Morphology of MitochondrialPermeability Transition:
Morphometric Volumetry inApoptotic Cells
ANTONIO SESSO,1* MARCIA M. MARQUES,2 MARIA M.T. MONTEIRO,3
ROBERT I. SCHUMACHER,4 ALISON COLQUHOUN,5 JOSE BELIZARIO,6
SERGIO N. KONNO,1 TAHIS B. FELIX,1 LUIS A.A. BOTELHO,1
VANESSA Z.C. SANTOS,1 GUILHERME R. DA SILVA,7
MARIA DE L. HIGUCHI,8 AND JOYCE T. KAWAKAMI1
1Laboratory of Immunopathology, Institute of Tropical Medicine, University of SaoPaulo, Sao Paulo, Brazil
2Department of Operative Dentistry, School of Dentistry, University of Sao Paulo,Sao Paulo, Brazil
3Department of Pathology, Faculty of Medicine, University of Sao Paulo, Sao Paulo, Brazil4Department of Biochemistry, Chemical Institute, University of Sao Paulo, Sao Paulo, Brazil
5Department of Histology, Biomedical Institute, University of Sao Paulo, Sao Paulo, Brazil6Department of Pharmacology, Biomedical Institute, University of Sao Paulo,
Sao Paulo, Brazil7Department of Preventive Medicine, Faculty of Medicine, University of Sao Paulo,
Sao Paulo, Brazil8Laboratory of Pathological Anatomy, Hearth Institute, University of Sao Paulo,
Sao Paulo, Brazil
ABSTRACTHere we report on the mitochondrial permeability transition (MPT), which refers to the morphology of mitochondria whose
inner membrane has lost its selective permeability. In all types of apoptotic cells so far examined, we found outer mitochondrialmembranes that had been ruptured. These mitochondria present a swollen matrix covered by an inner membrane herniating intothe cytoplasm through the breached outer membrane. Similarly ruptured outer mitochondrial membranes have been reported instudies on mitochondrial fractions induced to undergo MPT, carried out by others. Our observations were made on five types ofrat tissue cells and six different cultured cell lines in the early stages of apoptosis. Samples from the cell lines HL-60, HeLa,WEHI-164, and a special batch of PC-12 cells were subjected to various apoptogenic agents and analyzed morphometrically.Nonapoptotic companion cells with unaltered nuclear structure (CUNS) were also analyzed. The mitochondrial volume in �m3
Key words: morphometry; mitochondria; apoptosis; rupture of the outer mitochondrial membrane;mitochondrial permeability transition
The mitochondrial permeability transition (MPT) refersto an increase in the permeability of the inner mitochon-drial membrane caused by nonselective agents, in apopto-tic and necrotic cells (Lemasters et al., 1998). This condi-tion is associated with membrane depolarization andcollapse of the transmembrane potential (��m). Due tothe hyperosmolarity of the matrix and the loss of selectivepermeability of the inner membrane, the ensuing influx ofliquid to the mitochondrial matrix expands the matrix,promoting a large swelling of the mitochondria. The loss ofselective permeability was initially thought to be causedby defects in the lipid moiety of the membrane, consequent
Grant sponsor: Fundacao de Amparo a Pesquisa do Estado de SaoPaulo; Grant number: 00/06648-2; Grant sponsor: Conselho Nacionalde Pesquisas; Grant number: 520359/96-8; Grant sponsor: Pro-Reitoriade Pesquisa da University of Sao Paulo (Procontes 2001).
*Correspondence to: Antonio Sesso, Instituto de Medicina Trop-ical de Sao Paulo, Av: Eneas de Carvalho Aguiar, 500 Predio II 2°andar, CEP 05403-000 Sao Paulo, SP, Brasil. Fax: 55-11-3066-7065. E-mail: [email protected]
Received 3 March 2004; Accepted 20 July 2004DOI 10.1002/ar.a.20134Published online 5 November 2004 in Wiley InterScience(www.interscience.wiley.com).
THE ANATOMICAL RECORD PART A 281A:1337–1351 (2004)
to activation of phospholipase A2 by elevated Ca2� levels(Pfeiffer et al., 1979; Beatrice et al., 1980). The alternativehypothesis, that MPT results from the opening of a pore ormegachannel, referred to as the permeability transitionpore (PTP) transversing both mitochondrial membranes(Hunter and Haworth, 1979; Zoratti and Szabo, 1995;Bernardi, 1999), has been extensively investigated and iscurrently widely accepted.
It was thought that, after the onset of MPT, expansionof the swollen matrix would cause the mitochondrial outermembrane to rupture due to the limited capacity for dis-tension of this membrane. The inner membrane may ex-pand much more because of its continuity with the mem-brane of the cristae (Petit et al., 1998). The possibility thatthe rupture of the outer membrane would allow the re-lease of cytochrome c and other mitochondrial intermem-brane proteins (Petit et al., 1998) is one of various alter-native mechanisms found in the literature to explain howthese protein inducers of apoptosis reach the cytoplasm.Another hypothesis is that the proteins are released to thecytoplasm by passing through the outer membrane (De-sagher and Martinou, 2000). The idea that the release ofmitochondrial proteins occurs either through permeabili-zation of (Basanez et al., 1999; Belzacq et al., 2002; Rav-agnan et al., 2002) or the formation of supramolecularopenings in (Antonsson et al., 2001; Kuwana et al., 2002)the outer membrane has recently gained momentum.
In in vitro systems, the proteins of the PTP may interactwith proapoptotic proteins, such as Bax and Bid, promot-ing the permeabilization of the outer mitochondrial mem-brane to cytochrome c (Zamzami and Kroemer, 2003).MPT has been described in necrotic (Nieminen et al.,1995, 1997; Kim et al., 2003) and the majority of apoptoticcells. There have been reports of cases where the mito-chondria of apoptosis-induced cells release cytochrome cwithout exhibiting permeability transition (PT) (VanderHeiden et al., 1997; Goldstein et al., 2000); however, this isnot clearly understood (Tafani et al., 2001). In the major-ity of reports (Belzacq et al., 2002; Castedo et al., 2002),MPT is directly associated with the initiation of the apo-ptotic process.
Once in the cytoplasm, cytochrome c assembles with twoother proteins, the apoptotic protease-activating factor 1(Apaf-1) and procaspase 9 to form a complex, the apopto-some. The ensuing activation of caspase 9 leads the cell tothe execution phase of apoptosis.
