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Apoptosis detection: an overview
Experimental Gerontology, Vol. 33, No. 6, pp. 525–533, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0531-5565/98 $19.00 1 .00
PII S0531-5565(98)00031-X
MINI-REVIEW
APOPTOSIS DETECTION: AN OVERVIEW
R. SGONC1 and J. GRUBER2 1
Institute for General and Experimental Pathology, and 2Department of Internal Medicine, University of Innsbruck, Medical School, Innsbruck, Austria
Abstract—As the analysis of apoptosis is of interest in many basic and clinically oriented investigations, we intend to give a brief overview on the recently most-used methods for detection of apoptotic cells, including the study of morphology, analysis of DNA degradation, DNA end-labeling techniques, flow cytometric analysis, and nuclease assays. Features and advantages of the different methods are discussed. © 1998 Elsevier Science Inc. Key Words: apoptosis; flow cytometry; confocal laser scan microscopy; 3*OH end-labeling; TUNEL; annexin V; comet assay
INTRODUCTION APOPTOSIS PLAYS an important role in a variety of cellular events, for example, morphogenic death of cells during embryogenesis, in the course of normal tissue turnover, deletion of autoreactive T cells during T cell selection by glucocorticoid induced (early) and antigen induced (late) apoptosis, and clonal deletion of B cells (White, 1996; Nagata, 1997). These beneficial roles of apoptosis are counterbalanced by apoptotic cell death occurring in a variety of pathological conditions including cardiovascular and neurodegenerative diseases (Carson and Ribeiro, 1993; Wang, 1997). Consequently, the analysis of apoptosis is of interest in many basic and clinically oriented investigations. The present review discusses the most widely used methods to characterize and differentiate between apoptotic, necrotic and viable cells. MICROSCOPY The apoptotic process involves a sequence of cell shrinkage, increased cytoplasmic density, chromatin condensation, and segregation into sharply circumscribed masses that abut the
Correspondence address: Roswitha Sgonc, Ph.D., Institute for General and Experimental Pathology, University of Innsbruck, Medical School, A-6020 Innsbruck, Austria. Tel: 143-512-507-3104; Fax: 143-512-507-2867; E-mail: [email protected] (Received 16 April 1998; Accepted 8 May 1998) 525
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nuclear membrane and can form blister-like protrusions (“budding”). The latter then separate to produce membrane-bound apoptotic bodies. Initially, the mitochondria and the Golgi apparatus show no signs of swelling and the nuclear membrane remains intact for an extended period of time. In contrast, necrotic cells show chromatin clumping into ill-defined masses, gross swelling of organelles, and, at a later stage, membrane breakdown and the cell disintegration. These morphological changes can be detected with the electron microscope (Kerr et al., 1972; Gorman et al., 1996). The application of quantitative digital imaging techniques to electron microscopy to visualize fine gradations of gray levels enables the detection of even subtle changes in nuclear densities (Payne and Cromey, 1992). Budding and formation of apoptotic bodies can also be seen by simple phase contrast light microscopy. Another versatile tool is the confocal laser scanning microscopy, powerful for both morphological analysis and macromolecular localisation (Smith et al., 1991). Thus, condensed chromatin and nuclear fragmentation are easily determined by examining serial optical sections of cells stained with DNA dyes such as propidium iodide (PI) and hoechst 33342 (Ho342). These techniques are primarily used for qualitative determination of apoptosis. For the quantification of apoptotic cells flow cytometry is a more powerful technique. ANALYSIS OF DNA DEGRADATION The most striking biochemical event in apoptosis is the DNA cleavage between nucleosomes that produces fragments in multiples of approximately 185 bp (Wyllie, 1980). This phenomenon is most often analyzed by agarose gel electrophoresis, which measures DNA fragmentation in nuclear extracts showing the typical “DNA-ladder” configuration (Compton and Cidlowski, 1986) (Fig. 1). An improved method for the detection of DNA fragmentation visualizes the DNA after Southern blotting with a radiolabeled total cellular DNA probe instead of ethidium bromide staining, which increases the sensitivity (Facchinetti et al., 1991). Another method to measure low levels of DNA cleavage is based on the labeling of free 39OH end of nicked DNA with 32P-dATP or 32P-dCTP mediated by Klenow polymerase, followed by agarose gel electrophoresis and autoradiography (Rosl, 1992). These techniques have several disadvantages: they are time consuming, require a great number of cells, involve radioactivity in the more sensitive assays, and they are unable to determine the percentage of apoptotic nuclei or recognize the apoptotic cells in a heterogeneous cell population. The “comet assay,” a method of microelectrophoresis, allows measurement of DNA fragmentation in individual cells. In this assay, cells embedded in low gelling temperature agarose are lysed, either under neutral conditions to detect double-strand breaks or using an alkaline lysis solution for detection of single-strand breaks. DNA is allowed to migrate in an electric field, and after staining with a DNA-binding dye the cells are analyzed using a fluorescence microscope. The amount of DNA able to migrate depends on the number of strand breaks and the size of DNA fragments. This method is simple, but it is not easy to discriminate between apoptotic and necrotic cells. Differences in DNA conformation affecting DNA migration also complicate the analysis (Olive et al., 1992, 1998). Moreover, quantitative analysis is very time consuming and requires an image analysis system. A specific application of apoptosis detection is the analysis of cytotoxicity using the JAM test (Matzinger, 1991). This assay is based on the DNA fragmentation, which is an early event in the death of targets killed by cytotoxic T cells or NK cells. The target cells are labeled with [3H]-thymidine before setting up the lysis assay and harvested onto Fiberglas filters after incubation with cytotoxic T cells. Because DNA is essentially trapped in these filters rather than
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FIG. 1. Agarose gel electrophoresis showing DNA degradation (“ladder” pattern) of murine thymocytes. Lane 1: 1 kb molecular weight marker (Gibco, BRL). Lane 2: PBS-treated control. Lane 3, 4, and 5: 4-week-old healthy BALB/c and lupus-prone MRL/lpr and (NZB 3 NZW)F1 treated with 1 mg dexamethasone/kg body weight for eight hours.
