Chromatin association of replication protein A

Replication protein A (RPA) is the major single strand-specific DNA-binding protein in eukaryotic cells. We have investigated the distribution of RPA in nuclei of proliferating HeLa cells and found that only one-third of the detectable RPA appeared to be bound to DNA in chromatin, whereas the remainder was free in the nucleosol. This distribution did not significantly change when cells were released from a double thymidine block into the S phase of the cell cycle. Single strand-specific endonucleases failed to mobilize RPA bound to chromatin in G1 phase and S phase HeLa cells. In contrast, brief treatments with pancreatic DNase I or with micrococcal nuclease sufficed to release RPA from its chromatin-binding sites. Sucrose gradient analysis of soluble micrococcal nuclease digests showed that the released RPA sedimented free of mono- or oligonucleosomal chromatin fragments, possibly indicating that most of the detectable RPA may be associated with chromatin sites, which are more open to nuclease attack than bulk chromatin. The surprising conclusion is that the majority of the detectable RPA is, either directly or indirectly, associated with double-stranded DNA regions in chromatin from HeLa cells in G1 phase and in S phase.     

Frequency-shaped amplification changes the neural representation of speech with noise-induced hearing loss

Apoptosis is a mode of active cell death. We have examined whether 2-chloroethylethyl sulfide (CEES), a sulfur vesicating agent, triggers apoptosis as a cytotoxic mechanism. Incubation of thymocytes with CEES, resulted in an induction of apoptotic features of cell death. Treatment of cells with 100 microM CEES for 5 h increased DNA fragmentation to approximately 40% of control. The fragmentation of DNA was visualized by agarose gel electrophoresis. It showed ladder pattern of DNA fragmentation, which indicates internucleosomal cleavage of DNA. Further evidence of apoptosis was observed in morphological changes of nuclei by using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. The percentage of TUNEL positive cells was dependent upon CEES concentrations. CEES induced the classical morphological features of apoptosis in nucleus. These features were accompanied by condensation of chromatin, which arranged in sharply declined clumps and fragmentation of nucleus. To study requirement for synthesis of new protein in CEES-induced apoptosis, we studied the effect of cycloheximide for apoptotic activity. This protein synthesis inhibitor did not suppress the CEES-induced apoptotic activity. Taken together, these results suggest that CEES-induced apoptosis as a cytotoxicmechanism and this process occurs independent of synthesis of new protein.     

Comparison of the MOS short form-12 (SF12) health status questionnaire with the SF36 in patients with rheumatoid arthritis

Cultured HL-60, HeLa S3 and WiDr cells grown in male BALB/c nu/nu mice were studied by conventional and field-inversion DNA gel electrophoresis (FIGE), as well as by means of cytomorphological approaches, including TdT-mediated dUTP nick end labeling (TUNEL) assay. Chemosensitivity tests revealed HL-60 to be sensitive to vindesine (VDS), and HeLa S3 and WiDr to mitomycin C (MMC). Although VDS-treated HL-60 exhibited condensation of chromatin and a DNA ladder, MMC-exposed HL-60 cells showed apoptotic figures without typical DNA ladders. With MMC-treated WiDr cells, neither DNA ladders nor apoptotic figures were observed. Cells characterized by chromatin condensation were TUNEL-positive in both treated and untreated cases with the exception of the MMC-treated WiDr case, in which many TUNEL-positive cells were observed without cytomorphological changes. On FIGE, DNA fragments of approximately 50, 300 and 400 kbp were detected in groups treated with both effective and ineffective drugs, as well as in untreated controls. Furthermore, change of the time parameters in FIGE resulted in different sizes (550 and 850 kbp) of DNA fragments. These findings indicate that i) cell death is not always detectable in terms of apoptotic figures or DNA oligonucleosomal fragmentation, ii) only the TUNEL assay is a reliable tool to detect DNA damage and, iii) FIGE does not provide accurate size profiles of macromolecular DNA fragments.     

