Cell Death General Features and Morphological Aspects WALTER MALORNI AND GIANFRANCO DONELLI Department of Ultrastructures Istituto Superiore di Sanitri Kale Regina Elena 299 00161 Rome, Italy
Intracellular and plasma membrane modifications have been widely recognized as crucial factors in cell injury and death.’.* Changes in nuclear morphology and organelle structure or specific phenomena at the cell surface level, surface smoothing or surface blebbing, are often considered markers of cell p a t h ~ l o g y . ~In- ~addition, these structural findings are intimately related to a cascade of biochemical and physiological events leading to changes in cellular homeostasis, to the loss of cell volume regulation, to some modifications of macromolecule synthesis, and finally to the loss of cell ~ i a b i l i t y . ~ . ~ Although single intracellular events have been extensively studied using different biochemical or molecular biology approaches, little is known about the complex sequence of structural modifications that ultimately lead to cell death.’ As a general rule, two different pathways of cell death have been recognized: necrosis and apoptosis, or programmed cell death.27’ Main indicators of necrosis are the random degradation of DNA and the blockage of synthetic functions. In contrast, apoptosis is considered an active process of suicide that can also be triggered exogenously and that displays a characteristic, regular DNA double-strand cleavage and active protein ~ynthesis.’,~.~.~ However, morphological markers of such processes are still controversial,1*2,y but the finding that the subcellular effects induced by physical, chemical, and biological agents, such as stress-induced ion deregulation or free radical damage,’@-I2 are often similar, provides some clue to the general mechanisms underlying the structural modifications that lead to cell death. This process of cell damage can also be induced experimentally in vitro by specific “injury tools” and can be evaluated by immunochemical and ultrastructural analyses. The aim of this study, therefore, is to evaluate: (1) the possible relations between cell structure and function, such as that between morphological and biochemical findings, ( 2 ) the possibility of detecting target structures or organelles for precise stress types such as cell membranes and cytoskeleton, (3) the structural differences and homologies between various injury models, such as surface smoothing and blebbing, (4) the possible overlapping effects between different types of cell death (necrotic and apoptotic), (5) the ability of a cell to recover from a specific injury, and ( 6 ) the “point of no return” and the consequent detection of the boundary between cell injury and cell death. Specifically, ultrastructural effects of physical agents (temperature change and radiation), chemical agents (quinones and polar solvents), and biological agents (toxins and cytokines), and their particular modes of action on cells are reported. 218
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MATERIALS AND METHODS Cell Culture. Different human cell lines from colon carcinoma (HT29), epidermoid carcinoma (A431), melanoma (M14), leukemia (K562, HL60), and mouse embryo fibroblasts (MEF) in primary culture and epidermoid or fibroblastoid nontumor cell lines (McCoy, HF19, IEC-6) were used. Briefly, cells were grown at 37°C in RPMI 1640 supplemented with 10% fetal calf serum (Flow), 1% nonessential amino acids, L-glutamine (5 mM), penicillin (100 U/ml), and streptomycin (100 Pg 1ml). Treatment. The stressing agents considered thus far are summarized in TABLE 1, where the respective experimental conditions for each agent are also indicated. For
TABLE1. Physical, Chemical, and Biological Agents Inducing Cell Injury and Death Stress Agent Physical Mild hyperthermia Heat shock Cold shock Ionizing radiation EMFs Chemical Menadione N-methyl formamide Lonidamine Cycloheximide Biological Gliotoxin C. dificile toxin A C. dificile toxin B Cytochalasin B TNF ICMC
Cell Line
Experimental Conditions
Ref.
M14, K562 M14, K562 McCoy, HF19 A431, K562 K562, IEC-6
1-3 h/42.5"C 0.5 hi45"C 3-6 h/O"C 125 Gy" 48 h/5 mTb
13 13 14 15 16
A431, K562, MF M14, A431, HT29 Ehrlich, MI4 A431. HL-60
1-2 h/200 mM 1-2 h/400 mM 24-48 h/0.4 mM 2-3 h/4 KM
17 18 19 20
A431, K562 IEC-6 A431, K562 A431, K562 A431, HL-60 K562, HL-60
3-6 1-2 1-2 1-2 3-6 1-2
21 22 6 23 24 25
h/3 pM hi(1.5 pg/ml) h/(0.15 pglml) h/50 pM h/50 U/ml h /25 :1
"Gray. bh4illiTesla. ABBREVIATIONS: EMFs = electromagnetic fields; ICMC = immune cell-mediated cytotoxicity; TNF = tumor necrosis factor.