adherent, small pieces of fragmented DNA are washed through, leaving only the intact DNA from living cells. This method is more sensitive, less hazardous, quicker, and cheaper than the 51 Cr release assay, but is only suitable for proliferating targets. ANALYSIS OF ENDONUCLEASES Because DNA fragmentation is a primary event in apoptosis and may be the actual cause of cell death, the identification of the endonuclease(s), which is responsible for this cleavage, is also of interest in studies of apoptosis. Nuclease assays have been described that detected DNA-degrading activity in SDS-polyacrylamide gels in which a nonradioactive or 32P-labeled nucleic acid substrate was incorporated into the gel matrix, and nuclease activity was ascertained by loss of the substrate from the gel (Rosenthal and Lacks, 1977; Caron Leslie and Cidlowski, 1991). This assay is simple to perform, but lacks the specificity to detect internucleosomal DNA degradation. Another nuclease activity assay specifically developed to study nucleases cleaving
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DNA at internucleosomal sites (Schwartzman and Cidlowski, 1991; Compton, 1991) utilizes nuclei of cells not susceptible to glucocorticoid-induced lysis (HeLa or chicken red blood cells) as substrate to measure nuclease activity of glucocorticoid-treated thymocyte nuclear protein extracts. After incubation of HeLa or chicken red blood cell nuclei with nuclear protein extracts, DNA is isolated from the nuclei and analyzed by agarose gel electrophoresis, which allows differentiation between random DNA degradation and the internucleosomal cleavage that is typical during apoptosis. FLOW CYTOMETRIC ANALYSES Flow cytometric analyses based on the detection of morphological changes, DNA fragmentation, DNA loss, and membrane changes are increasingly used for quantitative investigations of apoptosis. Thus, the light scatter properties of cells can be used to analyse the changes in size and granularity. Apoptosis is accompanied by water loss, cell shrinkage, and nuclear fragmentation, whereas necrosis is not. Thus, apoptotic and necrotic cells differ in light scatter patterns (Carbonari et al., 1994); apoptotic cells give lower forward scatter and higher side scatter values than viable cells, reflecting the smaller size and different nucleus/cytoplasm consistency (Fig. 2A and B). In principle, flow cytometric analysis of morphological parameters is a very rapid and objective way of enumerating apoptotic cells but, unfortunately, these changes are not consistent in all cell types. Because activation of endonuclease(s) in apoptotic cells leads to DNA fragmentation and subsequently to extensive DNA loss, apoptosis can be determined by the simple measurement of DNA content using intercalating DNA dyes, such as propidium iodide (PI), ethidium bromide, and acridine orange, or dyes binding externally to DNA such as Hoechst 33342 (Ho342), DAPI (49-6-diaminido-2-phenylindole), and mithramycin (Telford et al., 1992). Fixed, permeabilized apoptotic cells or their isolated nuclei show a low DNA stainability resulting in a distinct, quantifiable region below the G0/G1 peak (Fig. 2C and D). The easiest and most rapid method for measuring apoptosis is DNA staining with a hypotonic PI solution (Nicoletti et al., 1991), which is particularly suitable for large scale in vitro studies. During apoptosis the plasma membrane undergoes multiple changes (Morris et al., 1984). The increased membrane permeability for Hoechst dyes, which diffuse through intact membranes, is reflected by a strong fluorescence staining after short exposure to low concentrations of Ho342 (Hardin et al., 1992) (Fig. 2E and F). The combination of staining with Ho342 and a PI/PBS solution that does not diffuse through intact cell membranes allows discrimination between, and quantification of, apoptotic, necrotic, and viable cells (Dive et al., 1992). The translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane during
™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ 3 FIG. 2. FACS analysis of BALB/c mouse thymocytes: left panel (A, C, E, G, I) untreated control, right panel (B, D, F, H, J) treated with 1026 M dexamethasone for four hours in vitro. (A, B) Morphological parameters. The apoptotic (Ap) population is characterized by a lower forward scatter (FSC) and higher side scatter (SSC) (B). This population is absent in untreated thymocytes (A). (C, D) Propidium iodide (PI) staining. Apoptotic cells are found in the “sub-G0/G1” peak. (E, F) Hoechst 33342 (Ho342) staining. Apoptotic cells show bright fluorescence. (G, H) Double staining with annexin V-FITC and PI. Early apoptotic cells (annexin V-positive/PI-negative) are localized in the lower right region, late apoptotic, and necrotic cells (annexin V-positive/PI-positive) in the upper right region, and vital cells (double negative) in the lower left region. (I, J): 39OH labeling by TUNEL. 10,000 cells were analyzed in each experiment.