Transition from caspase-dependent to caspase-independent mechanisms at the onset of apoptotic execution

We have compared cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway. Extracts from morphologically normal "committed stage" cells induce apoptotic morphology and DNA cleavage in substrate nuclei but require ongoing caspase activity to do so. In contrast, extracts from frankly apoptotic cells induce apoptotic events in added nuclei in a caspase-independent manner. Biochemical fractionation of these extracts reveals that a column fraction enriched in endogenous active caspases is unable to induce DNA fragmentation or chromatin condensation in substrate nuclei, whereas a caspase-depleted fraction induces both changes. Further characterization of the "execution phase" extracts revealed the presence of an ICAD/DFF45 (inhibitor of caspase-activated DNase/DNA fragmentation factor)- inhibitable nuclease resembling CAD, plus another activity that was required for the apoptotic chromatin condensation. Despite the presence of active caspases, committed stage extracts lacked these downstream activities, suggesting that the caspases and downstream factors are segregated from one another in vivo during the latent phase. These observations not only indicate that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than disassembling it themselves, but they also suggest that activation of the downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent to active phase of apoptosis.     

Resistance to DNA fragmentation and chromatin condensation in mice lacking the DNA fragmentation factor 45

The DNA fragmentation factor 45 (DFF45) is a subunit of a heterodimeric nuclease complex critical for the induction of DNA fragmentation in vitro. To understand the in vivo role of DFF45 in programmed cell death, we generated DFF45 mutant mice. DNA fragmentation activity is completely abolished in cell extracts from DFF45 mutant tissues. In response to apoptotic stimuli, splenocytes, thymocytes, and granulocytes from DFF45 mutant mice are resistant to DNA fragmentation, and splenocytes and thymocytes are also resistant to chromatin condensation. Nevertheless, development of the immune system in the DFF45 mutant mice is normal. These results demonstrate that DFF45 is critical for the induction of DNA fragmentation and chromatin condensation in vivo, but is not required for normal immune system development.     

Mechanisms of induction of apoptotic DNA fragmentation

PURPOSE: Despite its common use as an indicator of apoptosis, little is known about the mechanisms controlling apoptotic DNA fragmentation in irradiated cells. This review discusses the pathways of chromatin fragmentation, and the role of both nucleases and chromatin structure in this process. DEFINITIONS: DNA fragmentation linked to apoptosis is a combination of cleavage events excising both large DNA fragments within the range 0.4-1.0 Mbp and 50 kbp followed by random cuts within internucleosomal regions (i.e. DNA laddering). The first two cleavage steps can be detected in virtually all apoptotic cells, but DNA laddering is not ubiquitously observed. Endonucleases that mediate this cleavage of chromatin may be classified by substrate specificity, mode of DNA cleavage and their cofactor requirements. CONCLUSIONS: Three major pathways of DNA fragmentation are proposed and discussed: (1) upregulation of endonucleases, (2) their intranuclear/intracellular redistribution and (3) primary changes of chromatin structure.     

Assembly kinetics of replicating chromatin: isolation and characterization of prenucleosomal and nucleosomal DNA

Replicating chromatin is known to be more sensitive to micrococcal nuclease than bulk chromatin. We have used this property and a fractionation procedure based on the specific release of replicating material under mild micrococcal nuclease digestion, in order to analyse both the kinetics of maturation of newly replicated DNA into nucleosomes and the structure of the replicating material. As other authors, we initially observed that repetitive unit of newly replicated chromatin was shorter than that of bulk chromatin, however this result appears to be due to sliding of nucleosomes along the chromatin fibers close to the replicating fork. Replicative chromatin was fractionated and analysed. A prenucleosomal peak was observed and preliminary characterized.     

