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PubMed2001

Transient massive DNA fragmentation in nervous system during the early course of a murine neurodegenerative disease.

In neurodegenerative diseases, such as Alzheimer's disease or HIV encephalitis, neuronal DNA fragmentation has been observed at unexpected high frequencies, without definitive evidence for activation of an irreversible apoptotic pathway. The wobbler mouse is a suggested genetic model of neurodegenerative disease. The mutant mouse develops normally until the fourth week of age when atrophy and weakness of forelimb muscles become apparent. There is a slow progression of the disease and wobbler mice may survive for several months. Spinal cord examination reveals the presence of several motoneurons with perikaryal vacuolar degeneration. In this study, we observed, using terminal dUTP nick-end-labelling staining in mutant spinal cord sections, a massive although very transient DNA fragmentation in different cell types, including glial cells and motoneurons, before the apparition of any clinical symptoms. In older wobbler mice, this DNA fragmentation had completely disappeared and the majority of motoneurons survived. To our knowledge, this is the first example of a massive and transient DNA fragmentation in the central nervous system during the early course of a neurodegenerative disease.

Aging↗
PubMed1994

Differential roles of two types of the TNF receptor in TNF-induced cytotoxicity, DNA fragmentation, and differentiation.

Two different types of TNF receptors, the p60 receptor with a molecular mass of 60 kDa and the p80 receptor with a mass of 80 kDa, have been identified. TNF exhibits a wide variety of biologic actions, but which receptor is responsible for these biologic actions is not well characterized. In the present study, we examined the roles of the p60 and p80 receptors in three different TNF-induced biologic actions: 1) cytotoxicity; 2) DNA fragmentation; and 3) differentiation to macrophages. Analysis of TNF actions on various tumor cell lines revealed that TNF-induced cytotoxicity occurred in cells that expressed the p60 receptor, irrespective of expression of the p80 receptor. In contrast, DNA fragmentation and differentiation were observed only in cells that expressed both receptor types. Additionally, the specific Ab to each receptor were used to examine the roles of both receptors in the myelogenous leukemia cell line, ML-1a. Anti-p60 Ab alone showed cytotoxicity but little DNA fragmentation and differentiation, and anti-p80 Ab alone showed no effects. When these Abs were added in the presence of TNF, each independently, and almost completely, inhibited TNF-induced DNA fragmentation and differentiation. We also found that both Abs together synergistically induce differentiation and DNA fragmentation. These results indicate that signals through the p60 receptor are essential to induce cytotoxicity, but signals through both the p60 and p80 receptors are necessary, and act synergistically, for DNA fragmentation and differentiation.

Cell Differentiation↗
PubMed1994

Capillary electrophoresis as a technique to analyze sequence-induced anomalously migrating DNA fragments.

Sequence-induced anomalous migration of double-stranded (ds) DNA in native gel electrophoresis is a well known phenomenon. The retardation of migration is more obvious in polyacrylamide compared with agarose gels, and is greatly affected by the concentration of the gel and the temperature. This anomalous migration results in a difference between calculated and actual sizes of the affected DNA fragments. A low viscosity polymer solution (DNA Fragment Analysis Reagent) under investigation for use in dsDNA analysis by capillary electrophoresis is shown to be useful for the visualization of anomalies in migration of dsDNA fragments. Comparable with traditional slab gel systems, the retardation effect, indicative of bent or curved DNA, is strongly dependent on polymer concentration and separation temperature. These dependencies have implications on the accurate sizing of dsDNA fragments with unknown sequences and secondary structures.

