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PubMed2000

Prevention by vitamin E of DNA fragmentation and apoptosis induced by fumonisin B1 in C6 glioma cells.

Fumonisin B1 (FB1), produced by the fungus Fusarium moniliforme, belongs to a class of sphingosine analogue mycotoxins that occur widely in the food chain. Epidemiological studies have associated consumption of Fusarium moniliforme-contaminated food with human oesophageal cancer in China and South Africa. FB1 also causes equine leucoencephalomalacia. Evidence for induction of apoptosis by FB1 was first obtained when C6 glioma cells were incubated with fumonisin B1 (3-27 microM) causing DNA fragmentation profiles showing DNA laddering in gel electrophoresis and apoptotic bodies revealed by chromatin staining with acridine orange and ethidium bromide. Further confirmation experiments and comet assays have been performed under similar conditions. The results of the comet test show that FB1 at 9 and 18 microM induces respectively 50 +/- 2% and 40 +/- 1% of cells with a comet with an increased tail length of 93 +/- 9 microm and 102 +/- 17 microm respectively. Under these concentrations, FB1 induced DNA fragmentation and laddering and many apoptotic bodies. Pre-incubation of the cells with vitamin E (25 microM) for 24 h before FB1 (18 microM) significantly reduced DNA fragmentation and apoptotic bodies induced by FB1.

Animals↗
PubMed1978

Biochemical studies on bovine adenovirus type 3. IV. Transformation by viral DNA and DNA fragments.

By the calcium technique, intact DNA of bovine adenovirus type 3 (BAV3) was found to transform A31 cells, a clone of BALB/3T3. Transforming activity was resistant to RNase and Pronase but sensitive to DNase. The efficiency of transformation was approximately 5 to 10 foci per mug of DNA. Attempts were also made to test for transforming activity of BAV3 DNA fragments prepared with restriction endonucleases EcoRI and HindIII. The activity was found to associate exclusively with the EcoRI D fragment mapped in the region of 3.6 and 19.7 units (molecular weight, 3.9 x 10(6)). No transformation could be obtained with three HindIII fragments, J, E, and B, located at the left-hand end of the BAV3 genome. However, the enzymatic joining of J and E fragments (0 to 11.9 map units) with a ligase restored the transforming activity. These results suggest that all the genetic information of BAV3 required for transformation is located in the region between 3.6 and 11.9 units on the viral genome. Some properties of A31 cells transformed by BAV3 DNA EcoRI D fragment (TrD) and the ligated DNA of HindIII J and E fragments (TrJE), as well as those transformed by whole BAV3 DNA (Tr), were examined. As compared to untransformed A31 cells, all the transformed cell lines tested showed rapid growth, high saturation densities, and anchorage-independent growth. Moreover, they contained BAV3-specific T antigen and induced tumors in adult nude and BALB/c mice. These properties of Tr, TrD, and TrJE lines were similar to those of BAV3-transformed cells.

Adenoviridae↗
PubMed1982

Preparative scale, high resolution purification of low molecular weight DNA fragments.

A commercially available continuous electroelution system has been used to separate and purify low molecular weight DNA fragments from polyacrylamide gels. This method has several advantages over previously reported methods for the recovery of DNA fragments from polyacrylamide gels. This technique, which gives very high recovery rates (80-95%), can be carried out on a relatively large scale and in a way that is not labour intensive. Data are presented for the purification of DNA fragments with molecular weights in the range 1-4 x 10(5) (200-700 base-pairs), although the method is also applicable to larger molecular weight DNA fragments, RNAs and proteins.

Acrylic Resins↗
PubMed2001

Temporal and spatial patterns of DNA fragmentation following focally or systemically-evoked status epilepticus in rats.

Status epilepticus (SE) triggers neuronal degeneration comprised of both necrotic and apoptotic components. Here we determined whether internucleosomal DNA fragmentation reflects the severity of SE-induced neuronal damage. We utilized both a systemic (kainic acid) and a focally-induced model of SE in rats. DNA fragmentation was analyzed in rhinal cortex and hippocampus at various time points following SE episodes of varying durations (30-120 min). Radioactively labeled DNA fragments were analyzed by agarose gel electrophoresis and quantified by liquid scintillation counting. The spatial and temporal characteristics of the SE-evoked DNA fragmentation indicated that this marker of apoptosis appears as early as 8 h after SE and reaches peak expression at 48 h. This method permitted us to quantitatively monitor the evolution of the apoptotic component of cell death over the acute post-injury period (8-72 h). Moreover, in both models of SE, the DNA fragmentation varied directly as a linear function of the duration of SE between 30 and 120 min suggesting that this marker should be highly responsive to neuroprotective intervention.

