On the evolution of erythrocyte programmed cell death: apoptosisof Rana esculenta nucleated red blood cells involves cysteine proteinase

activation and mitochondrion permeabilization

Daniela Bratosin a, Jérôme Estaquier b, Christian Slomianny c, Jean-Pierre Tissier d,Brigitte Quatannens e, Tatiana Bulai f, Laura Mitrofan g, Alexandru Marinescu g,

Ioana Trandaburu g, Jean-Claude Ameisen h, Jean Montreuil i,*a Institutul National de Cercetare-Dezvoltare pentru Stiinte Biologice, Splaiul Independentei no. 296, P.O. Box 17-16, 77748 Bucuresti, Romania

b Institut Pasteur, Unité de Physiopathologie des Infections Lentivirales, 28, rue du Dr. Roux, 75015 Paris, Francec Laboratoire de Physiologie Cellulaire, Université des Sciences et Technologies de Lille 1, 59655 Villeneuve d’Ascq cedex, France

d Domaine du CERTIA, Station de l’INRA, 269, rue Jules Guesde, 59650 Villeneuve d’Ascq cedex, Francee Institut de Biologie de Lille, UMR 8526 du CNRS, 1, rue Calmette, 59019 Lille cedex, France

f Universitatea “Al. I. Cuza”, Facultatea de Chimie, Bdul Copou no. 11, 6600 Iasi, Romaniag Institutul de Biologie al Academiei Române, Spl. Independentei no. 296, P.O. Box 56-53, 79651 Bucuresti, Romania

h EMI–U 9922, INSERM-Université Paris 7, IFR 02, CHU Bichat, 16, rue H.Huchard, 75018 Paris, Francei Université des Sciences et Technologies de Lille 1 (USTL), Laboratoire de Chimie Biologique, UMR USTL/CNRS no. 8576,

59655 Villeneuve d’Ascq cedex, France

Received 6 October 2003; accepted 5 March 2004

Available online 12 April 2004

Abstract

Batracian Rana esculenta erythrocytes cell death induced by either calcium influx, or staurosporine, involves typical apoptotic phenotype.Our data reveal: (i) a drastic modification of the cell morphology with loss of the ellipsoidal form as assessed by phase contrast microscopy andscanning electron microscopy; (ii) an exposure of the phosphatidylserine residues in the outer leaflet of the cell membrane; (iii) acaspase-3-like activity; (iv) a mitochondrial membrane potential (DWm) loss; and (v) a chromatin condensation and fragmentation.Erythrocyte chromatin condensation and fragmentation are prevented by caspase and calpain peptide inhibitors. These inhibitors also preventDWm loss supporting the idea that mitochondria is a central sensor for Rana erythrocytes cell death. Our observations highlight theconservation of the programmed cell death machinery in erythrocytes across kingdom.© 2004 Elsevier SAS. All rights reserved.

Keywords: Apoptosis; Cell death; Red blood cells; Mitochondria; Calpain; Caspase; Amphibians

1. Introduction

Vertebrate erythrocytes have a finite programmed life-span in the circulation which varies between species but is

exceptionally constant with species: 900 days for frog,700 for turtle, 120 for human and 30 for chicken, for instance[1]. However, related to the life span of individuals humanerythrocytes have the shortest lifespan. The precision of sucha biological clock has intrigued many investigators whoattempted to identify the signals that mark the RBCs assenescent and give rise to their removal from circulation byresident macrophages.

During the last decades active studies were focused on themechanism of human erythrocyte programmed cell death(PCD) (for review see [2]). Apoptosis is one of the mainphenotype of the PCD. We and others have recently shownthat PCD of human erythrocyte is related to an apoptosis-likemechanism operating in the absence of mitochondria and

Abbreviations: Ac-DEVD-cmk, Ac-Asp-Glu-Val-L-Asp-chloromethylketone; Z-VAD-fmk, Z-Val-Ala-L-Asp-fluoromethylketone; Tosyl-cmk,tosyl-Lys-chloromethylketone; DiOC6, 3,3′-dihexyloxacarbocyanine-iodide; FITC, fluoresceylisothiocyanate; FSC, forward squatter; SSC, sidescatter; HEPES, N-[2-hydroxyethyl] piperazine-N′-ethane sulfonic acid buf-fer pH 7.4; OsO4, osmic acid; PBS, phosphate buffered saline solution pH7.4; RBC, red blood cell; PCD, programmed cell death; PS, phosphatidyl-serine; STX, staurosporine; PI, propidium iodide.