detailed methods (e.g., toxin purification, radiation exposure, and preparation of effectors from the immune system), the references provided used the same experimental design. Fluorescence Microscopy. Cytoskeletal analysis: For actin, alpha-actinin, filamin, spectrin, tubulin, cytokeratin, and vimentin detection, cells grown on coverslips and substrate-detached cells collected from the supernatant were fixed with 3.7% formaldehyde in phosphate-buffered saline solution (PBS), pH 7.4, for 10 minutes at room temperature. After being washed in the same buffer, the cells were permeabilized with 0.5% Triton X-100 (Sigma) in phosphate buffer, pH 7.4, for 10 minutes at room temperature. For actin detection, cells were stained with fluoresceinphalloidin (1500) at 37°C for 30 minutes. For labeling of other cytoskeletal elements, monoclonal antibodies directed against alpha-actinin (1:200), tubulin (a mixture of
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monoclonal antibodies against alpha- and beta-tubulin 1:l at a working dilution of 1:200), cytokeratin (1:20), and vimentin (1:40) were used. After 30 minutes at 37"C, cells were washed and then incubated with a sheep anti-mouse IgG rhodamine- or fluorescein-linked whole antibody for 30 minutes at 37°C. For filamin detection, cells were incubated with goat polyclonal antifilamin antibodies (1:lOO) at 37°C for 30 minutes. After washing, the cells were incubated with rabbit anti-goat IgG fluoresceinlinked antibody for 30 minutes at 37°C. Cytoplasmicand membrane antigens: To detect surface antigens epidermal growth factor receptor (EGFr), transferrin receptor (TFr), and B2-microglobulin(a subunit of human leukocyte antigen, HLA), cells were incubated with monoclonal antibodies anti-EGF receptor and anti-TFr (1:lOO) anti anti-B2 microglobulin (1:20) polyclonal antibodies at 4°C for 30 minutes. After washing in phosphate buffer, cells were fixed with 3.7% formaldehyde in the same buffer for 30 minutes at 4°C and incubated with a sheep anti-mouse and a goat anti-rabbit IgG rhodamine-linked antibody, respectively, at 37°C for 30 minutes. Surface glycoproteins recognized by the fluoresceinlinked lectin from Triticum vulgaris (wheat germ agglutinin at a working dilution of 1:20) were labeled by direct fluorescence at 4°C as performed for cell surface proteins. Wheat germ agglutinin mainly recognizes glycoproteins carrying N-acetylglucosamine carbohydrates. Scanning Electron Microscopy. Control and treated cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at room temperature for 20 minutes. Following postfixation in 1% osmium tetroxide for 30 minutes, cells were dehydrated through graded ethanols, critical point dried in COz, and gold coated by sputtering. Samples were examined with a Philips 515 scanning electron microscope. TransmissionElectron Microscopy. For ultrathin sectioning, cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) postfixed with 1% osmium tetroxide in the same buffer for 1 hour, dehydrated through graded ethanols, and embedded in Agar 100 resin. Ultrathin sections were stained with uranyl acetate and lead citrate. For freeze-fracturing, cell suspensions were fixed with 2.5% glutaraldehyde immediately after treatment added directly to the medium. After fixation for 20 minutes, cells were centrifuged for 10 minutes at 1,000 rpm and then resuspended in 25% glycerol in Hanks' balanced salt solution. The suspensions were then centrifuged at 1,000 rpm for 10 minutes and the pellets were put on carriers and quickly frozen in Freon 22, partially solidified by cooling with liquid nitrogen. The mounted carriers were then transferred into a Balzers BAF 300 freeze-etch unit, cleaved at - 100°C at a pressure of 2 to 4 x 10 mbar, shadowed with 2.5 nm of Pt-C (at an angle of 457, and replicated with 20 nm of carbon. Cells were digested overnight by chlorox, and replicas were mounted on naked 300-mesh grids. Both sections and replicas were observed with a Zeiss EMlOC electron microscope.
RESULTS In vitro exposure to different physical agents (heat shock, mild hyperthermia, cold shock, ionizing radiation, and nonionizing radiation, that is, electromagnetic fields), chemicals (menadione, N-methylformamide, lonidamine, and cycloheximide), or biological agents (Clostridium dificile toxins A and B, cytochalasin B, gliotoxin, tumor necrosis factor alpha, and immune cell-mediated cytotoxicity) resulted in numerous morphological and ultrastructural modifications represented mainly by cell surface or intracellular modifications. Some examples are provided in FIGURES 1-6. In particular, general cell morphology analyzed by scanning electron microscopy
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FIGURE 1. Scanning electron microscopy of HL-60 treated cells. Morphological modifications induced by cycloheximide (A) are mainly represented by the loss of microvilli and cell “smoothing.” In contrast, the tumor necrosis factor exposure induces the blebbing phenomenon (B).
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FIGURE 2. Transmission electron microscopy of A431 epithelial cells treated with cycloheximide (A) and tumor necrosis factor (B).Nuclear foldings, chromatin condensation as well as organelle changes are observable after cycloheximide exposure (A). Surface blebbing is easily appreciable in the early stages of cells intoxicated with TNF (B).
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FIGURE 3. Transmission electron microscopy of A431 cells exposed to toxin B from Clostridium dificile (A) and N-methylforrnamide (B). No organelles are detectable in the potocytotic toxin-induced blebs (A). By contrast, mitochondria are present in zeiotic N-methylformamideinduced blebs (B).
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FIGURE 4. Fluorescence microscopy of K562 cells stained with fluorescein phalloidin. Actin microfilaments appear to be absent in menadione-induced blebs (B and the correspondent brightfield, A) and present in N-methylformamide- and gliotoxin-treated cells (C and D, respectively). In particular, polarization of F-actin is detectable after gliotoxin exposure (D).
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FIGURE 5. lmmunofluorescence microscopy of W62 (A and B) and A431 cells (C-F). Surface antigens are often detected over the plasma membrane of treated cells including the blebbing regions. Transferrin receptors as well as EGF receptors are observable after menadione (A and B) and gliotoxin (C and D) exposure. However, expression of some surface antigens can be compromised as for B2-microglobulin in A431 cells treated with menadione (E and F).
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FIGURE 6. Transmission electron microscopy. Freeze-fracture of K562 cells treated with menadione. The redistribution of intramembrane particles on both protoplasmic (A) and exoplasmic (B) fracture faces can be observed.
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displayed surface smoothing or surface blebbing as induced by cycloheximide (FIG. 1A) or tumor necrosis factor alpha (FIG. lB), respectively. Such changes were paralleled with transmission electron microscopic studies that showed several intracellular organelle modifications after cycloheximide exposure (FIG. 2A) as well as characteristic surface blebbing typical of apoptotic cell death after tumor necrosis factor alpha treatment (FIG.2B). Surface blebs can be of the potocytotic type (no organelles inside the bleb cytoplasm), as in toxin B-exposed cells (FIG.3A), or of the zeiotic type, as induced by N-methylformamide (FIG.3B). Actin filaments appeared to play a role in cell injury in general and in surface blebbing in particular. The filaments were modified by exposure to stressing agents, resulting in different patterns of rearrangement of actjn microfilaments detectable in cells exposed to menadione (FIG.4A and B), to N-methylformamide (FIG. 4C), or to gliotoxin (FIG. 4D). Surface proteins were also affected; they can be expressed over the entire cell surface, including the bleb regions, as for transferrin receptor or epidermal growth factor receptor if evaluated in menadione- or gliotoxin-exposed cells (FIG.5A and B and 5C and D, respectively), or they can be absent in the plasma membrane of the regions undergoing blebbing as demonstrated by the lack of B2-microglobulin in menadione-treated cells (FIG.5E and F). Finally, this rearrangement of membrane proteins can also be visualized by the freeze-fracture technique which, after menadione exposure, showed an intramembrane particle redistribution on both protoplasmic (PF in FIG.6A) and exoplasinic (EF in FIG.6B) fracture faces. Thus, the various morphological changes that occur after specific treatment examined by ultrastruc2, 3, and 4 where these tural methods are highly heterogeneous, as seen in TABLES variations are summarized.
