Chromatin condensation and sensitivity of DNA in situ to denaturation during cell cycle and apoptosis - a confocal microscopy study

The goal of this study was to construct high resolution 3D confocal images of regions of condensed and extended chromatin in cell nuclei and individua l chromosomes. It has been shown previously that sensitivity of DNA in situ to denaturation correlates with chromatin condensation and varies during c ell cycle and apoptosis. Thus, detection of DNA which was partially denatur ed in situ provided a means to image areas of condensed chromatin. DNA dena turation was detected using a metachromatic dye acridine orange (AO) which differentially stains single stranded (ss) and double-stranded (ds) DNA sec tions. Early studies of denaturability of cellular DNA utilized flow cytome try and standard fluorescence microscopy. These techniques could not reveal small local differences in DNA denaturability within cell nucleus or in in dividual chromosomes. For instance, it was not possible to detect the initi al points of chromosome condensation in G2-phase of the division cycle or i n 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 denaturat ion of DNA with acid cells were stained with AO. Green (530 nm) and red (64 0 nm) fluorescence (exc. 457 nm) of non-denatured and denatured DNA was ima ged 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 implemente d. Nuclei of interphase cells exhibited predominantly green fluorescence re presenting AO binding to ds DNA. Punctuate areas of red fluorescence repres enting AO binding to denatured DNA and most likely associated with local re gions of condensed chromatin were also present in all interphase nuclei. Th e proportion of denatured DNA increased in cells entering mitosis. In proph ase 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 uni form susceptibility to denaturation. In telophase chromosomes contained pre dominantly denaturation-resistant DNA again and denaturated regions were si gnificantly less abundant. At cytokinesis some decondensing chromosomes wer e still resolved. At this stage almost all regions of denatured DNA were lo cated close to nuclear envelope. These regions may correspond to pockets of heterochromatin reforming at nuclear periphery. In early apoptosis condens ation of chromatin appeared to commence in several distinct regions within nucleus. Some apoptotic bodies contained condensed chromatin surrounding ce ntral regions of extended chromatin. At late stages of apoptosis the whole volume of apoptotic bodies was occupied by condensed chromatin. (C) 2001 El sevier Science Ltd. All rights reserved.