* Corresponding author. Tel.: +33-3-20-43-48-84;fax: +33-3-20-43-65-55.

E-mail address: jean.montreuil@univ-lille1.fr (J. Montreuil).

Biochimie 86 (2004) 183–192

www.elsevier.com/locate/biochi

© 2004 Elsevier SAS. All rights reserved.doi:10.1016/j.biochi.2004.03.003

lacking the terminal effectors of nuclear apoptosis. Thisregulated form of PCD was prevented by cysteine proteaseinhibitors that allowed erythrocyte survival in vitro and invivo [3,4]. Thus, the counter intuitive hypothesis, in regardswith the lifespan of erythrocyte, would be a priori that batra-cian erythrocytes are devoid of PCD machinery. However, incontrast to human, batracian erythrocytes possess bothnucleus and mitochondria, the sensor of the PCD machinery.Moreover, different groups have reported a chromatin con-densation and fragmentation of nucleated erythrocytes of thebatracians Rana castebeiana [5,6] and Xenopus laevis [7,8],and of chicken [9,10]. While nuclear fragmentation ofchicken erythrocytes has been reported by Weil et al. [10]PCD of chicken erythrocyte involved a caspase-independentcell death pathway. These findings raised the fascinatingquestion of the PCD conservation in erythrocytes acrosskingdoms. We assessed in Rana esculenta erythrocyte PCDmachinery using similar methodology that we applied tostudy human erythrocyte PCD [3]. Here we found that PCDcan be rapidly induced in Rana mature erythrocytes by cal-cium influx and staurosporine that can be prevented by cys-teine protease inhibitors. This PCD involves mitochondrialmembrane potential (DWm) loss, caspase-3-like activity andchromatin condensation and fragmentation. These findingsindicate that batracian erythrocyte PCD displays the typicalcascade of events that characterizes mammalian apoptosis.

2. Materials and methods

2.1. Reagents

Fluorescein-conjugated annexin V and propidium iodidewere from PharMingen (San Diego, CA, USA), ionophore A23187 of Streptomyces chartreusis from Calbiochem (LaJolla, CA, USA), calpain inhibitor Ac-Leu-Leu-Arg-CHO(leupeptin), caspase inhibitors Ac-DEVD-cmk, Z-VAD-fmkand Ac-YVAD-cmk as well as the serine proteinase inhibitortosyl-cmk were from Bachem France (Voisins-le-Bretonneux, France). Caspase-3 activity was assessed usingPhiPhiLux-G1D2 (green fluorescence) and G2D2 (red fluo-rescence) from OncoImmunin (Gaithersburg, MA, USA).Staurosporin from Streptomyces and human serum albuminprotease free were from Sigma Chemicals (San Diego, CA,USA) and 3,3′-dihexyloxacarbocyanine-iodide (DiOC6)from Molecular Probes (Interchim, Montluçon, France). Theflow cytometer was a Becton-Dickinson FACScan apparatus(San Jose, CA, USA) with LYSIS softwares for acquisitionand analysis.