Animals↗
PubMed1997

A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.

Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.

DNA↗
PubMed1996

Histochemical detection of intranuclear DNA fragmentation and its relation to the expression of bcl-2 oncoprotein in human prostatic cancer.

OBJECTIVE: To assess the incidence of intranuclear DNA fragmentation and the expression of the bcl-2 oncoprotein in prostatic carcinoma, both of which are related to programmed cell death. PATIENTS, MATERIALS AND METHODS: Specimens of tumour obtained from 17 patients with newly diagnosed prostatic carcinoma and 16 with hormone-treated prostatic carcinoma undergoing total prostatectomy were evaluated. DNA fragmentation was detected using the terminal-labelling method (d-uridine triphosphate conjugated with digoxigenin) and the expression of bcl-2 was detected immunohistochemically. RESULTS: There was a high incidence of intranuclear DNA fragmentation in 14 of 17 untreated tumours and 15 of 16 hormone-treated tumours. There were no differences between the positive cases in hormone-treated tumours and untreated tumours. There was significantly greater expression of bcl-2 in tumours treated with non-steroidal anti-androgen drugs (eight of nine were positive) than in those untreated (seven of 17) or treated with other drugs (one of seven) (P < 0.05). There was a consistent and marked dissociation between DNA fragmentation and bcl-2 positivity; most of the cells positive for bcl-2 showed no DNA fragmentation. CONCLUSION: The results indicated that cells positive for bcl-2 might potentially be hormone resistant and that the administration of non-steroidal anti-androgen drugs might have a role in the induction of hormone-resistant cells.

Aged↗
PubMed2000

DNA fragmentation in apoptosis.

Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro. Genetic studies in mice support the importance of DFF in DNA fragmentation and possibly in apoptosis in vivo. Recent work also suggests the existence of additional endonucleases for DNA degradation. Understanding the roles of individual endonucleases in apoptosis, and how they might coordinate to degrade DNA in different tissues during normal development and homeostasis, as well as in various diseased states, will be a major research focus in the near future.

Animals↗
PubMed1998

Bcl-2-mediated resistance to apoptosis is associated with glutathione-induced inhibition of AP24 activation of nuclear DNA fragmentation.

Studies on the mechanism of apoptosis in this laboratory support a model in which signal transduction involving caspase 3 leads to activation of a serine protease called Mr 24,000 apoptotic protease (AP24), which then induces internucleosomal DNA fragmentation in the nucleus. This study examined the effect of Bcl-2 overexpression on activation of AP24 and the induction of DNA fragmentation by AP24 in isolated nuclei. It was demonstrated that overexpression of Bcl-2 in either HL-60 or PW leukemia cell lines suppressed activation of AP24 induced by either tumor necrosis factor or UV light and protected cells from apoptosis. Furthermore, nuclei isolated from Bcl-2-overexpressing cells were relatively resistant to internucleosomal DNA fragmentation induced by AP24 isolated from apoptotic cells. Bcl-2-overexpressing cells that were nutritionally depleted of glutathione (GSH) became sensitive to tumor necrosis factor- or UV light-induced activation of AP24 and underwent apoptotic cell death. Moreover, nuclei isolated from Bcl-2-overexpressing cells that were depleted of GSH became sensitive to AP24-induced DNA fragmentation. The addition of exogenous GSH blocked the proteolytic activity of AP24, as well as its ability to induce DNA fragmentation in normal isolated nuclei. These results indicate that Bcl-2 can attenuate at least two events in the AP24 apoptotic pathway: activation of AP24 and induction of DNA fragmentation by activated AP24. Furthermore, agents that deplete intracellular levels of GSH may have therapeutic use in the sensitization of Bcl-2-overexpressing cancer cells to apoptotic cell death.

Apoptosis↗
PubMed1999

Electron microscopic demonstration of nick end-labeled DNA fragments during capsaicin-induced apoptosis of trigeminal primary neurons in neonatal rats.