Spermatogenic cell apoptosis induced by mitomycin C in the mouse testis

Spermatogenic cell degeneration in the mature mammalian testis occurs both spontaneously during normal spermatogenesis and in response to cytotoxic agents. Mitomycin C (MC) is an antibiotic that affects DNA synthesis. In the present study, we examined the induction of mouse spermatogenic cell apoptosis by MC, using TdT-mediated dUTP-biotin nick end labeling (TUNEL) to detect high levels of DNA fragmentation in situ, transmission electron microscopy (TEM) to observe nuclear chromatin condensation, and molecular methods to detect DNA ladders. This study shows that in the testis of MC-treated mice: (i) apoptotic cell death with fragmentation of nuclear DNA is induced by MC dose-dependently, (ii) apoptotic cell death is most commonly found in the spermatogonia and less frequently in spermatocytes, and (iii) apoptotic cell death induced by MC is not specific for the seminiferous stage of the tubules. The present study suggests that the spermatogenic cell apoptosis induced by MC might be involved in its testicular toxicity.     

Impairment of forearm vasodilatation to acetylcholine in hypercholesterolemia is reversed by aspirin

To study the intracellular apoptotic signaling pathways, we have established a cell-free system, in which DNA fragmentation of the isolated mouse liver nuclei was induced with lysates from the Fas-activated cells. We have found that the inactive nuclease present in the intact cell cytosol is activated by a caspase-3-like protease and the activated nuclease induces the nucleosomal DNA fragmentation. We attempted the purification of the inactive nuclease from bovine liver cytosol. The partially purified nuclease was activated by recombinant caspase-3, and to a lesser extent by caspase-6. The activated nuclease was able to digest plasmid DNA in addition to induce the DNA fragmentation of nuclei. DFF-45, which is a subunit of heterodimeric protein leading to DNA fragmentation upon its digestion by caspase-3, is found to inhibit the activity of the activated nuclease. These results suggest that the inactive nuclease in cytoplasm is converted to the active form by caspases, and the activated nuclease enters into nucleus to induce the DNA fragmentation. It is suggested that DFF-45 may function as an inhibitory factor of the caspase-sensitive nuclease in vivo.     

Purification and characterization of a Mg2+-dependent endonuclease (AN34) from etoposide-treated human leukemia HL-60 cells undergoing apoptosis

An important biochemical hallmark of apoptosis is the cleavage of chromatin into oligonucleosomal fragments. Here, we purified a Mg2+-dependent endonuclease from etoposide-treated HL-60 cells undergoing apoptosis. High levels of Mg2+-dependent endonuclease activity were detected in etoposide-treated HL-60 cells, and this activity increased in a time-dependent manner following etoposide treatment. Such an activity could not be detected in untreated cells or in cells treated with etoposide in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone (zVAD-fmk) or the serine protease inhibitor tosyl-L-phenylalanine chloromethyl ketone (TPCK). This Mg2+-dependent endonuclease was purified by a series of chromatographic procedures. The enzyme preparation showed a single major protein band with Mr 34,000, determined by SDS-PAGE. The presence of the Mr 34,000 Mg2+-dependent endonuclease was also confirmed by activity gel analysis. The enzyme required only Mg2+ for full activity. pH optimum was in the range of 6.5-7.5. This enzyme introduced single- and double-strand breaks into SV40 DNA and produced internucleosomal DNA cleavage in isolated nuclei from untreated cells. The DNA breaks were terminated with 3'-OH, consistent with characteristic products of apoptotic chromatin fragmentation. We propose to designate this Mr 34,000 Mg2+-dependent endonuclease AN34 (apoptotic nuclease Mr 34,000).     

Ethnic diversity and disease surveillance: Guinea worm among the Fulani in a predominantly Yoruba district of Nigeria

The effects of 3-aminobenzamide (3ABm) and benzamide (BAm), known specific inhibitors of poly(ADP-ribose) polymerase (PARP), on actinomycin D (Act D)-induced apoptosis in HL-60 cells were examined. These inhibitors had no appreciable effect on apoptotic DNA fragmentation, chromatin condensation or PARP restriction cleavage, but clearly inhibited morphological changes, especially nuclear fragmentation and apoptotic-body formation, in a dose-dependent manner. These results suggest that the synthesis of ADP-ribose polymers is not essential for the progression of apoptotic DNA fragmentation and chromatin condensation, but is required in the processes leading to nuclear fragmentation and the subsequent apoptotic-body formation during apoptosis in HL-60 cells.     