2.2. Cells and culture conditions

Erythrocytes from batracian species Rana esculenta bloodcollected under ether anesthesia on heparin were sedimentedby centrifugation (1000 g, 4 °C, 5 min) and washed threetimes with Dulbecco’s phosphate buffered saline solution pH7.4 (PBS: 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4

and 1.5 mM KH2PO4). Apoptosis of erythrocytes was in-duced in conditions varying with the nature of the inductor.Using HEPES buffer pH 7.4 containing 2.5 mM calciumchloride and supplemented with 0.1% human serum albu-min, incubations were carried out either at 37 °C (under 5%CO2 atmosphere) for 6 h and then at 20 °C for 24 h in theabsence of calcium ionophore A 23187 or at 37 °C for 4 h inthe presence of the ionophore. Induction of apoptosis bystaurosporine (10 µM) in solution in PBS buffer pH7.4 supplemented with 0.1% serum albumin was carried outby incubation of erythrocytes at 37 °C (under 5% CO2 atmo-sphere) for 6 h and then at 20 °C. Incubations were carriedout in the absence or presence of pretreatment for 30 min at20 °C with the following cysteine-protein inhibitors in solu-tion in DMEM culture medium: Ac-DEVD-cmk (200 µM),leupeptin (200 µM), a mixture of both (200 µM each) andZ-VAD-fmk (50 µM).

2.3. Flow cytometric analysis

Flow cytometric analysis was performed on a FACScanflow cytometer (Becton Dickinson, San Jose, CA, USA),using the LYSIS software for acquisition and analysis. Thelight-scatter channels were set on linear gains and the fluo-rescence channels on a logarithmic scale, a minimum of5000 cells being analysed in each condition. Erythrocyte sizeand density were assessed using forward and side-anglescatters (FSC vs. SSC). For analysis of FITC–annexin V andpropidium iodide labeling, erythrocytes were washed twicewith cold PBS buffer pH 7.4 and resuspended (1 × 106 cells)in HEPES buffer pH 7.4 containing 2.5 mM calcium chloridefor 15 min at room temperature in the dark with 10 µl (0.1 µg)of FITC–annexin V and 10 µl (50 µg/ml) of propidium iodidesolution. After incubation, cells were directly gated for bi-parametric histograms FL1 vs. FL2. Changes of mitochon-dria membrane potential were assessed using the cationiclipophilic fluorochrome DiOC6. Cells were incubated at37 °C for 30 min in the presence of DiOC6 (40 µM in PBSbuffer pH 7.4). Caspase-3 activity was assessed using thefluorogenic substrates PhiPhiLux G1D2 (green fluorescence)and G2D2 (red fluorescence) for multiparameter analysis.Erythrocytes (1 × 106) were labeled with 10 µl of PhiPhiLuxG1D2 (10 µM) or G2D2 (10 µM) substrate solutions. Thesuspensions were incubated in 5% CO2 at 37 °C for 1 h. Afterwashings, they were analyzed by flow cytometry. For doublestaining, the PhiPhiLux-labeled cells were then incubated inthe presence of either annexin V–FITC or propidium iodideas described above.

2.4. Scanning electron microscopy

Erythrocytes were fixed for 4 h with a 1.25% glutaralde-hyde solution in 0.1 M sodium cacodylate buffer pH 7.2 andpost-fixed for 4 h in 1% osmium tetraoxide in the samebuffer. The suspensions were then filtered onto 0.2 µ Anodiscfilters and dehydrated in an ethanol series. After drying withcarbon dioxide by the critical point method and sputter-

184 D. Bratosin et al. / Biochimie 86 (2004) 183–192

coating with gold, samples were examined on a 35 CF JEOLscanning electron microscope.

2.5. Fluorescence microscopy analysis

Cells were labeled either with PhiPhiLux G1D2 in thepresence or absence of propidium iodide, or with DiOC6 asdescribed above. After washing three times with PBS bufferpH 7.4, cells were visualized using a fluorescence microscop(Zeiss Axiophot).

