PMID- 11334733
OWN - NLM
STAT- MEDLINE
DCOM- 20010816
LR  - 20220409
IS  - 0968-4328 (Print)
IS  - 0968-4328 (Linking)
VI  - 32
IP  - 7
DP  - 2001 Oct
TI  - Chromatin condensation and sensitivity of DNA in situ to denaturation during cell 
      cycle and apoptosis--a confocal microscopy study.
PG  - 645-52
AB  - The goal of this study was to construct high resolution 3D confocal images of 
      regions of condensed and extended chromatin in cell nuclei and individual 
      chromosomes. It has been shown previously that sensitivity of DNA in situ to 
      denaturation correlates with chromatin condensation and varies during cell cycle 
      and apoptosis. Thus, detection of DNA which was partially denatured in situ 
      provided a means to image areas of condensed chromatin. DNA denaturation was 
      detected using a metachromatic dye acridine orange (AO) which differentially 
      stains single stranded (ss) and double-stranded (ds) DNA sections. Early studies 
      of denaturability of cellular DNA utilized flow cytometry and standard 
      fluorescence microscopy. These techniques could not reveal small local 
      differences in DNA denaturability within cell nucleus or in individual 
      chromosomes. For instance, it was not possible to detect the initial points of 
      chromosome condensation in G2-phase of the division cycle or in apoptosis. In 
      order to achieve this goal we have recently extended these studies by applying 
      confocal microscopy. We investigated DNA denaturability in normal human 
      fibroblasts and HL-60 leukemic cells, at different stages of cell cycle and 
      apoptosis. Following removal of RNA and partial denaturation of DNA with acid 
      cells were stained with AO. Green (530 nm) and red (640 nm) fluorescence (exc. 
      457 nm) of non-denatured and denatured DNA was imaged by confocal microscopy. 
      Blind deconvolution was used to further improve the quality of 3D images. 
      Photobleaching of AO fluorescence was minimized and a correction for chromatic 
      aberration and register shift was implemented. Nuclei of interphase cells 
      exhibited predominantly green fluorescence representing AO binding to ds DNA. 
      Punctuate areas of red fluorescence representing AO binding to denatured DNA and 
      most likely associated with local regions of condensed chromatin were also 
      present in all interphase nuclei. The proportion of denatured DNA increased in 
      cells entering mitosis. In prophase individual condensing chromosomes exhibited 
      varied proportions of green and red fluorescence indicating different content of 
      denatured chromatin. In some chromosomes bands of denatured and 
      denaturation-resistant chromatin were clearly resolved. In metaphase and anaphase 
      chromosomes exhibited red fluorescence along all length of their arms indicating 
      the highest and uniform susceptibility to denaturation. In telophase chromosomes 
      contained predominantly denaturation-resistant DNA again and denaturated regions 
      were significantly less abundant. At cytokinesis some decondensing chromosomes 
      were still resolved. At this stage almost all regions of denatured DNA were 
      located close to nuclear envelope. These regions may correspond to pockets of 
      heterochromatin reforming at nuclear periphery. In early apoptosis condensation 
      of chromatin appeared to commence in several distinct regions within nucleus. 
      Some apoptotic bodies contained condensed chromatin surrounding central regions 
      of extended chromatin. At late stages of apoptosis the whole volume of apoptotic 
      bodies was occupied by condensed chromatin.
FAU - Dobrucki, J
AU  - Dobrucki J
AD  - Laboratory of Confocal Microscopy, Department of Biophysics, Institute of 
      Molecular Biology, Jagiellonian University, Al. Mickiewicza 3, 31-120, Krakow, 
      Poland.
FAU - Darzynkiewicz, Z
AU  - Darzynkiewicz Z
LA  - eng
GR  - R01 CA028704/CA/NCI NIH HHS/United States
GR  - R01 CA028704-24/CA/NCI NIH HHS/United States
GR  - R01 CA 28 704/CA/NCI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - England
TA  - Micron
JT  - Micron (Oxford, England : 1993)
JID - 9312850
RN  - 0 (Chromatin)
RN  - 9007-49-2 (DNA)
RN  - F30N4O6XVV (Acridine Orange)
SB  - IM
MH  - Acridine Orange/pharmacology
MH  - Apoptosis/*physiology
MH  - Cell Cycle/*physiology
MH  - Cells, Cultured
MH  - Chromatin/*ultrastructure
MH  - DNA/*chemistry
MH  - Humans
MH  - *Microscopy, Confocal
MH  - Nucleic Acid Conformation/drug effects
MH  - Nucleic Acid Denaturation
EDAT- 2001/05/04 10:00
MHDA- 2001/08/17 10:01
CRDT- 2001/05/04 10:00
PHST- 2001/05/04 10:00 [pubmed]
PHST- 2001/08/17 10:01 [medline]
PHST- 2001/05/04 10:00 [entrez]
AID - S0968-4328(00)00069-X [pii]
AID - 10.1016/s0968-4328(00)00069-x [doi]
PST - ppublish
SO  - Micron. 2001 Oct;32(7):645-52. doi: 10.1016/s0968-4328(00)00069-x.