DISCUSSION The in vitro studies reported here focus on the mechanisms underlying cell injury induced by numerous physical, chemical, or biological stressing agents and are carried out by morphological and ultrastructural techniques. Several subcellular parameters that have been identified can be used to evaluate the cytopathological changes that lead to cell death. Generally, the plasma membrane, or membranes in general, the cytoskeleton, the mitochondria, and the nucleus seem to be the most important morphological targets of exogenous agent^.^ Spontaneous cytopathological modifications, to some extent, seem to cause the same changes induced by these agents8 Therefore, some chemical or biological stresses can induce subcellular modifications closely resembling those observed in cell aging. Hence, general features can be found in cells undergoing death. These changes can easily be induced in numerous in witro cultured cells. This approach, which has its roots in histopathology and anatomy, was used following the methods previously employed by some investigators.2”28 Cell death generally can be a physiological process of cell number regulation in tissues or it can be the result of exogenous or endogenous injuries.27 However, intriguing hypotheses were recently suggested on the role of injured/dying cells in n e o p l a ~ i aand ~ ~ human immunodeficiency virus (HIV) infection.30 In fact, it was suggested that malignancy can be related to a defective apoptosis (or programmed cell death) in which some repair mechanisms to restore the cell cycle can trigger neoplastic p r ~ l i f e r a t i o n .Furthermore, ~~ recent insights into gene involvement in
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some cell death processes have been provided by some investigators. In humans, high levels of expression of a gene (bcl-2) have been associated with impaired programmed cell death, leading to inhibition of cell Thus, active and programmed cell death could provide an additional means of regulating cell number, and unlike simple degeneration (necrosis), it could also depend on some intracellular components that can be exogenously activated (as well as inactivated, e.g., bcl-2). Hence, aberrant cell survival due to inhibition of cell death can effectively contribute to oncogenesis. Conversely, diverse anticancer drugs may induce a mode of cell death characteristic of apoptosis. Thus, drug-induced tumor regression could result from a simple triggering in the commitment to programmed cell death.D2Finally, a role for cell death in HIV infection has also been hypothesized in which viral particles appear able to induce apoptotic cell death in CD4 antigen-bearing lymphocytes.30 This could partially contribute to the understanding of the pathogenesis of the disease.
TABLE 2. Main Features of Induced Cell Injury Stress Agent Physical Mild hyperthermia Heat shock Cold shock Ionizing radiation EMFs Chemical Cycloheximide Lmidamine Menadione N-methylformamide Biological Gliotoxin C. difficile toxin A C. dificile toxin B Cytochalasin B TNF ICMC
Recovery Ability
Bleb Type
F-Actin in Blebs
Cell Death MorpholoRy A N A A N
I P P
z
AIN N N N A A A N A AIN
ABBREVIATIONS: A = apoptosis; EMFs = electromagnetic fields; ICMC = immune cell-mediated cytotoxicity; N = necrosis; P = potocytotic; TNF = tumor necrosis factor; Z = zeiotic.
Recently, efforts have been made by several investigators to describe morphologically the cytopathological aspects of spontaneous or induced cell death in an attempt to confirm previous biochemical findings and to detect a series of useful “diagnostic” tools in cell biology studies2’ In our experience, the complex morphological and ultrastructural picture derived from the analysis of numerous cell-injury models still appears insufficient and inexhaustive, but it seems to answer, at least partially, some of the questions proposed. From a horizontal analysis of our in vitro models, the most important markers of injured/dying cells are represented by the plasma membrane and the cytoskeleton. Changes at the plasma membrane level can lead to modifications in the expression of specific surface antigens (e.g., receptors) or to a general rearrangement (e.g., clustering) of membrane proteins. These changes can be
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TABLE 3. Subcellular Modifications Associated with Induced Cell Injury Type of Change
Recovery -
Karyorrhexis Nuclear pyknosis Karyolysis Nuclear folding Nuclear segmentation Eu- + Ethero-chromatin Nucleolar changes ER and Golgi dilatation Lysosomal enzyme release Mitochondria1 injury Qtoskeletal changes Surface antigen changes Intramembrane Particle rearrangement Surface blebbing Surface smoothing
+ + + ++ + + + + +
Necrosis
+ + + Rare -
-
Apoptosis -
+ -
+ + +
+ +
Rare
+ +
Frequent Frequent Frequent
t
Rare Rare
Rare
-
+ +
visualized and evaluated by different light and electron microscopic approaches and can represent precise cytopathological injury markers for discrete, sublethal lesions that can be due to (or generate) ion deregulation, free radical formation, oxidative stress, depletion of protein thiols, disruption of intracellular calcium homeostatis, and so on.11,33-35 Structural analyses can also provide useful information on changes taking place in the cytoskeletal apparatus. The formation of surface protusions (potocytotic or zeiotic blebbing) does not necessarily bring about cell death36and seems to be under the control of the cytoskeleton via a stress-induced rearrangement of specific elements.374 The presence or absence of organelles in the bleb cytoplasm, distinguishing potocytosis from zeiosis, can be related to actin filament rearrangement.'* Potocytotic blebs appear as an early pathological feature often associated with apoptosis, whereas cells undergoing necrosis can develop both potocytotic and
TABLE 4. Antigens Detectable in the Bleb Plasma Membrane or the Bleb Cytoplasm after Exposure to Menadione, N-Methylformamide, and C. dificile Toxin B An tigens Actin Alpha-actinin Filamin Tubulin Vimen tin Keratin Bz-microglobulin TFr EGFr WGA
Menadione
+ + + +a
-
+ + -
N-Methylformamide
+ + + +/+ + + + t
Toxin B -
+ + tl-
+ + + +
nMicrotubules are dep~lymerized.'~ ABBREVIATIONS: EGFr = epidermal growth factor receptor; TFr = transferrin receptor; WGA = wheat germ agglutinin.
