RNA synthesis and splicing interferes with DNA in situ end labeling techniques used to detect apoptosis

Abstract

The detection of DNA fragmentation by the use of the TUNEL technique has become a standard technique for the detection of apoptosis in tissue sections. DNA cleavage, detected by the TUNEL technique, is the last irreversible stage of the apoptosis cascade. When the nuclear DNA is cleaved in oligonucleosomal-sized fragments, processes such as transcription are completely abolished. The values of apoptotic cell death that were obtained for atherosclerotic plaques by the TUNEL technique ranged from less than 2% up to 60%. The latter value would indicate that plaques are in an imminent state of collapse, which is certainly not the case. Other factors that could result in TUNEL labeling have to be considered. Therefore, we developed a co-localization system and studied TUNEL labeling together with markers of RNA transcription and splicing. The present study demonstrates that, besides apoptotic nuclei, non-apoptotic nuclei that show signs of active gene transcription are labeled by the TUNEL technique. The fact that the TUNEL technique is not specific for the executive phase of apoptosis is not surprising, as the technique is only selective (rather than specific) for apoptotic nuclei as these contain a far greater degree of DNA fragmentation than non-apoptotic nuclei. Therefore, we think that the TUNEL technique should be combined with additional techniques, such as markers of transcription and morphological criteria.