3. Results

3.1. Erythrocyte cell death

As shown in Fig. 1A, incubation of Rana erythrocyteswith the calcium-ionophore A 23187 in the presence ofextracellular Ca2+ (2.5 mM) led to a rapid erythrocyte death.The kinetics of A 23187 induced erythrocyte death were dosedependent. A 23187 did not induce cell death in the absenceof extracellular Ca2+, nor in the presence of both Ca2+ and theCa2+ chelator EDTA (5 mM) (data not shown).As also shownin Fig. 1B, incubation of Rana erythrocytes with Ca2+

(2.5 mM) alone in the absence of A 23187, induced a muchslower death process and required 5 days to lead to thecomplete disappearance of erythrocytes. When erythrocyteswere incubated in PBS in the absence of extracellular Ca2+

the death was slowed down and 70% of the cells survivedduring the 6 day period (Fig. 1B). Staurosporine (10 µM) inthe absence of Ca2+ induced the death of erythrocytes at thesame level as Ca2+ (2.5 mM) alone (Fig. 1D). The addition ofCa2+ did not increase cell death (data not shown). In all thesubsequent experiments, the concentrations of Ca2+

(2.5 mM), A 23187 ionophore (0.025 µM) and staurosporine(10 µM) will be used.

We and others have previously reported [3,4] that inhibi-tors of cysteine proteinases (caspases and calpain inhibitors)are capable to prevent human erythrocytes PCD. Here, wefound that the addition of cysteine-protease inhibitors, Ac-DEVD-cmk and leupeptin, delayed the death of Rana eryth-rocytes at the concentration of 200 µM, but with differentrates of inhibition. As depicted in Fig. 1B–D, leupeptinshowed the highest preventive effect as compared to Ac-DEVD-cmk treatment. Leupeptin, at day 4, prevented around50% of erythrocyte cell death mediated by Ca2+ (Fig. 1B),around 70% of the cells in response to A 23187 after 30 h ofincubation (Fig. 1C) and around 80% at day 6 in response tostaurosporin treatment (Fig. 1D). The mixture of both inhibi-tors (200 µM each) almost completely prevented the erythro-cyte cell death (Fig. 1B–D). As control, tosyl-cmk, an inhibi-tor of the serine proteases, at the same concentration, has nopreventive effect (data not shown). We also used a pan-caspase inhibitor, Z-VAD-fmk, but it does not enhance thepreventive effect in comparison to DEVD-cmk treatment(data not shown). Altogether these results led us to use themixture of both caspase and calpain inhibitors in our furtherexperiments.

Visualization of the morphological changes induced bystaurosporine, Ca2+ and ionophore A 23187 treatment in thepresence of Ca2+ was assessed by scanning electron micros-copy and compared to native erythrocytes (Fig. 2). The eryth-rocytes lost their biconvex disk-like shape with an ellipsoidcontour and displayed cell shrinkage, membrane blebblingand formation of apoptotic bodies following apoptoticstimuli. Pretreatment of the cells with the cysteine-proteaseinhibitors prevented erythrocyte morphological changes andallowed to maintain the discoid shape of the erythrocytes(Fig. 2c,e,g).

3.2. Erythrocyte cell death displays apoptotic features

Rana erythrocytes incubated in the presence of Ca2+, A23187 with Ca2+ and staurosporine were analyzed by flowcytometry for phosphatidylserine exposure (annexin-V la-beling, Ax+) and membrane permeabilization (propidiumiodide labeling, PI+). Phosphatidylserine residues are ex-posed in the external leaflet of cell membrane early duringthe process of apoptosis whereas the uptake of propidiumiodide indicates a disrupted cellular membrane integrity gen-erally observed during late apoptosis and cell necrosis.

Fig. 3A,B showed that the number of live cells (Ax– PI–)decreased drastically after treatment with Ca2+ alone, Ca2+ inpresence of A 23187 ionophore and in a lesser extend in thepresence of staurosporine. The percentages of annexin-Vpositive and propidium iodide negative cells (Ax+ PI–) weresignificantly increased (67%, 34% and 51%, respectively) aswell as the percentages of annexin-V positive and propidiumiodide positive cells (Ax+ PI+) indicating late apoptosis(30%, 47% and 11%, respectively) as compared to untreatedand in PBS incubated RBCs (9% and 9%, respectively). Inthe presence of cysteine-protease inhibitors the percentagesof dying cells (Ax+ PI–) drastically decreased.