Bacteriophage phi X 174↗
PubMed1995

Induction of DNA fragmentation after 10 to 120 minutes of focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: The induction of neuronal necrosis has been studied after various durations of transient middle cerebral artery (MCA) occlusion in the rat. The objective of the present study was to measure the numbers and anatomic distribution of cells exhibiting apoptotic bodies as an indication of DNA fragmentation and apoptotic cell death as a function of duration of transient MCA occlusion in the rat. METHODS: The MCA of male Wistar rats (n = 24) was occluded for 10, 20, 30, 60, 90, and 120 minutes (n = 4 per group) with the use of an intraluminal monofilament, and reperfusion was instituted for 48 hours. DNA fragmentation was measured in paraffin sections with the use of a terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) method. Adjacent sections were stained with hematoxylin and eosin for analysis of ischemic cell damage, and immunohistochemical double staining methods were used for cell identification. Sham-operated rats (n = 4) and normal rats not subjected to any surgical procedure (n = 4) were used as controls for apoptosis detection. RESULTS: Within 5-microns-thick coronal sections, DNA fragmentation was present in 0 to 3 apoptotic cells in each hemisphere of normal, sham-operated rats as well as in the contralateral hemisphere of ischemic rats. After 10 to 20 minutes of MCA occlusion, apoptotic cells exhibiting DNA fragmentation (10 to 20) increased in the regions of selective neuronal necrosis in the preoptic area and in the striatum. After 30 to 60 minutes of ischemia, scattered apoptotic cells (30 to 60) exhibited DNA fragmentation and expanded into areas of selective neuronal necrosis in the cortex. After 90 to 120 minutes of occlusion, groups of apoptotic cells (70 to 200, > 95% neurons) were primarily localized to the inner boundary zone of the infarct. CONCLUSIONS: A range of mild to severe ischemia-reperfusion stimuli induce internucleosomal DNA cleavage. The presence and anatomic location of apoptotic cells exhibiting DNA fragmentation after transient cerebral occlusion indicate that apoptosis accompanies neuronal necrosis.

Animals↗
PubMed2000

Detection of high molecular weight DNA fragments characteristic of early stage apoptosis in cerebellar granule cells exposed to glutamate.

In cerebellar granule cells a rapid necrotic cell death has been observed during and immediately after glutamate exposure, followed by a delayed apoptotic type of neuronal death in a subpopulation of the surviving neurons. In some experimental models the DNA fragmentation characteristic of apoptosis is readily detected. In other systems apoptosis may occur only in a limited number of cells, rendering DNA fragmentation undetectable using conventional DNA-staining techniques (e.g., ethidium bromide). We have used a sensitive and non-radioactive method for labeling, detection, and quantification of high molecular weight (HMW) DNA fragments. This method is based on the introduction of thymine dimers into DNA after separation by pulse field gel electrophoresis, followed by detection with thymine dimer specific antibodies. Applying this method to cerebellar granule cells in culture, we detected an increase in the amount of HMW DNA fragments characteristic of apoptosis as early as 4 h after glutamate exposure. The N-methyl-D-aspartic acid (NMDA)-receptor antagonist MK801 protected against the fragmentation, whereas no protection was observed using the non-NMDA-receptor antagonist CNQX.

Animals↗
PubMed1985

A general method for saturation mutagenesis of cloned DNA fragments.

A new procedure for generating and isolating random single-base substitutions in cloned DNA fragments is presented. The mutations are generated by treatment of single-stranded DNA with various chemicals, followed by the synthesis of the complementary strand with reverse transcriptase. Misincorporation frequently occurs when the enzyme encounters a damaged base in the mutagenized template DNA. The resulting duplex DNA fragments containing random single-base substitutions are cloned, amplified as a population, and isolated from wild-type DNA by preparative denaturing gradient gel electrophoresis. The physical separation of mutant DNA fragments makes it possible to isolate and characterize large numbers of site-directed single-base substitutions in the absence of a phenotypic selection. This procedure should be generally applicable to the fine-structure genetic analysis of regulatory and protein-coding sequences.

Animals↗
PubMed2003

Enhancement of immune responses to the hepatitis B virus core protein through DNA vaccines with a DNA fragment encoding human IL-1beta 163-171 peptide.

DNA vaccines have been widely used as effective means of eradicating a variety of viruses, parasites, bacteria as well as means of alleviating allergic and autoimmune diseases and tumors. As interleukin 1 (IL-1) plays an essential role in augmenting both cellular and humoral immune responses to foreign antigens, it may represent a good candidate for an adjuvant to DNA vaccines. Since the inflammatory activity of IL-1 may have a restricted application to DNA vaccines, we explored the possibility of augmenting immune response without unwanted inflammatory effect using IL-1beta 163-171 peptide, which is essential for IL-1 receptor 1 binding. A DNA fragment encoding the human IL-1beta 163-171 peptide of concern was fused to the Hepatitis B virus (HBV) core DNA vaccine, and injected into mice to analyze its immune responses. Compared with the control mice which received hepatitis B virus core antigen (HBcAg) alone, significant increase in not only the HBcAg-specific antibody response but also in T cell proliferation was observed in mice which received IL-1beta 163-171-HBcAg. These results suggest that the DNA fragment encoding the IL-1beta polypeptide of aa 163-171 might represent a good candidate for an adjuvant of DNA vaccines.

Animals↗
PubMed1986

Affinity chromatographic procedure for the quantitative recovery of DNA fragments from agarose gels.