In addition to cytochrome c, other intermembrane mi-tochondrial proteins, such as Smac/DIABLO and Omi/Htra2, are able to induce or enhance the activation ofcaspases. The intermembrane proteins AIF and endonu-clease G may act independently of caspase activation (Ku-wana and Newmeyer, 2003). According to the cell type,caspases 2, 3, and 9 may be added to the list of mitochon-drial intermembrane proteins that enhance the apoptoticprocess when they are liberated into the cytoplasm (Susinet al., 1999a, 1999b).
When apoptosis is induced by the other major route ofinduction, occupation of the TNF receptors, there aremany cases in which the mitochondrial pathway is respon-sible for the manifestation of apoptosis. This seems tooccur when relatively low levels of caspase 8 are activatednear the cytoplasmic portion of the occupied TNF receptor.In this instance, procaspase 3 cannot be directly activatedby caspase 8 to implement the late stages of the apoptoticprogram. However, caspase 8 can cleave the cytosolic pro-
tein Bid, giving rise to the truncated form of Bid, tBid,which translocates to the outer mitochondrial membrane,where it will interact with Bax and other proteins topromote the release of the mitochondrial intermembranedeath-inducing proteins (Scaffidi et al., 1998; Antonssonet al., 2001). These facts emphasize the extensive partic-ipation of mitochondria in the enhancement of the apopto-tic program.
Examination using transmission electron microscope(TEM) sections from various cell types undergoing pro-grammed cell death identified many mitochondria withruptured outer membranes. Through the breach, the swol-len mitochondrial matrix, covered by an expanding innermembrane, herniates into the cytoplasm. This structuralchange in the mitochondria is identical to that previouslydescribed by Angermuller et al. (1998). The generalizedrupture of the outer mitochondrial membrane revealed awide range of microscopic profiles. Some of these profilesresemble in vitro TEM experiments in which isolated mi-tochondria swelled to various degrees of induction of thepermeability transition. These results will be commentedon below. Based on these observations, it seemed of inter-est to evaluate the magnitude and latitude of mitochon-dria undergoing the permeability transition in apoptoticcells from various cultured lineages induced with variousapoptogenic agents. We have obtained estimates of thevolume per cell and the cytoplasmic volume fraction ofmitochondria with ruptured and nonruptured outer mem-branes in apoptotic and companion nonapoptotic culturedcells (CUNS). The cellular volume of mitochondria is theproduct of the number of mitochondria per cytoplasm andthe average individual mitochondrial volume. Thus, byanalyzing how these parameters changed, we hope to ob-tain an indirect insight into how the number of mitochon-dria was affected by the early stages of apoptosis. In thefour cell lines studied morphometrically, we observed thatthe volume of mitochondria with ruptured outer mem-brane in apoptotic cells represents from 47% to 89% of themeasured cell total mitochondrial volume. In all studiedsamples, we noticed that a fraction of the CUNS alsopossessed mitochondria with a breached outer membrane.In these cells, MPT preceded the activation of the caspasesthat induce the apoptotic nuclear phenotype. Althoughthese results do not prove that the rupture of the outermitochondrial membrane is the mechanism by which theintermembrane proteins are released into the cytoplasm,it is a most likely possibility.
MATERIALS AND METHODSElectron Microscopy
The organ fragments and cultured cells were processedas previously described (Sesso et al., 1999). Silver sectionsstained with uranyl acetate and lead citrate (both fromLadd Research Industries) were observed using a Jeol1010 electron microscope or a Philips 301 at 80 kV. Whenit was necessary, to check whether the outer mitochon-drial membrane was actually in the section but could notbe observed due to an unfavorable sectioning angle, weperformed a 24° tilt on either side of the normal plane ofobservation. This plane is at 0° tilt.
Rat Tissue CellsCells obtained from rat tissue were as follows: secretory
epithelial cells from the ventral lobe of the prostate gland
from rats killed daily in the 2–10 days following castration(Kyprianou and Isaacs, 1988); plasma cells from the gran-ulation tissue of an experimentally induced scar in thedorsal skin of adult rats; macrophage, from the samegranulation tissue; pancreatic acinar cells from pancreaticglands that had undergone ligature of the excretory ducts(Gukovskaya et al., 1996) 2–4 days previously or from ratsmaintained on a protein-depletion diet and receiving dailyinjections (40 mg/100 g of body weight) of dl-ethionine(Sigma) for 5 days (Walker et al., 1993); and secretorymammary cells from female rats subjected to interruptedlactation with daily gland removal from days 1 to 10.
Cultured CellsPC-12 cells were kindly supplied by Dr. Paulo Lee Ho
from the Institute Butantan in Sao Paulo. These cellswere derived from PC-12 cells obtained directly fromATTC (pheochromocytoma; rat; ATCC CRL-172). Theywere grown in Dulbecco’s modified Eagle’s medium(DMEM; Life Technologies) supplemented with 10% FCS(Cultilab) at 37°C in a humid atmosphere in 5% CO2. Afterbeing stabilized in these growth conditions (Ho and Raw,1992), it is no longer necessary to add poly-L-lysine to theunderlying support as the cells adhere more easily thanthe original PC-12 cells. The adapted cells used here arereferred to as PC-12* cells. The PC-12* cells were serum-deprived for 3 and 8 hr or exposed to brefeldine A (BFA 2.0�M; Calbiochem; which blocks the anterograde vesiculartraffic from the ER to the Golgi, but not the retrogradetraffic), to 0.5 �M of staurosporine (STS; Calbiochem; aprotein kinase C inhibitor), and to BFA � STS in the sameconcentrations, with an exposure time of 16 hr.
WEHI-3 cells (myelomonocyte leukemia; mouse; ATCCTIB-68) maintained in RPMI-1640 (Sigma) plus 10% calfserum were exposed for 5 hr to 20 �g/ml of the teneposideVM 26 inhibitor of topoisomerase II; 0.4 �g/ml vimblastine(Calbiochem; which prevents tubulin polymerization; 400�g/ml of the antibiotic novobiocin (Sigma); 0.5 nM okadaicacid (Sigma), a potent inhibitor of protein phosphatases 1and 2A; and 0.5 �M STS.
K-562 cells (chronic myelogenous leukemia; human;ATCC (CCL-243) exposed for 5 hr to vimblastine 0.4 �g/ml; oligomycin (Sigma), a highly specific mitochondrialATP-synthase inhibitor (5 nM); VM 26, 20 �g/ml; novobio-cin (Sigma) 400 �g/ml; nigericin (Calbiochem), a proton-ionophore, 10 �M; and BFA 2.0 �M.
