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PubMed1996

Death of germinal center B cells without DNA fragmentation.

During the selection of B cells within germinal centers (GC) on the basis of their affinity for T-dependent antigen, B cells not positively selected are eliminated within GC. This process of B cell death has been considered to be apoptosis. In a recent study, we have reported that, although a substantial number of thymocytes were considered to be dead because of their extremely small cell size and heavy chromatin condensation even though they were not yet phagocytosed (pyknosis), they were devoid of DNA fragmentation, the most characteristic feature for apoptosis. In this study, we examined in vivo the mechanism of B cell death within GC by using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) method to detect DNA double-strand breaks. TUNEL+ B cells were scattered throughout the upper dark and the light zones of GC. Double staining of the sections by the TUNEL method and acid phosphatase (AcP) activities showed that all the TUNEL+ B cells were phagocytosed by macrophages. Light microscopic and ultrastructural studies revealed the presence of small unphagocytosed B cells within the light zone. These cells are undoubtedly dead because they were much smaller than surrounding lymphoid cells and have a heavy chromatin condensation. Furthermore, ultrastructural detection of DNA fragmentation confirmed that these small unphagocytosed B cells were TUNEL-, implying that DNA fragmentation is not primarily involved in the cell death process of these small dead B cells. These results indicate that most B cells, not positively selected and thus destined to be eliminated, die within GC without DNA fragmentation, and are subsequently phagocytosed by macrophages and become TUNEL+. Typical apoptosis, characterized by DNA fragmentation in situ, is not the predominant type of cell death that occurs during the selection of B cells in GC.

Animals↗

PubMed1996

DNA fragmentation in mouse organs during endotoxic shock.

The systemic inflammatory response syndrome has still an unpredictable outcome, and patients often die of multiple organ failure despite circulatory stabilization therapy. The still incompletely understood pathophysiological mechanisms include organ damage due to direct toxic actions of cytokines elicited by overactivation of the host response. To study this process of organ failure in experimental septic shock, we injected mice with a lethal dose of endotoxin and examined apoptotic and necrotic tissue damage biochemically, histologically, and ultrastructurally. Endotoxin administration caused oligonucleosomal as well as random DNA fragmentation in liver, lung, kidney, and intestine. In the liver, DNA fragmentation was not restricted to hepatocytes but also occurred in nonparenchymal cells. The DNA fragmentation was mediated by tumor necrosis factor and attenuated by endogenous nitric oxide release. Unlike the situation in D-galactosamine-sensitized mice, in which injection or release of tumor necrosis factor causes massive hepatocyte apoptosis, liver failure due to high doses of endotoxin was characterized by single-cell necrosis, a low incidence of apoptosis, and simultaneous damage to nonparenchymal cells. We conclude that, even though endotoxin causes cytokine-mediated DNA fragmentation in several organs including the liver, hepatocyte apoptosis itself seems to be a minor phenomenon in high-dose endotoxic shock in mice.

Animals↗

PubMed2001

Rotational dynamics of curved DNA fragments studied by fluorescence polarization anisotropy.

The rotational dynamics of short DNA fragments with or without intrinsic curvature were studied using time-resolved phase fluorimetry of intercalated ethidium with detection of the anisotropy. Parameters determined were the spinning diffusion coefficient of the DNA fragments about the long axis and the zero-time ethidium fluorescence anisotropy. We find a significant decrease in the spinning diffusion coefficient for all curved fragments compared to the straight controls. This decrease is likewise evident in rotational diffusion coefficients computed from DNA structures obtained by a curvature prediction program for these sequences. Using a hinged-cylinder model, we can identify the change in rotational diffusion coefficient with a permanent bend of 13-16 degrees per helix turn for the sequences studied. Moreover, for some of the curved fragments an increased flexibility has to be assumed in addition to the permanent bend in order to explain the data.

Algorithms↗

PubMed1995

Proteinases are involved in both DNA fragmentation and membrane damage during CTL-mediated target cell killing.

