Search

A number of methods have been developed to examine the morphologic, biochemical, and molecular changes that happen during the DNA damage response that may ultimately lead to death of cells through various mechanisms that include apoptosis. When cells are exposed to ionizing radiation or chemical DNA-damaging agents, double-stranded DNA breaks (DSB) are generated that rapidly result in the phosphorylation of histone variant H2AX. Because phosphorylation of H2AX at Ser 139 correlates well with each DSB, phospho-H2AX is a sensitive marker to used to examine the DNA damage and its repair. Apoptotic cells are characterized on the basis of their reduced DNA content and morphologic changes, including nuclear condensation, which can be detected by flow cytometry (sub-G1 DNA content), trypan blue, or Hoechst staining. The appearance of phosphatidylserine on the plasma membrane with annexin V-fluorochrome conjugates indicates the changes in plasma membrane composition and function. By combining it with propidium iodide staining, this method can also be used to distinguish early versus late apoptotic or necrotic events. The activation of caspases is another well-known biochemical marker of apoptosis. Finally, the Bcl-2 family of proteins and the mitochondria that play a critical role in DNA damage-induced apoptosis can be examined by translocation of Bax and cytochrome c in and out of mitochondria. In this chapter, we discuss the most commonly used techniques used in our laboratory for determining the DNA damage response leading to apoptosis.

A number of methods have been developed to identify the cells that undergo apoptosis by analyzing the morphological, biochemical, and molecular changes that take place during this universal biological process. The best recognized biochemical hallmark of both early and late stages of apoptosis is the activation of cysteine proteases (caspases). Detection of active caspase-3 in cells and tissues is an important method for apoptosis induced by a wide variety of apoptotic signals. Most common assays for examining caspase-3 activation include immunostaining, immunoblotting for active caspase-3, colorimetric assays using fluorochrome substrates, as well as employing the fluorescein-labeled CaspaTag pan-caspase in situ detection kit.

A large number of methods devoted to the identification of apoptotic cells and the analysis of the morphological, biochemical, and molecular changes that take place during this universal biological process have been developed. Apoptotic cells are recognized on the basis of their reduced DNA content and morphological changes that include nuclear condensation and which can be detected by flow cytometry (sub-G1 DNA content), Trypan Blue, or Hoechst staining. Changes in plasma membrane composition and function are detected by the appearance of phosphatidylserine on the plasma membrane, which reacts with Annexin V-fluorochrome conjugates. Combined with propidium iodide (PI) staining, this method can distinguish between the early and late apoptotic events. The best-recognized biochemical hallmarks of apoptosis are the activation of cysteine proteases (caspases), condensation of chromatin, and fragmentation of genomic DNA into nucleosomal fragments. Recognized by a variety of assays, activated caspases cleave many cellular proteins and the resulting fragments may serve as apoptosis markers. Finally, the mitochondria and the Bcl-2 family proteins play an important role in this process that can be recognized by translocation of apoptogenic factors, such as Bax and cytochrome c, in and out of mitochondria.