HeLa cells (epithelioid carcinoma; cervix; human; ATCCCCL-2) in medium devoid of serum were exposed to 0.5�M STS plus 2.0 �M BFA or to 0.5 �M STS for 16 hr.
WEHI-164 cells (mouse; methylcholanthrene-inducedfibrosarcoma; ATCC CRL-1751) were exposed to BFA, toSTS, and to BFA � STS in the same conditions previouslyemployed.
HL-60 cells (human; peripheral blood; promyelocyticleukemia; ATCC CCL-240) were exposed for 16 hr to BFA2 �M � human TNF 100 ng/ml � 2 �M camptothecin(CAMP; Sigma; this drug is a topoisomerase I drug thatacts primarily on cells in the S-phase of the proliferativecycle). WEHI-3, K-562, and HeLa cells were cultured inRPMI-1640 with 10% FBS (Cultilab) in a humid atmo-sphere at 37°C in 5% CO2.
The apoptogenic agents for all samples were added tothe cultures in fresh medium when the cells were about50–60% confluent. In earlier phases of this study, weexamined cultures of WEHI-3, K-562, L 929 (mouse fibro-
sarcoma; CLL-1.1), LLC-WRC 256 (carcinoma Walker;rat; ATCC CCL-38), WEHI-164, HL-60, and PC-12 cells(pheochromocytoma; rat; CRL-172). The PC-12 cells wereobtained from the American Type Culture Collection inRockville in 1992. Samples from all these cell lineageswere analyzed under TEM in the early stages of exponen-tial growth and not exposed to apoptogenic drugs.
Morphometric ProceduresThe morphometric study was carried out on the four
possible sectional profiles of mitochondria exhibiting rup-ture of the outer membrane (Fig. 1). Mitochondria with anintact outer membrane in apoptotic and CUNS cells fromthe same culture are referred to as type 1 mitochondrionand their sections are named type 1 profiles (Fig. 1A). Tofacilitate the identification of these profiles, we refer to themitochondrion with a breached outer membrane as a type2 mitochondrion (Fig. 1B). When it is sectioned unequiv-ocally, we refer to it as a type 2 mitochondrial profile(schematized in Fig. 1D). A type 2 mitochondrion may besectioned in such a way as to create mitochondrial profileswith both membranes (Fig. 1C) or only one membrane(Fig. 1E and F). The vesicular profiles covered only by theinner mitochondrial membrane are unequivocally recog-nized as type 3 profiles when they contain the remnants ofmitochondrial cristae (Fig. 1E). Mitochondria covered byonly the inner membrane and containing no cristae arereferred to as type 4 profiles (Fig. 1F). We measured onlyprofile types 1, 2, and 3. When a cell exhibits types 1 and2 mitochondria, we cannot be sure from which of these twotypes a given type 1 profile originates (Fig. 1C).
Estimates of the cytoplasmic volume and the volumesassociated with the mitochondrial profile types 1, 2, and 3in apoptotic cells and the type 1 profile in nonapoptoticcells. These parameters were obtained by point-countingvolumetry as indicated by Aherne and Dunnill (1982),Sesso et al. (1999), and Gundersen et al. (1988).
The equivalence of area fraction (AA) and volume frac-tion (Vv) by which AA � Vv is a fundamental concept of allmorphometry (Aherne and Dunnill, 1982). Areas are esti-mated with a known degree of accuracy by counting howmany regularly spaced points of a test system fall insidethe area (hits). A given area value at the microscopicmagnification chosen is associated with each point. Thisapproach was used to estimate the areas of the cell sec-tions from which we needed to obtain the radii. We willbriefly survey the complete morphometric proceduresused. The area fraction, AA, and, by extension, the volumefraction, Vv, also referred to as volume density, occupiedby the mitochondria in the cytoplasm, is obtained bycounting hits over the mitochondrial profiles (be they 1, 2,or 3) and over the cytoplasm including the previous countsover the mitochondrial profiles. The ratio between thesetwo counts is AA � Vv. In order to obtain the cytoplasmicvolume, the percentage of the cell volume the cytoplasmrepresents and its absolute value in �m3 must be calcu-lated. To estimate the cellular volume, the distribution ofradii of the cell sections is obtained. The cytoplasmic vol-ume fraction of the cell is obtained by counting hits overthe nuclear profiles and over the cytoplasm. The ratiobetween the number of hits over the cytoplasm and overthe entire cell profile (the sum of hits over nuclei andcytoplasm) is the parameter procured. The cellular volumeis obtained independently by measuring the radii of thecell sections.
The cellular and cytoplasmic volume of each culturedcell type was estimated using measurements from 10 to 20enlarged prints (2,000 � 2.5). Each recorded microscopicfield possessed 2–8 profiles of CUNS and at least oneprofile of a cell in explicit apoptosis. To be considered as acell undergoing apoptosis, in addition to the nuclear phe-notype of apoptosis, it had to have most of its perimetercovered by the cell membrane and no signs of advancedproteolysis. Some 15–30 and 70–100 profiles of apoptoticand CUNS cells, respectively, were measured. The testsystem superimposed over the prints was composed ofthree types of hit marker, regularly spaced small crossesand the extremities of two different types of regularlyspaced segments of straight lines (Gundersen et al., 1988).Estimates of the volume density (Vv) of the nucleus andcytoplasm of the cells were thus obtained [Vv of the cyto-plasm (Vvc) � number of hits over the cytoplasm/numberof hits over the nucleus plus the number of hits over thecytoplasm]. Since the cell sections are fairly circular, thenumber of hits over each cell section gives an estimate ofthe corresponding area (�r2). In each sample, the radiifrom apoptotic and cells with unaltered nuclear structurewere thus obtained. A 10-class distribution of radii wasconstructed. Employing the procedure of Bach (1963),used either in an HP machine (Arcon et al., 1980) or in aPC, the main parameters, including the mean sphere vol-ume of the corresponding distribution of radii, were ob-tained. Since the mean sphere volume is an estimate ofthe cellular volume (vcell), the cytoplasmic volume is vcyt �vcell � Vvcyt.