A simple and reliable method for the recovery of specific fragments of DNA from agarose gels is presented. The electroelution of the DNA onto the NENSORB cartridge matrix with the subsequent elution of the bound DNA by a methanol (50% v/v) wash has been shown to result in the quantitative recovery of the restriction fragment. Of importance is the fact that the DNA purified by this procedure is a viable substrate for further digestion by a second restriction endonuclease. The method does not require either phenol extraction or extensive desalting of the sample.

Bacteriophage phi X 174↗
PubMed1989

Alloreactive cytotoxic T cells induce DNA fragmentation in peritoneal macrophages: evidence for target cell killing by cytotoxic T cells in vivo.

This report addresses the question whether cytotoxic T cells can cause target cell death in vivo by examining target cell DNA fragmentation. The results show that alloreactive cytotoxic T cells induced significant DNA fragmentation in peritoneal macrophages in vivo and that the DNA fragment was a multiple of 180 +/- 30 bp. Furthermore, the effector cells which caused this characteristic DNA fragmentation were CD8+ T cells. These results demonstrate that cytotoxic T cells can cause target cell death in vivo.

Animals↗
PubMed2004

Promoting effects of polyunsaturated fatty acids on chromosomal giant DNA fragmentation associated with cell death induced by glutathione depletion.

Glutamate and buthionine sulfoximine (BSO) both reduce intracellular glutathione (GSH) concentration but by different mechanisms, and thereby induce cell death in C6 rat glioma cells. The effects of lipid peroxidation on chromosomal DNA damage during the GSH depletion-induced cell death were assessed. Polyunsaturated fatty acids (PUFA), such as arachidonic acid (AA), gamma-linolenic acid and linoleic acid enhanced lipid peroxidation, induced a loss of membrane integrity and consequently promoted 1-2 Mbp giant DNA fragmentation under both glutamate- and BSO-induced GSH-depletion. Treated C6 cells had 3'-OH termini in their DNA which were recognized by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) analysis. Antioxidants capable of scavenging reactive oxygen species and lipid radicals and iron or copper scavengers inhibited both lipid peroxidation and 1-2 Mbp giant DNA fragmentation, consequently protecting against cell death under GSH depletion. These results suggest that GSH depletion induces lipid peroxidation and leads to 1-2 Mbp giant DNA fragmentation; and that PUFAs can promote giant DNA fragmentation and 3'-OH termini in chromosomal DNA enhancing lipid peroxidation of C6 cells.

Animals↗
PubMed2000

Complement mediated cell death is associated with DNA fragmentation.

In this study, we demonstrate for the first time that complement attack of target cells, in the presence of suitably high levels of serum, can induce the oligonucleosomal DNA fragmentation characteristic of apoptosis. This phenomenon requires membrane permeabilisation induced by formation of the complete membrane attack complex and relies on physiologically relevant levels of serum. TUNEL analysis detected complement mediated DNA fragmentation as early as 30 min after the addition of serum and electron microscopy confirmed that chromatin became condensed after complement attack. Various experiments implicate serum DNase I as the mediator of this DNA fragmentation. Intriguingly, membrane permeability induced by melittin gave rise to similar serum dependent DNA fragmentation. The implications of these results for the study of apoptosis in vitro and in vivo are discussed.

Animals↗
PubMed1986

Digestion of the chicken beta-globin gene chromatin with micrococcal nuclease reveals the presence of an altered nucleosomal array characterized by an atypical ladder of DNA fragments.

The structure of the chicken adult beta-globin gene chromatin in immature and mature erythrocyte nuclei has been analysed using micrococcal nuclease digestion. The resulting DNA fragments were blotted onto DBM-papers and probed with labelled DNA fragments spanning the adult beta-globin gene and its 5'- and 3'-flanking regions. The structure of the nucleosomes within and in the regions flanking the adult beta-globin gene appears to be altered in at least two ways in erythrocyte chromatin, when compared with either bulk or inactive ovalbumin gene chromatin. First, oligomeric DNA fragments containing the beta-globin gene are released faster than those of either bulk or ovalbumin gene chromatin. Second, although the difference in size of the liberated oligomeric DNA fragments is similar to the nucleosomal repeat length of bulk and ovalbumin gene chromatin, the individual oligomers are approximately 100 bp shorter than their bulk or ovalbumin gene counterparts, most noticeably when the nuclease digestion is performed at 37 degrees C. This results in an atypical ladder of approximately 300, 500, 700, 900 bp instead of the canonical chicken erythrocyte ladder which is an integral multiple of 207 bp. The same ladder was obtained from immature erythrocytes, in which the beta-globin gene is actively transcribed, and from mature erythrocytes, in which it is considered to be inactive with RNA polymerase molecules clustered in the 5' moiety of the gene. This indicates that the alteration of the nucleosomal structure is not due to transcription per se.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Alkaline elution analysis of DNA fragmentation induced during apoptosis.