A number of inhibitors with different specificities were used to probe the involvement of proteinases in the mechanism of cytotoxic T lymphocyte (CTL)-mediated lysis. N-Acetyl-L-tyrosine ethyl ester (ATEE) and N-benzoyl-L-arginine ethyl ester (BAEE) are reversible substrate inhibitors of proteinases with trypsin-like and chymotrypsin-like specificities, respectively. BAEE did not prevent either the chromium release or DNA fragmentation induced in mouse tumor target cells by a mixed lymphocyte population. In contrast, ATEE inhibited both processes. The irreversible proteinase inhibitor 3,4-dichloroisocoumarin (DCI) also blocked both chromium release and DNA fragmentation, but at significantly lower concentrations than ATEE. More importantly, chromium release was more susceptible to inhibition by DCI than DNA fragmentation. Addition of a combination of the endonuclease inhibitor aurintricarboxylic acid plus DCI resulted in virtually complete inhibition of both DNA fragmentation and chromium release when the drugs were added at the beginning of the incubation period. In contrast addition of DCI 15 or 30 min following initiation of the lytic cycle abolished the affect of DCI on fragmentation, but not lysis. A model which suggests a dual role for the proteinases in CTL-mediated target cell death is presented. First, proteinases are involved in the initiation of DNA fragmentation. Second, they have an ongoing function in membrane damage.

Animals↗

PubMed1990

DNA fragmentation induced in macrophages by gliotoxin does not require protein synthesis and is preceded by raised inositol triphosphate levels.

We have shown that the immunomodulating agent gliotoxin induces DNA fragmentation in macrophages characteristic of programmed cell death or apoptosis (Waring, P., Eichner, R. D., Mullbacher, A., and Sjaarda, A. (1988) J. Biol. Chem, 263, 18493-18499). In addition, morphological changes and DNA fragmentation characteristic of apoptosis are induced in 48 h concanavalin A-stimulated T blasts by gliotoxin and these changes are inhibited by Zn2+ (Waring, P., Egan, M., Braithwaite, A., Mullbacher, A., and Sjaarda, A. (1990) Int. J. Immunopharmacol., in press). We have studied the effects of actinomycin D and the protein synthesis inhibitor cycloheximide on apoptosis induced by gliotoxin in these cells, and these studies demonstrate no effect on apoptosis induced by gliotoxin. Cycloheximide and actinomycin D alone induce DNA fragmentation in these cells. Gliotoxin itself proved to be a potent inhibitor of protein synthesis. The fragmentation caused by cycloheximide correlated with the extent of protein synthesis inhibition. The toxin ricin also induced DNA fragmentation in T blasts characteristic of apoptosis. These results indicate that protein synthesis is not required for induction of apoptosis in macrophages or T blasts by gliotoxin. Gliotoxin caused elevated levels of inositol triphosphate in treated macrophages which may be related to mobilization of Ca2+ levels during apoptosis.

Animals↗

PubMed1997

The characterization of composite agarose/hydroxyethylcellulose matrices for the separation of DNA fragments using capillary electrophoresis.

Mixtures of the polysaccharide derivatives, 19% hydroxyethylated SeaPrep agarose (SP-AG) and hydroxyethylcellulose (HEC), in aqueous buffer solutions are applied for the first time to the separation of DNA fragments using capillary electrophoresis (CE). These matrices form unique size-sieving networks that allow the separation of a wide size range of DNA fragments in a single analysis. Relative to their homogeneous counterparts, the composite separation matrices provide enhanced selectivity properties of DNA fragments, especially for fragments greater than 1000 base pairs (bp) in length. Additionally, the effects on separation performance of capillary temperature, the incorporation of a DNA intercalator, and applied field strength are demonstrated. Solution viscosity measurements of the homogeneous and composite matrix solutions were made in order to establish the entanglement threshold concentrations for the unique size-sieving solutions. The relatively low solution viscosities of the composite separation matrices allow reproducible replacement of the separation matrix between analyses. The mechanism of separation of DNA fragments for the composite matrices is proposed.

Buffers↗

PubMed1996

Anatomical studies of DNA fragmentation in rat brain after systemic kainate administration.

Rats treated systemically with kainate develop stereotyped epileptic seizures involving mainly limbic structures that may last for hours. This model of limbic status epilepticus has been widely studied using classical neuropathological techniques. We used in situ nick translation histochemistry to examine patterns of DNA fragmentation in this model. We found a stereotyped and reproducible pattern of neuronal populations that demonstrate evidence of DNA fragmentation from 24 h to one week after kainate treatment. Neither blockade of new protein synthesis nor blockade of the N-methyl-D-aspartate-type glutamate receptors significantly altered this response. Moreover, we saw no evidence of the regular internucleosomal cleavage of DNA that produces a characteristic laddered appearance of 180-200 bp DNA fragments after gel electrophoresis in samples obtained from microdissected affected regions. These studies suggest that DNA fragmentation after systemic kainate-induced seizures is not the result of programmed cell death. This assay may be useful for quantitative testing of both neuroprotective agents and mechanistic hypotheses.

Animals↗

PubMed2005

Portable capillary electrophoresis system with potential gradient detection for separation of DNA fragments.