Volumes of Mitochondria Types 1 and 2
Again, the area fraction was estimated by the point-counting procedure. The volume fraction is more com-monly referred to as volume density (Vv). Having thecytoplasmic volume vcyt and, for example, the Vvmit or the
AAmit of the mitochondria in the cytoplasm, one obtainsthe total mitochondrial volume or area (vtm or Atm). Thus,vtm or Atm � Vvmit (or AAmit) � vcyt. Since a randomlysectioned type 2 mitochondrion furnishes profile types 1,2, 3, and 4, of which only the initial three profiles weremeasured, the sum of the obtained volumes for profiletypes 2 and 3 gives the best possible estimate of thevolume of type 2 mitochondria. This volume, however, isforcibly underestimated on two accounts. In apoptoticcells, the type 1 profiles derived from type 2 mitochondriacannot be scored as such. Likewise, all type 4 profiles arenot counted. Therefore, the mitochondrial volume derivedfrom evaluations carried out on type 1 profiles in apoptoticcells represents the aggregated volume of actual type 1and some type 2 mitochondria. The estimates of the cyto-plasmic volume (or area) associated with mitochondrialtypes 1, 2, and 3 profiles in apoptotic cells were carried outusing prints enlarged to 20,000� (8,000 � 2.5). The cyto-plasm from both CUNS and cells undergoing apoptosiswere examined for each type of cell culture.
At least 10 micrographs were obtained from each group.In the apoptotic cells, we often found the three types ofmitochondrial profiles. In CUNS, particularly from cul-tures with high apoptotic indexes, in addition to profiletype 1, we also observed mitochondrial profile type 2and/or 3. These mitochondrial profiles, except for the casein line 2 of Table 1, were not point-counted as explainedabove. In the enlarged (20,000�) micrograph, each type ofmitochondrial profile was marked with a fine ink dis-penser. The number of hits falling within each profile type(H1, H2, and H3, for mitochondrial profile types 1, 2, and3, respectively) was scored. The sum of all mitochondrialhits (H � H1 � H2 � H3) and the total number of hits (h)over the cytoplasm was also scored (h � the sum of hitsover mitochondrial and nonmitochondrial cytoplasmicstructures). The volume density of the total mitochondrialtypes in the cytoplasm (Vvtm) is Vvtm � H/h; and the Vv of
Fig. 1. A schematic drawing illustrating how different angles of thesectioning plane will furnish different profiles in normal type 1 mitochon-drion (A) and mitochondrion with a ruptured outer membrane (type 2mitochondrion; B and D). Type 1 profiles can be generated from type 1
or 2 mitochondrion as demonstrated in C. The generation of profile types3 and 4 is demonstrated in E (containing cristae) and F (without cristae),respectively. In all micrographs, the bars measure 500 nm.
each mitochondria type in apoptotic cells is Vvi � Hi/h,where i refers to mitochondrial profile types 1, 2, and 3and � 1, 2, and 3. The respective total mitochondrialvolume (vtm) is vtm � Vvtm � vcyt and the volume associ-ated with each mitochondrial profile type is vmi � Vvi �vcyt.
Total Mitochondrial Surface Area on a Per-CellBasis in Apoptotic Cells and CUNS: Evaluationof the Average Surface-to-Volume Ratio ofMitochondria
The hits (hi) made by the extremities of the regularlyspaced segments [of length (l) in �m at the given magni-fication] of the test system, in the interior of the mitochon-drial profiles (1, 2, or 3 in apoptotic cells) and also over theremaining cytoplasm, were scored. The number of timesthe segments from the test system crossed (C) the limitingmembranes of the mitochondrial profiles (1, 2, or 3 inapoptotic cells and 1 profiles in CUNS) WAS also detected.The mitochondrial surface density (Sv) is Sv � 4C/l � hi,where C and hi are the total number of times the segmentsof the test system cross the mitochondrial borders and thenumber of hits the segment extremities superimpose onthe whole cytoplasm, including the mitochondrial profiles.
Sv is the mitochondrial surface area in �m2 per �m3 ofcytoplasm. The total mitochondrial membrane surfacearea per cell (TMMSA) is obtained by multiplying Sv byvcyt. The average cellular mitochondrial surface-to-volumeratio (s/v) is s/v � TMMSA/vtm.
Error Associated With Area Estimation
The coefficient of error associated with the estimation ofmitochondrial areas, circular or elliptical, was kept belowthe 0.05 level and was determined as described by Gun-dersen and Jensen (1987).
Apoptotic Index
With magnifications of 2,000 � 10 and 5,000 � 10, atleast 50–100 cells sections were randomly picked and thepercentage of apoptotic cells were evaluated. The charac-teristic nuclear alterations of the apoptotic cells are un-mistakably recognized using TEM. The apoptotic cellswere selected for morphometric studies if they possessedboth a typical apoptotic nucleus and a cell membrane atthe cytoplasmic border. For the estimation of the apoptoticindexes (AIs), all sections of cells with the nuclear pheno-type of apoptosis were counted. Therefore, cells in various
TABLE 1. Estimates of the volume density (Vv) and of the volume (v) of types 1 and 2 mitochondria inapoptotic (AP) cells and in CUNS*
*In CUNS exempted for the data in line 2 and column 2, only type 1 mitochondrial profiles were sampled.aPercentage of the estimated volume in relation to the total mitochondrial volume in AP cells.bPercentage of the estimated volume in relation to the total mitochondrial volume in AP cells.cIn this sample, the volumes of types 2 and 3 mitochondrial profiles were evaluated in CUNS and were 19 and 0.3 �m3,respectively.dExtreme percentual values in the columns.
1341MITOCHONDRIAL PERMEABILITY TRANSITION
advanced stages of cytoplasmic proteolysis were alsocounted.
RESULTSDetection of Apoptotic Cells by TransmissionElectron Microscopy
Among the biochemical and structural changes ob-served in cells undergoing apoptosis, the most prominentand an essential element in the identification of this typeof cell death are the structural alterations of the nucleus.Our observations confirm that the various forms of apo-ptotic nuclear phenotype are represented in both rat tis-sue cells and in immortalized cultured cells. Nuclear con-traction is coincident with hypercondensation of thechromatin (i.e., exceeding that seen in normal heterochro-matin) and the clumping of the chromatin into massesthat adhere along the nuclear membrane. Such massesvary in appearance from large spherical-like (Fig. 2A) todemilune-like conformations (Fig. 2B and C). The frag-mentation of these large masses, which often occupy theentire nuclear profile, into various smaller spheroid bodiesalso occurs. This particular nuclear phenotype corre-sponds to what the former cytologists called karyorrhexis.Additional phenotypes of apoptotic cells nuclei as well asof mitochondria in both CUNS and apoptotic cells may beexamined at http://www.sebepa.cjb.net/; the password is“sessoimt.”
