An ultrastructural approach to the study of apoptotic double strand DNA cleavage

Apoptotic DNA cleavage initially produces large fragments (50 kbp), followed by the formation of nucleosomic/ oligonucleosomic ones. On the other hand, apoptosis without DNA fragmentation, at least the nucleosomic one, has been described. To study the correlation between the DNA cleavage and the well known chromatin behavior, we applied TUNEL to electron microscopy, by using a gold-conjugated antidigoxigenin antibody, on U937 and Molt-4 cells, both exposed to UVB or staurosporine. Gold particle density in the different domains of apoptotic nuclei was statistically evaluated. Gold labelling was more intense in dense apoptotic chromatin than in the diffuse one. U937 cells, which evidenced in vitro oligonucleosomic fragmentation after both treatments, revealed a significantly higher gold particle density, when compared with Molt-4, which did not, even if showing larger DNA fragments in vitro. DNA fragment sizes, characterized by gel electrophoresis and by FIGE, appeared closely correlated to gold particle density on apoptotic chromatin domains. TUNEL applied to electron microscopy is an useful tool to highlight mechanisms underlying apoptotic chromatin condensation and DNA cleavage patterns