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Ultrastructure of cells undergoing apoptosis
VITAMINS
Ultrastructure
AND HORMONES,
VOL. 58
of Cells Undergoing
Apoptosis
KENJI KIMURA,* HIRONOBU SASANO,” TOORU SHIMOSEGAWA,? SHIZUE MOCHIZUKI,” HIROSHI NAGURA,” AND TAKAYOSHI TOYOTA? “Department of Pathology and i-Department of Medicine, Medicine, 2-l Seiryou-machi, Aoba-ku, Sendai-shi,
I. Overview of Apoptosis II. Ultrastructural Features of Cells III. Correlation between Ultrastructural Undergoing Apoptosis IV. Conclusion References
Undergoing Apoptosis and Biochemical
I. OVERVIEW
Tohoku University School Miyagi-ken, Japan
Features
of
of Cells
OF APOPTOSIS
Analysis of cell turnover, the balance between cell proliferation and cell death, can provide important information about our understanding of the biological characteristics of various tissues and their functions. Apoptosis or programmed cell death is a selective process of physiologic cell deletion (Kerr et al., 1972; Wyllie et al., 1980; Russel, 1981; Wyllie, 1981; Duvall and Wyllie, 1986; Smith et al., 1989). The process of apoptosis plays a critical role in development and/or morphogenesis, in determining the size and shape of developing tissues, in maintenance of dynamic steady state in turnover of certain cell lineages in normal tissues, and in defining the number of tumor cell populations in the course of neoplastic development. Apoptosis is also important in various pathologic conditions, such as the response to irradiation (Yamada and Ohyama, 1998) stimulation by glucocorticoid (Wyllie,1980), withdrawal of glucocorticoid (Kyprianou and Isaacs, 19881, cytotoxic damages to gastric epithelial cells following Helicobacter pylori infection (Yabuki et al., 1997), and changes in some kinds of growth hormones (Solviter et al., 1993; Wyllie, 1993). Apoptosis is generally considered to be a form of cell death that occurs as a result of intentional suicide based on a genetic mechanism (Majno and Joris, 1995; Sasano, 1995; Kimura et al., 1997). Apoptosis is also a form of cell death characterized by morphologic as well as biochemical criteria and can be considered as a counterpart of mitosis (Majno and Joris, 1995). Apoptosis is characterized morphologically by 257
Copyright 0 2000 by Academic Press. All rights of reproduction in any form reserved. 0083-67!29/00 $30.00
258
KENJIKIMURAetal.
shrinkage of the cells resulting in cytoplasm condensation and the fragmentation of the cell nucleus, and membrane blebbing. Apoptosis is also defined biochemically by increased endogenous endonuclease activity, chromatin cleavage, and DNA fragmentation, that is, the DNA is broken down into segments that are multiples of approximately 185 base pairs, due to specific cleavage between nucleosomes (Kerr et al., 1987). In contrast, accidental cell death or oncosis or necrosis, which represents all cell death by murders, is generally defined as cell death caused by various cytotoxic agents or a pathologic environment, and is morphologically characterized by cytoplasmic swelling without the nuclear changes just described (Majno and Joris, 1995). It is also important to note that the whole process of apoptosis or programmed cell death is under genetic control and can be initiated by an internal clock or by extracellular agents. In addition, the duration of the slow process of apoptosis can be very fast, even in minutes (Majno and Joris, 1995). Apoptosis has been demonstrated to be induced by glucocorticoids, ceramide, wild-type ~53, various monoclonal antibodies against cell-surface antigens including Apo-1 or Fas, and others to be inhibited by bcl-2 protein encoded by the protooncogene bcl-2 (Majno and Joris, 1995). Biochemical detection of DNA cleavage into oligonucleosomal fragments of multiples of 180-200 base pairs, or the ladder pattern of nucleosomal DNA fragments on agarose gel electrophoresis, has been generally recognized as the hallmarks of apoptosis (Wyllie, 1980; Wyllie et al., 1984; Majno and Joris, 1995). However, as expected, this method can by no means identify the individual cells that are undergoing apoptosis. In addition, it has been recently demonstrated that these ladder patterns are not necessarily considered as specific for the process of apoptosis (Rink et al., 1995). On the other hand, the concept of “apoptosis” itself, which Kerr and Wyllie originally proposed was based on morphologic and ultrastructural examination (Kerr et al., 1972; Wyllie et al., 1980; Wyllie, 1981). It is therefore very important for those involved in the study of apoptosis to obtain sufficient knowledge of ultrastructural features of the cells undergoing apoptosis and incorporate morphologic observation or examination, especially that using electron microscopy into the study of apoptosis in his or her laboratory. These features are discussed in the following sections.
