Optimization and Validation of RT-PCR as a Tool to Analyze Gene Expression During Apoptosis

Apoptosis is a mode of cell death that has been defined largely on the basis of morphology (Wyllie et al, 1980; Clarke, 1990). The primary criteria used to delineate cell death as “apoptotic” are the condensation of nuclear chromatin and the fragmentation of nuclear DNA into oligonucleosomal-sized fragments (multiples of about 180 bp). After the stimulus that initiates cell death, cells undergoing apoptosis typically show few early morphological alterations. The polyribosomes then disintegrate and may form semicrystal-line arrays concurrent with decreased global protein synthesis. The cell atrophies, but mitochondria and other organelles remain physically intact. Cells transiently show blebbing of the plasma membrane. Subsequent to the nuclear changes described above, cytoplasmic organelles start to degenerate and both nucleus and cytoplasm become compartmentalized into membrane-bound apoptotic bodies that are engulfed by phagocytic cells in intact tissues. A common, but not universal, characteristic of apoptosis is its dependence on continued RNA and protein synthesis.