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zeiotic blebs. In addition, zeiosis can also represent a physiological process by which discrete regions of the cytoplasm can be extruded (a kind of exocytosis as in UV-exposed melanocytes), without compromising cell viability. An actin crosslinking can impair organelle movement through the cell cytoplasm (and inside the bleb cytoplasm in potocytosis) and can contribute to surface blebbing by a “mechanical constriction” of the cytoplasmic region underlying the plasma ~ n e m b r a n e . ~ ~ , ~ ~ Moreover, in potocytotic blebs, impairment of the link between actin and alphaactinin (or other membrane-associated actin-binding proteins) has been hypotheTABLE 5. Main Morphological and Ultrastructural Features of Different Stages of the Cell Death Process Stage
Plasma Membrane
I (injury)
Small blebs Cell volume increase
I1 (injury)
Blebs Loss of microvilli Altered IMP distribution
I11 (commitment)
Large multiple blebs Smoothing Heavy IMP clustering Surface antigen changes
IV (death)
Pores and membrane ruptures Loss of normal cell shape
Cytoplasm Mitochondiral matrix rarefaction Vesicle dilatation Minor cytoskeletal rearrangement (e.g., adhesion plaque changes) Mitochondria1 cristae distortion Cvoskeletal changes (e.g., actin filament cross-links) Large mitochondria1 damage with swelling Cytoskeletal rearrangement (e.g., large patches of filaments, depolymerization) Lysosomal enzyme release Evident mitachondrial swelling with disruption of cristae Cytoskeletal disassembly Cytoplasmic matrix rarefaction
Nucleus Normal
Nucleolar changes Nuclear foldings
Chromatin condensation and segregation Nuclear segmentation
Karyorrhexis Pyknosis Karyolysis
whereas a probable, reversible modification of the relations between the whole microfilament system and the remaining cytoskeleton seems to occur in the zeiotic process. Surface antigen expression (rearrangement or redistribution in the bleb regions) can also depend on cytoskeletal element rearrangement (leading to intramembrane particle clustering) and on a modification of the link between the antigen and the cytoskeletal elements. Furthermore, cytopathological features seem to confirm that apoptosis, unlike necrosis, can be considered an active process in which organelles (e.g., mitochondria) generally appear well preserved. Finally,
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nuclear changes are characteristic as well, as the electrondensity and localization of chromatin are morphologically different in the two types of cell death. In conclusion, the cascade of cytopathological modifications that lead to one precise cell injury pattern has still to be elucidated, and additional specific markers must be found.41However, the cytopathological changes described in this report can be considered representative of injured but not yet dead cells. Two points emerge from our studies: (1) the importance of establishing, at least from a cytopathological point of view, what is really a dying cell, and (2) the difficulties in pointing out specific morphological markers that distinguish necrosis from apoptosis. Concerning the determination of a dying cell, we think that different “stages” in the cell damage process have to be recognized for specific injuries. TABLE5 summarizes the structural findings obtained in our studies and in the literature on the general features of free radical-induced cytopathology. Four stages are proposed for the process which can lead to both types of cell death, necrosis and apoptosis. We believe only stages I1 and 111 are of interest for studies of cell injury and death. Concerning the distinguishing morphological features, only chromatin clumping seems to represent a specific morphological marker that allows us to differentiate apoptosis from necrosis, because other structural parameters (e.g., surface smoothing or blebbing) are observable in both cell death processes. The morphology as well as the physiology of cell death also displays discrepancies that have to be understood. For instance, the postulate that programmed cell death is an active process involving protein synthesis has to be better clarified. Paradoxically, the protein synthesis blocker cycloheximide has been demonstrated to induce apoptosis when administered alone32or to facilitate tumor necrosis factor-induced a p o p t o s i ~ .Thus, ~ ~ programmed cell death is not always associated with active synthesis, and the existence of a third cell death pathway could be hypothesized. This third pathway could display specific structural and physiological features such as potocytosis or other “passive” processes. Other investigators have already suggested the existence of a third pattern of cell death represented by type B dark cells.42 Alternatively, apoptosis and programmed cell death could be considered separate phenomena, programmed cell death being the unique active cell suicide process. In this case, what today is called apoptosis could become an alternative cell death pathway to necrosis, not involved in ischemic or toxic injury (as necrosis) but characteristic of cell aging and turnover in tissues. On these bases, both questions just raised remain partially unanswered. However, molecular, biochemical, and morphological investigations, if performed in parallel, may provide in the future new insights into this highly complex cellular mechanism.
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26. WYLLIE,A. H., J. F. R. KERR & A. R. CURRIE.1980. Cell death: The significance of apoptosis. Int. Rev. Cytol. 6 8 251-306. 27. POTTEN,C. S. (Ed.) 1987. Perspectives on Mammalian Cell Death. Oxford University Press. oxford. L. D. & F. 0. COPE.(Eds.) 1991. Molecular Basis of Cell Death. Cold Spring 28. TOMEI. Harbor Laboratory Press. New York. C. E., K. J . LESTER & L. D. TOMEI. 1985. Tumor promoters: An overview of 29. WENNER, membrane-associated alterations and intracellular events. I n Molecular Basis of Cancer. P. B. Farmer & J. M. Walker, eds. Vol. 175A: Macromolecular Structure, Carginogens, and Oncogenes. A. R. Liss. New York. A. & J. C. AMEISEN.1991. Cell dysfunction and depletion in AIDS: The 30. CAPRON, programmed cell death hypothesis. Immunol. Today 4 102-105. G. T. 1991. Programmed cell death: Apoptosis and oncogenesis. Cell 65: 109731. WILLIAMS, 1098. 32. DIVE,C. & J. A. HICKMAN.1991. Drug-target interaction: Only the first step in the commitment to a programmed cell death? Br. J. Cancer 6 4 192-196. H. THOR,P. NICOTERA& S. ORRENIUS. 1984. Menadione33. DI MONTE,D., G . BELLOMO, induced cytotoxicity is associated with protein thiol oxidation and alterations in intracellular calcium homeostasis. Arch. Biochem. Biophys. 235: 343-350. G. & S. ORRENIUS. 1985. Altered thiol and calcium homeostasis in oxidative 34. BELLOMO, hepatocellular injury. Hepatology 5: 876-882. W., F. Iosr, S. MESCHINI, S. PARADISI & G. DONELLI. 1991. Cytophatological 35. MALORNI, features of cell suffering and death: Role of plasma membrane and cytoskeleton. Cytotechnology 5: 67-70. D. & J. E. ERICKSSON. 1990. Morphological changes and cytotoxicity in isolated 36. TOIVOLA, hepatocytes. Scand. Cell Toxicol. 1: 181-186. G., F. MIRABELLI, P. RICHELMI, W. MALORNI, F. 10%& S. ORRENIUS. 1990.The 37. BELLOMO, cytoskeleton as a target in quinone toxicity. Free Rad. Res. Commun. 8 391-399. W., F. IOSI,F. MIRABELLI & G. BELLOMO. 1991. Cytoskeleton as a target in 38. MALORNI, menadione-induced oxidative stress in cultured mammalian cells: Alterations underlying surface bleb formation. Chem. Biol. Interact. 8 0 217-236. D. B., L. A. SKLAR,B. BOHE,I. SCHRAUFSTATTER, P. A. HYSLOP, M. W. Rossi, 39. HINSHAW, R. G. SPRAGG& C. G. COCHRANE. 1986. Cytoskeletal and morphologic impact of cellular oxidant injury. Am. J. Pathol. 123: 454464. J. J., J. D. GIUSEPPI, A. L. NIEMINEN & B. HERMAN. 1987. Blebbing, free 40. LEMASTERS, calcium and mitochondria1 membrane potential preceding cell death in hepatocytes. Nature 325: 78-80. M. 1991. Death by a thousand cuts. Current Biol. 3: 140-142. 41. COLLINS, N. I., B. V. HARMON, G . C. GOBE'& J. F. R. KERR.1988. Patterns of cell death. 42. WALKER, Methods Achiev. Exp. Pathol. 13: 18-54. '
.