II. ULTRASTRUCTURALFEATURESOFCELLSUNDERGOINGA~OPTOSIS Kerr implied in 1971, shrinkage necrosis or apoptosis as a distinct mode of cellular death. The major morphologic feature of the cells un-
ULTRASTRUCTURE
OF CELLS
UNDERGOING
APOF’TOSI’S
259
FIG. 1. Ultrastructures of acinar cells in the pancreas of adrenalectomized rats treated with cerulein. Male Wistar strain rats weighing approximately 250 g underwent bilateral adrenalectomy and received 5 kg/kg per hour cerulein, intravenously, for 6 h. The cell shrinks and nuclei condense. The cytoplasm becomes dense but various intracellular organellae including mitochondria and endoplasmic reticula are still discernible. Bar = 1 urn.
dergoing apoptosis is the shrinkage of the cells. The cell becomes small in size and the cytoplasm becomes dense but various cell organellae including mitochondria are relatively normal (Fig. 1). Progression of the cytoplasmic condensation then results in marked crowding of organellae, which characteristically retain their integrity. However, the most characteristic ultrastructural feature of the cells undergoing apoptosis is chromatin condensation. The chromatin becomes very pyknotic, aggregating in the periphery of the nucleus to form dense smooth masses applied against the nuclear membrane (Fig. 2). These well-delimited dense masses of various shapes and sizes have been morphologically described as half-moon-, horseshoe-, lancet-, and navicular-shaped nuclei. The nucleus itself sometimes breaks up, resulting in one or two fragments of these dense smooth intranuclear masses (Fig. 2). These nuclear changes may be discernible in light microscopic examination but the need for ultrastructural confirmation of light microscopic identification of dying cells is required. The other ultrastructural features detected in the cells undergoing
260
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KIMUFtA
et al.
FIG. 2. Ultrastructures of acinar cells in the pancreas of adrenalectomized rats treated with cerulein. The arrow indicates apoptotic acinar cells. In the nucleus, condensed chromatin aggregates close to the nuclear membrane and demonstrates an appearance of crescent-like clumping (arrow). The nucleus also breaks up and forms relatively dense smooth intranuclear masses (double arrows). Zymogen granules are preserved in these cells. The polarity of zymogen granules in adjacent nonapoptotic acinal cells is relatively preserved. Bar = 1 km.
apoptosis is formation of cytoplasmic blebs and so-called “apoptotic bodies.” These processes emitted by the cells often contain pyknotic nuclear fragments (the budding phenomenon). These apoptotic bodies also contain cytoplasms and densely packed cell organellae. Sometimes, these apoptotic bodies may contain only cytoplasmic elements. These apoptotic bodies may be phagocytized by adjacent macrophages or parenchyma1 cells. Therefore, the cells undergoing apoptosis can be deleted from the tissue through these processes and the adjacent cells may migrate or proliferate to replace the space occupied by the deleted apoptotic cells. In contrast to these ultrastructural features of the cells undergoing apoptosis or programmed cell death, the cells undergoing the process of oncosis or accidental cell death may demonstrate cellular swelling, organellae swelling, and blebbing not accompanied by the nuclear changes described earlier (Majno and Joris, 1995).
ULTRASTRUCTUREOFCELLSUNDERGOINGAPOPTOSIS
III.