Apoptotic Cells in Rat TissuesThe finding of mitochondrial profile types 2 or 3, or
both, reveals the presence of a type 2 mitochondrion. Inpopulations with an elevated percentage of apoptoticcells, this finding is frequent. All apoptotic tissue cellswe examined possessed type 2 mitochondria (arrows inFigs. 3 and 4B). The low-magnification image of theapoptotic cell in Figure 3A reveals a common structuralalteration seen in both apoptotic cells and in CUNSundergoing MPT in a less stretched cytoplasmic form.We observe the absence of organelles in some sectors ofthe cytoplasm (2 in Fig. 3A) and their clustering inothers (3 in Fig. 3A).
Rat prostate secretory cells. The lack of male hor-mones induces a marked involution of the prostrate glandconsequent to death of the secretory epithelial cells byapoptosis. Ten days after castration, the gland mass isreduced to some 15% of the original weight. We havestudied glands removed 4–6 days after orchiectomy morethoroughly (Fig. 3A); these exhibit a high incidence ofapoptotic epithelial cells.
Plasma cell and macrophage. A distinct increasein the number of cells at the granulation tissue occurs 4–6days after scar formation. At day 5, we found more apo-ptotic plasma cells (Fig. 2B) and macrophages (Fig. 2C)than at other time intervals. This is coincident with theproliferation time of fibroblasts, plasma cells, and macro-phage.
Pancreatic acinar cells. Both procedures used toinduce apoptosis were highly effective. After ligature ofthe excretory ducts, the pancreas gland regions thatsuffered a lack of flowing secretion with consequentcompression of the cells exhibited generalized apoptosisof the acinar cells. In one of these glands, we observed
a nonapoptotic cell with a mitochondrion exhibitinga ruptured outer membrane (arrow in Fig. 4A). Inthe majority of apoptotic cells, mitochondria undergo-ing permeability transition could be recognized (Fig.4B).
Mammary gland secretory epithelial cells. Theabrupt removal of the suction stimulus from the breastpromotes a hormonal imbalance that induces apoptosis ofthe secretory cells (Walker et al., 1989) and consequentinvolution of the mammary glands. The incidence of apo-ptosis in the secretory cells is revealed by the rapid in-crease of intraepithelial macrophage filled with apoptoticbodies containing remnants of the secretory cell cyto-plasm. The percentage of macrophage in the epitheliumrose by day 3 after the interruption of lactation and re-mained elevated until day 9. We found type 2 mitochon-dria in the apoptotic bodies inside epithelial cells from agland obtained at day 3 after separation of the littermatesfrom the nursing female.
Apoptotic Cultured CellsExamining the various cell cultures maintained un-
der normal conditions without the addition of apopto-genic agents, in no instance did we find type 2 mitochon-dria in the nonapoptotic cells. The L 929 and the PC-12cells from ATCC were also examined after treatmentwith apoptogenic agents. TNF� was added to cultures ofL 929 cells while apoptosis was induced in the PC-12cells by sera removal. In these particular experiments,no apoptosis was detected in the L 929 cells. To confirmthis negative finding, a thorough search for apoptosis aswell as type 2 mitochondria in CUNS was carried outwith no success. After sera removal from the PC-12cells, samples were collected at various time intervalsbetween 2 and 48 hr. While the apoptotic cells exhibitedtype 2 mitochondria in all time intervals, in no instancedid we find mitochondria with a ruptured outer mem-brane in CUNS. We have examined more than 600CUNS profiles looking for type 2 mitochondria. Thesera-deprived PC-12 cells were the most thoroughly ex-amined as we analyzed samples sectioned serially andsemiserially for other studies (Sesso et al., 1999). In themodified PC-12* cells from the Butantan Institute, wefound mitochondria with a ruptured outer membrane inCUNS and also in apoptotic cells in the very first sam-ples deprived of serum. These observations will be pre-sented and discussed in a follow-up study.
WEHI-3 and K-562 were among the first cell culturesexamined and subjected to various apoptogenic sub-stances. We observed type 2 mitochondria in apoptoticWEHI-3 cells exposed to STS, novobiocin, VM 26, andvimblastin and in apoptotic K-562 cells treated with BFA,VM 26, and thapsigargin. All dead cells from culturessubjected to apoptogenic agents so far tested using mor-phometric studies possessed a cell membrane and nucleiwith the typical apoptotic phenotype.
It is common to observe mitochondria with sphericalprofiles along with the normal elongated mitochondria inCUNS. The incidence of these profiles was influenced bythe degree of cell death occurring in the cell culture. In theexperiments presented in Table 1, the percentage of cellsundergoing apoptosis varied from 6% to 66%. The higherthis apoptotic index, the more frequently one could findCUNS with spherical mitochondria, many of which exhib-
Fig. 2. A: Apoptotic PC-12* cell treated with BFA (2 �M for 16 hr).The compact mass of hypercondensed chromatin (1) appears detachedfrom the nuclear membrane (2), apparently leaving clear the regionwhere the nuclear lamina dwells (the small black dot at the extremity ofthe line marked 3 at the opposite end covers the region where thenuclear lamina reside, adherent to the nuclear membrane). Nuclearpores are indicated by arrows. Dilation of the endoplasmic reticulum (ER)cisternae, a common effect of BFA, is also shown (4). B: WEHI-164 cells
exposed to staurosporine (0.5 �M for 16 hr). Arrows indicate two apo-ptotic cells. Chromatin blocks can be visualized adhering to the nuclearmembrane (1). One of these blocks, in the cell on the left, has demiluneprofile (2). C: Apoptotic HeLa cell exposed to BFA (2 �M) plus stauro-sporine (0.5 �M), both for 16 hr. Peripheral disposition of the chromatinmasses in the nucleus (1) and dilation of the ER cisternae (2) can beobserved.
1343MITOCHONDRIAL PERMEABILITY TRANSITION
ited incipient swelling. From 1% to 17% of the CUNS inthe various samples possessed mitochondria undergoingthe permeability transition. In these cases, many type 2 or3 mitochondrial profiles could be found.
