A novel method for detecting apoptosis shows that hepatocytes undergo a time dependent increase in DNA cleavage and chromatin condensation which is augmented after TGF-beta 1 treatment.
This study describes a new method for quantitating apoptosis in hepatocyte monolayers in which nuclei were isolated from the cells and DNA strand breaks detected by in situ end-labeling and flow cytometry. Most (97%) nuclei from untreated hepatocytes had low end-labelling and were derived from non-apoptotic cells. Approximately 2-3% of the nuclei had high end-labelling and originated from apoptotic hepatocytes. The numbers of these nuclei increased linearly from 3 to 85% between 0 and 48 h after treatment with transforming growth factor-beta 1 (TGF-beta 1). However, a morphological assessment of apoptosis with Hoechst H33258 showed that the proportion of apoptotic nuclei plateaued at 18-19% between 24 and 48 h after TGF-beta 1 treatment. Thus, the in situ end-labeling technique also detected DNA cleavage in nuclei which did not have an obvious apoptotic morphology. Confocal microscopy of low and high end-labelled nuclei which had been separated by fluorescent cell sorting showed that nuclei with high levels of end-labeling exhibited a wide diversity of morphologies. These included nuclei with little or no chromatin condensation and nuclei with characteristic apoptotic morphology. In addition, nuclei from untreated hepatocytes contained low levels of DNA cleavage, which were localized in areas of condensed chromatin and increased according to the time in culture. Thus, hepatocytes undergo a progressive and cumulative process of DNA cleavage/chromatin condensation which is markedly enhanced by TGF-beta 1.
Cain
K
K
MRC Toxicology Unit, Leicester, United Kingdom.
Inayat-Hussain
S H
SH
Couet
C
C
Qin
H M
HM
Oberhammer
F A
FA
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002467:Cell Nucleus; D002478:Cells, Cultured; D002843:Chromatin; D004249:DNA Damage / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods*; D008099:Liver / Q000166:cytology / Q000502:physiology*; D008297:Male; D018613:Microscopy, Confocal; D051381:Rats; D011916:Rats, Inbred F344; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D002843:Chromatin; D016212:Transforming Growth Factor beta