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FIG. 3. Confocal laser scan microscopic images of various thymic cell preparations from an untreated (A, D) and a four-week-old BALB/c mouse treated with 25 mg dexamethasone/kg body weight for eight hours (B, C) showing overlays of transmission and fluorescence scans: apoptotic cells reveal green-stained nuclei after 39OH labeling by the TUNEL technique; intact nuclei are counterstained with propidium iodide (C, D). (A) Viable thymocyte. (B) Thymocyte undergoing apoptosis. Note the nuclear fragmentation and “budding” (arrow). (C) Macrophage (arrow) with two engulfed apoptotic bodies, vital thymocyte (arrow head). (D) Thymic nurse cell harboring an apoptotic thymocyte (arrow). Original magnification 633 oil, 803 zoom (A, B); 633 oil, 403 zoom (C, D).
FIG. 4. Apoptosis detection on frozen, paraformaldehyde-fixed skin sections of a two-week-old C57Bl/6 1pa/1pa mouse. (A) Apoptotic cells were detected by the TUNEL technique using FITC-dUTP for DNA labeling, an anti-FITC-alkaline phosphatase antibody, fast red for visualization, and counterstaining with haemalaun. At this age, apoptotic cells (pink nuclei) are found in some hair follicles (arrows) and the epidermis (arrow head). (B) Simultaneous detection of apoptotic cells with FITC-dUTP (green nuclei) and endothelial cells with anti-von Willebrand factor and TRITC conjugate (red). Original magnification 2003.
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apoptosis can be detected by annexin V, which binds preferentially to PS in the presence of Ca21 (Koopman et al., 1994). To discriminate between apoptotic and necrotic cells the annexin V-FITC binding assay should be combined with PI vital staining (Fig. 2G and H). Ho342 staining and the annexin V assay are both very rapid and simple methods for detection of early apoptosis. But due to increased membrane permeability late apoptotic cells cannot be discriminated from necrotic cells. Early during apoptosis a loss of mitochondrial membrane potential (DCm) occurs. This permeability transition (PT), which is under bcl-2 control, leads to the release of the so-called apoptosis inducing factor (AIF), causing caspase activation and finally cell death. DCm changes can be assessed by mitochondria staining with cationic, lipophilic fluorochromes such as 5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazolcarbocyanine iodide (JC-1), 3,39-dihexyloxacarbocyanine iodide [DiOC6(3)], or rhodamine 123 and flow cytometric analysis. Cells undergoing apoptosis show a reduction in the incorporation of DCm-sensitive dyes (Vayssiere et al., 1994; Zamzami et al., 1995). DCm analysis seems to be a reliable method for studying apoptosis. For the staining of mitochondria in living cells the fluorescent probe should be deliberately chosen as DiOC6(3), for example, is not only sensitive to DCm but also to changes in the plasma membrane potential that might mimic alterations in DCm (Salvioli et al., 1997). DNA strand breaks occurring in apoptotic cells as a result of endonuclease(s) activation can be labeled in situ in individual fixed, permeabilized cells or in tissue sections by in situ nick translation (ISNT) (Fehsel et al., 1991; Gold et al., 1993) or the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technique (Gavrieli et al., 1992; Sgonc et al., 1994, 1996). In both methods, the 39-hydroxyl ends of DNA fragments are labeled either with biotin-dUTP, digoxigenin-dUTP, or FITC-dUTP mediated by DNA polymerase I (ISNT) or by terminal deoxynucleotidyl transferase (TUNEL). In our own hands, indirect labeling with biotin-dUTP or digoxigenin-dUTP and subsequent detection by streptavidin-FITC or fluoresceinated antidigoxigenin resulted in higher background staining compared to direct labeling with FITC-dUTP (unpublished data). Comparison of TUNEL and ISNT shows a more pronounced distinction of apoptotic cells based on the intensity of green fluorescence of labeled vs. unlabeled cells (signal:noise ratio) and a higher sensitivity of the TUNEL technique (Gorczyca et al., 1993). Major advantages of the 39OH end labeling technique include the ability to reveal early DNA breaks during apoptosis, quantitation of the percentage of apoptotic cells by flow cytometric analysis (Fig. 2I and J), the capacity for standard multiple immunofluorescence staining for cell surface markers to characterize cells undergoing apoptosis within heterogenous populations, and its utility in morphological studies (Figs. 3 and 4). In addition, TUNEL can be combined with in situ detection of mRNA expression (Strater et al., 1996). CONCLUDING REMARKS In summary, flow cytometric analysis of PI stained nuclei complemented with light scatter analysis is very useful for simple detection of apoptosis, especially in large-scale experiments. The annexin V binding assay in combination with PI vital staining is a rapid method for detection of early apoptosis. For characterization of cells undergoing apoptosis, simultaneous determination of cell markers and apoptosis by TUNEL is highly recommended. Acknowledgments—This work was supported by the Austrian Research Council (project No. 12715). We thank Heidrun Recheis for CLSM pictures, and Guenther Boeck for flow cytometric analysis.