The successive morphological changes exhibited bythe apoptotic cells in the cultures we have examinedsuggest that apoptotic cells undergo caspase-orches-trated lysis of all membrane-bound and cytoskeletal
Fig. 3. A: An apoptotic cell from the secretory epithelium of theprostate gland of a rat castrated 4 days previously is seen in the centralpart of the micrograph. The nucleus is in the process of fragmentation(1). The cytoplasm of the region at right is devoid of organelles (2), someof which are well preserved and clustered at the opposite pole of theprofile (3). The arrow points to the region enlarged in the inset. B:Apoptotic plasma cell from the granulation tissue of a scar experimen-tally induced 5 days previously in the dorsal skin of an adult rat. Besides
the two mitochondria exhibiting permeability transition (indicated byarrows), a relatively large cluster of 50 nm microvesicles can beobserved (1) (Sesso et al., 1999). C: Apoptotic macrophage from thesame granulation tissue referred to in B. A sector of the nucleus can beobserved in the upper right corner (1). The type 2 mitochondrion in thelower central part (2) possesses a relatively small breach of the outermitochondrial membrane, indicated by an arrow.
cytoplasmic structures. We observed dense chromatinmasses, often with circular profiles, in apoptotic nucleiof cells in advanced stages of cytolysis. The cytoplasm ofthese cells was either almost completely devoid of or-ganelles or contained some dense or swollen type 2mitochondria as residual organelles. When caspase-driven proteolysis and the action of nucleases was ad-
vanced, it was often difficult to be precise about theformer morphology of the nucleus and cytoplasmic or-ganelles, all then appearing as remnants. Thesechanges occurred along with the complete rupture of thecell membrane. When this happened, either in an apo-ptotic cell or in very rare, perhaps necrotic, nonapop-totic ones, the cytoplasm was fragmented and the or-
Fig. 4. A: Nonapoptotic pancreatic acinar cell from a pancreas thathad undergone ligature of the excretory ducts 2 days earlier. This cellwas close to others that were undergoing generalized cell death. Notethe normal texture of the chromatin (1) and the type 2 mitochondria
indicated by an arrow. B: Apoptotic pancreatic acinar cell from the samegland referred to in A. Type 2 mitochondrion (indicated by the arrow) canbe visualized and, near the nucleus with compacted chromatin (1), acluster of 50 nm microvesicles (2) is noted.
1345MITOCHONDRIAL PERMEABILITY TRANSITION
ganelles exposed to the culture medium became swollenand disintegrated.
Two (PC-12* and WEHI-164 cells) of the four cell lines(the two others are HL-60 and HeLa cells) in which themitochondria were studied morphometrically were alsosubjected to various apoptogenic agents. In all eight sam-ples, type 2 mitochondria were found in apoptotic cells(Fig. 5) and in some of the cells with normal nuclei. Fre-quently, one observed type 2 mitochondrial profiles with arelatively small site of rupture at the outer membrane(lower type 2 profile in Fig. 3C and profile with an asteriskin Fig. 5A). In the apoptotic cells, type 2 and 3 mitochon-drial profiles may be present in the cytoplasm even inadvanced stages of cellular proteolysis. These cell profilesexhibited a few scattered chromatin blocks and scarcetype 2 and 3 mitochondrial profiles in a cytoplasm virtu-ally devoid of organelles. The main organelles in PC-12*cells in advanced stages of apoptosis when the cell was
being segmented into apoptotic bodies were mitochondrialprofile types 2 and 3.
The configuration of the type 2 mitochondrial profilesmay vary considerable with regard to the degree ofswelling of the mitochondrial matrix. In Figure 6A and6C, distinct differences in magnification are presentedto demonstrate that, in the smaller images (Fig. 6B and6D), it is not easy to perceive whether the regions indi-cated by arrows actually have one or two membranes. Inthese markedly swollen mitochondrial profiles (arrowsin Fig. 6B and D), two apposite poles are visible. Oneappears as a dense membrane with fragments of cristaenearby. Opposite to this pole, the mitochondrial matrixis limited by a thin covering membrane. The region ofthe profile with mitochondrial cristae and a dense mem-brane is where the profile exhibits both mitochondrialmembranes. At the opposite pole, the matrix is coveredonly by the inner membrane.
Fig. 5. A and B are from PC-12* CUNS cells treated with BFA (2 �M)and STS (0.5 �M) for 16 hr. The types of mitochondrial profiles are indicatedby numbers. In the upper left corner of A, a type 2 profile is sectioned atvarious levels orthogonally to the plane of the image. Planes AB and CDdemonstrate type 2 and 3 profiles, respectively; plane EF, which contains
no cristae remnants, presents a type 4 profile. The arrows indicate unimem-branous vesicles with homogeneous contents that do not resemble thecontents of the swollen part of profiles 2 that are devoid of cristae. These dono look like type 4 profiles. In A, an asterisk marks a type 3 profile with arelatively small breach of the outer membrane.
1346 SESSO ET AL.
Surface-to-Volume Ratio of Type 1 and 2Mitochondria in Apoptotic Cells and inCompanion Cells With Normal Nuclei
Measurements of the total surface area of the mitochon-dria carried out in the eight studied samples allowed us toobtain the mitochondrial surface-to-volume ratio in allapoptotic cells and CUNS examined (data not shown). Theratio between the mitochondria of apoptotic cells and inCUNS did not vary significantly (P 0.05), indicatingthat no important change in the average mitochondrialvolume between the two groups was actually detected.Therefore, in these two groups, the mitocondrial volumesare probably also a measure of the number of mitochon-dria per cell.
Evaluation of the volume, in �m3, and of the volumefraction Vv, occupied by mitochondria types 1 and 2 in thecytoplasm of apoptotic cells and in the correspondingCUNS of the same cell culture, is presented in Table 1.The data in the second line of Table 1 represent the bestestimates of the average volume per cell of types 1 and 2mitochondria in PC-12* apoptotic cells and CUNS. Themitochondria of the CUNS occupied 10% of the total cyto-plasmic area or volume (second line/second column) andhad an average global volume per cell of 28 �m3. In apo-ptotic cells, mitochondrial profile type 1 and the sum oftypes 2 and 3 occupied 2.3% and 3.8% (second line/col-umns 3 and 6, respectively) of the cytoplasm, with vol-umes of 6 and 10 �m3, respectively. In the PC12* apopto-
Fig. 6. Apoptotic HeLa and WEHI-164 cells, both exposed to STS(0.5 �M) for 16 hr. These images illustrate that at relatively low magni-fications, it is not possible to perceive that the dense parts of themitochondrial profiles close to the cristae and pointed by arrows in B
and D actually possess two mitochondrial membranes as shown in Aand C. The opposite pole of each enlarged profile is covered by a thininner mitochondrial membrane.