DNA fragmentation was induced in the trigeminal ganglion of newborn rats by subcutaneous capsaicin injection (50 mg/kg). Twenty-four hours later, numerous roundish profiles were intensely labeled by both a DNA polymerase I-mediated nick translation method and a terminal transferase-mediated tailing method. Direct electron microscopic examination of labeled profiles indicated that the labeled profiles were neurons at earlier stages of apoptosis. DNA fragmentation signal was first detected in the nucleoplasm and later spread to the cytoplasm. The cell finally disintegrated forming many small apoptotic bodies. DNA fragmentation signal in the apoptotic bodies was readily labeled by the tailing but not the translation method.

Analysis of Variance↗
PubMed1999

Copper-zinc superoxide dismutase prevents the early decrease of apurinic/apyrimidinic endonuclease and subsequent DNA fragmentation after transient focal cerebral ischemia in mice.

BACKGROUND AND PURPOSE: DNA damage and its repair mechanism are thought to be involved in ischemia/reperfusion injury in the brain. We have previously shown that apurinic/apyrimidinic endonuclease (APE/Ref-1), a multifunctional protein in the DNA base excision repair pathway, rapidly decreased after transient focal cerebral ischemia (FCI) before the peak of DNA fragmentation. To further investigate the role of reactive oxygen species in APE/Ref-1 expression in vivo, we examined the expression of APE/Ref-1 and DNA damage after FCI in wild-type and transgenic mice overexpressing copper-zinc superoxide dismutase. METHODS: Transgenic mice overexpressing copper-zinc superoxide dismutase and wild-type littermates were subjected to 60 minutes of transient FCI by intraluminal blockade of the middle cerebral artery. APE/Ref-1 protein expression was analyzed by immunohistochemistry and Western blot analysis. DNA damage was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL). RESULTS: A similar level of APE/Ref-1 was detected in the control brains from both groups. APE/Ref-1 was significantly reduced 1 hour after transient FCI in both groups, whereas the transgenic mice had less reduction than that seen in wild-type mice 1 and 4 hours after FCI. DNA laddering was detected 24 hours after FCI and was decreased in transgenic mice. Double staining with APE/Ref-1 and TUNEL showed that the neurons that lost APE/Ref-1 immunoreactivity became TUNEL positive. CONCLUSIONS: These results suggest that reactive oxygen species contribute to the early decrease of APE/Ref-1 and thereby exacerbate DNA fragmentation after transient FCI in mice.

Animals↗
PubMed1995

Relationships between DNA fragmentation, chromatin condensation, and changes in flow cytometry profiles detected during apoptosis.

The determination of whether a cell dies by apoptosis as opposed to necrosis is usually best made on the basis of distinct structural changes in the chromatin. These changes include extensive condensation of the chromatin and DNA fragmentation. We have shown that the cytotoxic drug bleomycin (BLM) is able to cleave the DNA between the nucleosomes when it enters into the cell. If sufficient amounts of BLM are internalized, the nuclear morphological changes characteristic of apoptosis are detected. In this work, we describe the nuclear changes that occurred after DNA fragmentation as a function of the number of DNA double-strand breaks generated per cell and of the time after their generation. Our results show that DNA fragmentation and degradation of higher-order DNA structure were directly responsible for the nuclear morphological changes associated with apoptosis. During apoptosis reduced fluorescence with respect to the G0/G1 cell cycle region (the sub-G1 region) is often detected if fixed cells from cultures undergoing apoptosis are analyzed by flow cytometry. We demonstrate here that, depending on the extent of the DNA fragmentation and on ulterior changes in chromatin structure, the content of the fluorescent sub-G1 region can be either soluble pieces of DNA or apoptotic bodies or cells depleted in the DNA content by partial loss of fragmented DNA dissolved in the washing media and/or by the release of apoptotic bodies.

Animals↗
PubMed1997

Capillary electrophoresis of DNA fragments in 9 to 20% uncrosslinked polyacrylamide gels: unique separating capacity hypothetically related to maintenance of random-coil DNA conformation independently of gel concentration.