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REFERENCES CARBONARI, M., CIBATI, M., CHERCHI, M., SBARIGIA, D., PESCE, A.M., DELL’ANNA, L., MODICA, A., and FIORILLI, M. Detection and characterization of apoptotic peripheral blood lymphocytes in human immunodeficiency virus infection and cancer chemotherapy by a novel flow immunocytometric method. Blood 83, 1268 –1277, 1994. CARON, LESLIE, L. A.and CIDLOWSKI, J.A. Similar actions of glucocorticoids and calcium on the regulation of apoptosis in S49 cells. Mol. Endocrinol. 5, 1169 –1179, 1991. CARSON, D.A. and RIBEIRO, J.M. Apoptosis and disease. Lancet 341, 1251–1254, 1993. COMPTON, M.M. and CIDLOWSKI, J.A. Rapid in vivo effecst of glucocorticoids on the integrity of rat lymphocyte genomic deoxyribonucleic acid. Endocrinology 118, 38 – 45, 1986. COMPTON, M.M. Development of an apoptosis endonuclease assay, DNA Cell Biol. 10, 133–141, 1991. DIVE, C., GREGORY, C.D., PHIPPS, D.J., EVANS, D.L., MILNER, A.E., and WYLLIE, A.H. Analysis and discrimination of necrosis and apoptosis (progarmmed cell death) by multiparameter flow cytoemtry. Biochim. Biophys. Acta 1133, 275–285, 1992. FACCINETTI, A., TESSAROLLO, L., MAZZOCCHI, M. KINGSTON, R., COLLAVO, D., and BIASI, G. An improved method for the detection of DNA fragmentation. J. Immunol. Methods 136, 125–131, 1991. FEHSEL, K., KOLB BACHOFEN, V., and KOLB, H. Analysis of TNF alpha-induced DNA strand breaks at the single cell level. Am. J. Pathol. 139, 251–254, 1991. GAVRIELI, Y., SHERMAN, Y., and BEN SASSON, S.A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493–501, 1992. GOLD, R., SCHMIED, M., ROTHE, G., ZISCHLER, H., BREITSCHOPF, H., WEKERLE, H., and LASSMANN, H. Detection of DNA fragmentation in apoptosis: Application of in situ nick translation to cell culture systems and tissue sections. J. Histochem. Cytochem. 41, 1023–1030, 1993. GORCZYCA, W., GONG, J., and DARZYNKIEWICZ, Z. 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, 1993. GORMAN, A., McCARTHY, J., FINUCANE, D., REVILLE, W., and COTTER, T. Morphological assessment of apoptosis. In: Techniques in Apoptosis. A User’s Guide, Cotter, T.G. and Martin, S.J. (Editors), pp. 1–20, Portland Press Ltd, London, 1996. HARDIN, J.A., SHERR, D.H., DEMARIA, M., and LOPEZ, P.A. A simple fluorescence method for surface antigen phenotyping of lymphocytes undergoing DNA fragmentation. J. Immunol. Methods 154, 99 –107, 1992. KERR, J.F.R., WYLLIE, A.H., and CURRIE, A.R. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239 –257, 1972. KOOPMAN, G., REUTELINGSPERGER, C.P., KUIJTEN, G.A., KEEHNEN, R.M., PALS, S.T. and VAN OERS, M.H. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84, 1415–1420, 1994. MATZINGER, P. The JAM test. A simple assay for DNA fragmentation and cell death. J. Immunol. Methods 145, 185–192, 1991. MORRIS, R.G., HARGREAVES, A.D., DUVALL, E., and WYLLIE, A.H. Hormone-induced cell death 2. Surface changes in thymocytes undergoing apoptosis. Am. J. Pathol. 115, 426 – 436, 1984. NAGATA, S. Apoptosis by death factor. Cell 88, 355–365, 1997. NICOLETTI, I., MIGLIORATI, G., PAGLIACCI, M.C., GRIGNANI, F., and RICCARDI, C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139, 271–279, 1991. OLIVE, P.L., WLODEK, D., DURAND, R.E., and BANa´ TH, J.P. Factors influencing DNA migration from individual cells subjected to gel electrophoresis. Exp. Cell Res. 198, 259 –267, 1992. OLIVE, P.L., JOHNSTON, P.J., BANa´ TH, J.P., and DURAND, R.E. The comet assay: A new method to examine heterogeneity associated with solid tumors. Nat. Med. 4, 103–105, 1998. PAYNE, C.M. and CROMEY, D.W. An image analysis workstation designed for multiple users: Application of quantitative digital imaging techniques to electron microscopy. Ultrastruct. Pathol. 16, 147–154, 1992. ROSENTHAL, A.L. and LACKS, S.A. Nuclease detection in SDS-polyacrylamide gel electrophoresis. Anal. Biochem. 80, 76 –90, 1977. ROSL, F. A simple and rapid method for detection of apoptosis in human cells. Nucleic Acids Res. 20, 5243, 1992. SALVIOLI, S., ARDIZZONI, A., FRANCESCHI, C., and COSSARIZZA, A. JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess DC changes in intact cells: Implications for studies on mitochodnrial functionality during apoptosis. FEBS Lett. 411, 77– 82, 1997.
