Differential staining of DNA strand breaks in dying cells by non-radioactive in situ nick translation

Abstract

Various lines of evidence indicate the involvement of DNA strand breaks (DSB) in the regulation of physiological states of cells, especially in cell death. Currently, cell death is divided into two categories, apoptosis and necrosis. As lysosomal integrity is maintained in apoptosis, while disrupted in necrosis, it is possible to assume that necrotic chromatin is exposed to digestion by various lysosomal enzymes. We have therefore investigated whether apoptotic DSB and necrotic DSB can be discriminated by in situ nick translation (INT) under various conditions of protease pretreatment. Used models of apoptosis and necrosis were the rat thymus with an intraperitoneal injection of hydrocortisone (10 mg/100 g body weight (b.w.)) and rat liver with an intraperitoneal injection of CCl4 (100 microliters/g b.w.), respectively. As results, we found that necrotic DSB was readily detected by INT without protein digestion, whereas apoptotic ones were not. These results indicate that the environment around DSB and/or the nature of DSB in apoptosis differs from that of necrosis, and that INT is a convenient molecular histochemical tool to discriminate both types of cell death in frozen sections.