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Journal of Cell Science, Vol 104, Issue 2 317-326, Copyright © 1993 by Company of Biologists
JOURNAL ARTICLES |
F Oberhammer, G Fritsch, M Schmied, M Pavelka, D Printz, T Purchio, H Lassmann and R Schulte-Hermann
Institute of Tumorbiology-Cancer Research, Department of Toxicology, University of Vienna, Austria.
A current hypothesis holds that chromatin fragmentation into oligonucleosomal patterns is an early event during apoptosis. In contrast, induction of apoptosis in cultured hepatocytes by TGF-beta 1 was not associated with DNA fragmentation into oligonucleosomes in hepatocyte monolayers and apoptotic fragments. For a more rigorous test of the hypothesis we performed a number of experiments. We compared nuclear changes resulting from TGF-beta 1 with those induced by Ca2+, a known activator of endonuclease. The morphology of apoptotic and Ca(2+)-treated nuclei was different as judged by DNA staining with Hoechst 33258. Likewise, electron microscopy of apoptotic nuclei showed characteristic condensation of the chromatin as well as dissolution of the nucleolar structure and nuclear fragmentation, changes not seen after Ca2+ treatment, after three hours of incubation. Analysis of DNA fluorescence of nuclei by FACS revealed that treatment with Ca2+ reduced the signal by 20%. In contrast, nuclei from TGF-beta 1-treated hepatocytes did not exhibit a reduced signal and after sorting by FACS, apoptotic nuclei remained in the 2N and 4N fractions. The absence of detectable DNA fragmentation in apoptotic nuclei was further verified by in situ nick translation, not only in hepatocytes but also in a mouse lymphoma cell line. From these findings we conclude that activation of an endonuclease is not an early event on the pathway to morphologically recognizable apoptosis.
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