1347MITOCHONDRIAL PERMEABILITY TRANSITION
tic cells, the volume of type 3 mitochondria is of 6 �m3,representing 37.5% of the total mitochondrial volume ofthese apoptotic cells (6/16 � 100 � 37.5%); in this case, thevalue 16 �m3 (second line/column 7). The values of type 1mitochondrial profiles found in apoptotic cells (column 3)lied in the range of 14% (fifth line 3/22 � 100) to 53%(sixth line 60/113 � 100) of the total mitochondrial volumefrom these cells. In the various cell lineages analyzed, thevolumes associated with type 2 mitochondrial profiles var-ied from 25% to 77% of the estimated total mitochondrialvolume in apoptotic cells (column 4). For the apoptoticPC-12* cells (second line/column 4), this value is 4/16 �100 � 25%. The percentages of the mitochondrial volumeof the apoptotic cells occupied by the sum of mitochondrialprofiles types 2 and 3 are presented in column 6. In theapoptotic PC-12* cells of the second line, this percentage is10/16 � 100 � 63%. In the various samples, this param-eter varied from 47% (sixth line) to 89% (fourth line), withan average of 69%. This important information conveyedby the data of column 6 reveals that the majority of themitochondria of the apoptotic cells when captured underthe TEM are or were in the state of permeability transi-tion. It was common to observe some 1–5 type 2 and some2–6 type 3 mitochondrial profiles in different sections ofapoptotic PC-12* cells.
The total mitochondrial volume of the apoptotic cells (vin column 7) in six out of eight cases is lower than that ofthe CUNS (lines 1, 2, 5, 6, 7, and 8 and columns 2 and 7 ofTable 1), while in the other two samples the mitochondrialvolume in apoptotic cells and in CUNS are about equal. Inthe third line, the cited volume in apoptotic cells is 66�m3, while in the corresponding CUNS (column 2), it is 47�m3. If the 19 �m3 (see footnote to Table 1) of the CUNSwith type 2 mitochondria is included, the total amount isequal to that of the apoptotic cells. In the fourth line, bothvalues of v (columns 2 and 7) for the mitochondrial volumeare equal (38 �m3).
As already mentioned, the incidence of type 2 mitochon-dria in cells with normal nuclei was quite variable (itvaried from 1% to 17%) and they would not always beadequately sampled when 10 random (range, 10–20) sec-tions of cells with normal nuclei are taken for measure-ments. For this reason, with one exemption, presented asan example (line 3 and footnote), we have not includedsuch cells in the nonapoptotic group when estimating mi-tochondrial volumes. None of the mitochondrial volumesof the various columns of Table 1 correlate significantlywith the corresponding AIs. These varied from 6% to 68%.
Cytoplasmic Sectors Devoid of Organelles inApoptotic Cells and in CUNS
In many of the CUNS possessing a ruptured outer mi-tochondrial membrane, the cytoplasmic distribution of or-ganelles was altered. The membrane-bound structureswere concentrated in certain sectors of the cytoplasmwhile absent from others. This clustering of organelles ismore frequently found in apoptotic cells than in CUNS(Fig. 3A). This observation suggests that the cytoskeletonis undergoing alterations before the nuclear changes ap-pear (data not shown). A literature search reveals that incells induced to undergo programmed death, alterations ofthe distribution and morphology of the actin microfila-ments is an early apoptotic event.
DISCUSSIONWe decided to use transmission electron microscopy to
identify apoptotic cells as it seems to be the most reliableprocedure to accomplish this task (Yasuhara et al., 2003).Morphological data to distinguish between necrotic andapoptotic cells have long since been identified (Kerr et al.,1995). A literature search revealed that the TUNEL reac-tion gives high false positive rates while DNA ladder as-say lacks sensitivity (Yasuhara et al., 2003). The bindingof annexin V at the cell surface and positive staining withpropidium iodide occur in both apoptotic and necroticcells. In cultured cells, annexin V marks early stages ofapoptosis when the cell is still covered by a continuousmembrane that keeps the propidium iodide out of the cell.As apoptosis progresses, various successive portions ofthis membrane are removed, allowing the entrance ofpropidium iodide that stains the cell. This apoptotic cell ispositive for both annexin V and propidium iodide, as arenecrotic cells (Vermes et al., 1995).
The results presented in this article clearly demonstratethe high incidence of morphometric change in the mito-chondria of apoptotic cells. From our data, we calculatethat at least 47–89% (average 69%) of the mitochondriafrom these cells have a ruptured outer membrane. Oncethe outer mitochondrial membrane has ruptured, the in-ner membrane, covering an expanding swollen matrix,passes through the formed hole and spreads into the sur-rounding cytoplasm. Since the continuous expansion ofthe mitochondrial matrix needs to be membrane-bound inorder not to rupture, the membrane from the cristae areincorporated into the inner membrane.
It is difficult to evaluate the magnitude of the underes-timations of the actual volume of type 2 mitochondria. Wewould need to know what fraction of the measured type 1profiles derive from type 2 mitochondria and how theunimembranous vesicles derived from type 2 mitochon-dria, but devoid of cristae, the type 4 profiles are numer-ically related to type 3 profiles. It was also evident duringthe collection of morphometric data that the frequency ofthe four profile types varied according to the morphologi-cal changes of the forming and developing type 2 mito-chondria. The change in size of the unimembranous-boundpart of type 2 mitochondria will affect the yield of type 3and 4 profiles when the mitochondria are randomly sec-tioned.
It is curious that, besides the publications of Angermul-ler et al. (1998) and of Kwong et al. (1999) in apoptotichepatocytes and secretory epithelial cells from the pros-tate gland of castrated rats, respectively, no other reportsof rupture of the outer mitochondrial membrane in apo-ptotic cells have been published.
As mentioned, in six samples out of eight, the mitochon-drial volume of the apoptotic cells was less than that of theCUNS. Since we do not know the magnitude of underes-timation of the volume of type 2 mitochondria, we cannotbe sure of a possible reduction of this volume in apoptoticcells. It must be noted that these results may simplyrepresent the process of organelle disassembly in many ofthe cells analyzed. It is unclear whether the numericalreduction of mitochondria detected in cells with typicalapoptotic nuclei had begun before the changes in nuclearstructure.
One of the facts that must have determined the lack ofstatistical difference between the surface-to-volume ratio
of the mitochondria of the apoptotic cells and of the CUNSis that, in cultures subjected to apoptogenic agents, theCUNS often exhibit type 1 mitochondria with a sphericalshape (examples in Figs. 10–15; Figs. 36, 72, 73, and 75 ofthe site http://www.sebepa.cjb.net/). Not rarely a variablepart of these profiles, mainly in cultures with high AIs,may appear swollen. It is possible that these swollen mi-tochondria found in CUNS, namely, those exhibiting rup-tured outer membrane, are revealing that such cells are inline to start showing the nuclear signs of apoptosis.
