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 developed to detect DNA fragmentation in apoptosis at the cellular level. However, since DNA fragmentation also occurs in cellular necrosis, we studied to which extent the use of DNA polymerase (nick translation) or terminal transferase (tailing) allows the differentiation between internucleosomal DNA degradation, typical for apoptosis, and the more random DNA destruction in necrosis.


METHODS
We compared these techniques on in vitro and in vivo models for apoptotic or necrotic cell death. Apoptosis of thymocytes in vitro was induced by gamma-irradiation, necrosis by the cytotoxic action of antibody and complement. Cell death in vivo was examined on paraffin-embedded tissue material from animals with autoimmune encephalomyelitis that served as a model for apoptosis, or in kainic acid-induced nerve cell degeneration as a model for necrosis.


RESULTS
DNA fragmentation was visualized by the incorporation of labeled nucleotides into the nuclei of affected cells utilizing tailing or nick translation techniques. In the early stages of cell degeneration in vitro, cells undergoing apoptosis were preferentially labeled by tailing, whereas necrotic cells were identified by nick translation. Similarly, early stages of necrosis in vivo were preferentially detected by nick translation, whereas tailing was slightly more sensitive for the detection of apoptosis. Results obtained with these enzymatic techniques were in accord with the assessment of cell death by morphologic criteria. Both techniques could be applied in tissue samples even after prolonged fixation in paraformaldehyde if the sections were pretreated with proteinase K digestion.


CONCLUSIONS
Our studies show that both in situ nick translation and in situ tailing are useful in detecting DNA fragmentation in cell suspensions and tissue sections. These techniques may help to define the molecular mechanisms leading to cell death in experimental conditions and eventually in human tissue.
Gold
R
R
Department of Neurology, Julius-Maximilians-Universität Würzburg.
Schmied
M
M
Giegerich
G
G
Breitschopf
H
H
Hartung
H P
HP
Toyka
K V
KV
Lassmann
H
H
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D016923:Cell Death / Q000235:genetics; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase; D005434:Flow Cytometry; D005821:Genetic Techniques; D008297:Male; D009336:Necrosis; D009474:Neurons / Q000473:pathology; D051381:Rats; D011917:Rats, Inbred Lew; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D013950:Thymus Gland / Q000473:pathology; D014166:Transferases
D014166:Transferases; D004259:DNA-Directed DNA Polymerase