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-β1 treatment

Kelvin Cain, Salmaan H. Inayat-Hussain, Carole Couet, Hong Min Qin, Franziska A. Oberhammer

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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-β1 (TGF-β1). 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-β1 treatment. Thus, thein 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-β1.

Original languageEnglish
Pages (from-to)312-321
Number of pages10
JournalCytometry
Volume23
Issue number4
DOIs
Publication statusPublished - 1 Apr 1996
Externally publishedYes

Fingerprint

DNA Cleavage
Transforming Growth Factors
Chromatin
Hepatocytes
Apoptosis
DNA Breaks
Confocal Microscopy
Flow Cytometry

Keywords

  • Apoptosis
  • End labelling
  • Monolayers
  • Morphological assessment

ASJC Scopus subject areas

  • Hematology
  • Cell Biology
  • Pathology and Forensic Medicine
  • Biophysics
  • Endocrinology

Cite this

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-β1 treatment. / Cain, Kelvin; Inayat-Hussain, Salmaan H.; Couet, Carole; Qin, Hong Min; Oberhammer, Franziska A.

In: Cytometry, Vol. 23, No. 4, 01.04.1996, p. 312-321.

Research output: Contribution to journalArticle

Cain, Kelvin ; Inayat-Hussain, Salmaan H. ; Couet, Carole ; Qin, Hong Min ; Oberhammer, Franziska A. / 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-β1 treatment. In: Cytometry. 1996 ; Vol. 23, No. 4. pp. 312-321.
@article{8e4b81bd479a4d10bef52f69869bb965,
title = "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-β1 treatment",
abstract = "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-β1 (TGF-β1). 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-β1 treatment. Thus, thein 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-β1.",
keywords = "Apoptosis, End labelling, Monolayers, Morphological assessment",
author = "Kelvin Cain and Inayat-Hussain, {Salmaan H.} and Carole Couet and Qin, {Hong Min} and Oberhammer, {Franziska A.}",
year = "1996",
month = "4",
day = "1",
doi = "10.1002/(SICI)1097-0320(19960401)23:4<312::AID-CYTO7>3.0.CO;2-I",
language = "English",
volume = "23",
pages = "312--321",
journal = "Cytometry",
issn = "0196-4763",
publisher = "John Wiley and Sons Inc.",
number = "4",

}

TY - JOUR

T1 - 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-β1 treatment

AU - Cain, Kelvin

AU - Inayat-Hussain, Salmaan H.

AU - Couet, Carole

AU - Qin, Hong Min

AU - Oberhammer, Franziska A.

PY - 1996/4/1

Y1 - 1996/4/1

N2 - 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-β1 (TGF-β1). 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-β1 treatment. Thus, thein 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-β1.

AB - 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-β1 (TGF-β1). 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-β1 treatment. Thus, thein 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-β1.

KW - Apoptosis

KW - End labelling

KW - Monolayers

KW - Morphological assessment

UR - http://www.scopus.com/inward/record.url?scp=0029871999&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029871999&partnerID=8YFLogxK

U2 - 10.1002/(SICI)1097-0320(19960401)23:4<312::AID-CYTO7>3.0.CO;2-I

DO - 10.1002/(SICI)1097-0320(19960401)23:4<312::AID-CYTO7>3.0.CO;2-I

M3 - Article

C2 - 8900474

AN - SCOPUS:0029871999

VL - 23

SP - 312

EP - 321

JO - Cytometry

JF - Cytometry

SN - 0196-4763

IS - 4

ER -