261
CORRELATIONBETWEENULTRASTRUCTURALANDBIOCHEMICAL FEATURESOFTHECELLSUNDERGOINGAPOPTOSIS
When examining the presence or absence of the cells undergoing apoptosis in the tissue specimen or cultured cells, it is very important to study these tissues and cells using election microscopy in order to examine whether ultrastructural features described earlier are detected or not. However, it is also true that these ultrastructural features of the cells undergoing apoptosis or programmed cell death are observed only for a limited period in the course of apoptosis (Arends et al., 1990; Ansari et al., 1993; Gorczyca et al., 1993; Hockenbery, 1995). In particular, these ultrastructural features may not be discernible in the early stages of cells undergoing apoptosis. In addition, examination by electron microscopy is not necessarily practical when screening for the presence or absence of apoptosis in tissue specimens or cell lines due to the labor-intensive and time-consuming nature of the technique. Apoptosis has been recognized as an essential component of tissue dynamics for many years. However, the relative difficulty in assessing apoptotic cells has prevented its study in both normal and pathologic processes (Ansari et al., 1993). Consequently, the development of other in situ methods to study apoptosis has been desired. In 1992, Gavrieli et al. demonstrated that DNAfragmentation can be detected in situ by labeling 3’-OH ends with biotinylated deoxyuridine triphosphate (dUTP) through the action of terminal deoxynucleotidyl transferase (TdT). Since then, this method, subsequently termed 3’-OH nick end labeling, or TdT-mediated dUTP-biotin nick end labeling (TUNEL), has been widely used to detect cells with DNA fragmentation, especially in surgical pathology materials of human disorders. The relatively good correlation of results obtained by the TUNEL method and DNA ladder formation by agarose gel electrophoresis has been demonstrated (Ansari et al., 1995; Abe et al., 1995). The TUNEL method has been widely applied to the analysis of apoptosis in a wide variety of materials including tissue specimens and cell lines (Hiraishi et al., 1993; Sasano et al., 1995; Kimura et al., 1998). With the introduction of various commercially available staining kits, the TUNEL method can now be easily and rapidly performed in properly fixed and processed tissue materials. The TUNEL method is now being incorporated into many pathology laboratories throughout the world. However, increasing evidence suggests that the TUNEL method is by no means specific for the detection of apoptosis (Ansari et al., 1995) and this method may also detect cells that are committed to, but not yet in the process of, apoptosis (Wyllie et al., 1984; Ansari et al., 1995). Therefore it becomes
262
KENJI
KIMURAet
al.
very important to examine the ultrastructural or morphologic features of TUNEL-positive cells. We have recently examined ultrastructural features of TUNELpositive cells in continuous mirror image semithin and ultrathin sections of Epon-Araldite-embedded tissues of pancreas from bilaterally adrenalectomized rats with cerulein-induced pancreatitis (Kimura et al., 1997). Application of this method through continuous mirror sections can directly demonstrate ultrastructural features of the TUNELpositive cells, although the sensitivity of the TUNEL method may be lower in Epon-Araldite-embedded tissue sections than in regular 10% formalin or 8% paraformaldehyde fixed and paraffin-embedded tissue sections possibly due to the interruption of the binding of dUTP to nick by Epon-Araldite plastic and osmium. Results of this study revealed that all ultrastructurally discernible apoptotic cells were positive by the TUNEL method (Fig. 3), but de-
FIG. 3. iA) A TUNEL-positive nucleus of an acinar cell in an Epon-Araldite semithin section. The arrow indicates an intensely labeled acinar cell nucleus. The other nuclei are stained by hematoxylin (X 300). (B) Ultrastructural features of the same acinus in a serial adjacent ultrathin section. The arrow indicates condensed chromatin with creascentic clumping. Bar = 2 km. (C)At higher magnification, mitochondria and zymogen granules are observed to be almost intact. Rough endoplasmic reticulum revealed a vesicular pattern. Bar = 2 pm.
ULTRASTRUCTURE
OF CELLS
FIG.
UNDERGOING
3. Continued
APOPTOSIS
263
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et al.
