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PubMed2000

Histone H2B phosphorylation in mammalian apoptotic cells. An association with DNA fragmentation.

Histone phosphorylation was investigated in several mammalian cells undergoing apoptosis (human HL-60 and HeLa, mouse FM3A and N18 cells, and rat thymocytes). Among the four nucleosomal core histones (H2A, H2B, H3, and H4), H2B, which is not usually phosphorylated in quiescent or growing cells, was found to be phosphorylated after treatment with various apoptotic inducers. The H2B was phosphorylated around the time when nucleosomal DNA fragmentation was initiated and, like this fragmentation, was completely blocked with Z-Asp-CH(2)-DCB, an inhibitor of ICE or ICE-like caspase. The involved single phosphopeptide of H2B proved to be phosphorylatable in vitro with a protein kinase C, and the site Ser-32 was tentatively identified. Despite typical apoptotic chromatin condensation, the H3 phosphorylation was at a low level, and the sites where phosphorylation did occur did not include any mitosis-specific phosphopeptides. Phosphorylation of H4 was increased, but the other two histone proteins (H1 and H2A) were not appreciably changed. These observations imply that 1) H2B phosphorylation occurs universally in apoptotic cells and is associated with apoptosis-specific nucleosomal DNA fragmentation, 2) chromatin condensation in apoptosis occurs by a different biochemical mechanism from those operating during mitosis or premature chromosome condensation, and 3) this unique phosphorylation of H2B is a useful biochemical hallmark of apoptotic cells.

Animals↗
PubMed1995

Induction of apoptotic cell DNA fragmentation in human cells after treatment with hyperthermia.

The biological significance of apoptosis is becoming increasingly clear. Its relevance in tumor response to treatment as well as recent evidence for its important function as a regulating mechanism in tumorigenesis has also been demonstrated. One of the most prominent biological features of apoptosis is nucleosomal DNA fragmentation. In this communication, we present a study of DNA fragmentation in Raji cells which have been subjected to hyperthermia treatment to induce apoptosis. We found that the induction and onset of fragmentation is swift, and consistent with previous reports that fragmentation must be a rapid event.

Apoptosis↗
PubMed1972

RNA-linked nascent DNA fragments in Escherichia coli.

Nucleic acid that is extracted from E. coli labeled by a brief pulse of [(3)H]dT and depatured by treatment with heat, formamide, or formaldehyde bands in a region with a density higher than that of single-stranded E. coli DNA in a Cs(2)SO(4) equilibrium density gradient. If treated with alkali or RNase, it then exhibits the density of single-stranded DNA. These results suggest the presence of a short strand of RNA covalently linked to the nascent DNA. Evidence for the presence of covalently linked RNA-DNA molecules is also obtained by pulse labeling with [(3)H]U. Analyses of nascent nucleic acids from cells pulse labeled for various times, and of the molecules with different sizes, support the hypothesis that the short DNA fragments are formed by extension of even shorter RNA chains, which are synthesized on the parental DNA strands and are removed before ligation of the DNA fragments. The synthesis of the RNA segment of the RNA-DNA molecule is much less sensitive to rifampicin than is the synthesis of bulk RNA.

Carbon Isotopes↗
PubMed1984

Rapid identification of cloned HSV2 DNA fragments.

Randomly generated herpes simplex virus type 2 (HSV2) DNA fragments can be localized on the viral genome by digestion with SmaI and subsequent comparison of the resulting fragment patterns with a reference picture of SmaI-digested recombinant plasmids that contain inserts representing the entire HSV2 (strain 333) genome as adjacent fragments. The feasibility of the technique, which we call 'Sma fingerprinting' and which is much faster than fragment identification by blot hybridization, is demonstrated.

Cloning, Molecular↗
PubMed2001

Ebselen reduces cytochrome c release from mitochondria and subsequent DNA fragmentation after transient focal cerebral ischemia in mice.