Caspase-3 activation was assessed by flow cytometricanalysis using the fluorogenic caspase-3 substrate PhiPhiLuxG2D2. As shown in Fig. 4A, a significant cleavage of thefluorogenic substrate was detected in Ca2+-treated Ranaerythrocytes (Peak 2). Caspase-3-like activity was greatlydecreased in erythrocytes pretreated with the mixture ofAc-DEVD-cmk and leupeptin. The fluorescence intensity oferythrocytes incubated in presence of these inhibitors (Peak3) is close to the fluorescence intensity observed in untreatederythrocytes (Peak 1). Similar results were obtained in re-sponse to Ca2+ in presence of A 23187 (data not shown). Aspreviously described [3] no caspase-3 activity was detectedin human erythrocytes (data not shown) indicating that acti-vated caspases characterize batracian erythrocyte cell death.

These results were confirmed by phase contrast and fluo-rescence microscopic analysis. Fig. 4B shows micrographsof PhiPhiLux G1D2 labeled Rana esculenta erythrocytesincubated in presence of Ca2+ alone (2.5 mM calcium chlo-ride) with or without pretreatment of a mixture of Ac-DEVD-cmk and leupeptin (200 µM each). Phase contrast micros-copy revealed the major changes of cellular morphology in

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Fig. 1. Rana esculenta erythrocyte cell death. (A) Freshly isolated erythrocytes were incubated at 37 °C in HEPES buffer pH 7.4 with various concentrations ofA 23187 in the presence of Ca2+ (2.5 mM). Erythrocytes were also incubated in the presence of Ca2+ (2.5 mM) alone (B) A 23187 (0.025 µM) in the presenceof Ca2+ (2.5 mM) (C) and staurosporine (STX) 10 µM (D). In B, C and D, cells were also pretreated in absence or presence of Ac-DEVD-cmk 200 µM (DEVD),leupeptine 200 µM (Leup) or a mixture of both (200 µM each) (DEVD + Leup). Incubation in PBS serves as control. Values indicate the percentages oferythrocytes remaining in the culture at the indicated time points. Each experiment shown is representative of four experiments performed giving similar results.

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dying cells (Fig. 4B(c)) associated with detectable caspase-3activity (Fig. 4B(d)). Cysteine-protease inhibitors preventedboth morphological changes (Fig. 4B(e)) and caspase-3 ac-tivity (Fig. 4B(f)). In addition, we found that dying erythro-cytes observed by contrast and fluorescence microscopyanalyses also displayed fragmented nuclei associated withcaspase-3-like activity (Fig. 5).

It is now well admitted that mitochondria is a centralsensor in PCD [11]. In contrast to human erythrocyte, batra-cian erythrocytes possess mitochondria. Since apoptosis dis-played loss in mitochondrial membrane potential (DWm), weassess whether changes in DWm characterize death of Ranaerythrocytes. As depicted in Fig. 6A, erythrocytes incubatedwith Ca2+ alone showed a significant decrease of DWm (Peak2) in comparison to untreated erythrocytes (Peak 1). DWm

loss was decreased by the pretreatment with a mixture ofAc-DEVD-cmk and leupeptin (200 µM each) (Peak 3). Simi-larly, treatment with A 23187 plus Ca2+ or staurosporine alsoinduced DWm loss (data not shown). Fluorescence micros-copy confirmed these observations (data not shown). Finally,a time course was carried out which revealed that death oferythrocytes is associated with an early depolarization of themitochondria membrane potential (Fig. 7) that precedesphosphatidylserine externalization.