generated nuclei that may represent the end stage of cell death, either programmed or accidental, are also strongly labeled by the TUNEL method. Ansari et al. (1993) suggested that the TUNEL method may identify not only the apoptotic nuclei, but also the nuclei in necrotic tissues; the staining of necrotic cells could be explained either by the activation of an apoptotic pathway in damaged cells by the degradation of DNA by lysozomal enzymes. However, these two patterns can be distinguished by the distribution of TUNEL-positive nuclei; apoptotic nuclei are usually scattered, without localized aggregation. Several investigators have proposed the possibility that cells which have not yet begun the process of apoptosis and which lack its morphologic features can also be detected by the TUNEL method (Gorczyca et al., 1993; Migheli et al., 1995), which may therefore be able to detect apoptotic cells much earlier than electron microscopy. In our study employing continuous mirror image semithin and ultrathin sections, however, nearly all TUNEL-positive cells exhibited the characteristic ultrastructural features of apoptosis. Migheli et al. (1995) also applied the TUNEL method at the electron microscopic level, using an immunogold staining technique in embryonic mouse dorsal root ganglia. They reported that nuclear localization of immunogold was observed not only in typical apoptotic cells at various stages of cell death, but also in some apparently normal cells, which were structurally indistinguishable from adjacent unstained cells. Morphologically viable TUNEL-positive cells, including the “pre-apoptotic” cells (Migheli et aZ., 1995) may represent experimentally induced artefactual DNAdamage, leading to TUNEL positivity. The possibility that the relatively rapid process of apoptosis in pancreatic acinar cells in a cerulein-induced pancreatitis model made it difficult to detect preapoptotic cells with TUNEL cannot be completely ruled out. However, it is also true that ultrastructural examination of TUNEL-positive cells in continuous mirror image semithin and ultrathin sections of EponAraldite-embedded tissue sections employed in our study Wimura et al., 1997) has the following technical advantages over the immunogold technique reported by Migheli et al. (1995): (1) Much more ultrastructural detail can be obtained than in a single sections, (2) TUNEL-positive cells can be identified in a much wider area by light microscopy, and (3) TUNEL-positive cells are much more clearly visualized due to the calorimetric reaction in contrast to relatively small dots in the immunogold electron microscopy method. Despite these minor differences, results of our study and Migheli’s clearly demonstrated that the ultrastructural features of apoptosis are unequivocally associated with DNA fragmentation and support the close association of DNA frag-
265
ULTRASTRUCTUREOFCELLSUNDERGOINGAPOF'TOSIS
mentation tosis.
detected by TUNEL
with ultrastructurally
identified
apop-
IV. CONCLUSION Ultrastructural examination using conventional electron microscopy is still a very important method of analyzing apoptosis or programmed cell death. It is therefore very important for those involved in the study of tissue dynamics or cell proliferation and death to be familiar with these ultrastructural features of apoptosis. REFERENCES Abe,
R., Shimosegawa, T., Kimura, K., Abe, T., Kashimura, J., Koizumi, M., and Toyota, T. (1995). The role of endogenous glucocorticoids in rat experimental models of acute pancreatitis. Gastroenterology 109,933-943. Ansari, B., Coates, P. J., Greenstein, B. D., and Hall, P. A. (1993). In situ end-labeling detects DNAstrand breaks in apoptosis and other physiological and pathological states. J. Puthol. 170, 1-8. Arends, M. J., Morris, R. G., and Wyllie, A. H. (19901. Apoptosis. The role of endonuclease Am. J. Pathol. 136,593-600. Duvall, E., and Wyllie, A. H. (1986). Death and the cell. Zmmunol. Today 7, 115-119. Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. (1992). Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493-501. Gorczyca, W., Gong, J., and Darzynkiewicz, Z. (1993). Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res. 53, 1945-1951. Hiraishi, K., Suzuki, K., Hakomori, S., and Adachi, M. (1993). LeY antigen expression is correlated with apoptosis (programmed cell death). Glycobiology 3,381-390. Hockenbery, D. (1995). Review: Defining apoptosis. Am. J. Puthol. 146,16-19. Kerr, J. F. R. (1971). Shrinkage necrosis: A distinct mode of cellular death. J. Pathol. 105, 13-20. Kerr, J. F. R., Wyllie, A. H., and Currie, A. R. (1972). Apoptosis: Basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239-
257. Kerr,
J. F. R., Searle, J., Harmon, B. V., and Bishop, C. J. (1987). Apoptosis in perspectives on mammalian cell ing of death. In “OxfordAgeing” (C. S. Potten, ed.), pp. 9398. Oxford University Press, Oxford. Kimura, K., Sasano, H., Shimosegawa, T., Kato, K., Noguchi, T., Mochizuki, S., Sawai, T., Koizumi, M., Toyota, T., and Nagura, H. (19971. Ultrastructural and confocal laser scanning microscopic examination of tunel-positive cells. J. Pathol. 181, 235-242. Kimura, K., Shimosegawa, T., Sasano, H., Abe, R., Satoh, A., Masamune, A., Koizumi, M., Nagura, H., and Toyota, T. (1998). Endogenous glucocorticoids decrease the acinar cell sensitivity to apoptosis during cerulein pancreatitis in rats. Gastroenterology
114,372-381. Kyprianou, ventral
N., and prostate
Isaacs, J. T. (1988). Activation after castration. Endocrinology
of programmed (Baltimore)
cell death
122,552-562.