BACKGROUND AND PURPOSE: The seleno-organic compound ebselen has both antioxidant and anti-inflammatory properties. Although ebselen has been shown to protect the brain against stroke, it is unclear how ebselen provides neuroprotection. In the present study the authors examined whether ebselen inhibits neuronal apoptosis resulting from transient focal cerebral ischemia in mice. The cytochrome c release and DNA fragmentation, both of which are biochemical markers of apoptosis, were compared between vehicle- and ebselen-treated mice. METHODS: Cerebral ischemia was induced by transient middle cerebral artery occlusion for 30 minutes in ICR mice under halothane anesthesia. Ebselen (10 mg/kg) was given orally twice, 30 minutes before ischemia and 12 hours after reperfusion. By Western blot analysis, we examined release of mitochondrial cytochrome c. To evaluate brain damage, the brain sections were treated for terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) and Nissl staining. Prolonged neuroprotective efficacy of ebselen was determined by counting neuronal nuclei (NeuN) immunopositive cells at 21 days after ischemia. RESULTS: - Cytochrome c release was detected in the ischemic hemisphere at 3 to 24 hours after ischemia. Ebselen treatment diminished the cytochrome c release at 12 and 24 hours. In addition, ebselen decreased both DNA fragmentation determined by TUNEL and brain damage volume at 3 days after ischemia. Furthermore, ebselen increased the number of NeuN immunopositive cells at 21 days after ischemia. CONCLUSIONS: These results indicate that ebselen attenuates ischemic neuronal apoptosis by inhibiting cytochrome c release. Ebselen may be a potential compound in stroke therapy.

Animals↗
PubMed1986

In vivo competition of delta-crystallin gene expression by DNA fragments containing a GC box.

Expression of the chicken delta-crystallin gene 1 injected into the nuclei of mouse cells is lens specific. Coinjection of GC box-containing DNA fragments from delta-crystallin, simian virus 40 early, and herpes simplex virus type 1 tk promoters effectively suppressed delta-crystallin expression in the lens, but coinjection with DNA fragments not containing the GC box did not. This suppression was likely due to the competition of an Sp1-like transcription factor(s) and indicates involvement of the apparently ubiquitous factor(s) in the tissue-specific expression of the delta-crystallin gene.

Animals↗
PubMed2001

Quantification of apoptotic DNA fragmentation in a transformed uterine epithelial cell line, HRE-H9, using capillary electrophoresis with laser-induced fluorescence detector (CE-LIF).

Apoptotic cell death of uterine epithelial cells is thought to play an important role in the onset of menstruation and the successful implantation of an embryo during early pregnancy. Abnormal apoptosis in these cells can result in dysmenorrhoea and infertility. In addition, decreased rate of epithelial apoptosis likely contributes to endometriosis. A key step in the onset of apoptosis in these cells is cleavage of the genomic DNA between nucleosomes, resulting in polynucleosomal-sized fragments of DNA. The conventional technique for assessing apoptotic DNA fragmentation uses agarose (slab) gel electrophoresis (i.e. DNA laddering). However, recent technological advances in the use of capillary electrophoresis (CE), particularly the introduction of the laser-induced fluorescence detector (LIF), has made it possible to perform DNA laddering with improved automation and much greater sensitivity. In the present study, we have further developed the CE-LIF technique by using a DNA standard curve to quantify accurately the amount of DNA in the apoptotic DNA fragments and have applied this new quantitative technique to study apoptosis in a transformed uterine epithelial cell line, the HRE-H9 cells. Apoptosis was induced in the HRE-H9 cells by serum deprivation for 5, 7 and 24 h, resulting in increased DNA fragmentation of 2.2-, 3.1- and 6.2-fold, respectively, above the 0 h or plus-serum controls. This ultrasensitive CE-LIF technique provides a novel method for accurately measuring the actions of pro- or anti-apoptotic agents or conditions on uterine epithelial cell lines.

Animals↗
PubMed2002

Influence of an 8-oxoadenine lesion on the structural and dynamic features of a 30-mer DNA fragment with and without a mismatch.