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SCHAWRTZMAN, R.A. and CIDLOWSKI, J.A. Internucleosomal deoxyribonucleic acid cleavage activity in apoptotic thymocytes: Detection and endocrine regulation. Endocrinology 128, 1190 –1197, 1991. SGONC, R., BOECK, G., DIETRICH, H., GRUBER, J., RECHEIS, H., and WICK, G. Simultaneous determination of cell surface antigens and apoptosis. Trends Genet. 10, 41– 42, 1994. SGONC, R., GRUSCHWITZ, M.S., DIETRICH, H., RECHEIS, H., GERSHWIN, M.E., and WICK, G. Endothelial cell apoptosis is a primary pathogenetic event underlying skin lesions in avian and human scleroderma. J. Clin. Invest. 98, 785–792, 1996. SMITH, G.J., BAGNELL, C.R., BAKEWELL, W.E., BLACK, K.A., BOULDIN, T.W., EARNHARDT, T.S., HOOK, G.E., and PRYZWANSKY, K.B. Application of confocal scanning laser microscopy in experimental pathology. J. Electron. Microsc. Technol. 18, 38 – 49, 1991. STRATER, J., WALCZAK, H., KRAMMER, P.H., and MOLLER, P. Simultaneous in situ detection of mRNA and apoptotic cells by combined hydridization and TUNEL. J. Histochem. Cytochem. 44, 1497–1499, 1996. TELFORD, W.G., KING, L.E., and FRANKER, P.J. Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry. Cytometry 13, 137–143, 1992. VAYSSIERE, J.L., PETIT, P.X., RISLER, Y., and MIGNOTTE, B. Commitment to apoptosis is associated with changes in mitochondrial biogenesis and activity in cell lines conditionally immortalized with simian virus 40. Proc. Natl. Acad. Sci. USA 91, 11752–11756, 1994. WANG, E. Regulation of apoptosis resistance and ontogeny of age-dependent diseases. Exp. Gerontol. 32, 471– 484, 1997. WHITE, E. Life, death, and the pursuit of apoptosis. Gene Dev. 10, 1–15, 1996. WYLLIE, A.H. Glucocorticoid-induced thymocyte apoptosis is associated with endogeneous endonuclease activation. Nature 284, 555–556, 1980. ZAMZAMI, N., MARCHETTI, P., CASTEDO, M., ZANIN, C., VAYSSIERE, J.L., PETIT, P.X., and KROEMER, G. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181, 1661–1672, 1995.