4. Discussion

We found that PCD of nucleated Rana esculenta erythro-cytes mediated by either calcium influx, ionophore A23178 or staurosporine, shares typical features of apoptosis

Fig. 2. Scanning electron microscopic analysis of Rana esculenta erythrocyte morphological changes. Freshly isolated erythrocytes were incubated (forconditions see Section 2) either with PBS buffer (a) or staurosporine (10 µM) in the absence (b) or presence (c) of a mixture of cysteine protease inhibitorsAc-DEVD-cmk and leupeptin (200 µM each). Erythrocytes were also incubated in HEPES buffer containing calcium chloride 2.5 mM in the absence (d) orpresence (e) of inhibitors or in HEPES buffer containing calcium ionophore A 23187 (0.025 µM) in the absence (f) or presence (g) of inhibitors. Short arrows:membrane blebs; long arrows: apoptotic bodies.

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including cell shrinkage, plasma membrane vesiculation, PSexposure, caspase-3-like activity, mitochondrial membranepotential loss and chromatin condensation and fragmenta-tion. Our findings also indicated that this form of PCD isprevented by in vitro treatment with cysteine proteinase in-hibitors as we recently found in human erythrocytes which

are devoided of mitochondria, nucleus and other organelles[3]. These observations may be crucial for understandinghow PCD regulates erythrocyte survival and question aboutthe conservation of the PCD machinery across kingdom.

Mammalian fetal erythrocytes [12] and erythrocytes fromamphibians, reptiles and birds [5–10] which contain a

Fig. 3. Flow cytometric analysis of Rana esculenta cell death. (A) Flow cytometric analysis using annexin V–FITC (FL1) and propidium iodide (FL2) to assessphosphatidylserine exposure and cell membrane integrity, respectively. Untreated: freshly isolated erythrocytes (standard of reference). PBS, Ca2+, STX:erythrocytes incubated (37 °C for 6 h, then 20 °C for 48 h) in PBS buffer, HEPES buffer containing calcium chloride (2.5 mM) and staurosporine (10 µM),respectively. A 23187: erythrocytes incubated (37 °C for 4 h) in the presence of Ca2+ (2.5 mM) and of A 23187 ionophore (0.025 µM). Inh–, Inh+: erythrocytespretreated for 30 min in absence (Inh–) or presence (Inh+) of a mixture of Ac-DEVD-cmk and leupeptin (200 µM each). LL quadrant: viable cells (annexin V andpropidium iodide negative cells); LR quadrant: apoptotic cells (annexin V positive and propidium iodide negative cells); UR quadrant: dead cells (annexin V andpropidium iodide positive cells). Numbering refers to the cell percentage of each population. Number of counted cells: 10,000. (B) Percentages of erythrocytesdisplaying PS exposure. Data represent the means ± S.D. of four independent experiments. Cells were treated as in A in the absence (–) or presence of leupeptin(L), Ac-DEVD-cmk (D) or the mixture Ac-DEVD-cmk and leupeptin (L + D) (200 µM each).

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nucleus undergo typical apoptosis. However, the main com-ponents of the death machinery were not clearly defined inthese different models. Chicken erythrocytes PCD inducedby staurosporin plus cycloheximide either in the absence orpresence of serum have been reported to display cell shrink-age, PS exposure and nuclear chromatin condensation andfragmentation [10]. However, chicken erythrocytes PCD,contrary to Rana PCD, was devoided of caspase activation asdemonstrated by Weil et al. [10]. These authors also foundthat this process of cell death was not prevented by a broadsynthetic caspase inhibitor (Z-VAD-fmk). Here, we foundthat DEVD-cmk and leupeptin prevented Rana erythrocyte.DEVD-cmk, however, was less efficient to prevent cell deathas compared to leupeptin (Fig. 2). The mixture of both had anadditive effect and prevented all hallmark of apoptosis. Al-though the caspases are key components in apoptosis, numer-ous results suggest the presence of other cell death pathways.Those included Ca2+-dependent serine proteases, DNAse I,DNAse II and DNAse c [13–17]. Initial study had revealedthe presence in the cytosol of Rana [6] of a DNAse I, a Ca2+-Mg2+-dependent endonuclease which is a 30 kDa protein,

