Apoptosis is an evolutionarily conserved form of physiological cell death by which redundant and damaged cells are eliminated during embryonic development and tissue homeostasis. In diseased tissue the process may be disturbed in such a way that accumulation of cells or increased cell loss may occur. Rapid elimination of the dying cell, without evoking an inflammatory response, is accomplished by disintegration of the cell into apoptotic bodies, which will then be phagocytosed (1). During this process, in which the plasma membrane remains intact, cleavage of cytoskeletal and nucleoskeletal proteins occurs by caspases, a family of cysteine proteases that cleave key proteins at conserved aspartic amino acid residues. The cleavage of cell-cell adhesion molecules leads to loss of contact with neighbouring cells (2), proteolysis of nuclear proteins facilitates nuclear disassembly (3), and the reorganization of actin filaments plays a role in the formation of apoptotic bodies (4). Proteolysis of fodrin may play a role in the loss of plasma membrane lipid asymmetry, resulting in exposure of phosphatidylserine (PS) at the outer leaflet of the cell membrane (5). Apoptosis can be detected on the basis of the characteristic morphological changes, such as chromatin aggregation and formation of apoptotic bodies. Furthermore, techniques have been developed which are based on apoptosis-specific biochemical changes (e.g. DNA fragmentation), expression of apoptosis-associated proteins, proteolysis of apoptosis-specific substrates, generation of apoptosis-specific neo-epitopes, or exposure of specific phospholipids at the outer plasma membrane. However, the individual techniques detect specific events in the process of apoptosis and therefore every technique has its limitations. In this chapter we describe methods to study changes in cyto- and nucleoskeletal proteins during apoptosis by means of immunocytochemistry. For detection of apoptotic cells the annexin V affinity assay and the TUNEL assay is used. The applications and limitations of the detection systems will be discussed.

0 0

Post a comment