in the
rat
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Majno, G., and Joris, I. (1995). Apoptosis, oncosis, and necrosis. An overview of cell death. Am. J. Pathol. 146,3-15. Migheli,A.,Attanasio,A., and Shiffer, D. (1995). Ultrastructural detection of DNA strand breaks in apoptotic numeral cells by in situ end-labeling techniques. J. Pathol. 176, 27-35. Rink, A., Fung, K. M., Trajanowski, J. Q., Lee, Y. M., Neugebauer, E., and McIntosh, T. K. (1995). Evidence of apoptotic cell death after experimental traumatic brain injury in the rat. Am. J. Pathol. 147, 1575-1583. Russel, J. H. (1981). Internal disintegration model of cytotoxic lymphocyte-mediated cytotoxicity. Biol. Rev. Cambridge Philos. Sot. 56, 153-197. Sasano, H. (1995). Zn situ end labeling and its application to the study of endocrine disease: How can we study programmed cell death in surgical pathology materials? Endoer: Pathol. 6, l-3. Sasano, H., Imatani, A., Shizawa, S., Suzuki, T., and Nagura, H. (1995). Cell proliferation and apoptosis in normal and pathologic human adrenal. Mod. Pathol. 8, 11-17. Smith, C. A., Williams, G. T., Kingston, R., Jendinson, E. J., and Owen, J. J. (1989). Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature (Londoni 337, 181-184. Solviter, R. A., Sollas, A. L., and Dean, E. (1993). Adrenalectomy-induced granule cell degeneration in the rat hippocampal dentate gyrus: Characterization of an in vitro model of controlled neuronal death. J. Camp. Neural. 330,324-336. Wyllie, A. H. (1980). Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature (London) 284,555-556. Wyllie, A. H. (1981). Cell death: A new classification separating apoptosis from necrosis. In “Cell Death in Biology and Pathology” (I. D. Bowen and R. A. Lockshin, eds.) pp. 9-34. Chapman & Hall, London. Wyllie, A.H. (1993). Apoptosis. (The 1992 Frank Rose Memorial Lecture) Br. J. Cancer 67, 205-208. Wylie, A. H., Kerr, J. F. R., and Currie, A. R. (1980). Cell death: The significance of apoptosis. Znt. Rev. Cytol. 68, 251-306. Wylie, A. H., Morris, R. G., Smith, A. L., and Dunlop, D. (1984). Chromatin cleavage in apoptosis: Association with condensed chromatin morphology and dependence on macromolecular synthesis, J. Pathol. 142,67-77. Yabuki, N., Sasano, H., Tobita, M., Imatani, A., Hoshi, T., Kato, K., Ohara, S., Asaki, S., Toyota, T., and Nagura, H. (1997). Analysis of cell damage and proliferation in Helicobacter pylori-infected human gastric mucosa from patients with gastric adenocarcinema. Am. J. Pathol. 151, 821-829. Yamada, T., and Ohyama, H. (1998). Radiation-induced interphase death of rat thymocytes is internally programmed (apoptosis). Znt. J. Radial. Biol. 53,65-75.
Ultrastructure
AND HORMONES,
VOL. 58
of Cells Undergoing
Apoptosis
KENJI KIMURA,* HIRONOBU SASANO,” TOORU SHIMOSEGAWA,? SHIZUE MOCHIZUKI,” HIROSHI NAGURA,” AND TAKAYOSHI TOYOTA? “Department of Pathology and i-Department of Medicine, Medicine, 2-l Seiryou-machi, Aoba-ku, Sendai-shi,
I. Overview of Apoptosis II. Ultrastructural Features of Cells III. Correlation between Ultrastructural Undergoing Apoptosis IV. Conclusion References
Undergoing Apoptosis and Biochemical
I. OVERVIEW
Tohoku University School Miyagi-ken, Japan
Features
of
of Cells
OF APOPTOSIS
Analysis of cell turnover, the balance between cell proliferation and cell death, can provide important information about our understanding of the biological characteristics of various tissues and their functions. Apoptosis or programmed cell death is a selective process of physiologic cell deletion (Kerr et al., 1972; Wyllie et al., 1980; Russel, 1981; Wyllie, 1981; Duvall and Wyllie, 1986; Smith et al., 1989). The process of apoptosis plays a critical role in development and/or morphogenesis, in determining the size and shape of developing tissues, in maintenance of dynamic steady state in turnover of certain cell lineages in normal tissues, and in defining the number of tumor cell populations in the course of neoplastic development. Apoptosis is also important in various pathologic conditions, such as the response to irradiation (Yamada and Ohyama, 1998) stimulation by glucocorticoid (Wyllie,1980), withdrawal of glucocorticoid (Kyprianou and Isaacs, 19881, cytotoxic damages to gastric epithelial cells following Helicobacter pylori infection (Yabuki et al., 1997), and changes in some kinds of growth hormones (Solviter et al., 1993; Wyllie, 1993). Apoptosis is generally considered to be a form of cell death that occurs as a result of intentional suicide based on a genetic mechanism (Majno and Joris, 1995; Sasano, 1995; Kimura et al., 1997). Apoptosis is also a form of cell death characterized by morphologic as well as biochemical criteria and can be considered as a counterpart of mitosis (Majno and Joris, 1995). Apoptosis is characterized morphologically by 257
Copyright 0 2000 by Academic Press. All rights of reproduction in any form reserved. 0083-67!29/00 $30.00
258
KENJIKIMURAetal.