activated in p13 and p11 kDa subunits. In thymocytes, a18 kDa nuclease (NUC-18) has been also described requiringCa2+ and Mg2+ for optimal activity to induce 50 kb pair DNAfragmentation [18–20]. DNAse I which cuts genomic DNAis activated following its release from actin cleavage. Inciliary neurons deprived of neurotrophic factors (CNTF) andthymocytes, calpain inhibitors prevent DNA fragmentationby inhibiting actin cleavage. An hypothetical model for RanaPCD could be that following Ca2+ influx calpain is activatedwhich in turn cleaves cytoskeleton and induces the release ofDNAse I in an active form. However, cleavage of actin is notonly mediated by calpain [21,22]. Some other groups haveproposed that actin may be a substrat of caspase-1 [21,23–

Fig. 4. Caspase-3-like activity in Rana esculenta erythrocytes. (A)Caspase-3 activity assessed using PhiPhiLux G2D2 fluorescent substrate byflow cytometric analysis. (1) Fluorescence of untreated RBCs. Erythrocytesincubated (37 °C for 6 h, then 20 °C for 24 h) in HEPES buffer pH7.4 containing calcium chloride (2.5 mM) in the absence (2) or presence (3)of a mixture of Ac-DEVD-cmk and leupeptin (200 µM each). Abcissae:fluorescence (FL2). Ordinates: cell numbers. Number of counted cells:10,000. Similar results have been obtained with erythrocytes incubated inpresence of A 23187 (0.025 µM) or staurosporine (10 µM). (B) Phasecontrast (a, c, e) and fluorescence (b, d, f) microscopic analysis. Caspase-3activity of Rana esculenta erythrocytes was assessed using PhiPhiLuxG1D2. Caspase-3 positive cells are green colored. (a and b) native erythro-cytes (arrows indicate apoptotic cells); (c to f) erythrocytes incubated (37 °Cfor 6 h, then 20 °C for 24 h) in HEPES buffer pH 7.4 containing 2.5 mMcalcium chloride in absence (c and d) or presence (e and f) of a mixture ofAc-DEVD-cmk and leupeptin (200 µM each). Bar (in a): 30 µm. Theexperiments shown are representative of three independent experimentsgiving similar results.

Fig. 5. Changes in nuclear morphology of Rana esculenta erythrocytes.Phase contrast (A) and fluorescence (B) micrographs of Rana esculentaerythrocytes incubated (37 °C for 6 h, then 20 °C for 24 h) in HEPES bufferpH 7.4 containing 2.5 mM calcium chloride. Erythrocytes were double-labeled with PhiPhiLux G1D2 (in green) and propidium iodide (in red).Yellow color corresponds to apoptotic cells. Short arrows indicate chromatincondensation and nucleus fragmentation. Long arrows are caspase positivecells. Bar: 30 µm.

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26] but remains controversial [27,28]. Active caspase-3 hasbeen also proposed to cleave the spectrin, an integral proteinof the cytoskeleton [3,4]. Here, we found caspase-3-likeactivity in dying erythrocytes and that the death diminishedafter in vitro treatment with DEVD-cmk. Altogether, theseobservations may underline the possible interaction that mayexist between cleavage of cytoskeleton and protease activa-tion during Rana PCD.

However, how these different pathways are involved inmitochondria membrane potential loss? We observed duringRana PCD that mitochondrial depolarization precedes PSexposure and cell death (Fig. 7). Consecutive to apoptoticstimuli, members of the Bcl-2 family have a crucial role inthe control of mitochondria disruption [29]. Recent worksrevealed that calpain cleaves not only Bid which in turntargets Bid to the mitochondria in inducing cytochrome crelease [30,31] but also cleaves Bax that has a cytochrome creleasing activity [32–35]. However, does Rana PCD involvethe cleavage of Bid or Bax remains an open question.