Ca2+ channel blockers verapamil and nifedipine inhibit apoptosis induced by 25-hydroxycholesterol in human aortic smooth muscle cells

We have characterized the death of human aortic smooth muscle cells induced by 25-hydroxycholesterol, an oxidation product of cholesterol. Chromatin condensation characteristic of apoptosis was observed by enzymatic (TUNEL) staining of chromatin, and by electron microscopy. Fourteen percent of cells treated with 5 microg/ml of 25-hydroxycholesterol for 24 h displayed chromatin degradation as determined by positive TUNEL staining. Addition of TNF alpha (10 ng/ml) and IFN gamma (20 ng/ml) increased the proportion of TUNEL positive cells to 30%, whereas the cytokines alone were without effect. After 48 h, 40% of the cells treated with 5 microg/ml of 25-hydroxycholesterol were TUNEL positive, and 21% of the cells displayed chromatin condensation. Oligonucleosomal DNA fragmentation typical of apoptosis was demonstrated by agarose gel electrophoresis. Furthermore, activation of the ICE-like protease caspase 3 (CPP32) was observed in cells treated with 25-hydroxycholesterol. Addition of the Ca2+ entry blockers verapamil or nifedipine to the culture medium inhibited apoptosis by more than 70% and reduced cytotoxicity, while removal of Ca2+ from culture medium reduced apoptosis by 42%. Within a few minutes after addition, 25-hydroxycholesterol induced intracellular Ca2+ oscillations with a frequency of approximately 0.3-0.4 min(-1). Thus it appears that Ca2+ influx through plasma membrane channels is an important signal in oxysterol-induced apoptosis. Addition of TNF alpha and IFN gamma enhanced cytotoxicity and resulted in a higher proportion of apoptotic cells, suggesting that inflammatory cytokines can increase the cytotoxicity of lipid oxidation products.     

Effect of naftidrofuryl (LS129) on axonal growth

Wistar rats, eight days old, were subjected to permanent bilateral forebrain ischemia, followed by hypoxia for 15 minutes. A cerebral infarct, mainly involving the cerebral neocortex, hippocampus, amygdala, striatum and subcortical white matter was produced. Neurons and glia showing punctate chromatin condensation and karyorrhectic cells were observed 12 hours after hypoxia-ischemia. Their number increased during the first two days and recruitment of cells with degenerating nuclei occurred until day five. In situ labeling of nuclear DNA fragmentation stained many normal-appearing nuclei, as well as punctate chromatin condensations and nuclear fragments in karyorrhectic cells. Delayed neuronal death in the CA1 area of the hippocampus was observed after 20 minutes of transient forebrain ischemia in the adult gerbil. In situ labeling of nuclear DNA fragmentation demonstrated stained punctate chromatin condensation in a few degenerating cells at 48 hours post-ischemia. Substantial labeling of CA1 neurons occurred in the fourth day. Agarose gel electrophoresis of extracted brain DNA from ischemic infant rats and adult gerbils showed a ladder-type pattern which is typical of nuclear DNA fragmentation into oligonucleosomal fragments (internucleosomal cleavage). These findings suggest that endonuclease(s) activation may play a role in cell death induced by different forms of hypoxia-ischemia.     