It is as yet unclear what causes the rupture of the outermitochondrial membrane in CUNS. The involvement ofterminal caspases such as caspase 3 can be excluded dueto the absence of nuclear alterations; these alterations area consequence of the activation of this caspase. It is alsounclear whether caspase 3 may influence the appearanceof type 2 mitochondria once the nuclear alterations havebegun. One cannot rule out the possibility that in many ofthe observed cases an initiatory caspase, such as caspase8, activates the mitochondrial-dependent apoptotic path-way (Scaffidi et al., 1998, 1999) and is a causative agent ofthe rupture of the outer mitochondrial membrane inCUNS. In this hypothesis, the type 2 mitochondria inCUNS would be actively involved in causing the explicitstructural changes of apoptosis.
We suggest that type 2 mitochondria seems to be anindicator of the MPT since the morphology of these swol-len mitochondria could only be attained if the inner mito-chondrial membrane had lost its selective permeability.MPT has also been described in necrotic cells. However,we do not know whether rupture of the outer mitochon-drial membrane also occurs in necrotic cultured cells. Inrare identifiable necrotic cells found in our studies, thecytoplasm was fragmented and the organelles, mitochon-dria included, in the process of disintegration. An answerto this question may be obtained by performing TEMstudies in cells induced to undergo necrosis by an abruptand severe reduction in the cellular ATP levels (Kim et al.,2003).
To associate the TEM images of type 2 mitochondriaundergoing MPT with the concept of a permeability tran-sition pore or megachannel, it is necessary to envisagethat once the permeability transition pore is fully open, amassive influx of fluids would occur between both mito-chondrial membranes in the region of the pore, i.e., in arestricted sector of the outer membrane. We have micro-scopic data that will be presented in a follow-up study,supporting this assertion. As the punctual accumulationof liquid progresses, it would promote a small focal rup-ture of the outer membrane. The depolarization of theinner membrane would then rapidly spread from the ini-tial point where the pore was. In such a manner, themitochondrial matrix would accumulate fluid from thecytoplasm causing the swelling observed. The frequentfinding of type 2 mitochondrial profiles with relativelysmall breaches in the outer membrane (see also results onour Web site) supports this conjecture.
A clear-cut demonstration of rupture of outer mitochon-drial membrane was obtained in mitochondria isolatedfrom cortical neurons and induced to undergo permeabil-ity transition. The release of cytochrome c from the inter-membranous space appeared to be dependent of the rup-ture of the outer mitochondrial membrane (Brustovetskyet al., 2002). A second occurrence of ruptured outer mito-chondrial membrane in mitochondrial fractions induced to
undergo permeability transition may be observed with amagnifying lens in the right superior quadrant of Figure4B from Petronilli et al. (1993) directly on the journalpage. Three extremely swollen and unequivocal type 2mitochondrial profiles, joined two by two, may be seen.The connection between the mitochondrial configurationin apoptotic cells, and the results in isolated mitochondriaexpressing permeability transition, substantiates theview that the structurally altered mitochondria we areobserving in apoptotic cells and also in companion cul-tured cells with normal nuclei are actually expressing thestate of permeability transition.
Possibly in connection with the just cited observationsin isolated mitochondria, it is of relevance to mention thatthe low-power images of type 2 mitochondrial profilesshown in Figure 6B and 6D resemble transmission elec-tron micrographs, similarly imaged, of pelleted swollenmitochondrial induced to undergo MPT [Figs. 8B–D and9A, C, and D in Beatrice et al. (1982); Fig. 4B and C inIgbavboa and Pfeiffer (1988); Fig. 4B in Petronilli et al.(1993); Fig. 3B in Jung et al. (1997)]. Ours and thesemitochondrial profiles have in common a dense region inone pole of variable curved length. This dense region israpidly recognized by the presence of mitochondrial cris-tae close to or contacting its inner surface. In associationwith a variable amount of cristae, the swollen isolatedmitochondria exhibit a more or less empty matrix with avariable amount of cristae. These profiles opposite to theirdense region are covered by a thin membrane.
In order to clarify whether the type 2 mitochondria weobserve in CUNS and apoptotic cells actually release cy-tochrome c into the cytoplasm, we will carry out essays inwhich the time-course incidence of type 2 mitochondriawill be correlated to the amount of cytosolic cytochrome cin the cells under analysis. Parallel evaluation of theactivity of caspases 8, 9, and 3 will also be performed.
The following observations strongly suggest that therupture of the outer mitochondrial membrane we describeis the mechanism by which intermembrane mitochondrialproteins are released into the cytoplasm.
One, the morphology of the type 2 mitochondrial profilesreveals exposure of the external surface of the inner mem-brane and therefore the intermembrane and membranousmitochondrial proteins associated with this surface to thecytoplasm. This indicates that there may be consequencesto the apoptotic program if cytochrome c is exposed tocytoplasm containing the Apaf 1. If cytochrome c is indeedloosely attached to the inner membrane (Lemasters et al.,1998), it will be not only exposed, but actually progres-sively released into the cytoplasm as the inner membranetransverses the breached outer membrane.
Two, various agents can open the permeability transi-tion pore and promote loss of the inner mitochondrialmembrane selective permeability. The appearance of theMPT is accompanied by the release of intermembranemitochondrial proteins that trigger the activation of pro-caspases (Tafani et al., 2001; de Giorgi et al., 2002; Koko-szka et al., 2004).
Three, the simultaneous occurrence of MPT and alteredorganelle distribution in the cytoplasm of the CUNS isanother circumstantial element, compatible with the viewthat in these cells the apoptotic program was alreadybeginning to take place.
Four, of the drugs we have employed to induce apoptosis(listed in Table 1), staurosporine (Tafani et al., 2001), BFA
(Ito et al., 2001), TNF (Vikhanskaya et al., 2002a), andcamptothecin (Stefanis et al., 1999; Sanches-Alcazar etal., 2000) also promote the cytoplasmic release of cyto-chrome c.
ACKNOWLEDGMENTSThe authors thank Angela Batista Gomes dos Santos,
Maria Cecılia dos Santos L. Marcondes and Marcelo AlvesFerreira of the Department of Pathology of the Faculty ofMedicini of Sao Paulo, for their technical support andMiguel da Silva Passos Junior from the Department ofSurgery for doing the photographic work.
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