PURPOSE: To elucidate the influence of the oxidative lesion 7,8-dihydro-8-oxoadenine (8-oxoA) on the structural and dynamic features of a 30-mer DNA fragment, and to understand if differences occur when C is positioned opposite 8-oxoA instead of T. MATERIALS AND METHODS: Two 30-mer DNA oligomers with or without the 8-oxoA and two complementary oligomers with C or T base opposite the lesion site were synthesized and annealed. Duplexes named AT, A*T, AC and A*C were characterized by means of circular dichroism and UV denaturation measurements. gamma-Ray footprinting experiments were performed to give insight into their fine three-dimensional structure. Elastic torsional constants were derived by following the decay of the fluorescence polarization anisotropy (FPA) of the ethidium-DNA complexes measured by multifrequency-phase fluorometry. RESULTS: The introduction of one oxidative lesion in a 30-mer DNA oligomer with and without a mismatch did not cause relevant changes in their overall conformation and slightly modified their elastic properties. Small energetic differences were revealed by thermodynamic analysis in the sample bearing both the oxidative lesion and the mismatch. Minor variations in the cleavage pattern due to the hydroxyl radicals in the A*T sample were observed and present along the entire DNA fragment length. In the A*C sample, by contrast, there was a major modification in the cleavage pattern extending for about 11 bases around the lesion, especially towards the 5'-end. CONCLUSIONS: Differences in the fine structure and in the elastic properties between the A*T and A*C samples were observed, while their overall conformation was unchanged. The results are consistent with the hypothesis that the observed local changes of the double helix structure in A*C are due to the pairing of the oxidized adenine in a syn conformation with the cytosine.

Adenine↗
PubMed2003

Using apoptosis for targeted cancer therapy by a new gonadotropin releasing hormone-DNA fragmentation factor 40 chimeric protein.

PURPOSE: GnRH-based chimeric proteins have been shown to specifically target and kill adenocarcinomas both in vitro and in vivo. The purpose of this study is to construct a new GnRH-based chimeric protein for the treatment of adenocarcinomas in humans. RESULTS: In this study, we constructed and characterized a new chimeric protein, GnRH-DFF40, composed of a new human killing moiety: the apoptotic DNase-DFF40 (DNA fragmentation factor), known also as caspase-activated DNase (CAD). GnRH-DFF40 exhibits DNase activity in vitro. We found that this chimeric protein can target and kill adenocarcinoma cells. Such death occurs via apoptotic pathways, resulting in an increase in the sub-G(1) population, DNA fragmentation, terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-positive cells, and morphology typical of apoptotic cells. These apoptotic events involve the mitochondria because we found cytochrome c depletion and caspase-9 and caspase-3 activation. Preliminary in vivo results showed that treatment of colon adenocarcinoma xenografts in nude mice with the new chimeric protein caused a reduction in tumor weight. CONCLUSIONS: Because GnRH-DFF40 is a whole human-based chimeric protein when applied to humans, the nonspecific toxicity and immunogenicity seen with bacterial/plant-based chimeric proteins should be avoided. Thus, GnRH-DFF40 is a promising candidate for the treatment of adenocarcinomas in humans.

Adenocarcinoma↗
PubMed1987

DNA fragments of Bombyx mori nuclear polyhedrosis virus containing the promoters active in prokaryotes.

Using a promoter probe plasmid in E. coli called pHE5, eight different HindIII and one SalI DNA fragments of Bombyx mori nuclear polyhedrosis virus, directing the expression of the tetracycline resistance gene, have been cloned and isolated. The tetracycline resistance levels of the strains containing the recombinant plasmids were measured. Among them, the highest level of the resistance to tetracycline was 30 micrograms/ml. Part of the nucleotide sequence of a DNA fragment was determined. A sequence similar to the E. coli promoter was found.

