al., 2005), we have not observed a sig-nificant redistribution of GFP–Np until the end ofthe blebbing stage. Moreover, by fluorescence recov-ery after photobleaching experiments, we have ob-served that this recombinant protein is soluble anddiffuses rapidly throughout the nucleus of A375 cells(R. Andrade, unpublished data), suggesting that itsnuclear location during apoptosis is not due to chro-matin attachment, but rather to maintenance of afunctional nuclear envelope. The organization of nuc-leoli during apoptosis, followed in our experimentsby the accumulation of DsRed2–Nuc in A375 cells,shows a rearrangement and fusion of nucleoli, as hasbeen described previously at the ultrastructural level(Reipert et al., 1999; Biggiogera et al., 2004). Inspite of this nucleolar reorganization, the distribu-tion of DsRed2–Nuc was always nuclear during theblebbing stage. The accessibility of vital selectivedyes that bind to DNA corroborates the maintenanceof the permeability of cellular membranes during theblebbing stage, as both DAPI or PI (data not shown)did not label DNA until the end of this period. Onthe other hand, the permeant dye Hoechst 33342,which stains condensed chromatin of living cells,did not exit the cell until it exploded via secondarylysis.

There is emerging evidence that annexin V is notpresent during the initial blebbing stage (McCarthyet al., 1997; Willingham, 1999; Lane et al., 2005;present study). Using time-lapse microscopy in apop-totic cultures, only a minor proportion of cells whichhad ceased blebbing exhibited PS on the outer leafletof the plasma membrane. The rest of the annexin-V-positive cells had already been permeabilized viasecondary lysis, as their DNA was also stained withthe vital dye PI. This result was independent of theagent used to induce apoptosis, as both resveratrolplus butyrate or the use of Fas ligand showed absenceof PS in blebbing cells. We believe that this observa-tion should be taken into account when extrapolated

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to the field of flow cytometry, where annexin V isalways considered as a reference of early events inapoptosis.

Time-lapse microscopy reveals that there is a largemorphological variability in apoptotic cells, depend-ing on the cell type or apoptotic stimuli. We haveobserved that the majority of apoptotic A375 cellsusually remain in a single apoptotic body, lackingcellular fragmentation. This result seems to be in-dependent of the agent used to induce apoptosis, asapoptosis induced by Fas or camptothecin in A375cells also exhibited profound blebbing and no cell dis-gregation. Preliminary studies using several celllines reveal that apoptosis induced by resveratrolplus butyrate generates different effects dependingon cellular fragmentation, cell detachment or en-gulfment of apoptotic bodies by neighbouring cells(R. Andrade, unpublished data). Similar differencesin the blebbing phase (Mills et al., 1999; McCarthyet al., 1997; Willingham, 1999; Lane et al., 2005) orin the fragmentation pattern depending on the celltype (Moss et al., 2006) or the apoptotic stimulus(Buendia et al., 1999) have been observed, but themeaning of this observation remains unresolved. Itis possible that the cellular mechanism used to ac-complish apoptotic fragmentation is specific to eachcell type, depending on its embryonic origin or onthe capability of the cells to phagocytose apoptoticbodies.

In spite of the several apoptotic morphologies ob-served in different experimental approaches, the out-come is always the end of cell viability. It is conceiv-able that the slight variations in the timing of theexecution phase, the presence or absence of cellularfragmentation and exposure of apoptotic signals maybe of great importance in vivo, where factors such astissue renewal turnover or the rate of phagocytosis byadjacent cells mediate tissue homoeostasis.

Materials and methodsCell culture and treatmentsA375 melanoma cells were obtained from the A.T.C.C. (A.T.C.C.number CRL-1619). Cells were maintained in DMEM (Dul-becco’s modified Eagle’s medium) containing 10% FBS (fetalbovine serum) and 1% penicillin/streptomycin at 37◦C in 5%CO2. Cells were treated with 30 μM resveratrol or 2 mM sodiumbutyrate or the combination of both drugs for up to 48 h. Res-veratrol (Sigma) stock solution was prepared at 12 mM in DMSOand kept at −20◦C. Sodium butyrate (Sigma) was dissolved inPBS (phosphate-buffered saline) at 0.2 M and sterilized by fil-

tration. Fas-induced apoptosis was carried out using 0.5 μg/mlof anti-(human CD95) antibody (Biosource) for 24 h.