PII S0531-5565(98)00031-X
MINI-REVIEW
APOPTOSIS DETECTION: AN OVERVIEW
R. SGONC1 and J. GRUBER2 1
Institute for General and Experimental Pathology, and 2Department of Internal Medicine, University of Innsbruck, Medical School, Innsbruck, Austria
Abstract—As the analysis of apoptosis is of interest in many basic and clinically oriented investigations, we intend to give a brief overview on the recently most-used methods for detection of apoptotic cells, including the study of morphology, analysis of DNA degradation, DNA end-labeling techniques, flow cytometric analysis, and nuclease assays. Features and advantages of the different methods are discussed. © 1998 Elsevier Science Inc. Key Words: apoptosis; flow cytometry; confocal laser scan microscopy; 3*OH end-labeling; TUNEL; annexin V; comet assay
INTRODUCTION APOPTOSIS PLAYS an important role in a variety of cellular events, for example, morphogenic death of cells during embryogenesis, in the course of normal tissue turnover, deletion of autoreactive T cells during T cell selection by glucocorticoid induced (early) and antigen induced (late) apoptosis, and clonal deletion of B cells (White, 1996; Nagata, 1997). These beneficial roles of apoptosis are counterbalanced by apoptotic cell death occurring in a variety of pathological conditions including cardiovascular and neurodegenerative diseases (Carson and Ribeiro, 1993; Wang, 1997). Consequently, the analysis of apoptosis is of interest in many basic and clinically oriented investigations. The present review discusses the most widely used methods to characterize and differentiate between apoptotic, necrotic and viable cells. MICROSCOPY The apoptotic process involves a sequence of cell shrinkage, increased cytoplasmic density, chromatin condensation, and segregation into sharply circumscribed masses that abut the
Correspondence address: Roswitha Sgonc, Ph.D., Institute for General and Experimental Pathology, University of Innsbruck, Medical School, A-6020 Innsbruck, Austria. Tel: 143-512-507-3104; Fax: 143-512-507-2867; E-mail: [email protected] (Received 16 April 1998; Accepted 8 May 1998) 525
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nuclear membrane and can form blister-like protrusions (“budding”). The latter then separate to produce membrane-bound apoptotic bodies. Initially, the mitochondria and the Golgi apparatus show no signs of swelling and the nuclear membrane remains intact for an extended period of time. In contrast, necrotic cells show chromatin clumping into ill-defined masses, gross swelling of organelles, and, at a later stage, membrane breakdown and the cell disintegration. These morphological changes can be detected with the electron microscope (Kerr et al., 1972; Gorman et al., 1996). The application of quantitative digital imaging techniques to electron microscopy to visualize fine gradations of gray levels enables the detection of even subtle changes in nuclear densities (Payne and Cromey, 1992). Budding and formation of apoptotic bodies can also be seen by simple phase contrast light microscopy. Another versatile tool is the confocal laser scanning microscopy, powerful for both morphological analysis and macromolecular localisation (Smith et al., 1991). Thus, condensed chromatin and nuclear fragmentation are easily determined by examining serial optical sections of cells stained with DNA dyes such as propidium iodide (PI) and hoechst 33342 (Ho342). These techniques are primarily used for qualitative determination of apoptosis. For the quantification of apoptotic cells flow cytometry is a more powerful technique. ANALYSIS OF DNA DEGRADATION The most striking biochemical event in apoptosis is the DNA cleavage between nucleosomes that produces fragments in multiples of approximately 185 bp (Wyllie, 1980). This phenomenon is most often analyzed by agarose gel electrophoresis, which measures DNA fragmentation in nuclear extracts showing the typical “DNA-ladder” configuration (Compton and Cidlowski, 1986) (Fig. 1). An improved method for the detection of DNA fragmentation visualizes the DNA after Southern blotting with a radiolabeled total cellular DNA probe instead of ethidium bromide staining, which increases the sensitivity (Facchinetti et al., 1991). Another method to measure low levels of DNA cleavage is based on the labeling of free 39OH end of nicked DNA with 32P-dATP or 32P-dCTP mediated by Klenow polymerase, followed by agarose gel electrophoresis and autoradiography (Rosl, 1992). These techniques have several disadvantages: they are time consuming, require a great number of cells, involve radioactivity in the more sensitive assays, and they are unable to determine the percentage of apoptotic nuclei or recognize the apoptotic cells in a heterogeneous cell population. The “comet assay,” a method of microelectrophoresis, allows measurement of DNA fragmentation in individual cells. In this assay, cells embedded in low gelling temperature agarose are lysed, either under neutral conditions to detect double-strand breaks or using an alkaline lysis solution for detection of single-strand breaks. DNA is allowed to migrate in an electric field, and after staining with a DNA-binding dye the cells are analyzed using a fluorescence microscope. The amount of DNA able to migrate depends on the number of strand breaks and the size of DNA fragments. This method is simple, but it is not easy to discriminate between apoptotic and necrotic cells. Differences in DNA conformation affecting DNA migration also complicate the analysis (Olive et al., 1992, 1998). Moreover, quantitative analysis is very time consuming and requires an image analysis system. A specific application of apoptosis detection is the analysis of cytotoxicity using the JAM test (Matzinger, 1991). This assay is based on the DNA fragmentation, which is an early event in the death of targets killed by cytotoxic T cells or NK cells. The target cells are labeled with [3H]-thymidine before setting up the lysis assay and harvested onto Fiberglas filters after incubation with cytotoxic T cells. Because DNA is essentially trapped in these filters rather than
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FIG. 1. Agarose gel electrophoresis showing DNA degradation (“ladder” pattern) of murine thymocytes. Lane 1: 1 kb molecular weight marker (Gibco, BRL). Lane 2: PBS-treated control. Lane 3, 4, and 5: 4-week-old healthy BALB/c and lupus-prone MRL/lpr and (NZB 3 NZW)F1 treated with 1 mg dexamethasone/kg body weight for eight hours.