A portable capillary electrophoresis (CE) system with a novel potential gradient detection (PGD) was utilized to separate DNA fragments. For the first time it was demonstrated that separation of DNA fragments in polymer solution could be detected by a portable CE system integrated with PGD, with a limit of detection (LOD) comparable to that of the CE-ultraviolet (UV) method. Effects of buffer solution, sieving medium, and applied voltage were also investigated. The portable CE-PGD system shows several potential advantages, such as simplicity, cost effectiveness, and miniaturization.

Bacteriophage phi X 174↗

PubMed2005

Oligomerization state of the DNA fragmentation factor in normal and apoptotic cells.

The caspase-activated DNase (CAD) is the primary nuclease responsible for oligonucleosomal DNA fragmentation during apoptosis. The DNA fragmentation factor (DFF) is composed of the 40-kDa CAD (DFF40) in complex with its cognate 45-kDa inhibitor (inhibitor of CAD: ICAD or DFF45). The association of ICAD with CAD not only inhibits the DNase activity but is also essential for the co-translational folding of CAD. Activation of CAD requires caspase-3-dependent proteolysis of ICAD. The tertiary structures of neither the inactive nor the activated DFF have been conclusively established. Whereas the inactive DFF is thought to consist of the CAD/ICAD heterodimer, activated CAD has been isolated as a large (>MDa) multimer, as well as a monomer. To establish the subunit stoichiometry of DFF and some of its structural determinants in normal and apoptotic cells, we utilized size-exclusion chromatography in combination with co-immunoprecipitation and mutagenesis techniques. Both endogenous and heterologously expressed DFF have an apparent molecular mass of 160-190 kDa and contain 2 CAD and 2 ICAD molecules (CAD/ICAD)2 in HeLa cells. Although the N-terminal (CIDE-N) domain of CAD is not required for ICAD binding, it is necessary but not sufficient for ICAD homodimerization in the DFF. In contrast, the CIDE-N domain of ICAD is required for CAD/ICAD association. Using bioluminescence resonance energy transfer (BRET), dimerization of ICAD in DFF was confirmed in live cells. In apoptotic cells, endogenous and exogenous CAD forms limited oligomers, representing the active nuclease. A model is proposed for the rearrangement of the DFF subunit stoichiometry in cells undergoing programmed cell death.

Animals↗

PubMed1990

Effects of sequence selective drugs on the gel mobility of a bent DNA fragment.

The effects of various drugs on the structure of a bent DNA fragment have been investigated by studying DNA mobility in polyacrylamide gels. This DNA fragment has an anomalously slow rate of migration on account of its phased runs of adenines. Nogalamycin and echinomycin increase the gel mobility of kinetoplast DNA suggesting that the bending has been removed. Mithramycin, actinomycin, distamycin and ethidium have either no effect or cause a further reduction in mobility. These results are compared with other, non-bent DNA species which always show a decrease in gel mobility in the presence of DNA binding drugs.

Adenine↗

PubMedDate not supplied

A comparison of distinct modes of tumor cell death in Hodgkin's disease using morphology and in situ DNA fragmentation.

The study examined the morphology and frequency of cell death occurring spontaneously in lymph nodes from patients with Hodgkin's disease. In addition to necrosis, which was infrequent and usually in patches, we document two cell types showing features of individual cell death: mummy cells end apoptotic cells. Mummy cells present no evidence of DNA fragmentation, but show electron microscopic features of "dark cells." Apoptotic Hodgkin-Reed-Sternberg cells are found frequently and are easier to demonstrate by in situ and labeling of fragmented DNA than by light microscopy only. In many cases phagocytosis of apoptotic cells is also documented. The significance of these findings to the limited number of Hodgkin-Reed-Sternberg cells in most cases of Hodgkin's disease is discussed.

Cell Death↗

PubMed2002

TNF-alpha is involved in activating DNA fragmentation in skeletal muscle.

Intraperitoneal administration of 100 microg kg(-1) (body weight) of tumour necrosis factor-alpha to rats for 8 consecutive days resulted in a significant decrease in protein content, which was concomitant with a reduction in DNA content. Interestingly, the protein/DNA ratio was unchanged in the skeletal muscle of the tumour necrosis factor-alpha-treated animals as compared with the non-treated controls. Analysis of muscle DNA fragmentation clearly showed enhanced laddering in the skeletal muscle of tumour necrosis factor-alpha-treated animals, suggesting an apoptotic phenomenon. In a different set of experiments, mice bearing a cachexia-inducing tumour (the Lewis lung carcinoma) showed an increase in muscle DNA fragmentation (9.8-fold) as compared with their non-tumour-bearing control counterparts as previously described. When gene-deficient mice for tumour necrosis factor-alpha receptor protein I were inoculated with Lewis lung carcinoma, they were also affected by DNA fragmentation; however the increase was only 2.1-fold. These results suggest that tumour necrosis factor-alpha partly mediates DNA fragmentation during experimental cancer-associated cachexia.

