Apoptotic DNA fragmentation can be revealed in situ: An ultrastructural approach

Abstract

A common pattern of apoptotic death is DNA cleavage, initially producing large fragments (50 kbp), followed by the production of nucleosomic/oligonucleosomic fragments. Nevertheless, apoptosis without DNA fragmentation, at least of the nucleosomic type, has been reported. To investigate the spatial relationship between DNA cleavage and chromatin condensation, we applied the TUNEL technique to the ultrastructural analysis of apoptotic cells. A modified method, utilizing a gold-conjugated antidigoxigenin antibody, was carried out on U937 versus Molt-4 cells, both exposed to UVB radiation or staurosporine treatment. Gold particle density in the different domains of apoptotic cells was evaluated by a four-way ANOVA test. Gold labelling was more strongly localised in condensed chromatin than in the diffuse chromatin. U937 cells, which evidenced in vitro oligonucleosomic fragmentation after both UVB and staurosporine treatments, revealed a significantly higher gold particle density, when compared with Molt-4, which did not show, on the other hand, oligonucleosomic cleavage even in the presence of < or = 50 kbp cleavage. Thus, a correlation between DNA fragment sizes and gold particle density appears. TUNEL applied to electron microscopy is an effective approach to study the relationship between apoptotic chromatin condensation and DNA cleavage. Both these events indeed appear in the apoptotic nucleus, but their reciprocal correlation is still greatly unknown. Microsc. Res. Tech. 2009. (c) 2009 Wiley-Liss, Inc.