Annexin V labelling and terminal transferase-mediated DNA end labelling (TUNEL) assay in human arrested embryos

Confocal laser scanning microscopy was used to observe human arrested and fragmented preimplantation embryos obtained by in-vitro fertilization. Observation of the cellular actin cortex and chromatin showed a high frequency of embryos with blastomeres exhibiting two or more nuclei, while others had nuclei displaying chromatin condensation and fragmentation patterns. Many of the abnormal chromatin images could be due to the process of programmed cell death (apoptosis). The possible link between abnormalities of the blastomeres and apoptosis was investigated using two detection methods for cells undergoing apoptosis. Detection of phosphatidylserine exposure was performed using annexin V; the chromosomal breakdown preceding the nuclear collapse of apoptotic nuclei was tested using the terminal transferase-mediated DNA end labelling (TUNEL) assay. Annexin V staining was observed in all arrested and/or fragmented human embryos, but not in cryopreserved embryos which continued to develop normally after thawing. The TUNEL assay was positive in 30% (15/50) of arrested embryos, all of which had cytoplasmic fragments. In contrast, embryos showing regular size blastomeres without fragments were TUNEL negative.     

Light-induced apoptosis: differential timing in the retina and pigment epithelium

Apoptosis is a genetically regulated form of cell death. Individual cells show condensed nuclear chromatin and cytoplasm, and biochemical analysis reveals fragmentation of the DNA. Ensuing cellular components, apoptotic bodies, are removed by macrophages or neighboring cells. Genes involved in the regulation of apoptosis as well as stimuli and signal transduction systems, are only beginning to be understood in the retina. Therefore, we developed a new in vivo model system for the investigation of events leading to apoptosis in the retina and the pigment epithelium. We induced apoptosis in retinal photoreceptors and the pigment epithelium of albino rats by exposure to 3000 lux of diffuse, cool white fluorescent light for short time periods of up to 120 minutes. Animals were killed at different time intervals during and after light exposure. The eyes were enucleated and the lower central retina was processed for light- and electron microscopy. DNA fragmentation was analysed in situ by TdT-mediated dUTP nick-end labeling (TUNEL) or by gel electrophoresis of total retinal DNA. We observed that the timing of apoptosis in the photoreceptors and pigment epithelium was remarkably different, the pigment epithelium showing a distinct delay of several hours before the onset of apoptosis. In photoreceptors, apoptosis was induced within 90 minutes of light exposure, with the morphological appearance of apoptosis preceding the fragmentation of DNA. In the pigment epithelium, the morphological appearance of apoptosis and DNA fragmentation were coincident. Different regulative mechanisms may lead to apoptotic cell death in the retinal photoreceptors and pigment epithelium. This in vivo model system will allow measurement of dose-responses, a potential spectral dependence and the molecular background of apoptotic mechanisms in the retina.     

Mobilization of chromatin-bound Mcm proteins by micrococcal nuclease

Mcm (minichromosome maintenance) proteins are important components of the eukaryotic replication initiation apparatus. We investigate the binding of human Mcm proteins to HeLa cell chromatin using micrococcal nuclease as a tool. In previous work we prepared chromatin under low ionic strength conditions. The use of a low salt buffer was necessary to prevent the dissociation of Mcm proteins. Here we use chromatin prepared at more physiological salt concentrations (100 mM NaCl) following the procedure of Fujita et al. (J. Biol. Chem. 272, 10928-10935; 1997) who had shown that ATP stabilizes the interaction of Mcm proteins with chromatin. We show here that micrococcal nuclease released Mcm proteins early during the digestion process suggesting that Mcm proteins reside on chromatin sites which are more open to nuclease attack than bulk chromatin. Released Mcm proteins sedimented through glycerol gradients as a multiprotein complex comprising several of the six known human Mcm proteins.     