I
Intracellular and plasma membrane modifications have been widely recognized as crucial factors in cell injury and death.’.* Changes in nuclear morphology and organelle structure or specific phenomena at the cell surface level, surface smoothing or surface blebbing, are often considered markers of cell p a t h ~ l o g y . ~In- ~addition, these structural findings are intimately related to a cascade of biochemical and physiological events leading to changes in cellular homeostasis, to the loss of cell volume regulation, to some modifications of macromolecule synthesis, and finally to the loss of cell ~ i a b i l i t y . ~ . ~ Although single intracellular events have been extensively studied using different biochemical or molecular biology approaches, little is known about the complex sequence of structural modifications that ultimately lead to cell death.’ As a general rule, two different pathways of cell death have been recognized: necrosis and apoptosis, or programmed cell death.27’ Main indicators of necrosis are the random degradation of DNA and the blockage of synthetic functions. In contrast, apoptosis is considered an active process of suicide that can also be triggered exogenously and that displays a characteristic, regular DNA double-strand cleavage and active protein ~ynthesis.’,~.~.~ However, morphological markers of such processes are still controversial,1*2,y but the finding that the subcellular effects induced by physical, chemical, and biological agents, such as stress-induced ion deregulation or free radical damage,’@-I2 are often similar, provides some clue to the general mechanisms underlying the structural modifications that lead to cell death. This process of cell damage can also be induced experimentally in vitro by specific “injury tools” and can be evaluated by immunochemical and ultrastructural analyses. The aim of this study, therefore, is to evaluate: (1) the possible relations between cell structure and function, such as that between morphological and biochemical findings, ( 2 ) the possibility of detecting target structures or organelles for precise stress types such as cell membranes and cytoskeleton, (3) the structural differences and homologies between various injury models, such as surface smoothing and blebbing, (4) the possible overlapping effects between different types of cell death (necrotic and apoptotic), (5) the ability of a cell to recover from a specific injury, and ( 6 ) the “point of no return” and the consequent detection of the boundary between cell injury and cell death. Specifically, ultrastructural effects of physical agents (temperature change and radiation), chemical agents (quinones and polar solvents), and biological agents (toxins and cytokines), and their particular modes of action on cells are reported. 218
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MATERIALS AND METHODS Cell Culture. Different human cell lines from colon carcinoma (HT29), epidermoid carcinoma (A431), melanoma (M14), leukemia (K562, HL60), and mouse embryo fibroblasts (MEF) in primary culture and epidermoid or fibroblastoid nontumor cell lines (McCoy, HF19, IEC-6) were used. Briefly, cells were grown at 37°C in RPMI 1640 supplemented with 10% fetal calf serum (Flow), 1% nonessential amino acids, L-glutamine (5 mM), penicillin (100 U/ml), and streptomycin (100 Pg 1ml). Treatment. The stressing agents considered thus far are summarized in TABLE 1, where the respective experimental conditions for each agent are also indicated. For
TABLE1. Physical, Chemical, and Biological Agents Inducing Cell Injury and Death Stress Agent Physical Mild hyperthermia Heat shock Cold shock Ionizing radiation EMFs Chemical Menadione N-methyl formamide Lonidamine Cycloheximide Biological Gliotoxin C. dificile toxin A C. dificile toxin B Cytochalasin B TNF ICMC
Cell Line
Experimental Conditions
Ref.
M14, K562 M14, K562 McCoy, HF19 A431, K562 K562, IEC-6
1-3 h/42.5"C 0.5 hi45"C 3-6 h/O"C 125 Gy" 48 h/5 mTb
13 13 14 15 16
A431, K562, MF M14, A431, HT29 Ehrlich, MI4 A431. HL-60
1-2 h/200 mM 1-2 h/400 mM 24-48 h/0.4 mM 2-3 h/4 KM
17 18 19 20
A431, K562 IEC-6 A431, K562 A431, K562 A431, HL-60 K562, HL-60
3-6 1-2 1-2 1-2 3-6 1-2
21 22 6 23 24 25
h/3 pM hi(1.5 pg/ml) h/(0.15 pglml) h/50 pM h/50 U/ml h /25 :1
"Gray. bh4illiTesla. ABBREVIATIONS: EMFs = electromagnetic fields; ICMC = immune cell-mediated cytotoxicity; TNF = tumor necrosis factor.