GFP–Np plasmid construction and transfectionsThe full-length nucleoplasmin cDNA sequence was obtainedby PCR from pET-11b using N-terminal (5′-CAGTAGATCT-ATGGCCTCTACCGTCAGCAA-3′) and C-terminal (5′-TT-AAGAATTCTCACTTCTTAGCAGCCGGCTT-3′) primers.The amplified fragment was digested with BglII and EcoR1restriction enzymes respectively (underlined), and the fragmentwas ligated into eGFP-C1 (Clontech) to yield vector pGFP-Np.Competent Escherichia coli (JM 109) were transformed, theplasmid was re-isolated and the sequence verified by sequencing.

A375 melanoma cells (10 000 cells per well) were culturedovernight in 24-well plates in DMEM with 10% FBS in a5% CO2 incubator at 37◦C. Afterwards, cells were transfectedwith eGFP-C1, pDsRed2-Nuc (Clontech) or pGFP-NP usingFuGENETM 6 (Roche), according to the manufacturer’s instruc-tions. Stable DsRed2–Nuc, GFP and GFP–Np A375 cell lineswere generated by clonal selection. Maintenance of the culturewas performed by adding G-418 to the growth medium.

Proliferation and viability assaysCells were grown in triplicate in 24-well plates at adensity of 3 × 104 cells per well and were treated with2 mM butyrate and 30 μM resveratrol alone or in combin-ation for 0, 12, 24 and 48 h. Proliferation was measuredwith the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay. Briefly, MTT (0.5 mg/ml) was addedto each well and cells were incubated for 3 h at 37◦C. Formazancrystals were dissolved in DMSO and the colour intensity wasmeasured using an ELISA plate reader at a wavelength of 570 nm.

Cell viability was evaluated by flow cytometry using the PIexclusion assay. Briefly, floating and adherent cells were collected,washed and incubated with PI (2 μg/ml) for 5 min in the dark.The percentage of cell death was determined by the ratio ofPI-positive cells to all cells.

Cell-cycle analysis by flow cytometryCells were seeded in 75 cm2 tissue culture flasks and were cul-tured with or without drug treatments for 24 and 48 h. Float-ing and adherent cells were combined, washed in PBS (pH 7.4)and 106 cells were fixed and permeabilized in 70% ethanolfor 1 h. Cells were washed twice in PBS, treated with RNaseA (300 μg/ml; Boehringer Mannheim) and stained with PI at20 μg/ml for 1 h in the dark. DNA content of at least 10 000events per sample was then analysed on a flow cytometer (EPICSElite ESP; Coulter). Cell cycle and apoptosis were analysed usingthe Weasel software (WEHI Biotechnology Centre, Bundoora,Victoria, Australia).

Scanning electron microscopyCells were grown on glass coverslips in Petri dishes and treatedwith resveratrol or butyrate as indicated above. Cells were washedin PBS, fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer(pH 7.2), washed in cacodylate buffer with 4% sucrose, post-fixed in 1% osmium tetroxide and washed in cacodylate buffer.Samples were dehydrated through an ethanol series, immersedtwice in hexamethyldisilazane before air drying and being goldcoated in a JFC-1100 ion sputter (JEOL). Images were acquiredwith a JSM-35A scanning electron microscope (JEOL).

