DETECTION OF DNA FRAGMENTATION IN APOPTOSIS - APPLICATION OF INSITU NICK TRANSLATION TO CELL-CULTURE SYSTEMS AND TISSUE-SECTIONS

Since DNA fragmentation is a key feature of programmed cell death (PCD ) and also occurs in certain stages of necrosis, we have adapted the m ethodology of in situ nick-translation (ISNT) to detect DNA fragmentat ion on a single-cell level. We first established the technique for cel l preparations. Apoptosis was induced by gamma-irradiation on freshly isolated rat thymocytes. After fixation procedures, ISNT was performed by overnight incubation either with fluorescein-12-UTP or with digoxi genin-labeled 11-dUTP and DNA polymerase I. The enzymatic incorporatio n of labeled nucleotides at sites of DNA fragmentation was detected by flow cytometry either directly on indirectly with fluorescein-conjuga ted anti-digoxigenin. The quantitative results demonstrated dose corre lation with morphological essays for apoptosis, DNA gel electrophoresi s, and ISNT. Proliferating cells determined by bromodeoxyuridine immun ofluorescence were not labeled by ISNT. Immunocytochemistry for cell s urface antigens in combination with ISNT allowed the identification of specific cell types undergoing PCD. Furthermore, the simultaneous app lication of photolabeling techniques with ethidium monoazide and ISNT led to the identification of DNA fragmentation in cells with still int act membranes. Extending ISNT to tissue sections of paraformaldehyde-f ixed, paraffin-embedded material reliably revealed labeling of cells w ith typical morphological features of apoptosis. However, this techniq ue was not useful in detecting early stages of necrotic cell death.