Animals↗

PubMed2000

Column efficiency and separation of DNA fragments using slalom chromatography: hydrodynamic study and fractal considerations.

Novel equations (Guillaume Y. C.; et al. Anal. Chem. 2000, 72, 853) were developed to describe the large double-stranded DNA molecule retention in slalom chromatography (SC). These equations were applied for the first time to model both the "apparent selectivity" and the resolution between two eluted DNA fragments on a chromatogram. A study of the column efficiency corroborated the fact that slalom chromatography is not based on an adsorption or equilibrium phenomenon, but can be attributed to a hydrodynamic phenomenon. Using a combination of the dynamics of DNA fragment progression in the column and fractal considerations, it was shown that the apparent selectivity depends both on the DNA fragment sizes and mobile-phase flow rate and therefore a balance between two hydrodynamic regimes. A chromatographic response function was also used to obtain the most efficient separation conditions for a mixture of DNA fragments in a minimum analysis time. The chromatographic data confirmed that in SC the flow rate can increase or maintain the separation efficiency with an associated decrease in the analysis time. This constitutes an attractive outcome in relation to the classical chromatographic separation.

Chromatography↗

PubMed1995

Expression immunoassay. Antigen quantitation using antibodies labeled with enzyme-coding DNA fragments.

A novel immunoassay is reported which uses an enzyme-coding DNA fragment as label (expression immunoassay). The DNA label is determined with high sensitivity by measuring the enzymatic activity produced after expression. A DNA fragment encoding the firefly luciferase is biotinylated and complexed with streptavidin. Biotinylated, specific antibodies are used for quantitation of antigen immobilized on microtiter wells. After completion of the immunoreaction, streptavidin-DNA is bound to the immunocomplex. Subsequent expression of the solid phase-bound DNA, by an one-step (coupled) cell-free transcription/translation, produces luciferase. The enzyme catalyzes the luminescent reaction of luciferin with O2 and ATP. As few as 3000 molecules of DNA label can be detected. Also, 50,000 antigen molecules can be detected, and the luminescence is a linear function of the number of antigen molecules in a range extending over 3 orders of magnitude. The high sensitivity achieved is a result of the combined amplification due to transcription/translation and the substrate turnover.

Antigens↗

PubMed1992

Vincristine potentiates cytochalasin B-induced DNA fragmentation in vitro.

The cytochalasins are fungal metabolites that have previously been shown to have some chemotherapeutic potential. When various cell types are treated in vitro with both cytochalasin B and vincristine, the resultant DNA fragmentation is greater than the sum of that caused by each agent alone. The levels necessary to achieve this potentiation are obtainable in vivo. DNA fragmentation induced by cytochalasin E, an actin-specific agent, is potentiated by vincristine. Pretreatment of the mastocytoma line P815 with vincristine results in an enhancement of the ability of cytochalasin B to fragment DNA. These results indicate that cytochalasin B might be effective as a chemotherapeutic agent in the presence of vincristine.

Cytochalasin B↗

PubMed2000

Reduction of the DNA base excision repair protein, XRCC1, may contribute to DNA fragmentation after cold injury-induced brain trauma in mice.

The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in the DNA base excision repair pathway by interacting with DNA ligase III and DNA polymerase beta. The present study examined the protein expression of XRCC1 and DNA fragmentation before and after cold injury-induced brain trauma (CIBT) in mice, in which apoptosis is assumed to participate. Immunohistochemistry showed the nuclear expression of XRCC1 in the entire region of the control brains. Fifteen minutes after CIBT, nuclear immunoreactivity was predominantly decreased in the inner boundary of the lesion, followed by a significant reduction of XRCC1 in the entire lesion 4 h after CIBT. A characteristic 70-kDa band was detected in the non-traumatic area, and was markedly decreased after CIBT as shown by Western blot analysis. DNA fragmentation was also observed after CIBT, and double staining with XRCC1 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed a spatial relationship between XRCC1 loss and DNA fragmentation 24 h after CIBT. These data indicate that early decrease of XRCC1 and failure of the DNA repair mechanism may contribute to DNA-damaged neuronal cell death after CIBT.

