Early Necrotic DNA Degradation: Presence of Blunt-Ended DNA Breaks, 3′ and 5′ Overhangs in Apoptosis, but only 5′ Overhangs in Early Necrosis

Abstract

The structure of DNA breaks in early necrosis was analyzed and compared with apoptotic DNA degradation using in vivo and cell culture models. Early necrosis (1 hour after cell death) was produced in vivo by the freezing-thawing of rat thymus and in cell culture of Jurkat cells. Apoptosis was induced in the same cell types using dexamethasone for thymus and staurosporine for Jurkat cells. Selective detection of double-strand DNA breaks with blunt ends was performed by in situ ligation. Blunt-ended breaks bearing 5' phosphates were detected in apoptotic but not in early necrotic cells. Pretreatment of apoptotic and necrotic tissue with Klenow enzyme with or without added dNTPs reduced all 3' or 5' overhangs to blunt ends. Subsequent in situ ligation with blunt-ended probes revealed no 3' overhangs in necrotic cells. However double-strand cuts with 5' overhangs were abundant in necrotic DNA. 5' Overhangs were also detected in apoptotic cells. Presence of exclusively 5' overhangs in early necrosis with absence of a variety of possible DNA ends, suggests the existence of a specific orderly mechanism of DNA degradation.