Biochemical Assessment of Apoptosis

Many biochemical methods have been developed for the assessment of apoptosis. They are based on the analysis of cellular morphology, DNA fragmentation, or 

In normal viable cells, PS localizes predominantly in membrane leaflets facing the cytosol (S6). Fadok et al. established the fundamentals for a novel apoptosis detection methodology by showing that apoptotic cells expose PS at their outer leaflet of cell surface during early apoptosis (El). This change of exposure 

Propidium iodide can be used to assess plasma membrane integrity in annexin V apoptosis assays. It does not cross the plasma membrane of cells that are viable or in the early stages of apoptosis because of their plasma membrane integrity. In contrast, cells in the late stages of apoptosis or already dead have lost plasma membrane integrity and are permeable to PI for DNA staining (Fig. 5). In flow cytometric assays, another nucleic acid dye that can be used in place of PI for the exclusion of nonviable cells is 7-AAD. The advantage of 7-AAD over PI is its ability to be used in conjunction with phycoerythrin (PE)- and FITC-labeled monoclonal antibodies with minimal spectral overlap between the 7-AAD, PE, and FTTC fluorescence emissions. 

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) Assay 

The combination of annexin V binding assay and the TUNEL method can reveal the presence of three subpopulations of apoptotic cells in tissues (1) annexin V-positive/TUNEL-negative cells which are in the early phase of apoptosis, 

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Fig. 6. Apoptotic DNA ladder pattern of eosinophils treated with dexamethasone (Dexa, 2 (xM) for 18 h (Zl). DNA was extracted from cells with ethanol (P4) and electrophoresed on 1% agarose gel in 1 X TAE (Tris acetate-EDTA) buffer (pH 8.0). After electrophoresis, the gel was soaked in 1 x TAE buffer containing 0.5 /tg/ml ethidium bromide, and DNA was visualized by an ultraviolet illuminator. Reproduced with permission from Zhang, J. R, Wong, C. K., Lam, C. W. K., Ho, C. Y., and Hjelm, N. M., Biochemical assessment of apoptosis. Chinese J. Lab. Med. Clin. Sci. 1, 27-28 (2000).

Welsh, N. Assessment of apoptosis and necrosis in isolated islets of Langerhans Methodological considerations. Curr. Top. Biochem. Res. 2000, 3, 189-200. 

Additionally, patients with familial adenomatous polyposis, an autosomal-dominant disease characterized by numerous small intestinal and colonic polyps with a nearly universal progression to colon cancer, have a favorable response to NSAIDs. Administration of NSAID (usually sulindac) to patients with this disorder reduces the number and size of polyps (DuBois et al., 1996). Recent biochemical evidence indicates that colon polyps and colon cancer are frequendy associated with induction of Cox-2 in the lesion as assessed by expression of Cox-2 mRNA and protein. Such induction appears to correlate with growth of the lesion, and inhibition of Cox-2 correlates with apoptosis of the involved cells (Gupta and DuBois, 1998). 

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