detailed methods (e.g., toxin purification, radiation exposure, and preparation of effectors from the immune system), the references provided used the same experimental design. Fluorescence Microscopy. Cytoskeletal analysis: For actin, alpha-actinin, filamin, spectrin, tubulin, cytokeratin, and vimentin detection, cells grown on coverslips and substrate-detached cells collected from the supernatant were fixed with 3.7% formaldehyde in phosphate-buffered saline solution (PBS), pH 7.4, for 10 minutes at room temperature. After being washed in the same buffer, the cells were permeabilized with 0.5% Triton X-100 (Sigma) in phosphate buffer, pH 7.4, for 10 minutes at room temperature. For actin detection, cells were stained with fluoresceinphalloidin (1500) at 37°C for 30 minutes. For labeling of other cytoskeletal elements, monoclonal antibodies directed against alpha-actinin (1:200), tubulin (a mixture of
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monoclonal antibodies against alpha- and beta-tubulin 1:l at a working dilution of 1:200), cytokeratin (1:20), and vimentin (1:40) were used. After 30 minutes at 37"C, cells were washed and then incubated with a sheep anti-mouse IgG rhodamine- or fluorescein-linked whole antibody for 30 minutes at 37°C. For filamin detection, cells were incubated with goat polyclonal antifilamin antibodies (1:lOO) at 37°C for 30 minutes. After washing, the cells were incubated with rabbit anti-goat IgG fluoresceinlinked antibody for 30 minutes at 37°C. Cytoplasmicand membrane antigens: To detect surface antigens epidermal growth factor receptor (EGFr), transferrin receptor (TFr), and B2-microglobulin(a subunit of human leukocyte antigen, HLA), cells were incubated with monoclonal antibodies anti-EGF receptor and anti-TFr (1:lOO) anti anti-B2 microglobulin (1:20) polyclonal antibodies at 4°C for 30 minutes. After washing in phosphate buffer, cells were fixed with 3.7% formaldehyde in the same buffer for 30 minutes at 4°C and incubated with a sheep anti-mouse and a goat anti-rabbit IgG rhodamine-linked antibody, respectively, at 37°C for 30 minutes. Surface glycoproteins recognized by the fluoresceinlinked lectin from Triticum vulgaris (wheat germ agglutinin at a working dilution of 1:20) were labeled by direct fluorescence at 4°C as performed for cell surface proteins. Wheat germ agglutinin mainly recognizes glycoproteins carrying N-acetylglucosamine carbohydrates. Scanning Electron Microscopy. Control and treated cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at room temperature for 20 minutes. Following postfixation in 1% osmium tetroxide for 30 minutes, cells were dehydrated through graded ethanols, critical point dried in COz, and gold coated by sputtering. Samples were examined with a Philips 515 scanning electron microscope. TransmissionElectron Microscopy. For ultrathin sectioning, cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) postfixed with 1% osmium tetroxide in the same buffer for 1 hour, dehydrated through graded ethanols, and embedded in Agar 100 resin. Ultrathin sections were stained with uranyl acetate and lead citrate. For freeze-fracturing, cell suspensions were fixed with 2.5% glutaraldehyde immediately after treatment added directly to the medium. After fixation for 20 minutes, cells were centrifuged for 10 minutes at 1,000 rpm and then resuspended in 25% glycerol in Hanks' balanced salt solution. The suspensions were then centrifuged at 1,000 rpm for 10 minutes and the pellets were put on carriers and quickly frozen in Freon 22, partially solidified by cooling with liquid nitrogen. The mounted carriers were then transferred into a Balzers BAF 300 freeze-etch unit, cleaved at - 100°C at a pressure of 2 to 4 x 10 mbar, shadowed with 2.5 nm of Pt-C (at an angle of 457, and replicated with 20 nm of carbon. Cells were digested overnight by chlorox, and replicas were mounted on naked 300-mesh grids. Both sections and replicas were observed with a Zeiss EMlOC electron microscope.
RESULTS In vitro exposure to different physical agents (heat shock, mild hyperthermia, cold shock, ionizing radiation, and nonionizing radiation, that is, electromagnetic fields), chemicals (menadione, N-methylformamide, lonidamine, and cycloheximide), or biological agents (Clostridium dificile toxins A and B, cytochalasin B, gliotoxin, tumor necrosis factor alpha, and immune cell-mediated cytotoxicity) resulted in numerous morphological and ultrastructural modifications represented mainly by cell surface or intracellular modifications. Some examples are provided in FIGURES 1-6. In particular, general cell morphology analyzed by scanning electron microscopy
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FIGURE 1. Scanning electron microscopy of HL-60 treated cells. Morphological modifications induced by cycloheximide (A) are mainly represented by the loss of microvilli and cell “smoothing.” In contrast, the tumor necrosis factor exposure induces the blebbing phenomenon (B).
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FIGURE 2. Transmission electron microscopy of A431 epithelial cells treated with cycloheximide (A) and tumor necrosis factor (B).Nuclear foldings, chromatin condensation as well as organelle changes are observable after cycloheximide exposure (A). Surface blebbing is easily appreciable in the early stages of cells intoxicated with TNF (B).
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FIGURE 3. Transmission electron microscopy of A431 cells exposed to toxin B from Clostridium dificile (A) and N-methylforrnamide (B). No organelles are detectable in the potocytotic toxin-induced blebs (A). By contrast, mitochondria are present in zeiotic N-methylformamideinduced blebs (B).
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FIGURE 4. Fluorescence microscopy of K562 cells stained with fluorescein phalloidin. Actin microfilaments appear to be absent in menadione-induced blebs (B and the correspondent brightfield, A) and present in N-methylformamide- and gliotoxin-treated cells (C and D, respectively). In particular, polarization of F-actin is detectable after gliotoxin exposure (D).
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FIGURE 5. lmmunofluorescence microscopy of W62 (A and B) and A431 cells (C-F). Surface antigens are often detected over the plasma membrane of treated cells including the blebbing regions. Transferrin receptors as well as EGF receptors are observable after menadione (A and B) and gliotoxin (C and D) exposure. However, expression of some surface antigens can be compromised as for B2-microglobulin in A431 cells treated with menadione (E and F).
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FIGURE 6. Transmission electron microscopy. Freeze-fracture of K562 cells treated with menadione. The redistribution of intramembrane particles on both protoplasmic (A) and exoplasmic (B) fracture faces can be observed.