Fractionation of chromatin, released by nuclease digestion, on ECTHAM-cellulose. Separation of active and inactive chromatin

Chromatin released by two nucleases under various ionic conditions has been fractionated by chromatography on ECTHAM-cellulose. Mg2+ -soluble chromatin, which according to Gottesfeld and Partington is enriched in transcribed DNA sequences (Gottesfeld, J.M. and Partington, G.A., (1977) Cell 12, 953-962) and produced by DNAase II digestion at intermediate ionic strength, comprises material eluting from ECTHAM-cellulose at 80-100 mM Cl-, pH 6.8-7.0, whereas bulk, Mg2+ -insoluble chromatin comprises more tightly binding material. Free hnRNP particles elute at 30 mM Cl-, pH 6.8. Oligonucleosomes, which according to Dimitriadis and Tata are enriched in transcribed sequences (Dimitriadis, G.J. and Tata, J.R. (1980) Biochem. J. 187, 467-477) and produced by micrococcal nuclease digestion at physiological ionic strength, also elute predominantly at 80-100 mM Cl-, pH 6.8-7.0. When liver nuclei are digested with micrococcal nuclease at low ionic strength, the most rapidly released chromatin is enriched in nascent RNA and hnRNP particles, and binds weakly to ECTHAM-cellulose. More slowly solubilised chromatin, containing fewer hnRNP particles, binds much more strongly to ECTHAM-cellulose. In confirmation of results with mechanically sheared chromatin, the affinity of particular chromatin fractions is not dependent on the size of chromatin particles, rather it reflects the differing composition, and in particular the non-histone protein and hnRNP content, which, we propose, determines the conformation adopted by different chromatin fractions in the cation conditions used for elution from ECTHAM-cellulose.     

The HIV-1 gp120 causes ultrastructural changes typical of apoptosis in the rat cerebral cortex

We used transmission electron microscopy (TEM) and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labelling (TUNEL) techniques to study the neuropathological effects of intracerebroventricular (i.c.v.) injection of recombinant HIV-1 gp 120 in rats. In brain cortical tissue sections from rats treated with a single daily dose of gp120 (100 ng day-1 for 7 or 14 consecutive days) TEM analysis showed chromatin compaction and marginalization along the inner surface of the nuclear envelope followed by masses of condensed chromatin, ultrastructural signs demonstrating the occurrence of apoptotic cell death. These effects were paralleled by in situ DNA fragmentation, as revealed by application of TUNEL technique to cryostat brain tissue sections from rats treated likewise with the viral coat protein. In no instance was apoptosis seen in the brain cortex of control rats. The present data demonstrate that gp120 given i.c.v. produces apoptosis in the neocortex of rats.     

Native recombinant cyclophilins A, B, and C degrade DNA independently of peptidylprolyl cis-trans-isomerase activity. Potential roles of cyclophilins in apoptosis

Previous work in our laboratory (Montague, J., Gaido, M., Frye, C., and Cidlowski, J. (1994) J. Biol. Chem. 269, 18877-18880) has shown that human recombinant cyclophilins A, B, and C have sequence homology with the apoptotic nuclease NUC18 and that denatured cyclophilins can degrade DNA. We have now evaluated the nucleolytic activity of recombinant cyclophilins under native conditions. We show that nuclease activity inherent to cyclophilins is distinct from cis-trans-peptidylprolyl isomerase activity and is similar to that described for apoptotic nucleases. Cyclophilin nucleolytic activity is stimulated by Ca2+ and/or Mg2+, with a combination of the two being optimal for cyclophilins A and B. Mg2+ alone is sufficient for cyclophilin C nuclease activity. pH optimums are in the range of pH 7.5-9.5. Cyclophilins can degrade both single-stranded and double-stranded DNA. Additionally, cyclophilins produce 3'-OH termini in linear double-stranded substrates, suggesting the cuts produced are similar to those of apoptotic cells. Cyclophilins also display endonucleolytic activity, demonstrated by their ability to degrade supercoiled DNA. In the absence of ions, cyclophilins bind linearized DNA. When added to nuclei from nonapoptotic cells, cyclophilin C induces 50-kilobase pair DNA fragmentation but not internucleosomal fragmentation. Together, these data suggest that cyclophilins are involved in degradation of the genome during apoptosis.