DNA fragments (0.1 to 2 kb) were separated by capillary electrophoresis (CE) in 9 to 20% uncrosslinked polyacrylamide gels with a resolving power ranging from 3 to o.1 million theoretical plates/meter across that DNA size range. The unique feature of electrophoresis in 18 to 20% uncrosslinked polyacrylamide is that it provides a method capable of resolving charge isomeric species of DNA fragments (0.4 to 2 kb), confirming a previous report by Heiger et al. [Heiger DN, Cohen AS, Karger BL. J Chromatogr 516 (1990) 33-48]. A similarly unique resolving capacity of uncrosslinked polyacrylamide gels for DNA previously reported is that for heteroduplex DNA [Pulyaeva H, Zakharov SF, Garner MM, Chrambach A. Electrophoresis 15 (1994) 1095-1100] matched by crosslinked gels only in the presence of denaturants [Peeters AV, Kotze MJ. PCR Methods Appl 4 (1994) 188-190; Ganguly A, Rock MJ, Prockop DJ. Proc Natl Acad Sci USA 90 (1993) 10 325-10 329]. A clue as to the cause of that unique resolving capacity of crosslinked polyacrylamide is provided by the finding in the present study of a single, gel concentration independent KR [retardation coefficient, d(log mobility)/d(gel concentration)] for the DNA fragments, which contrasts with the decrease of KR with gel concentration observed for crosslinked polyacrylamide across a wide concentration range [Orban L, Chrambach A. Electrophoresis 12 (1991) 241-246; Tietz D, Chrambach A. Electrophoresis 14 (1993) 185-190]. Since the decrease of KR with gel concentration correlates with a decrease in equivalent molecular radius [Tietz D, Chrambach A. Electrophoresis 14 (1993) 185-190], it has been interpreted as being due to the transition from a random-coiled to a stretched DNA conformation upon passage through gels of increasing concentration. Since in uncrosslinked gels the decrease of KR does not occur, it is correspondingly assumed that the random-coil conformation of DNA is maintained in those gels in the investigated concentration range up to 20%. The maintenance of random-coil conformation [Tietz D, Chrambach A. Electrophoresis 14 (1993) 185-190]. The effect of denaturants in allowing for resolution of heteroduplex DNA in crosslinked gels [Peeters AV, Kotze MJ. PCR Methods Appl 4 (1994) 188-190; Ganguly A, Rock MJ, Prockop DJ, Proc Natl Acad Sci USA 90 (1993) 10 325-10 329] supports that hypothesis of the enhanced resolving power of electrophoresis in gels that maintain random-coiled DNA within the gel concentration range used.

Acrylic Resins↗
PubMed2005

Sustained transgene expression in rat kidney with naked plasmid DNA and PCR-amplified DNA fragments.

Recently, we developed a kidney-targeted gene transfer technique, in which naked DNA was injected into the renal vein while the renal vein and artery were clamped. Kidney-targeted DNA transfer with only the renal vein clamped is an important modification that may permit less invasive catheter-based gene transfer in future clinical applications. The preparation of PCR-amplified DNA fragments is less time-consuming than that of naked plasmid DNA. We examined rat erythropoietin (Epo) plasmid, pCAGGS-Epo, or PCR-amplified DNA fragment, fCAGGS-Epo, transfer into the rat kidney with only the renal vein clamped. The Epo level peaked at week 3 and then was sustained for 24 weeks, which resulted in significant erythropoiesis. This modified technique, allowing long-term expression of both PCR-amplified DNA fragments and naked plasmid DNA, could potentially be used for catheter-based gene transfer in humans, and could help determine the physiological functions of putative genes.

Animals↗
PubMed1997

Induction of tumor suppressor p53 and DNA fragmentation in organotypic hippocampal cultures following excitotoxin treatment.

The p53 tumor suppressor gene encodes a cell cycle regulatory protein that is induced by DNA damage and has been implicated in apoptosis. To investigate whether excitotoxic cell death due to kainic acid (KA) and cell death due to N-methyl-D-aspartate (NMDA) share similar molecular mechanisms, we studied p53 expression and DNA fragmentation in organotypic hippocampal slice cultures following excitotoxin treatment. Cellular analyses showed that both p53 induction and DNA fragmentation occurred only in injured neurons following exposure to either excitotoxin. The temporal profiles of these changes demonstrated that p53 induction preceded DNA fragmentation. The extent of regional alterations in p53 expression and DNA fragmentation correlated with drug-related toxicity (i.e., NMDA > KA). These results support the hypothesis that p53 is a marker of neuronal death in the CNS and suggest the possibility that excitotoxin-mediated neuronal death may occur through a p53-dependent pathway.

