Probing chromatin structure in the early phases of apoptosis

Abstract

A typical flow cytometric marker of apoptosis is the appearance of a hypodiploid peak. This phenomenon is related to the chromatin fragmentation and loss that occurs during the late stages of the process. We describe herein the changes occurring at the chromatin level in purified nuclei preparations obtained from human peripheral blood mononuclear cells in a time-course study, including the simultaneous evaluation of nuclear proteins and DNA stainability, light-scattering properties, and spectrophotometric determination of the protein content. An augmentation of fluoroscein isothiocyanate (FITC) stainability was noticed as early as 1 h after irradiation. As this phenomenon is not correlated to changes in actual protein content, one can conclude that modifications of basic protein accessibility occur from the early phases of the apoptotic process. Also DNA stainability augmented with time, generating the transient appearance of a hyperdiploid peak that preceded the appearance of the hypodiploid peak typical of the late stages of the process, and that shared with the latter the same light-scattering properties. Chromatin status was further explored by staining apoptotic nuclei using DNA probes with peculiar molecular weight. Propidium iodide (PI) and ethidium bromide (EB), but not the much bulkier 7-aminoactinomycin D (7-AAD), identified the nuclei with a transient increase in DNA stainability confirming that an increased dye accessibility to binding sites was responsible for the phenomenon. Remarkably, all dyes identified the same proportion of hypodiploid nuclei when an apoptotic nucleus shed its fragmented chromatin. Control experiments included differential interference contrast and fluorescence microscopy that showed the purity of nuclei preparations and the typical morphological apoptotic features. Finally, the simultaneous evaluation of DNA by PI and nuclear proteins by FITC in a time course study allowed a thorough assessment of changes occurring at the chromatin level in the diverse stages of apoptosis. It is suggested that proteolysis precedes endonucleolysis and probably renders it easier the final endonucleolytic step leading to DNA fragmentation and loss.

Nuclear chromatin texture and sensitivity to DNase I in human leukaemic CEM cells incubated with nanomolar okadaic acid

“…Previous studies indicate that a decondensation of chromatin would be a prerequisite change which permits subsequent access to the chromatin by factors involved in DNA replication or transcription ( Chadee et al . 1995 ), or apoptosis ( Ferlini et al . 1996 , Palissot et al .…”

Cell cycle checkpoint efficiency and cellular response to paclitaxel in prostate cancer cells

“…To investigate from which cell cycle phase cell death was initiated in taxane-treated cells, we performed a DNA analysis by LSC. This technique allows to detect DNA hyperchromicity that is typical of cells either in mitosis or in the earlier phases of cell death since an increased DNA stainability with intercalating dyes has been shown to precede DNA fragmentation [26,36]. The analysis of the whole cell population (adherent and non-adherent cells) evidenced DNA hyperchromicity in the 4N DNA region of both cell lines after 24 hr of exposure to the drug (Fig.…”

Measurement of Cell Death in Mammalian Cells

“…Annexin V binding Schutte et al, 1998;Cummings and Schnellmann, 2002 Activation of caspases Fluorometric substrate cleavage Expression of specific caspases Cytochrome c translocation Liu et al, 1996;Thornberry et al, 1997;Saraste and Pulkki, 2000 Maintenance of plasma and lysosomal membrane integrity PI staining Neutral red Ferlini et al, 1996;Singh, 2000 Cellular volume Cell size Bortner and Cidlowski, 2001 Release of intracellular contents LDH Mertens et al, 1995;Moran and Schnellmann, 1996 Inflammation Inflammatory cell infiltration or expression of markers of inflammation Jaeschke et al, 1996;Licht et al, 1999 Formation of cellular buds, fragments, or blebs Cell morphology Lemasters et al, 1987;Zhang et al, 1999 Chromatin condensation DAPI or Hoechst staining Lieberthal et al,1996 ATP levels HPLC analysis Groves and Schnellmann, 1996 DNA fragmentation DAPI or Hoechst staining Agarose gel electrophoresis DNA hypoploidy Singh, 2000;Cummings and Schnellmann, 2002 Ca 2+ gradients FURA-2 Lemasters, 1999;Lemasters et al, 1999 Mitochondrial function and integrity MTT JC-1 Tetramethylrhodamine Reers et al, 1995;Lemasters, 1999;Lemasters et al, 1999;Cummings and Schnellmann, 2002 Cellular and mitochondrial respiration O 2 consumption Schnellmann, 1994 death is membrane integrity. Plasma membrane permeability is assessed using propidium iodide or lactate dehydrogenase (see below), and lysosomal membrane integrity is determined using neutral red (Monks et al, 1988;Mertens et al, 1995).…”

“…Current Protocols in Pharmacology Supplement 56 Licht et al, 1999 PI staining b + -- Ferlini et al, 1996;Hamel et al, 1996 Neutral red exclusion c + -- Modha et al, 1993;Mertens et al, 1995 Trypan blue staining + -- Lash and Tokarz, 1989;Modha et al, 1993 Caspase activation -+ - Thornberry et al, 1997;Cummings and Schnellmann, 2002 LDH release b + -- Moran and Schnellmann, 1996 JC-1 transition d + + ? e Reers et al, 1995 Mitochondrial dehydrogenase activity Fanning et al, 1990;Yang et al, 1998 Cell shrinkage -+ + Trump et al, 1997 Cell swelling + -- Lieberthal et al, 1996;Trump et al, 1997 Loss of Ca 2+ gradients + -?…”