We report that alkaline elution analysis fails to provide a complete profile of DNA fragmentation induced by some genotoxic agents, particularly if these agents induce apoptotic cell death resulting in fragmentation of DNA into oligonucleosomal length multimers. It was questioned whether these oligonucleosome fragments are responsible for the more rapidly eluting component of DNA that is observed as a biphasic elution profile when cells treated with certain genotoxic agents are subjected to alkaline elution. The results of this study indicate that DNA fragmented during apoptotic cell death is eluted in fractions before alkaline denaturation that are normally discarded. Collection of fractions prior to alkaline denaturation is recommended for complete evaluation of the total spectrum of damage induced by genotoxic agents, especially when the compound is suspected of inducing cell death by apoptosis.

Alkalies↗
PubMed1996

Research communication copper-1,10-phenanthroline induces internucleosomal DNA fragmentation in HepG2 cells, resulting from direct oxidation by the hydroxyl radical.

In view of the current speculation regarding the possible role of reactive oxygen species (ROS) in apoptosis, both under physiological conditions and in response to chemicals that promote their intracellular formation, the present investigation was undertaken to examine whether DNA fragmentation during oxidative stress results from endonuclease activity (apoptosis) or from direct attack by ROS. We report that the incubation of HepG2 cells (a human-derived hepatoma cell line) with the copper(II) complex of 1,10-phenanthroline, CuII(OP)2, results in internucleosomal DNA fragmentation, which is widely recognized as being a hallmark of apoptosis. DNA fragmentation did not occur at low temperature, but activity was restored by the addition of ascorbic acid. It is proposed that DNA fragmentation results from the direct attack of hydroxyl radicals upon DNA. Hydroxyl radicals are produced from oxygen by the redox-cycling of CuII(OP)2, which is supported by metabolic processes at normal temperature. At low temperature ascorbic acid provides an artificial cellular reducing environment, thereby restoring hydroxyl radical formation. These findings were confirmed by the detection of internucleosomal DNA fragmentation following the exposure of isolated chromatin to a biomimetic CuII(OP)2 redox-cycling system. We conclude that DNA laddering, the widely employed hallmark of apoptosis, is not unique to endonuclease activity and may also result from direct attack upon DNA by the hydroxyl radical.

Cell Death↗
PubMed2002

Loss of heterozygosity in primary lung cancer using laser capture microdissection and WAVE DNA fragment analysis techniques.

BACKGROUND: A number of molecular changes observed by varied conventional methods, including loss of heterozygosity (LOH) on chromosome 3, have been associated with primary lung cancer. To further define the locus of chromosome 3p allele loss in lung cancer, we performed LOH study by using innovative laser capture microdissection and WAVE DNA Fragment Analysis. MATERIAL/METHODS: Thirty-eight paired specimens from patients with adenocarcinoma of the lung were used for this study. Formalin-fixed, paraffin-embedded tissue from normal stromal cells or lymphocytes and adenocarcinoma were collected using laser capture microdissection. DNA was extracted and amplified by PCR using six polymorphic DNA markers for chromosome 3. PCR products were analyzed by both gel electrophoresis and WAVE DNA Fragment Analysis. RESULTS: LOH at 3p22-24 was found in tumor cells from twelve out of thirty-eight patients (32%) when analyzed by WAVE DNA Fragment Analysis and LOH was found in tumor cells from nine out of thirty-eight patients (23%) when analyzed by gel electrophoresis. LOH was found in normal control from one out of thirty-eight patients. CONCLUSIONS: 1. Our results suggest putative tumor suppressor gene(s) is present in a region at 3p22-24, which may play a role in carcinogenesis of lung cancer. 2. Laser capture microdissection is essential tool for defined LOH studies. 3. WAVE DNA Fragment Analysis is an accurate, sensitive and automated tool for analysis of DNA fragments.

Adenocarcinoma↗
PubMed1999

Extent and limitation of the control of nuclear apoptosis by DNA-fragmenting factor.