Animals↗
PubMed2002

Random DNA fragmentation with endonuclease V: application to DNA shuffling.

The enzyme endonuclease V nicks uracil-containing DNA at the second or third phosphodiester bond 3' to uracil sites. I applied the enzyme to random fragmentation of DNA to revise the complex DNA shuffling protocol. The merit of using endonuclease V is that cleavage occurs at random sites and the length of the fragments can easily be adjusted by varying the concentration of dUTP in the polymerase chain reaction. Unlike the conventional method using DNase I, no partial digestion or gel separation of fragments is required. Therefore, labor is dramatically reduced and reproducibility ensured. I applied this method to recombine two truncated green fluorescent protein (GFP) genes and demonstrated successful DNA shuffling by the appearance of the fluorescent full-length GFP genes.

DNA↗
PubMed1993

P80 form of the human tumor necrosis factor receptor is involved in DNA fragmentation.

Two different types of TNF receptors with molecular masses of 60 kDa (p60) and 80 kDa (p80) have been identified. TNF is known to cause DNA fragmentation in certain tumor cell lines but the role of p60 and p80 in this action is not understood. In the present study, we examined the role of these receptors in TNF-induced DNA fragmentation. Treatment of U-937 cells with phorbol ester caused downregulation of both types of TNF receptors and this was accompanied by disappearance of the TNF-induced DNA fragmentation. The removal of phorbol ester led to two time-dependent events: (1) the rapid regeneration of the p80 form but not the p60 form of the TNF receptor; and (2) the reappearance of TNF-induced DNA fragmentation. These results suggest that the p80 receptor could mediate the TNF-induced DNA fragmentation.

Cells, Cultured↗
PubMed1999

Glutathione depletion induces giant DNA and high-molecular-weight DNA fragmentation associated with apoptosis through lipid peroxidation and protein kinase C activation in C6 glioma cells.

Glutathione (GSH) depletion caused by l-buthionine-(S,R)-sulfoximine (BSO) induced apoptosis that was recognized by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endo-labeling (TUNEL), nuclear DNA staining with fluorescence dye, and internucleosomal DNA fragmentation in C6 rat glioma cells. The BSO-induced cell death was associated with caspase-3 activation. Lipid peroxidation and protein kinase C (PK-C) activation were observed during the apoptosis of C6 cells, and these events were inhibited by antioxidants and iron chelators without affecting BSO-induced GSH depletion. Furthermore, approximately 2 Mbp giant DNA fragments were observed in the BSO-treated cells. The giant DNA fragmentation were followed by approximately 30-700 kbp and then less than 100 kbp, including internucleosomal DNA fragmentations. Such serial DNA degradation was prevented by the antioxidants, the iron chelators, and the PK-C inhibitors. These results suggest that during apoptosis induced by GSH-depletion caused by BSO, reactive oxygen species endogenously produced cause lipid peroxidation and that the lipid peroxidation induced PK-C activation, processes which are thought to be involved in the giant DNA, high-molecular-weight DNA, and the internucleosomal DNA fragmentations.

Animals↗
PubMed1999

DNA fragmentation factor 45-deficient cells are more resistant to apoptosis and exhibit different dying morphology than wild-type control cells.

The DNA fragmentation factor 45 (DFF45) is a subunit of a heterodimeric DNase complex critical for the induction of DNA fragmentation in vitro. To understand the in vivo role of DFF45 in programmed cell death, we measured the expression of DFF45 during mouse development and compared DNA fragmentation and viability of DFF45-deficient cells with wild-type control cells after activation of apoptosis. We found that DFF45 is ubiquitously expressed throughout mouse development. Moreover, DFF45-deficient thymocytes are resistant to DNA fragmentation with in vivo dexamethasone treatment. Furthermore, primary thymocytes from DFF45 mutant mice are also more resistant to apoptosis than wild-type control cells on exposure to several apoptotic stimuli. Dying DFF45-deficient thymocytes exhibit different morphology than wild-type control cells in that they show reduced degree of chromatin condensation, absent nuclear fragmentation, intranuclear cytoplasmic invagination, and striking nuclear chromatin conglutination after release from disintegrating cells. These results indicate that DFF45 is essential during normal apoptosis.

Animals↗