Our data raised major issue about the conservation of thedeath machinery in erythrocytes across kingdom. Amphib-ians which were the first terrestrian vertebrates possess themain components of the apoptotic machinery, like observedin mammalian nucleated cells. During evolution, the emer-gence of birds leads to the inactivation of caspase activity[10], whereas in vertebrates, during differentiation, erythro-cytes banish both nucleus and mitochondria and were de-voided of an active apoptosome (cytochrome c and APAF)

[3,4]. Although caspases may be important for erythrocytedifferentiation [36], progressively with evolution, compo-nents crucial for PCD machinery were lost. These observa-tions raise the following question: does the loss of mitochon-dria and nucleus represents a selective advantage forerythrocyte vertebrates? Since with evolution, erythrocyteshave been in contact with progressive increase concentrationof oxygen, an oxidative agent that accelerates the aging ofcells, the price to pay has been to eliminate mitochondriacontents which in turn shortness the lifespan of vertebrateerythrocytes. Indeed, although mitochondria and cytochromec have been proposed to be essential components of theapoptotic pathway, the mitochondria is crucial for energyproduction in aerobic organisms and for life [37,38]. Thus,perish or banish has been the crucial dilemma of erythrocytesto survive across the kingdoms.

Fig. 6. Flow cytometry of mitochondrial transmembrane potential (DWm).Freshly isolated Rana esculenta erythrocytes either untreated (1) or incuba-ted (37 °C for 6 h, then 20 °C for 24 h) in HEPES buffer pH 7.4 containingCa2+ (2.5 mM) in the absence (2) or presence (3) of a mixture of Ac-DEVD-cmk and leupeptin (200 µM each). Abcissae: DiOC6 fluorescence (FL1mode). Ordinates: cell number. Numbering refers to the cell percentage ofeach cell population. Number of counted cells: 10,000. Similar results wereobtained with erythrocytes incubated in presence of Ca2+ (2.5 mM) andionophore A 23187 (0.025 µM) or of staurosporine (10 µM). Data shown arerepresentative of four independent experiments.

Fig. 7. Kinetics of phosphatidylserine exposure and mitochondrial mem-brane potential loss in dying Rana esculenta erythrocyte. (A) Flow cytome-tric analysis of phosphatidylserine exposure (Annexine V–FITC, FL1) andmembrane permeability (PI, FL2). Freshly isolated erythrocytes were incu-bated at 37 °C in HEPES buffer pH 7.4 containing calcium chloride 2.5 mMin the absence or presence of a mixture of Ac-DEVD-cmk and leupeptine(200 µM each) (24 h + Inh). LL quadrant: viable cells (annexin V andpropidium iodide negative cells); LR quadrant: apoptotic cells (annexin Vpositive and propidium iodide negative cells); UR quadrant: dead cells(annexinV and propidium iodide positive cells). Numbering refers to the cellpercentage of each. (B): Mitochondrial membrane potential (DiOC6, FL1).Peak colors, yellow: 0 h, violet: 3 h, blue: 6 h, mauve: 16 h, green: 24 h; Redpeak, incubation for 24 h in the presence of inhibitors (24 h + Inh); %,percentage of DWm loss.

190 D. Bratosin et al. / Biochimie 86 (2004) 183–192

Acknowledgements

This work was supported in part by the Université desSciences et Technologies de Lille 1, the Ministère del’Enseignement Supérieur de la Recherche et de la Technolo-gie, the Centre National de la Recherche Scientifique (UMRno. 8576 du CNRS, Director: Dr. Jean-Claude Michalski),the Etablissement Régional de Transfusion Sanguine de Lille(Director: Dr. Jean-Jacques Huart), the MacoPharma Co.,Tourcoing, France (Director: Dr. Francis Goudaliez) and theRomanian Academy. Daniela Bratosin and Laura Mitrofanare fellows of MacoPharma Co. The authors are grateful withmany thanks to Dr. Jean-Jacques Huart and Hervé Dubly(Chairman of MacoPharma Co.) who provided a kind inter-est and financial support to this project and to VéroniqueLebret, Catherine Alonso, Jocelyne Celen and JacquelineDondeyne, for their skilful assistance. Authors are indebtedto the referees for their comments and criticisms, whichhelped them to improve the manuscript.

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