During apoptosis, changes to the nucleus of the dying cell include DNA degradation and structural collapse. These changes are accomplished by caspase-mediated cleavage of DNA-fragmenting factor DFF45, an inhibitor of the effector molecule DFF40. DFF45 and, more efficiently, a mutant lacking one caspase-cleavage site (DFF45m) inhibited nuclear changes in a cell-free system when apoptosis was initiated by adding caspase-3 to cell extracts. In primary tissues from several mammalian species, human caspase-3 activated and human DFF45m blocked nuclear apoptosis demonstrating evolutionary conservation of this step. However, DFF45m did not significantly inhibit DNA-fragmenting activity in extracts from staurosporine-treated cells from the human cell line Jurkat. In extracts from normal Jurkat cells, DFF45m blocked caspase-triggered DNA cleavage efficiently only if added within a short time of the addition of the caspase. At later time points, this inhibition by DFF45m was strongly reduced in efficiency while Zn2+ still completely blocked DNA fragmentation. These results demonstrate the evolutionary conservation of a linear pathway in apoptosis and suggest the existence of more complex events as final effector machinery.

Animals↗
PubMed2004

DNA fragmentation in V79 cells irradiated with light ions as measured by pulsed-field gel electrophoresis. II. Simulation with a generalized broken stick model.

PURPOSE: To characterize the differences among the experimental DNA fragmentation spectra induced in Chinese hamster V79 cells by gamma-rays, low-energy protons and alpha-particles through the use of a phenomenological model. MATERIALS AND METHODS: A model of DNA fragmentation was developed as a generalization of the broken-stick model, in which the double-strand breaks induced by radiation were considered randomly placed, but in which the manifestly non-random fragmentation of the control sample was fully taken into account and considered as the initial fragment distribution. Further, an analytical method was introduced that allowed an evaluation of the deviation from randomness of the fragmentation induced by radiation. RESULTS: The analysis of the experimental distribution of DNA fragments showed that there was a progressive departure from randomness in radiation-induced fragmentation going from gamma-rays to protons and then to alpha-particles. This deviation was characterized by an enhanced induction of fragments, and therefore by a larger correlation of double-strand breaks, in the experimental range of lower molecular weights. CONCLUSION: The analysis shows that low-energy light ions induce DNA fragmentation, at the loop level of the chromatin organization, that can be significantly non-random. The same analysis can readily be applied at different length scales, and thus it could offer a basis for the study of the link between DNA damage, correlated at various spatial scales and biological end-points.

Alpha Particles↗
PubMed1996

Subarachnoid injections of lysed blood induce the hsp70 stress gene and produce DNA fragmentation in focal areas of the rat brain.

BACKGROUND AND PURPOSE: Most experimental studies of subarachnoid hemorrhage have demonstrated little histological evidence of injury. In the present study we examined both the expression of the hsp70 heat-shock gene, a molecular marker of reversible neuronal injury, and DNA fragmentation, a marker of irreversible cell injury and death. METHODS: Lysed blood, whole blood, oxyhemoglobin, bovine serum albumin, and saline were injected into the cisterna magna of adult rats. The induction of hsp70 mRNA and HSP70 heat-shock protein was assessed with the use of in situ hybridization and immunocytochemistry, respectively. Fragmentation of genomic DNA was studied by DNA nick end- labeling with the use of terminal deoxynucleotidyl transferase and biotinylated dATP. RESULTS: Expression of the hsp70 gene was not induced in the brains of rats injected with whole blood, oxyhemoglobin, bovine serum albumin, or saline. Lysed blood injections, however, induced hsp70 mRNA at 6 and 24 hours in the cerebellar hemispheres and in focal regions of the basal forebrain. HSP70 protein was induced by 24 hours and persisted for at least 4 days in the same regions. HSP70 protein was localized to patches of glial cells and occasional neurons in the forebrain. In the cerebellum HSP70 was localized to Bergmann glial cells, granule cells, molecular layer stellate cells, and occasional Purkinje cells. DNA nick end-labeling showed patches of labeled cells in the basal forebrain that occurred in the same regions that hsp70 mRNA was induced. CONCLUSIONS: The results demonstrate focal stress gene induction and DNA fragmentation after subarachnoid hemorrhage. It is hypothesized that the focal areas of hsp70 induction may reflect ischemic injury due to vasospasm produced by lysed blood and/or injury mediated by direct toxic effects of the lysed blood. The hsp70 induction and DNA nick end-labeling in the same regions suggests that lysed blood produces a spectrum of injury from HSP70 protein-labeled, reversibly injured cells to dead cells with fragmented DNA. Induction of the hsp70 stress gene and DNA nick end-labeling may be useful for evaluating the causes of injury, the spectrum of injury, and potential pharmacological therapies in experimental models of subarachnoid hemorrhage.

Animals↗