adherent, small pieces of fragmented DNA are washed through, leaving only the intact DNA from living cells. This method is more sensitive, less hazardous, quicker, and cheaper than the 51 Cr release assay, but is only suitable for proliferating targets. ANALYSIS OF ENDONUCLEASES Because DNA fragmentation is a primary event in apoptosis and may be the actual cause of cell death, the identification of the endonuclease(s), which is responsible for this cleavage, is also of interest in studies of apoptosis. Nuclease assays have been described that detected DNA-degrading activity in SDS-polyacrylamide gels in which a nonradioactive or 32P-labeled nucleic acid substrate was incorporated into the gel matrix, and nuclease activity was ascertained by loss of the substrate from the gel (Rosenthal and Lacks, 1977; Caron Leslie and Cidlowski, 1991). This assay is simple to perform, but lacks the specificity to detect internucleosomal DNA degradation. Another nuclease activity assay specifically developed to study nucleases cleaving
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DNA at internucleosomal sites (Schwartzman and Cidlowski, 1991; Compton, 1991) utilizes nuclei of cells not susceptible to glucocorticoid-induced lysis (HeLa or chicken red blood cells) as substrate to measure nuclease activity of glucocorticoid-treated thymocyte nuclear protein extracts. After incubation of HeLa or chicken red blood cell nuclei with nuclear protein extracts, DNA is isolated from the nuclei and analyzed by agarose gel electrophoresis, which allows differentiation between random DNA degradation and the internucleosomal cleavage that is typical during apoptosis. FLOW CYTOMETRIC ANALYSES Flow cytometric analyses based on the detection of morphological changes, DNA fragmentation, DNA loss, and membrane changes are increasingly used for quantitative investigations of apoptosis. Thus, the light scatter properties of cells can be used to analyse the changes in size and granularity. Apoptosis is accompanied by water loss, cell shrinkage, and nuclear fragmentation, whereas necrosis is not. Thus, apoptotic and necrotic cells differ in light scatter patterns (Carbonari et al., 1994); apoptotic cells give lower forward scatter and higher side scatter values than viable cells, reflecting the smaller size and different nucleus/cytoplasm consistency (Fig. 2A and B). In principle, flow cytometric analysis of morphological parameters is a very rapid and objective way of enumerating apoptotic cells but, unfortunately, these changes are not consistent in all cell types. Because activation of endonuclease(s) in apoptotic cells leads to DNA fragmentation and subsequently to extensive DNA loss, apoptosis can be determined by the simple measurement of DNA content using intercalating DNA dyes, such as propidium iodide (PI), ethidium bromide, and acridine orange, or dyes binding externally to DNA such as Hoechst 33342 (Ho342), DAPI (49-6-diaminido-2-phenylindole), and mithramycin (Telford et al., 1992). Fixed, permeabilized apoptotic cells or their isolated nuclei show a low DNA stainability resulting in a distinct, quantifiable region below the G0/G1 peak (Fig. 2C and D). The easiest and most rapid method for measuring apoptosis is DNA staining with a hypotonic PI solution (Nicoletti et al., 1991), which is particularly suitable for large scale in vitro studies. During apoptosis the plasma membrane undergoes multiple changes (Morris et al., 1984). The increased membrane permeability for Hoechst dyes, which diffuse through intact membranes, is reflected by a strong fluorescence staining after short exposure to low concentrations of Ho342 (Hardin et al., 1992) (Fig. 2E and F). The combination of staining with Ho342 and a PI/PBS solution that does not diffuse through intact cell membranes allows discrimination between, and quantification of, apoptotic, necrotic, and viable cells (Dive et al., 1992). The translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane during
™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ 3 FIG. 2. FACS analysis of BALB/c mouse thymocytes: left panel (A, C, E, G, I) untreated control, right panel (B, D, F, H, J) treated with 1026 M dexamethasone for four hours in vitro. (A, B) Morphological parameters. The apoptotic (Ap) population is characterized by a lower forward scatter (FSC) and higher side scatter (SSC) (B). This population is absent in untreated thymocytes (A). (C, D) Propidium iodide (PI) staining. Apoptotic cells are found in the “sub-G0/G1” peak. (E, F) Hoechst 33342 (Ho342) staining. Apoptotic cells show bright fluorescence. (G, H) Double staining with annexin V-FITC and PI. Early apoptotic cells (annexin V-positive/PI-negative) are localized in the lower right region, late apoptotic, and necrotic cells (annexin V-positive/PI-positive) in the upper right region, and vital cells (double negative) in the lower left region. (I, J): 39OH labeling by TUNEL. 10,000 cells were analyzed in each experiment.