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displayed surface smoothing or surface blebbing as induced by cycloheximide (FIG. 1A) or tumor necrosis factor alpha (FIG. lB), respectively. Such changes were paralleled with transmission electron microscopic studies that showed several intracellular organelle modifications after cycloheximide exposure (FIG. 2A) as well as characteristic surface blebbing typical of apoptotic cell death after tumor necrosis factor alpha treatment (FIG.2B). Surface blebs can be of the potocytotic type (no organelles inside the bleb cytoplasm), as in toxin B-exposed cells (FIG.3A), or of the zeiotic type, as induced by N-methylformamide (FIG.3B). Actin filaments appeared to play a role in cell injury in general and in surface blebbing in particular. The filaments were modified by exposure to stressing agents, resulting in different patterns of rearrangement of actjn microfilaments detectable in cells exposed to menadione (FIG.4A and B), to N-methylformamide (FIG. 4C), or to gliotoxin (FIG. 4D). Surface proteins were also affected; they can be expressed over the entire cell surface, including the bleb regions, as for transferrin receptor or epidermal growth factor receptor if evaluated in menadione- or gliotoxin-exposed cells (FIG.5A and B and 5C and D, respectively), or they can be absent in the plasma membrane of the regions undergoing blebbing as demonstrated by the lack of B2-microglobulin in menadione-treated cells (FIG.5E and F). Finally, this rearrangement of membrane proteins can also be visualized by the freeze-fracture technique which, after menadione exposure, showed an intramembrane particle redistribution on both protoplasmic (PF in FIG.6A) and exoplasinic (EF in FIG.6B) fracture faces. Thus, the various morphological changes that occur after specific treatment examined by ultrastruc2, 3, and 4 where these tural methods are highly heterogeneous, as seen in TABLES variations are summarized.
DISCUSSION The in vitro studies reported here focus on the mechanisms underlying cell injury induced by numerous physical, chemical, or biological stressing agents and are carried out by morphological and ultrastructural techniques. Several subcellular parameters that have been identified can be used to evaluate the cytopathological changes that lead to cell death. Generally, the plasma membrane, or membranes in general, the cytoskeleton, the mitochondria, and the nucleus seem to be the most important morphological targets of exogenous agent^.^ Spontaneous cytopathological modifications, to some extent, seem to cause the same changes induced by these agents8 Therefore, some chemical or biological stresses can induce subcellular modifications closely resembling those observed in cell aging. Hence, general features can be found in cells undergoing death. These changes can easily be induced in numerous in witro cultured cells. This approach, which has its roots in histopathology and anatomy, was used following the methods previously employed by some investigators.2”28 Cell death generally can be a physiological process of cell number regulation in tissues or it can be the result of exogenous or endogenous injuries.27 However, intriguing hypotheses were recently suggested on the role of injured/dying cells in n e o p l a ~ i aand ~ ~ human immunodeficiency virus (HIV) infection.30 In fact, it was suggested that malignancy can be related to a defective apoptosis (or programmed cell death) in which some repair mechanisms to restore the cell cycle can trigger neoplastic p r ~ l i f e r a t i o n .Furthermore, ~~ recent insights into gene involvement in
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some cell death processes have been provided by some investigators. In humans, high levels of expression of a gene (bcl-2) have been associated with impaired programmed cell death, leading to inhibition of cell Thus, active and programmed cell death could provide an additional means of regulating cell number, and unlike simple degeneration (necrosis), it could also depend on some intracellular components that can be exogenously activated (as well as inactivated, e.g., bcl-2). Hence, aberrant cell survival due to inhibition of cell death can effectively contribute to oncogenesis. Conversely, diverse anticancer drugs may induce a mode of cell death characteristic of apoptosis. Thus, drug-induced tumor regression could result from a simple triggering in the commitment to programmed cell death.D2Finally, a role for cell death in HIV infection has also been hypothesized in which viral particles appear able to induce apoptotic cell death in CD4 antigen-bearing lymphocytes.30 This could partially contribute to the understanding of the pathogenesis of the disease.
TABLE 2. Main Features of Induced Cell Injury Stress Agent Physical Mild hyperthermia Heat shock Cold shock Ionizing radiation EMFs Chemical Cycloheximide Lmidamine Menadione N-methylformamide Biological Gliotoxin C. difficile toxin A C. dificile toxin B Cytochalasin B TNF ICMC
Recovery Ability
Bleb Type
F-Actin in Blebs
Cell Death MorpholoRy A N A A N
I P P
z
AIN N N N A A A N A AIN
ABBREVIATIONS: A = apoptosis; EMFs = electromagnetic fields; ICMC = immune cell-mediated cytotoxicity; N = necrosis; P = potocytotic; TNF = tumor necrosis factor; Z = zeiotic.
Recently, efforts have been made by several investigators to describe morphologically the cytopathological aspects of spontaneous or induced cell death in an attempt to confirm previous biochemical findings and to detect a series of useful “diagnostic” tools in cell biology studies2’ In our experience, the complex morphological and ultrastructural picture derived from the analysis of numerous cell-injury models still appears insufficient and inexhaustive, but it seems to answer, at least partially, some of the questions proposed. From a horizontal analysis of our in vitro models, the most important markers of injured/dying cells are represented by the plasma membrane and the cytoskeleton. Changes at the plasma membrane level can lead to modifications in the expression of specific surface antigens (e.g., receptors) or to a general rearrangement (e.g., clustering) of membrane proteins. These changes can be
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TABLE 3. Subcellular Modifications Associated with Induced Cell Injury Type of Change
Recovery -
Karyorrhexis Nuclear pyknosis Karyolysis Nuclear folding Nuclear segmentation Eu- + Ethero-chromatin Nucleolar changes ER and Golgi dilatation Lysosomal enzyme release Mitochondria1 injury Qtoskeletal changes Surface antigen changes Intramembrane Particle rearrangement Surface blebbing Surface smoothing
+ + + ++ + + + + +
Necrosis
+ + + Rare -
-
Apoptosis -
+ -
+ + +
+ +
Rare
+ +
Frequent Frequent Frequent
t
Rare Rare
Rare
-
+ +
visualized and evaluated by different light and electron microscopic approaches and can represent precise cytopathological injury markers for discrete, sublethal lesions that can be due to (or generate) ion deregulation, free radical formation, oxidative stress, depletion of protein thiols, disruption of intracellular calcium homeostatis, and so on.11,33-35 Structural analyses can also provide useful information on changes taking place in the cytoskeletal apparatus. The formation of surface protusions (potocytotic or zeiotic blebbing) does not necessarily bring about cell death36and seems to be under the control of the cytoskeleton via a stress-induced rearrangement of specific elements.374 The presence or absence of organelles in the bleb cytoplasm, distinguishing potocytosis from zeiosis, can be related to actin filament rearrangement.'* Potocytotic blebs appear as an early pathological feature often associated with apoptosis, whereas cells undergoing necrosis can develop both potocytotic and
TABLE 4. Antigens Detectable in the Bleb Plasma Membrane or the Bleb Cytoplasm after Exposure to Menadione, N-Methylformamide, and C. dificile Toxin B An tigens Actin Alpha-actinin Filamin Tubulin Vimen tin Keratin Bz-microglobulin TFr EGFr WGA
Menadione
+ + + +a
-
+ + -
N-Methylformamide
+ + + +/+ + + + t
Toxin B -
+ + tl-
+ + + +
nMicrotubules are dep~lymerized.'~ ABBREVIATIONS: EGFr = epidermal growth factor receptor; TFr = transferrin receptor; WGA = wheat germ agglutinin.
