Cytometric Analyses to Distinguish Death Processes

Home , Experimental pathology, Karyolysis, Karyorrhexis

Endocrine-Related Cancer (1999) 6 17-19 Cytometric analyses to distinguish death processes

W Gorczyca

Department of Pathology, Basic Science Building, New York Medical College, Valhalla, New York 10595, USA

Abstract The morphological changes typical of apoptosis, as well as the loss of integrity of the plasma membrane and the breakdown of nuclear DNA provide numerous features that permit recognition of apoptotic cell death by various methods. Flow cytometry (FCM) and laser scanning cytometry (LSC) allow for accurate and rapid measurement of apoptosis in both cultures and clinical samples (e.g. solid tumors, bone marrow aspirates, peripheral blood etc.). Furthermore, both FCM and LSC enable one to correlate the apoptosis with the position of the dying cell in the cell cycle. Discussion includes the cytometric identification and quantitation of apoptotic or necrotic cells, based on the analysis of a particular biochemical or molecular feature that is characteristic for either necrosis or apoptosis.

Endocrine-Related Cancer (1999) 6 17-19

Introduction Arends & Wyllie 1991, Wyllie 1992, Vaux 1993, Bowen et al. 1998) which, together with the role of apoptosis in Cell death can be divided into oncosis and apoptosis tumor progression, explain the wide interest in apoptosis (Majno & Joris 1995, Darzynkiewicz et al. 1997). Both of in recent years. Because of this interest, numerous them eventually lead into oncotic or apoptotic necrosis. techniques have been developed to study apoptosis and to Apoptosis occurs in untreated tumors as a consequence of differentiate it from oncosis (necrosis) (see reviews by many factors, e.g. deprivation of oxygen and other Darzynkiewicz et al. 1992, 1997). The morphological nutrients or individual cell targeting by hunter-killer cells criteria on which recognition of apoptosis is based still like macrophages or NK cells (Bowen et al. 1998). The remain the gold standard of analysis of this mode of cell tumor cells die by apoptosis during chemotherapy or death. The characteristic morphological features of radiotherapy and monitoring the level of apoptosis apoptosis are: reduced size due to cell shrinkage and (‘apoptotic index’) may prove useful in modulating cytoplasmic condensation, plasma membrane undulations treatment or in predicting the biological behavior of (‘blebbing’), condensation of chroma-tin beginning at the tumors following treatment (Gorczyca et al. 1993a, Fisher periphery of the nucleus followed by nuclear fragmen- 1994). In malignant neoplasms, the rate at which a tumor tation (karyorrhexis), dilatation of endo-plasmic reticulum grows depends on the cell proliferation and cell death and, finally, formation of apoptotic bodies (Arends et al. (Wyllie 1992, Bowen et al. 1998). Although these two 1990, Arends & Wyllie 1991). The cell organelles remain processes appear to be opposing each other, they are relatively unchanged in the early stages of apoptosis. The linked as is evident from light microscopical observation events described above usually occur within 30-60 min. of malignant tumors. Usually, the more aggressive tumors Necrotic cells (oncosis) are characterized by cell and with high mitotic rates are also noted to have a much more nuclear swelling, patchy chromatin condensation, swell- pronounced degree of cell death (see Dowsett et al. 1999 ing of mitochondria, vacuolization of cytoplasm and and Lipponen et al. 1999, both in this issue). On the plasma membrane rupture leading to the formation of molecular level, the relationship between proliferation and ‘ghost-like’ cells and, finally, dissolution of DNA cell death can be explained by the participation of the same (karyolysis). oncogenes in the regulation of those processes: oncogenes which force cells into cell cycle by omitting cell cycle checkpoints also induce apoptosis or ‘mitotic catas- Cytometric detection of apoptosis trophe’. The morphological changes typical of apoptosis, loss of Apoptosis is regulated by products of many oncogenes integrity of the plasma membrane and breakdown of or tumor suppressor genes, including c-myc, wt p53, and nuclear DNA provide several features that permit the family of bcl-2 and caspases (Arends et al. 1990, recognition of apoptotic cell death by a number of