Animals↗

PubMed1999

Regional and temporal alterations in DNA fragmentation factor (DFF)-like proteins following experimental brain trauma in the rat.

DNA fragmentation, an early event in neuronal death following traumatic brain injury, may be triggered by the 40-kDa subunit of DNA fragmentation factor (DFF40). DFF40 is typically bound to the 45-kDa subunit of DFF (DFF45), and activation of DFF40 may occur as a result of caspase-3-mediated cleavage of DFF45 into 30- and 11-kDa fragments. In this study, the intracellular distribution of DFF45 and DFF40 was examined following lateral fluid percussion brain injury of moderate severity (2.4-2.7 atm) in male Sprague-Dawley rats. In the cytosolic fraction (S1) of the injured cortex at 2 and 24 h postinjury, significant decreases in the intensities of DFF45-like proteins at 45- and 32-kDa bands and a concomitant increase in the 11-kDa bands were observed (p < 0.05 vs. uninjured controls). A significant decrease in the intensities of the 32-kDa band in the nuclear (P1) fraction of the injured cortex was observed at 30 min and 2 h postinjury (p < 0.01). Concomitantly, a decrease in DFF40 was observed in the cortical S1 fraction at 2 and 24 h (p < 0.05) and in the P1 fraction at 30 min and 2 h postinjury (p < 0.01). In the hippocampus, DFF45 decreased at 30 min in the P1 and 2 h in the S1 fraction (p < 0.05) and recovered by 24 h postinjury, whereas DFF40 was significantly decreased in the S1 and increased in the P1 fraction at both 2 and 24 h (p < 0.01), which indicated a translocation of DFF40 from cytosol to nucleus. These data are the first to demonstrate that changes in DFF proteins occur after brain trauma and suggest that these changes may play a role in apoptotic cell death via caspase-3-DFF45/DFF40-DNA cleavage observed following traumatic brain injury.

Animals↗

PubMed1999

Paclitaxel sensitivity correlates with p53 status and DNA fragmentation, but not G2/M accumulation.

PURPOSE: The antitumor agent paclitaxel (Taxol) has been shown to arrest cells in mitosis through microtubule stabilization and to induce apoptosis. The tumor suppressor gene p53 is implicated in the regulation of cell cycle checkpoints and can mediate apoptotic cell death. Although initial studies demonstrated that various DNA-damaging agents can induce p53, more recent studies have also shown p53 induction following nonDNA-damaging agents, including paclitaxel. We investigated the influence of p53 abrogation on paclitaxel-induced cell kill and correlated the extent of mitotic arrest and DNA fragmentation by paclitaxel with the drug's cytotoxic effect. MATERIALS AND METHODS: The parental human colorectal carcinoma cell line (RKO) with wild-type p53 alleles, and two transfected RKO cell lines with inactivated p53 (RKO.p53.13 with transfected mutant p53 and RC 10.3 with HPV-16-derived E6 gene) were exposed to graded doses of paclitaxel (1-100 nM) for 24-h intervals. The functional status of p53 in cells was assessed by thymidine and BrdU incorporation following exposure to ionizing radiation (4 Gy). Reproductive integrity following paclitaxel treatment was assessed by clonogenic assay. Immunolabeling and microscopic evaluation were used to assess mitotic accumulation and micronucleation. Apoptosis was assayed using DNA fragmentation analyses. RESULTS: A 4-fold increase in paclitaxel sensitivity was observed among RKO cells deficient in p53 function compared with wild-type RKO cells (IC 50: 4 nM, 1 nM, 1nM for RKO, RKO.p53.13, RC 10.3, respectively). The increased cytotoxic effect in RKO cells with inactive p53 correlated with an increased propensity towards micronucleation and DNA fragmentation following paclitaxel treatment. However, no significant difference in peak mitotic accumulation was observed among RKO cells with functional or abrogated p53. CONCLUSIONS: RKO cells lacking functional p53 demonstrate significantly enhanced sensitivity to paclitaxel compared with that of wild-type RKO cells. This response corresponded with increased micronucleation and DNA fragmentation in cells deficient in p53 function. Although previous published reports of enhanced paclitaxel sensitivity in p53-deficient cells correlated this finding with increased G2/M arrest, we did not observe any significant correlation between paclitaxel-induced cell kill and the degree of mitotic arrest. Our data suggest that apoptosis is the predominant mechanism of paclitaxel cytotoxicity in RKO cells and is likely mediated by a p53-independent process.

Antineoplastic Agents, Phytogenic↗