Flow cytometry of apoptotic cell death

Abstract

The term apoptosis or programmed cell death defines a genetically encoded cell death program, which is morphologically and biochemically distinct from necrosis or accidental cell death. The characteristic morphological signs of apoptosis (cellular shrinkage, membrane blebbing, nuclear condensation and fragmentation) are the final results of a complex biochemical cascade of events which is an integral part of physiological homeostasis. Techniques designed to identify, quantitate and characterize apoptosis are numerous, but flow cytometry (FCM) remains the methodology of choice to study the apoptotic cascade in relation to cell type, trigger and time. This review outlines the main stages of the apoptotic cascade together with current FCM methods. All FCM apoptosis assays described have a solid experimental basis and have been used successfully in basic research on molecular and biochemical mechanisms of apoptosis. In various clinical settings the ability to follow the apoptotic process in patient samples may offer the rationale for optimal treatment schedules.

Introduction

The term apoptosis or programmed cell death (PCD) defines a genetically encoded cell death program, which is morphologically, biochemically and molecularly distinct from necrosis. Apoptosis is distinguished from necrosis by unique events including the degradation of chromatin into internucleosomal fragments, a loss of cellular volume associated with cytoskeletal breakdown and blebbing of the plasma membrane. Apoptotic and necrotic cell death are different from a mechanistic point of view because necrosis is merely the passive result of cellular injury and apoptosis forms an integral part of normal physiological cell processes. Apoptosis ensures an equilibrium between cell proliferation and cell death and plays a regulatory role in the control of the size of cell populations and tissues (Kerr et al., 1972). Continuous signalling by growth factors, hormones, cytokines, cell–cell contacts and cell–matrix interactions are necessary for cells to refrain from undergoing apoptosis, keeping them alive. Cells which are most sensitive for survival signals stay alive and cells that can not compete with their more avid sister cells undergo apoptosis due to relative shortness of survival factors. Aberrations in cell death signalling, in membrane or cytoplasmic receptors or alterations in genes that govern apoptosis are involved in the pathogenesis of congenital malformations and many acquired diseases (Haanen and Vermes, 1996). Too little apoptosis may result in malignancies (Sturzbecher et al., 1990, Tomlinson and Bodmer, 1995), lymphoproliferative diseases (Nagata and Golstein, 1995), leukemias (Mapara et al., 1993, Lepelley et al., 1995, Sachs, 1996, Estrov and Talpaz, 1996), resistance to anticancer therapy (Fisher, 1994, D’Amico and McKenna, 1994, Pahor et al., 1996), persistence of viral infections (Chiou et al., 1994, Haanen and Vermes, 1995) or the occurrence of autoimmune diseases (Nagata and Suda, 1995, Tax et al., 1995). Too much apoptosis can result in immune deficiency (Groux et al., 1991, Ameisen, 1992, Meyaard et al., 1992, Kabelitz et al., 1993, TeVelde et al., 1996) and degenerative conditions (Griffith et al., 1995). It is therefore important to discriminate between necrosis and apoptosis in order to learn how to modulate apoptosis in view of its potential therapeutic use.

The very nature of the apoptotic cell death promotes the underestimation of this phenomenon, because apoptosis involves scattered single cells of which the early stages of the apoptotic process escape recognition, the apoptotic bodies are small and undergo rapid phagocytosis. The duration of the whole process takes not more than a few hours and finally, any inflammatory reaction is absent (Wyllie et al., 1980, Trump et al., 1982).

The various cell biological alterations, which occur during the process of apoptosis take place in an orderly sequence. A number of these can be exploited to discriminate vital and dying cells and to analyse the extent and the type of cell death (apoptosis or necrosis). In this review those cellular changes, which can be measured by flow cytometry (FCM), are discussed according to the sequence at which they occur. Apoptosis starts with cell shrinkage, expressed by changes in cell light-scatter (2) signals. Initially the integrity of the cell membrane (3) remains intact. Activation of caspases (4), leads to the transition of the mitochondrial membrane potential (5), accompanied by intracellular shifts in Ca²⁺ and pH (6). After loss of the mitochondrial membrane potential, the lysosomal membrane pumps (7) lose their function. At this stage, when the cell has passed the point of no return, endogenous endonucleases are activated, which is reflected by phosphatidylserine redistribution (11). Finally the cell disintegrates into apoptotic bodies (12) (Fig. 1). Detailed descriptions of cell features, which can be measured by FCM during apoptosis have recently been given in comprehensive reviews by Darzynkiewicz et al. (Darzynkiewicz et al., 1992, Darzynkiewicz et al., 1994, Darzynkiewicz et al., 1997), Telford et al. (1994), Vermes and Haanen (1994), Vermes et al. (1998), Gorczyca et al. (1998) and Robinson et al. (1998).