Quantitative Differentiation of Both Free 3′-OH and 5′-OH DNA Ends Between Heat-induced Apoptosis and Necrosis

Abstract

Cell death is roughly categorized as either apoptosis or necrosis. For better understanding of the differences in DNA cleavage between them, we performed quantitative analysis of both the 3′-OH and the 5′-OH ends of DNA strand breaks via in situ nick-end labeling (ISEL) combined with transmission electron microscopy (TEM) of both heat-induced apoptosis and necrosis in mouse B-cells derived from a lymphoma cell line. To detect the 5′-OH ends, the 3′-P ends located on the opposite side holding the 5′-OH ends were dephosphorylated into 3′-OH ends with alkaline phosphatase. As assessed by statistical analysis of both the 3′-OH and the 5′-OH ends, their labeling densities were significantly higher in both the apoptotic and the necrotic cells in the early stage than in control cells. The labeling densities increased during the apoptotic and necrotic processes, except for a decrease in the density of the 3′-OH ends in necrotic cells in the late stages. Therefore, DNA degradation in both necrosis and apoptosis provides early evidence for these processes, and both apoptosis and necrosis may share at least the first steps of DNA degradation pathways.