Assays for DNA fragmentation, endonucleases, and intracellular pH and Ca2+ associated with apoptosis

The methods described here facilitate assessment of the DNA digestion that was once considered a hallmark of apoptosis, but is now recognized as a common overdigestion of genomic DNA; less frequent breaks also lead to the morphological appearance of an apoptotic nucleus. Whatever level of DNA digestion occurs, it may be brought about by activity of one of a number of endonucleases. Whether one endonuclease is responsible in all cases of apoptosis or whether different endonucleases are responsible in various systems and circumstances remains unknown. In attempting to identify changes in ion concentrations that might activate these endonucleases, we have observed intracellular acidification to correlate with apoptosis consistently. This observation may have other implications since the acidification could be responsible for activating other proteins, such as proteases, which are also associated with apoptosis. The importance of such events to the onset of apoptosis remains to be fully established.