Animals↗
PubMed2000

Different patterns of DNA fragmentation and degradation of nuclear matrix proteins during apoptosis induced by radiation, hyperthermia or etoposide.

Several nuclear matrix proteins are substrates for proteolytic cleavage during apoptosis. Using Western blotting, the temporal patterns of cleavage of three nuclear matrix proteins (lamin B, NUMA and the nucleoporin TPR) were compared in HL60 cells induced to undergo apoptosis after irradiation, heat shock or treatment with etoposide. Flow cytometry was used to compare the kinetics of post-cleavage degradation of lamin B, NUMA and TPR after irradiation, and to correlate DNA fragmentation with protein degradation in cells induced to undergo apoptosis with different agents. During radiation-induced apoptosis, cleavage and subsequent degradation of lamin B, NUMA and TPR occurred with different kinetics. Low-molecular-weight DNA fragmentation occurred subsequent to the initiation of NUMA cleavage, coincided with lamin B cleavage, but occurred before more extensive degradation of lamin B and NUMA. A similar sequence was observed for cells treated with etoposide. However, during heat-induced apoptosis, cleavage of lamin B and NUMA occurred much sooner compared to other agents, with NUMA cleaved into multiple fragments within 15 min after heating. We conclude that the hierarchical sequence and kinetics of degradative events contributing to nuclear disassembly during apoptosis are highly dependent on the inducing agent. Furthermore, the nuclear pore complex, like the nuclear lamina and internal nuclear matrix, is a target for proteolytic cleavage.

Antigens, Nuclear↗
PubMed1993

Messenger RNA 3'-end formation of a DNA fragment from the human c-myc 3'-end region in Saccharomyces cerevisiae.

We have tested the functioning of the human c-myc polyadenylation signal in Saccharomyces cerevisiae. A DNA fragment containing the two AATAAA polyadenylation signals of the c-myc gene was inserted into a plasmid designed for the in-vivo testing of polyadenylation signals in yeast. The c-myc fragment had a partial capacity for directing mRNA 3'-end formation in yeast. The 3'-endpoints were 50-100 bp distant from the mRNA 3'-ends mapped in humans. This human DNA fragment is therefore unspecifically functional in yeast, indicating that other sequence elements than the human polyadenylation signal, AATAAA, are necessary for 3'-end formation.

Base Sequence↗
PubMed2004

Ultrastructural detection of DNA fragmentation in myonuclei of fatal reducing body myopathy.

Muscle biopsy specimens of two patients with fatal reducing body myopathy showed few necrotic or regenerating fibers. On the other hand, enlarged densely stained myonuclei, in addition to the presence of many reducing bodies, were striking on light microscopy, and chromatin condensation was seen on electron microscopy. To confirm the involvement of apoptosis, we performed the TUNEL method at the electron microscopic level on muscles of the two patients and five age-matched controls. The density of DNA fragments in myonuclei showing chromatin condensation was significantly higher in reducing body myopathy compared to that in control muscles. In addition, the myonuclei surrounded by many reducing bodies tended to show more intense chromatin condensation and a higher density of DNA fragments than those distant from the reducing bodies. These results suggest that apoptosis takes part in muscle fiber degeneration in fatal reducing body myopathy, although the precise relationship between reducing bodies and apoptosis is uncertain.

Apoptosis↗
PubMedDate not supplied

Ribosomal DNA fragments enhance the stability of transfected DNA in Entamoeba histolytica.

Genetic manipulation of Entamoeba histolytica is limited by the inability to express foreign genes at high levels. We tested whether sequences from the E. histolytica rDNA episome, present in 200 copies per cell, could act to stabilize the episomal transfection vector pTCV1. Ligation of the rDNA transcription unit, or sequences downstream of the rDNA transcription unit, increased pTCV1 copy number and stability and conferred additional zones of DNA replication. Sequences upstream of the rDNA transcription unit dramatically destabilized pTCV1. These experiments give additional insights into the mechanism of DNA replication and provide for E. histolytica a set of transfection vectors with unique properties.