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FIG. 3. Confocal laser scan microscopic images of various thymic cell preparations from an untreated (A, D) and a four-week-old BALB/c mouse treated with 25 mg dexamethasone/kg body weight for eight hours (B, C) showing overlays of transmission and fluorescence scans: apoptotic cells reveal green-stained nuclei after 39OH labeling by the TUNEL technique; intact nuclei are counterstained with propidium iodide (C, D). (A) Viable thymocyte. (B) Thymocyte undergoing apoptosis. Note the nuclear fragmentation and “budding” (arrow). (C) Macrophage (arrow) with two engulfed apoptotic bodies, vital thymocyte (arrow head). (D) Thymic nurse cell harboring an apoptotic thymocyte (arrow). Original magnification 633 oil, 803 zoom (A, B); 633 oil, 403 zoom (C, D).
FIG. 4. Apoptosis detection on frozen, paraformaldehyde-fixed skin sections of a two-week-old C57Bl/6 1pa/1pa mouse. (A) Apoptotic cells were detected by the TUNEL technique using FITC-dUTP for DNA labeling, an anti-FITC-alkaline phosphatase antibody, fast red for visualization, and counterstaining with haemalaun. At this age, apoptotic cells (pink nuclei) are found in some hair follicles (arrows) and the epidermis (arrow head). (B) Simultaneous detection of apoptotic cells with FITC-dUTP (green nuclei) and endothelial cells with anti-von Willebrand factor and TRITC conjugate (red). Original magnification 2003.
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apoptosis can be detected by annexin V, which binds preferentially to PS in the presence of Ca21 (Koopman et al., 1994). To discriminate between apoptotic and necrotic cells the annexin V-FITC binding assay should be combined with PI vital staining (Fig. 2G and H). Ho342 staining and the annexin V assay are both very rapid and simple methods for detection of early apoptosis. But due to increased membrane permeability late apoptotic cells cannot be discriminated from necrotic cells. Early during apoptosis a loss of mitochondrial membrane potential (DCm) occurs. This permeability transition (PT), which is under bcl-2 control, leads to the release of the so-called apoptosis inducing factor (AIF), causing caspase activation and finally cell death. DCm changes can be assessed by mitochondria staining with cationic, lipophilic fluorochromes such as 5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazolcarbocyanine iodide (JC-1), 3,39-dihexyloxacarbocyanine iodide [DiOC6(3)], or rhodamine 123 and flow cytometric analysis. Cells undergoing apoptosis show a reduction in the incorporation of DCm-sensitive dyes (Vayssiere et al., 1994; Zamzami et al., 1995). DCm analysis seems to be a reliable method for studying apoptosis. For the staining of mitochondria in living cells the fluorescent probe should be deliberately chosen as DiOC6(3), for example, is not only sensitive to DCm but also to changes in the plasma membrane potential that might mimic alterations in DCm (Salvioli et al., 1997). DNA strand breaks occurring in apoptotic cells as a result of endonuclease(s) activation can be labeled in situ in individual fixed, permeabilized cells or in tissue sections by in situ nick translation (ISNT) (Fehsel et al., 1991; Gold et al., 1993) or the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technique (Gavrieli et al., 1992; Sgonc et al., 1994, 1996). In both methods, the 39-hydroxyl ends of DNA fragments are labeled either with biotin-dUTP, digoxigenin-dUTP, or FITC-dUTP mediated by DNA polymerase I (ISNT) or by terminal deoxynucleotidyl transferase (TUNEL). In our own hands, indirect labeling with biotin-dUTP or digoxigenin-dUTP and subsequent detection by streptavidin-FITC or fluoresceinated antidigoxigenin resulted in higher background staining compared to direct labeling with FITC-dUTP (unpublished data). Comparison of TUNEL and ISNT shows a more pronounced distinction of apoptotic cells based on the intensity of green fluorescence of labeled vs. unlabeled cells (signal:noise ratio) and a higher sensitivity of the TUNEL technique (Gorczyca et al., 1993). Major advantages of the 39OH end labeling technique include the ability to reveal early DNA breaks during apoptosis, quantitation of the percentage of apoptotic cells by flow cytometric analysis (Fig. 2I and J), the capacity for standard multiple immunofluorescence staining for cell surface markers to characterize cells undergoing apoptosis within heterogenous populations, and its utility in morphological studies (Figs. 3 and 4). In addition, TUNEL can be combined with in situ detection of mRNA expression (Strater et al., 1996). CONCLUDING REMARKS In summary, flow cytometric analysis of PI stained nuclei complemented with light scatter analysis is very useful for simple detection of apoptosis, especially in large-scale experiments. The annexin V binding assay in combination with PI vital staining is a rapid method for detection of early apoptosis. For characterization of cells undergoing apoptosis, simultaneous determination of cell markers and apoptosis by TUNEL is highly recommended. Acknowledgments—This work was supported by the Austrian Research Council (project No. 12715). We thank Heidrun Recheis for CLSM pictures, and Guenther Boeck for flow cytometric analysis.
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