shrinkage of the cells resulting in cytoplasm condensation and the fragmentation of the cell nucleus, and membrane blebbing. Apoptosis is also defined biochemically by increased endogenous endonuclease activity, chromatin cleavage, and DNA fragmentation, that is, the DNA is broken down into segments that are multiples of approximately 185 base pairs, due to specific cleavage between nucleosomes (Kerr et al., 1987). In contrast, accidental cell death or oncosis or necrosis, which represents all cell death by murders, is generally defined as cell death caused by various cytotoxic agents or a pathologic environment, and is morphologically characterized by cytoplasmic swelling without the nuclear changes just described (Majno and Joris, 1995). It is also important to note that the whole process of apoptosis or programmed cell death is under genetic control and can be initiated by an internal clock or by extracellular agents. In addition, the duration of the slow process of apoptosis can be very fast, even in minutes (Majno and Joris, 1995). Apoptosis has been demonstrated to be induced by glucocorticoids, ceramide, wild-type ~53, various monoclonal antibodies against cell-surface antigens including Apo-1 or Fas, and others to be inhibited by bcl-2 protein encoded by the protooncogene bcl-2 (Majno and Joris, 1995). Biochemical detection of DNA cleavage into oligonucleosomal fragments of multiples of 180-200 base pairs, or the ladder pattern of nucleosomal DNA fragments on agarose gel electrophoresis, has been generally recognized as the hallmarks of apoptosis (Wyllie, 1980; Wyllie et al., 1984; Majno and Joris, 1995). However, as expected, this method can by no means identify the individual cells that are undergoing apoptosis. In addition, it has been recently demonstrated that these ladder patterns are not necessarily considered as specific for the process of apoptosis (Rink et al., 1995). On the other hand, the concept of “apoptosis” itself, which Kerr and Wyllie originally proposed was based on morphologic and ultrastructural examination (Kerr et al., 1972; Wyllie et al., 1980; Wyllie, 1981). It is therefore very important for those involved in the study of apoptosis to obtain sufficient knowledge of ultrastructural features of the cells undergoing apoptosis and incorporate morphologic observation or examination, especially that using electron microscopy into the study of apoptosis in his or her laboratory. These features are discussed in the following sections.
II. ULTRASTRUCTURALFEATURESOFCELLSUNDERGOINGA~OPTOSIS Kerr implied in 1971, shrinkage necrosis or apoptosis as a distinct mode of cellular death. The major morphologic feature of the cells un-
ULTRASTRUCTURE
OF CELLS
UNDERGOING
APOF’TOSI’S
259
FIG. 1. Ultrastructures of acinar cells in the pancreas of adrenalectomized rats treated with cerulein. Male Wistar strain rats weighing approximately 250 g underwent bilateral adrenalectomy and received 5 kg/kg per hour cerulein, intravenously, for 6 h. The cell shrinks and nuclei condense. The cytoplasm becomes dense but various intracellular organellae including mitochondria and endoplasmic reticula are still discernible. Bar = 1 urn.
dergoing apoptosis is the shrinkage of the cells. The cell becomes small in size and the cytoplasm becomes dense but various cell organellae including mitochondria are relatively normal (Fig. 1). Progression of the cytoplasmic condensation then results in marked crowding of organellae, which characteristically retain their integrity. However, the most characteristic ultrastructural feature of the cells undergoing apoptosis is chromatin condensation. The chromatin becomes very pyknotic, aggregating in the periphery of the nucleus to form dense smooth masses applied against the nuclear membrane (Fig. 2). These well-delimited dense masses of various shapes and sizes have been morphologically described as half-moon-, horseshoe-, lancet-, and navicular-shaped nuclei. The nucleus itself sometimes breaks up, resulting in one or two fragments of these dense smooth intranuclear masses (Fig. 2). These nuclear changes may be discernible in light microscopic examination but the need for ultrastructural confirmation of light microscopic identification of dying cells is required. The other ultrastructural features detected in the cells undergoing
260
KENJI
KIMUFtA
et al.