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Time-lapse confocal microscopyCells were grown on glass-bottomed dishes (Mattek Corporation)and observed by time-lapse microscopy with a Fluoview FV500confocal microscope (Olympus), using a 20× Plan APO 0.7 NA(numerical aperture) objective at 37◦C at 5% CO2. After 18 h oftreatment with mock, resveratrol, butyrate or a combination ofboth drugs, images were acquired every 30 s for up to 8 h. For dyepermeability assays, stock solutions of DAPI or Hoechst 33342were added to control and treated cells. Calcein/AM (MolecularProbes) was used according to the manufacturer’s instructions.Laser power was minimized via acousto-optical tunable filteringto avoid cell damage. Under these conditions, the power of eachlaser line (in μW) was as follows: 405 nm laser line, 23.1+−2.0;488 nm, 16.7+−1.2; 543 nm, 29.7+−2.3, as measured using aPower Meter Field Master GS (Coherent) with a LM-2-VISsensor. When using several fluorochromes, line sequential excita-tion and image acquisition were performed to avoid overlappingof fluorescence spectra. The longest pixel time was 12.2 μs, whichin this system corresponds to capturing three fluorochromes plustransmitted light images. Cultures of untreated cells examinedunder the same microscopy conditions did not show any signs ofblebbing, underwent cell division properly and were viable afterreturning them to the CO2 incubator (see Supplementary Movie12 at http:// www.biolcell.org/boc/102/boc1020025add.htm).

Annexin V assayThe annexin-V-FLUOS staining kit (Roche) was used, accordingto the manufacturer’s instructions, for staining of adherent cells.Cultures of living cells were observed by time-lapse confocalmicroscopy.

ImmunofluorescenceThe immunofluorescence in apoptotic A375 cells was performedas described previously (Andrade et al., 2003). The primaryantibody against cleaved caspase 3 (Cell Signalling Technology,Danvers, MA, U.S.A.) was used at 1:100 dilution. Samples werevisualized by confocal microscopy.

AcknowledgementsWe thank Raul Moreno (Laser Facility, University ofthe Basque Country) for valuable help measuring thelaser power of the confocal microscope.

FundingThis work was supported by the Spanish Ministryof Education and Science [grant number BFU 2007-66610]; the University of the Basque Country [grantnumber GIU08/04] (research group grant to J.A.);and the Juan Gangoiti Barrera Foundation, Bilbao,Spain (predoctoral fellowship to L.C.)

ReferencesAggarwal, B.B., Bhardwaj, A., Aggarwal, R.S., Seeram, N.P.,

Shishodia1, S. and Takada, Y. (2004) Role of resveratrol inprevention and therapy of cancer: preclinical and clinical studies.Anticancer Res. 24, 2783–2840

Andrade, R., Alonso, R., Pena, R., Arlucea, J. and Arechaga, J. (2003)Localization of importin α (Rch1) at the plasma membrane andsubcellular redistribution during lymphocyte activation.Chromosoma 112, 87–95

Atencia, R., Garcıa-Sanz, M., Perez-Yarza, G., Asumendi, A., Hilario,E. and Arechaga, J. (1997) A structural analysis of cytoskeletoncomponents during the execution phase of apoptosis,Protoplasma 198, 163–169

Biggiogera, M., Bottone, M.G., Scovassi, A.I., Soldani, C., Vecchio, L.and Pellicciari, C. (2004) Rearrangement of nuclearribonucleoprotein (RNP)-containing structures during apoptosisand transcriptional arrest. Biol. Cell 96, 603–615

Blumenstein, I., Keseru, B., Wolter, F. and Stein, J. (2005) Thechemopreventive agent resveratrol stimulates cyclic AMPdependent chloride secretion in vitro. Clin. Cancer Res. 11,5651–5656

Buendia, B., Santa-Maria, A. and Courvalin, J.C. (1999)Caspase-dependent proteolysis of integral and peripheral proteinsof nuclear membranes and nuclear pore complex proteins duringapoptosis. J. Cell Sci. 112, 1743–1753

Coleman, M.L. and Olson, M.F. (2002) Rho GTPase signallingpathways in the morphological changes associated withapoptosis. Cell Death Differ. 9, 493–504

Coleman, M.L., Sahai, E.A., Yeo, M., Bosch, M., Dewar, A. and Olson,M.F. (2001) Membrane blebbing during apoptosis results fromcaspase-mediated activation of ROCK I. Nat. Cell Biol. 3,339–341

