DIFFERENTIATION BETWEEN CELLULAR APOPTOSIS AND NECROSIS BY THE COMBINED USE OF IN-SITU TAILING AND NICK TRANSLATION TECHNIQUES

BACKGROUND: A number of enzymatic techniques have recently been develo ped to detect DNA fragmentation in apoptosis at the cellular level. Ho wever, since DNA fragmentation also occurs in cellular necrosis, we st udied to which extent the use of DNA polymerase (nick translation) or terminal transferase (tailing) allows the differentiation between inte rnucleosomal DNA degradation, typical for apoptosis, and the more rand om DNA destruction in necrosis. EXPERIMENTAL DESIGN: We compared these techniques on in vitro and in vivo models for apoptotic or necrotic c ell death. Apoptosis of thymocytes in vitro was induced by gamma-irrad iation, necrosis by the cytotoxic action of antibody and complement. C ell death in vivo was examined on paraffin-embedded tissue material fr om animals with autoimmune encephalomyelitis that served as a model fo r apoptosis, or in kainic acid-induced nerve cell degeneration as a mo del for necrosis. RESULTS: DNA fragmentation was visualized by the inc orporation of labeled nucleotides into the nuclei of affected cells ut ilizing tailing or nick translation techniques. In the early stages of cell degeneration in vitro, cells undergoing apoptosis were preferent ially labeled by tailing, whereas necrotic cells were identified by ni ck translation. Similarly, early stages of necrosis in vivo were prefe rentially detected by nick translation, whereas tailing was slightly m ore sensitive for the detection of apoptosis. Results obtained with th ese enzymatic techniques were in accord with the assessment of cell de ath by morphologic criteria. Both techniques could be applied in tissu e samples even after prolonged fixation in paraformaldehyde if the sec tions were pretreated with proteinase R digestion. CONCLUSIONS: Our st udies show that both in situ nick translation and in situ tailing are useful in detecting DNA fragmentation in cell suspensions and tissue s ections. These techniques may help to define the molecular mechanisms leading to cell death in experimental conditions and eventually in hum an tissue.