FIG. 2. Ultrastructures of acinar cells in the pancreas of adrenalectomized rats treated with cerulein. The arrow indicates apoptotic acinar cells. In the nucleus, condensed chromatin aggregates close to the nuclear membrane and demonstrates an appearance of crescent-like clumping (arrow). The nucleus also breaks up and forms relatively dense smooth intranuclear masses (double arrows). Zymogen granules are preserved in these cells. The polarity of zymogen granules in adjacent nonapoptotic acinal cells is relatively preserved. Bar = 1 km.
apoptosis is formation of cytoplasmic blebs and so-called “apoptotic bodies.” These processes emitted by the cells often contain pyknotic nuclear fragments (the budding phenomenon). These apoptotic bodies also contain cytoplasms and densely packed cell organellae. Sometimes, these apoptotic bodies may contain only cytoplasmic elements. These apoptotic bodies may be phagocytized by adjacent macrophages or parenchyma1 cells. Therefore, the cells undergoing apoptosis can be deleted from the tissue through these processes and the adjacent cells may migrate or proliferate to replace the space occupied by the deleted apoptotic cells. In contrast to these ultrastructural features of the cells undergoing apoptosis or programmed cell death, the cells undergoing the process of oncosis or accidental cell death may demonstrate cellular swelling, organellae swelling, and blebbing not accompanied by the nuclear changes described earlier (Majno and Joris, 1995).
ULTRASTRUCTUREOFCELLSUNDERGOINGAPOPTOSIS
III.
261
CORRELATIONBETWEENULTRASTRUCTURALANDBIOCHEMICAL FEATURESOFTHECELLSUNDERGOINGAPOPTOSIS
When examining the presence or absence of the cells undergoing apoptosis in the tissue specimen or cultured cells, it is very important to study these tissues and cells using election microscopy in order to examine whether ultrastructural features described earlier are detected or not. However, it is also true that these ultrastructural features of the cells undergoing apoptosis or programmed cell death are observed only for a limited period in the course of apoptosis (Arends et al., 1990; Ansari et al., 1993; Gorczyca et al., 1993; Hockenbery, 1995). In particular, these ultrastructural features may not be discernible in the early stages of cells undergoing apoptosis. In addition, examination by electron microscopy is not necessarily practical when screening for the presence or absence of apoptosis in tissue specimens or cell lines due to the labor-intensive and time-consuming nature of the technique. Apoptosis has been recognized as an essential component of tissue dynamics for many years. However, the relative difficulty in assessing apoptotic cells has prevented its study in both normal and pathologic processes (Ansari et al., 1993). Consequently, the development of other in situ methods to study apoptosis has been desired. In 1992, Gavrieli et al. demonstrated that DNAfragmentation can be detected in situ by labeling 3’-OH ends with biotinylated deoxyuridine triphosphate (dUTP) through the action of terminal deoxynucleotidyl transferase (TdT). Since then, this method, subsequently termed 3’-OH nick end labeling, or TdT-mediated dUTP-biotin nick end labeling (TUNEL), has been widely used to detect cells with DNA fragmentation, especially in surgical pathology materials of human disorders. The relatively good correlation of results obtained by the TUNEL method and DNA ladder formation by agarose gel electrophoresis has been demonstrated (Ansari et al., 1995; Abe et al., 1995). The TUNEL method has been widely applied to the analysis of apoptosis in a wide variety of materials including tissue specimens and cell lines (Hiraishi et al., 1993; Sasano et al., 1995; Kimura et al., 1998). With the introduction of various commercially available staining kits, the TUNEL method can now be easily and rapidly performed in properly fixed and processed tissue materials. The TUNEL method is now being incorporated into many pathology laboratories throughout the world. However, increasing evidence suggests that the TUNEL method is by no means specific for the detection of apoptosis (Ansari et al., 1995) and this method may also detect cells that are committed to, but not yet in the process of, apoptosis (Wyllie et al., 1984; Ansari et al., 1995). Therefore it becomes
262
KENJI
KIMURAet
al.