Croft, D.R., Coleman, M.L., Li, S., Robertson, D., Sullivan, T.,Stewart, C.L. and Olson, M.F. (2005) Actin–myosin-basedcontraction is responsible for apoptotic nuclear disintegration. J.Cell Biol. 168, 245–255

Daehn, I.S., Varelias, A. and Rayner, T.E. (2006) Sodium butyrateinduced keratinocyte apoptosis. Apoptosis 8,1379–1390

Earnshaw, W.C., Martins, L.M. and Kaufmann, S.H. (1999)Mammalian caspases: structure, activation, substrates, andfunctions during apoptosis. Annu. Rev. Biochem. 68,383–424

Faleiro, L. and Lazebnik, Y. (2000) Caspases disrupt thenuclear–cytoplasmic barrier. J. Cell Biol. 151, 951–959

Ferrando-May, E. (2005) Nucleocytoplasmic transport in apoptosis.Cell Death Differ. 12, 1263–1276

Ferrando-May, E., Cordes, V., Biller-Ckovric, I., Mirkovic, J., Gorlich,D. and Nicotera, P. (2001) Caspases mediate nucleoporincleavage, but not early redistribution of nuclear transport factorsand modulation of nuclear permeability in apoptosis. Cell DeathDiffer. 8, 495–505

Galfi, P., Jakus, J., Molnar, T., Neogrady, S. and Csordas, A. (2005)Divergent effects of resveratrol, a polyphenolic phytostilbene, onfree radical levels and typeof cell death induced by the histonedeacetylase inhibitors butyrate and trichostatin A. J. SteroidBiochem. Mol. Biol. 94, 39–47

Garvin, S., Ollinger, K. and Dabrosin, C. (2006) Resveratrol inducesapoptosis and inhibits angiogenesis in human breast cancerxenografts in vivo. Cancer Lett. 231, 113–122

Heerdt, B.G., Houston, M.A. and Augenlicht, L.H. (1997) Short-chainfatty acid-initiated cell cycle arrest and apoptosis of colonicepithelial cells is linked to mitochondrial function. Cell GrowthDiffer. 8, 523–532

Joe, A.K., Liu, H., Suzui, M., Vural, M.E., Xiao, D. and Weinstein, I.B.(2002) Resveratrol induces growth inhibition, S-phase arrest,apoptosis, and changes in biomarker expression in several humancancer cell lines. Clin. Cancer Res. 8, 893–903

Kihlmark, M., Imreh, G. and Hallberg, E. (2001) Sequentialdegradation of proteins from the nuclear envelope duringapoptosis. J. Cell Sci. 114, 3643–3653

34 C© The Authors Journal compilation C© 2010 Portland Press Ltd

Plasma membrane and nuclear envelope integrity in apoptosis Research article

Lane, J.D., Allan, V.J. and Woodman, P.G. (2005) Active relocation ofchromatin and endoplasmic reticulum into blebs in late apoptoticcells. J. Cell Sci. 118, 4059–4071

Marks, P., Rifkind, R.A., Richon, V.M., Breslow, R., Miller, T. and Kelly,W.K. (2001) Histone deacetylases and cancer: causes andtherapies. Nat. Rev. Cancer 3, 194–202

Mason, D.A., Shulga, N., Undavai, S., Ferrando-May, E., Rexach,M.F. and Goldfarb, D.S. (2005) Increased nuclear envelopepermeability and Pep4p-dependent degradation of nucleoporinsduring hydrogen peroxide-induced cell death. FEMS Yeast Res.12, 1237–1251

McCarthy, N.J., Whyte, M.K., Gilbert, C.S. and Evan, G.I. (1997)Inhibition of Ced-3/ICE-related proteases does not prevent celldeath induced by oncogenes, DNA damage, or the Bcl-2homologue Bak. J. Cell Biol. 136, 215–227

Mertens-Talcott, S.U. and Percival, S.S. (2005) Ellagic acid andquercetin interact synergistically with resveratrol in the induction ofapoptosis and cause transient cell cycle arrest in human leukemiacells. Cancer Lett. 218, 141–151