Downloaded from Bioscientifica.com at 09/23/2021 04:26:11PM via free access Gorczyca: Cytometric detection of apoptosis methods (Darzynkiewicz et al. 1992, Gorczyca et al. disintegration of the tumor when preparing samples for 1997). flow cytometry. Flow cytometric identification and quantitation of Several fluorochromes can serve as markers of ∆Ψ apoptotic or necrotic cells are generally based on the decreased mitochondrial transmembrane potential ( m), analysis of a particular biochemical or molecular feature which can be used in cytometric discrimination of viable that is characteristic for either necrosis or apoptosis versus dying cells. The most commonly used dyes are (Darzynkiewicz et al. 1992, Dive et al. 1992, Elstein & Rhodamine 123 (Rh123, green fluorescence) and 3,3’ Zucker 1994, Steck et al. 1996, Gorczyca et al. 1998, dihexiloxadicarbocyanine (DiOC6, green fluorescence). Gorczyca et al. 1994). The most commonly applied flow The mitochondrial changes are among the earliest changes cytometric methods are based on detection of endo- in apoptosis. When Rh123 or DiOC6 is applied together nucleolytic DNA degradation that results in extraction of with PI (red fluorescence), live cells stain with Rh123 or low molecular weight DNA from the cell; such cells are DiOC6 (green fluorescence, no PI staining because of then recognized by their fractional DNA content (sub-G1, intact membrane function). Cells with compromised peak, sub-diploid peak). When DNA extraction is plasma membrane (necrotic and late apoptotic cells) stain prevented by crosslinking via cell fixation with formal- only with PI, showing red fluorescence. The early dehyde, the in vivo presence of numerous DNA strand apoptotic cells in contrast to viable cells will have breaks in apoptotic cells can be detected by labeling their markedly diminished stainability with Rhl23 or DiOC6 3' OH termini with fluorochrome-conjugated nucleotides and will have dim green fluorescence. JC-1 is another ∆Ψ in a reaction utilizing exogenous terminal deoxy- probe which can serve to measure m. Its binding to nucleotidyl transferase (TdT assay, ‘TUNEL’, in situ end- mitochondria is detected by the shift in color of labeling) (Gorczyca et al. 1992, 1993b). TdT assay can use fluorescence from green, which is characteristic of its digoxigenin-biotin or directly fluorochrome-conjugated monomeric form, to orange which reflects its aggregation nucleotides, or can be based on incorporation of BrdUrd in mitochondria, driven by the transmembrane potential. and its subsequent detection by FITC-conjugated anti- During apoptosis there is marked decrease in orange, and BrdUrd antibody (Darzynkiewicz et al. 1997). to a lesser degree in green fluorescence of JC-1. One has Other methods are based on detection of apoptosis- to keep in mind, however, that the uptake of these cationic associated changes in distribution of plasma membrane probes mentioned above depends not only on the phospholipids or transport function of the membrane. In mitochondrial transmembrane potential, but also on live non-apoptotic cells, the plasma membrane phospho- transport of these molecules through the plasma lipids are asymmetrically distributed between inner and membrane. In multidrug resistant cells, these fluoro- outer leaflets of the plasma membrane, and phospha- chromes can be pumped out and their measurement will tidylserine (PS) is almost exclusively observed on the not reflect the exact status of the cell. The apoptosis- inner surface of the membrane. Early in apoptosis there is associated changes in cell size and granularity can be a breakdown of this asymmetry and PS undergoes detected by analysis of laser light scattered by the cell in translocation to the external leaflet of the plasma forward or side directions. Light scatter changes, however, membrane. Annexin V, a Ca2+-dependent anticoagulant are not characteristic for apoptosis and this method cannot protein, has high affinity for negatively charged PS and, be used to analyze apoptosis in heterogenous cell when conjugated with a fluorochrome (e.g. Annexin V- populations. FITC) can be used as a marker to identify apoptosis The major advantage of flow cytometry is that, like no (together with DNA staining with propidium iodide, PI). other methodology, it offers the possibility of multi- Non-apoptotic cells are Annexin V-negative and PI- parametric analysis of several cell attributes, including negative (negative green and red fluorescence), early cell cycle position. On the other hand, the major drawback apoptotic cells are Annexin V-positive but PI-negative, of flow cytometric methods stems from the fact that and late apoptotic cells as well as necrotic cells are stained identification of apoptotic or necrotic cells is not based on intensely with PI (only red fluorescence). The Annexin V morphology and cannot be correlated with morphological assay offers the possibility of detecting early phases of classification. Rather, it relies on a single parameter apoptosis before the loss of cell membrane integrity and reflecting the change in biochemical or molecular permits measurement of the kinetics of apoptotic death in attributes of the cell, presumed to represent either relation to the cell cycle. It is a very easy and fast method apoptosis or necrosis. Most of the flow cytometric and can be applied in the analysis of apoptosis in cell methods do not allow for a clear distinction between late cultures or leukemic cells from peripheral blood or bone apoptotic and necrotic cells. When in doubt, extensive marrow aspirates. It is not, however, very useful in DNA fragmentation detected by DNA gel electrophoresis evaluation of apoptosis in solid tumors, most likely due to or TdT assay may serve as a marker of apoptosis rather damage to the membrane during processing and/or than necrosis. Flow cytometric measurement of apoptosis,