Animals↗
PubMed1990

Electrophoresis and movements of fluorescence pattern after photobleaching of large DNA fragments in agarose gels.

By combining electrophoresis with movements of fluorescence pattern after photobleaching (MOFPAP), which is abbreviated as EMOFPAP, we are able to measure electrophoretic mobilities of large DNA fragments in an agarose gel within a fairly short time scale (about 10 min or even down to 1 min). The new method represents a significant improvement in experiment time when compared with the time (typically on the order of hours) required to determine the average electrophoretic mobility of large DNA fragments in agarose gels by means of either conventional gel electrophoresis or pulsed-field gel electrophoresis. In this article, we present the EMOFPAP experimental setup and consider optical conditions, including beam profile geometry and fluorescence pattern formation. A realistic formula that can explain the parameters governing the EMOFPAP method using our present optical setup has been derived. A comparison of results between experimental and computer simulation data is made, and an optimization of the EMOFPAP method is proposed.

DNA↗
PubMed2003

Cold-induced calcium elevation triggers DNA fragmentation in immature pig oocytes.

Fluo-4 loaded immature oocytes were cooled from 30 degrees C to various lower temperatures between 20 and 10 degrees C and changes in intracellular calcium (Ca(2+)) levels were measured. Pig oocytes cooled to 14 degrees C exhibited a clear biphasic Ca(2+) rise. Lower temperatures produced similar responses, while higher temperatures did not exert any effect. The Ca(2+) response appeared to rely on ryanodine dependent stores as removal of extracellular Ca(2+) and intracytoplasmic injection of heparin did not modify cold-induced Ca(2+) elevation, while procaine or ruthenium red virtually eliminated the response. Confocal analysis of subcellular Ca(2+) distribution during cooling revealed that the ion rises sharply within the nucleus. As Ca(2+) imbalance may activate nuclear endonucleases, DNA integrity of cooled pig oocytes was evaluated by TUNEL and comet assays. Most cooled oocytes showed clear signs of DNA fragmentation. Oocytes injected with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetracetic acid tetrapotassium salt (BAPTA), a Ca(2+) chelator, maintained their DNA integrity thus confirming that intracellular Ca(2+) is involved in triggering DNA fragmentation. The protective effect exerted by ruthenium red and/or procaine further confirmed this hypothesis. These data show that a moderate and transient cooling is sufficient to cause an intracellular Ca(2+) rise that leads to DNA damage. The addition of inhibitors of ryanodine dependent Ca(2+) stores may represent a valuable protective treatment to reduce chilling injuries.

Animals↗
PubMed1999

1,3-Selenazine derivatives induce cytotoxicity and DNA fragmentation in human HT-1080 fibrosarcoma cells.

The inhibitory effects of a series of 5,6-dihydro-4H-1,3-selenazine derivatives, 1,3-selenazole, and 5,6-dihydro-4H-1,3-thiazine derivatives on the proliferation of human HT-1080 fibrosarcoma cells were investigated. The compounds 4-ethyl-4-hydroxy-2-p-tolyl-5, 6-dihydro-4H-1,3-selenazine (TS-2) and 4-hydroxy-4-methyl-6-propyl-2-p-tolyl-5,6-dihydro-4H-1,3-selenazine++ + (TS-6) exhibited the strongest inhibitory effect among 1, 3-selenazine derivatives, and the EC(50) of TS-2 and TS-6 was 7.76 and 8.40 microM, respectively. On the other hand, 1,3-selenazole and 5,6-dihydro-4H-1,3-thiazines had no inhibitory effects. TS-2 and TS-6 inhibited the proliferation of HT-1080 cells time- and dose-dependently. They induced dose-dependent DNA fragmentation in HT-1080 cells, revealing a typical apoptosis characteristics. The present study demonstrated that TS-2 and TS-6 inhibited HT-1080 proliferation through the induction of DNA fragmentation.

Apoptosis↗