Mills, J.C., Stone, N.L., Erhardt, J. and Pittman, R.N. (1998)Apoptotic membrane blebbing is regulated by myosin light chainphosphorylation. J. Cell Biol. 140, 627–636

Mills, J.C., Stone, N.L. and Pittman, R.N. (1999) Extranuclearapoptosis: the role of the cytoplasm in the execution phase. J. CellBiol. 146, 703–707

Monneret, C. (2007) Histone deacetylase inhibitors for epigenetictherapy of cancer. Anticancer Drugs 18, 363–370

Moss, D.K., Betin, V.M., Malesinski, S.D. and Lane, J.D. (2006) Anovel role for microtubules in apoptotic chromatin dynamics andcellular fragmentation. J. Cell Sci. 119, 2362–2374

Park, J.W., Choi, Y.J., Jang, M.A., Lee, Y.S., Jun, D.Y., Suh, S.I.,Baek, W.K., Suh, M.H., Jin, I. and Kwon, T.K. (2001)Chemopreventive agent resveratrol, a natural product derived fromgrapes, reversibly inhibits progression through S and G2 phasesthe cell cycle in U937 cells. Cancer Lett. 163, 43–49

Reipert, S., Bennion, G., Hickman, J.A. and Allen, T.D. (1999)Nucleolar segregation during apoptosis of haemopoietic stem cellline FDCP-Mix. Cell Death Differ 3, 334–341

Rudolf, E. and Cervinka, M. (2005) Membrane blebbing in cancercells treated with various apoptotic inducers. Acta Med. 48, 29–34.

Schneider, Y., Vincent, F., Duranton, B., Badolo, L., Gosse, F.,Bergmann, C., Seiler, N. and Raul, F. (2000) Antiproliferative effectof resveratrol, a natural component of grapes and wine, on humancolonic cancer cells. Cancer Lett. 159, 85–91

Schwab, M., Reynders, V., Ulrich, S., Zahn, N., Stein, J. andSchroder, O. (2006) PPARγ is a key target of butyrate-inducedcaspase-3 activation in the colorectal cancer cell line Caco-2.Apoptosis 10, 1801–1811

Signorelli, P. and Ghidoni, R. (2005) Resveratrol as an anticancernutrient: molecular basis, open questions and promises. J. Nutr.Biochem. 16, 449–466

VanOosten, R.L., Earel, Jr, J.K. and Griffith, T.S. (2007) Histonedeacetylase inhibitors enhance Ad5-TRAIL killing ofTRAIL-resistant prostate tumor cells through increased caspase-2activity. Apoptosis 3, 561–571

Willingham, M.C. (1999) Cytochemical methods for the detection ofapoptosis. J. Histochem. Cytochem. 47, 1101–1109

Wolter, F. and Stein, J. (2002) Resveratrol enhances the differentiationinduced by butyrate in Caco-2 colon cancer cells. J. Nutr. 132,2082–2086

Received 13 May 2009/7 July 2009; accepted 27 July 2009

Published as Immediate Publication 27 July 2009, doi:10.1042/BC20090077

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Biol. Cell (2010) 102, 25–35 (Printed in Great Britain) doi:10.1042/BC20090077

Supplementary online data

Plasma membrane and nuclear envelope integrity during theblebbing stage of apoptosis: a time-lapse study

Ricardo Andrade*, Lorena Crisol†, Roberto Prado*, Marıa Dolores Boyano†, Jon Arluzea*† and Juan Arechaga*†1

*Biomedical Analytical and High Resolution Microscopy Facility, University of the Basque Country, E-48940 Leioa, Vizcaya, Spain, and

†Laboratory of Stem Cells, Development and Cancer, Department of Cell Biology and Histology, Faculty of Medicine and Dentistry,

University of the Basque Country, E-48940 Leioa, Vizcaya, Spain

Table S1 Summary of Supplementary Movies 1–12 (see http://www.biolcell.org/boc/102/boc1020025add.htm)