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zeiotic blebs. In addition, zeiosis can also represent a physiological process by which discrete regions of the cytoplasm can be extruded (a kind of exocytosis as in UV-exposed melanocytes), without compromising cell viability. An actin crosslinking can impair organelle movement through the cell cytoplasm (and inside the bleb cytoplasm in potocytosis) and can contribute to surface blebbing by a “mechanical constriction” of the cytoplasmic region underlying the plasma ~ n e m b r a n e . ~ ~ , ~ ~ Moreover, in potocytotic blebs, impairment of the link between actin and alphaactinin (or other membrane-associated actin-binding proteins) has been hypotheTABLE 5. Main Morphological and Ultrastructural Features of Different Stages of the Cell Death Process Stage
Plasma Membrane
I (injury)
Small blebs Cell volume increase
I1 (injury)
Blebs Loss of microvilli Altered IMP distribution
I11 (commitment)
Large multiple blebs Smoothing Heavy IMP clustering Surface antigen changes
IV (death)
Pores and membrane ruptures Loss of normal cell shape
Cytoplasm Mitochondiral matrix rarefaction Vesicle dilatation Minor cytoskeletal rearrangement (e.g., adhesion plaque changes) Mitochondria1 cristae distortion Cvoskeletal changes (e.g., actin filament cross-links) Large mitochondria1 damage with swelling Cytoskeletal rearrangement (e.g., large patches of filaments, depolymerization) Lysosomal enzyme release Evident mitachondrial swelling with disruption of cristae Cytoskeletal disassembly Cytoplasmic matrix rarefaction
Nucleus Normal
Nucleolar changes Nuclear foldings
Chromatin condensation and segregation Nuclear segmentation
Karyorrhexis Pyknosis Karyolysis
whereas a probable, reversible modification of the relations between the whole microfilament system and the remaining cytoskeleton seems to occur in the zeiotic process. Surface antigen expression (rearrangement or redistribution in the bleb regions) can also depend on cytoskeletal element rearrangement (leading to intramembrane particle clustering) and on a modification of the link between the antigen and the cytoskeletal elements. Furthermore, cytopathological features seem to confirm that apoptosis, unlike necrosis, can be considered an active process in which organelles (e.g., mitochondria) generally appear well preserved. Finally,
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nuclear changes are characteristic as well, as the electrondensity and localization of chromatin are morphologically different in the two types of cell death. In conclusion, the cascade of cytopathological modifications that lead to one precise cell injury pattern has still to be elucidated, and additional specific markers must be found.41However, the cytopathological changes described in this report can be considered representative of injured but not yet dead cells. Two points emerge from our studies: (1) the importance of establishing, at least from a cytopathological point of view, what is really a dying cell, and (2) the difficulties in pointing out specific morphological markers that distinguish necrosis from apoptosis. Concerning the determination of a dying cell, we think that different “stages” in the cell damage process have to be recognized for specific injuries. TABLE5 summarizes the structural findings obtained in our studies and in the literature on the general features of free radical-induced cytopathology. Four stages are proposed for the process which can lead to both types of cell death, necrosis and apoptosis. We believe only stages I1 and 111 are of interest for studies of cell injury and death. Concerning the distinguishing morphological features, only chromatin clumping seems to represent a specific morphological marker that allows us to differentiate apoptosis from necrosis, because other structural parameters (e.g., surface smoothing or blebbing) are observable in both cell death processes. The morphology as well as the physiology of cell death also displays discrepancies that have to be understood. For instance, the postulate that programmed cell death is an active process involving protein synthesis has to be better clarified. Paradoxically, the protein synthesis blocker cycloheximide has been demonstrated to induce apoptosis when administered alone32or to facilitate tumor necrosis factor-induced a p o p t o s i ~ .Thus, ~ ~ programmed cell death is not always associated with active synthesis, and the existence of a third cell death pathway could be hypothesized. This third pathway could display specific structural and physiological features such as potocytosis or other “passive” processes. Other investigators have already suggested the existence of a third pattern of cell death represented by type B dark cells.42 Alternatively, apoptosis and programmed cell death could be considered separate phenomena, programmed cell death being the unique active cell suicide process. In this case, what today is called apoptosis could become an alternative cell death pathway to necrosis, not involved in ischemic or toxic injury (as necrosis) but characteristic of cell aging and turnover in tissues. On these bases, both questions just raised remain partially unanswered. However, molecular, biochemical, and morphological investigations, if performed in parallel, may provide in the future new insights into this highly complex cellular mechanism.
REFERENCES
1. SATO,C. & S. YONEI.1987. Membrane changes. In Perspectives on Mammalian Cell Death. C. S. Potten, ed.: 1-17. Oxford University Press, Oxford. A. R., D. J. FAWTHROP & D. S. DAVIES. 1989. Mechanisms of cell death. TIPS 2. BOOBIS, 10 275-280. 3. THANK, D. W., W. EN-SHINN & W. W. WEBB.1982. Enhanced molecular diffusibility in muscle membrane blebs: Release of lateral constraints. J. Cell Biol. 9 8 207-212. 4. BORRELLI, M. J., R. L. L. WONG& W. C. DEWEY.1986. A direct correlation between hyperthermia-induced membrane blebbing and survival in synchronous G1 CHO cells. J. Cell Physiol. 126: 181-190. 5. BIGNOLD, L. P. & A. FERRANTE. 1988. Effects of cytochalasin B, N-formyl peptide and plasma on polarization, zeiosis (blebbing) and degranulation of polymorphonuclear leukocytes in suspension. Cell Biol. Int. Rep. 12 195-203.
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