very important to examine the ultrastructural or morphologic features of TUNEL-positive cells. We have recently examined ultrastructural features of TUNELpositive cells in continuous mirror image semithin and ultrathin sections of Epon-Araldite-embedded tissues of pancreas from bilaterally adrenalectomized rats with cerulein-induced pancreatitis (Kimura et al., 1997). Application of this method through continuous mirror sections can directly demonstrate ultrastructural features of the TUNELpositive cells, although the sensitivity of the TUNEL method may be lower in Epon-Araldite-embedded tissue sections than in regular 10% formalin or 8% paraformaldehyde fixed and paraffin-embedded tissue sections possibly due to the interruption of the binding of dUTP to nick by Epon-Araldite plastic and osmium. Results of this study revealed that all ultrastructurally discernible apoptotic cells were positive by the TUNEL method (Fig. 3), but de-
FIG. 3. iA) A TUNEL-positive nucleus of an acinar cell in an Epon-Araldite semithin section. The arrow indicates an intensely labeled acinar cell nucleus. The other nuclei are stained by hematoxylin (X 300). (B) Ultrastructural features of the same acinus in a serial adjacent ultrathin section. The arrow indicates condensed chromatin with creascentic clumping. Bar = 2 km. (C)At higher magnification, mitochondria and zymogen granules are observed to be almost intact. Rough endoplasmic reticulum revealed a vesicular pattern. Bar = 2 pm.
ULTRASTRUCTURE
OF CELLS
FIG.
UNDERGOING
3. Continued
APOPTOSIS
263
264
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KIMURA
et al.
generated nuclei that may represent the end stage of cell death, either programmed or accidental, are also strongly labeled by the TUNEL method. Ansari et al. (1993) suggested that the TUNEL method may identify not only the apoptotic nuclei, but also the nuclei in necrotic tissues; the staining of necrotic cells could be explained either by the activation of an apoptotic pathway in damaged cells by the degradation of DNA by lysozomal enzymes. However, these two patterns can be distinguished by the distribution of TUNEL-positive nuclei; apoptotic nuclei are usually scattered, without localized aggregation. Several investigators have proposed the possibility that cells which have not yet begun the process of apoptosis and which lack its morphologic features can also be detected by the TUNEL method (Gorczyca et al., 1993; Migheli et al., 1995), which may therefore be able to detect apoptotic cells much earlier than electron microscopy. In our study employing continuous mirror image semithin and ultrathin sections, however, nearly all TUNEL-positive cells exhibited the characteristic ultrastructural features of apoptosis. Migheli et al. (1995) also applied the TUNEL method at the electron microscopic level, using an immunogold staining technique in embryonic mouse dorsal root ganglia. They reported that nuclear localization of immunogold was observed not only in typical apoptotic cells at various stages of cell death, but also in some apparently normal cells, which were structurally indistinguishable from adjacent unstained cells. Morphologically viable TUNEL-positive cells, including the “pre-apoptotic” cells (Migheli et aZ., 1995) may represent experimentally induced artefactual DNAdamage, leading to TUNEL positivity. The possibility that the relatively rapid process of apoptosis in pancreatic acinar cells in a cerulein-induced pancreatitis model made it difficult to detect preapoptotic cells with TUNEL cannot be completely ruled out. However, it is also true that ultrastructural examination of TUNEL-positive cells in continuous mirror image semithin and ultrathin sections of EponAraldite-embedded tissue sections employed in our study Wimura et al., 1997) has the following technical advantages over the immunogold technique reported by Migheli et al. (1995): (1) Much more ultrastructural detail can be obtained than in a single sections, (2) TUNEL-positive cells can be identified in a much wider area by light microscopy, and (3) TUNEL-positive cells are much more clearly visualized due to the calorimetric reaction in contrast to relatively small dots in the immunogold electron microscopy method. Despite these minor differences, results of our study and Migheli’s clearly demonstrated that the ultrastructural features of apoptosis are unequivocally associated with DNA fragmentation and support the close association of DNA frag-
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mentation tosis.
detected by TUNEL
with ultrastructurally
identified
apop-
IV. CONCLUSION Ultrastructural examination using conventional electron microscopy is still a very important method of analyzing apoptosis or programmed cell death. It is therefore very important for those involved in the study of tissue dynamics or cell proliferation and death to be familiar with these ultrastructural features of apoptosis. REFERENCES Abe,
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