Supplementary Movie(duration, min) Apoptotic inductor

Fluorochrome/fluorescent protein Observations

1 (142) Resveratrol plus butyrate None Panorama of cells undergoing blebbing

2 (58) Resveratrol plus butyrate Annexin V and PI Blebbing cells do not show PS exposure

3 (168) FAS ligand Annexin V and PI Blebbing cells do not show PS exposure

4 (249) Resveratrol plus butyrate GFP GFP remains inside cell until final lysis

5 (119) Camptothecin GFP GFP remains inside cell until final lysis

6 (59) Camptothecin Calcein/AM Calcein/AM remains inside cell until final lysis

7 (119) Resveratrol plus butyrate Calcein/AM Calcein/AM remains inside cell until lysis

8 (349) Resveratrol plus butyrate GFP–Np GFP–Np remains nuclear until final cell lysis

9 (373) Resveratrol plus butyrate DsRed2–Nuc DsRed2–Nuc fuse and remain nuclear until finalcell lysis

10 (499) Resveratrol plus butyrate GFP and Hoechst33342

GFP and Hoechst 33342 remain inside cell untilfinal lysis

11 (351) Resveratrol plus butyrate GFP-Np and DAPI GFP–Np remains nuclear. DAPI enters nucleusat the end of the blebbing phase

12 (179) None (control) None Healthy untreated cells divide properly undertime-lapse microscopy conditions

1To whom correspondence should be addressed (email juan.arechaga@ehu.es).

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R. Andrade and others

Figure S1 Synergistic effect of resveratrol and butyrate on melanoma cell death(A) The synergistic induction of apoptosis by resveratrol and butyrate was assayed measuring cell viability using PI exclusion by

flow cytometry. The percentage of cell death after 24 h or 48 h treatment with resveratrol (Rsv), butyrate (But) or a combination

of both drugs (Rsv+But) in A375 cells revealed a synergistic effect of both drugs after 24 h, where 40% of the cell population

was PI-positive. (B) Similar results were obtained by MTT assays, where the combined treatment completely abolished cell

proliferation at 24 h. (C) Evaluation of the effect of resveratrol and butyrate on the cell cycle of A375 human melanoma cells by

flow cytometry. We observed that both drugs blocked cells on different phases of the cell cycle. Thus the percentage of cells in

S-phase after resveratrol treatment increased to 50% after 24 h and to 56% after 48 h. However, butyrate treated cells exhibited

a G0/G1 block of 70% after 24 h. This percentage decreased to 47% at 48 h due to the large amount of apoptotic cells (39%),

shown by the sub-G0/G1 peak. Interestingly, the concomitant use of both resveratrol and butyrate induced a clear synergistic

effect in the induction of apoptosis in A375 cells, which reached up to 38% of the total population after 24 h. However, the

amount of apoptosis was 10% in resveratrol-treated cells and only 5% after butyrate treatment at the 24 h time period. (D) To

evaluate the reversibility of the different drug treatments on cell cycle detention, we performed 24 h treatments with resveratrol

or butyrate alone and, afterwards, we added fresh culture medium with DMSO or PBS (Mock) and waited for another 24 h. Our

results showed that both treatments exerted neither a cell-cycle arrest nor evident signs of apoptosis. However, after sequential

treatments with resveratrol or butyrate (Rsv→But or But→Rsv) for 24 h and subsequent exchange of both drugs for the next

24 h, the amount of apoptosis was nearly half of that of the 24 h combined treatment. Moreover, we observed that, in these

conditions, blocking of the cell cycle was due to the effect of the drug added during the last 24 h. Hence, 24 h resveratrol

treatment followed by butyrate for the next 24 h blocked cells in G0/G1, but an initial exposure to butyrate and then to resveratrol

arrested them in S-phase.

Received 13 May 2009/7 July 2009; accepted 27 July 2009

Published as Immediate Publication 24 July 2009, doi:10.1042/BC20090077

C© The Authors Journal compilation C© 2010 Portland Press Ltd