Downloaded from Bioscientifica.com at 09/23/2021 04:26:11PM via free access Endocrine-Related Cancer (1999) 6 17-19 regardless of the method used, is associated with selective Dowsett M, Archer C, Assesrshon L, Gregory RK, Ellis PA, loss of apoptotic cells during sample preparation. Salter JM, Chang J, Mainwaring P, Boeddinghaus I, Johnston Laser scanning cytometry (LSC) is a microscope- SRD et al. 1999 Clinical studies of apoptosis and proliferation based cytofluorimeter that combines the advantages of in breast cancer. Endocrine-Related Cancer 6 25-28. flow cytometry and image analysis (Kamentsky & Elstein KH & Zucker RM 1994 Comparison of cellular and Kamentsky 1991). Fluorescence of individual cells is nuclear flow cytometric techniques for discriminating measured rapidly, with high sensitivity and accuracy apoptotic subpopulations. Experimental Cell Research 211 comparable to that of flow cytometry. Since the cells are 322-333. prepared and measured on a slide, LSC is especially useful Fisher DE 1994 Apoptosis in cancer therapy: crossing the when clinical material is limited, as in fine needle aspirates threshold. Cell 78 539-542. or touch smears. The major advantage of LSC, however, Gorczyca W, Bruno S, Darzynkiewicz RJ, Gong J & is that each cell’s coordinates (X-Y position) are recorded Darzynkiewicz Z 1992 DNA strand breaks occurring during together with fluorescence values, so that any selected apoptosis: their early in situ detection by the terminal cell(s) can be relocated for visual examination (light or deoxynucleotidyl transferase and nick translation assays and fluorescence microscope) or to capture its image by CCD prevention by serine protease inhibitors. International camera (‘CompuSorting’). Almost all of the methods Journal of Oncology 1 639-648. developed for identification of apoptotic and necrotic cells Gorczyca W, Bigman K, Mittelman A, Ahmed T, Gong J, can be modified and adapted for LSC. LSC measurement Melamed MR & Darzynkiewicz Z 1993a Induction of DNA of total nuclear or cellular fluorescence is done by strand breaks associated with apoptosis during treatment of integration of light intensity of individual pixels over the leukemia. Leukemia 7 659-670. area of nucleus and/or cytoplasm, measuring both the Gorczyca W, Gong J & Darzynkiewicz Z 1993b Detection of intensity of individual pixels as well as the fluorescence DNA strand breaks in individual apoptotic cells by the in vivo area (number of pixels). Maximal pixel intensity within terminal deoxynucleotidyl transferase and nick translation the measured area is also measured. Because apoptotic assays. Cancer Research 52 1945-1951. cells, similarly to mitotic cells, have strongly condensed Gorczyca W, Tuziak T, Kram A, Melamed MR & Darzynkiewicz chromatin, they can be identified based on values of the Z 1994 Detection of apoptosis-associated DNA strand breaks maximal pixels of DNA-associated fluorescence. This in fine-needle-aspiration biopsies by in situ end labeling of approach, however, cannot differentiate between apop- fragmented DNA. Cytometry 15 169-175. totic and mitotic cells. Gorczyca W, Darzynkiewicz Z & Melamed MR 1997 Laser The choice of method used to study apoptosis depends scanning cytometry in pathology of solid tumors. Acta on many factors, including type of specimen (cell cultures Cytologica 41 98-108. versus solid tumors), the stage of cell death (early versus Gorczyca W, Melamed MR & Darzynkiewicz Z 1998 Analysis late apoptosis) and the technical possibilities available to of apoptosis by flow cytometry. In Methods in Molecular the laboratory. Biology. 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