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PubMed1998

3,4-Dichloroisocoumarin serine protease inhibitor induces DNA fragmentation and apoptosis in susceptible target cells.

3,4-Dichloroisocoumarin (DCI) inhibition of serine proteases generates reactive intermediates that have been theorized to affect apoptosis. To examine this possibility various target cells were treated with different concentrations of DCI and assessed for intracellular nuclear DNA fragmentation and apoptosis. DCI treatment caused oligonucleosomal DNA fragmentation in cell lines expressing high levels of protease activity (LAK cells, NK-92, CTLL-2, L929, 3T3). This DNA breakdown characteristic of apoptosis occurred in a dose-dependent fashion within 4-6 hr of treatment and was confirmed by electron microscopy. In cell lines expressing low levels of protease activity (unstimulated human peripheral blood mononuclear (PBMN) cells, YAC-1 cells), DCI effectively inhibited protease activity without inducing oligonucleosomal DNA fragmentation. ZN2+ ions significantly inhibited DCI-induced DNA degradation. The mixture of DCI and BLT esterase active NK cell lysate triggered DNA fragmentation in isolated YAC-1 nuclei. Degree of DNA fragmentation in YAC-1 nuclei was proportional to the level of BLT esterase activity. Cell lysate protease activity, initially inhibited by DCI acylation, was restored by hydroxylamine deacylation, thus preventing DCI-mediated DNA fragmentation. Our results suggest that DCI treatment of cells expressing high levels of protease activity generates toxic levels of acyl-enzyme intermediates. These intermediates may trigger nuclear DNA breakdown and apoptosis by activating endogenous endonucleases. This effect may compromise the analysis of apoptosis in experimental systems using high concentrations of DCI for extended periods.

3T3 Cells↗
PubMed1989

Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements.

Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing [3H]triamcinolone acetonide [( 3H]TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, [3H]TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins prior to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. When similar experiments were conducted under conditions of large receptor excess, using 3' [32P]-MMTV LTR DNA, the trace quantity of DNA formed a stable 10-14S complex with DNA-cellulose pretreated cytosols or with untreated cytosols in the presence of excess Escherichia coli competitor DNA. If trace quantities of the 3' [32P]-MMTV LTR DNA were incubated with untreated crude cytosols, much larger complexes were formed, indicating the association of other cytosolic proteins with the MMTV LTR DNA fragment. Activated [3H]TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA, but with lower apparent affinities in the order MMTV LTR DNA fragment much greater than pBR322 fragment containing a single GRE DNA consensus sequence greater than non-GRE-containing pBR322 fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2001

Effect of temperature on the separation of long DNA fragments in polymer solution.

Electrophoresis of long DNA fragments in polymer solutions is still attractive when performed in short capillaries. Then the separations can be accomplished in minutes rather than hours as is usual in various slab electrophoresis techniques. In this paper we focused on the behavior of large DNA fragments in pulsed field capillary electrophoresis under various temperature conditions. The mobility dependence of fragments of lambda-DNA single-cut mixture on various frequencies at three different temperatures showed that the antiresonance mobility minima are shifted to higher frequencies at higher temperatures. This interesting result is explained in terms of the geometration model of DNA motion.

DNA↗
PubMed1997

The cleavage of nuclear DNA into high molecular weight DNA fragments occurs not only during apoptosis but also accompanies changes in functional activity of the nonapoptotic cells.

In this paper we demonstrate that apoptosis in primary culture of murine thymocytes and in continuously growing human cells is associated with the progressive disintegration of nuclear DNA into high molecular weight (HMW)-DNA fragments of about 50-150 kb. We also show that the formation of similarly sized HMW-DNA fragments takes place in the same cells in the absence of apoptotic inducers. Unlike an apoptotic fragmentation of nuclear DNA, the formation of HMW-DNA fragments in nonapoptotic cells is rapidly induced, has no correlation with the cell death, and is not associated with the development of oligonucleosomal "ladder" or apoptotic changes in nuclear morphology. The disintegration of DNA into HMW-fragments is also observed in nuclei isolated from healthy, nonapoptosizing tissues of various eukaryotes. We show that the formation of HMW-DNA fragments in the absence of apoptotic inducers is strongly dependent on the ionic detergents, is responsive to the topoisomerase II-specific poison, teniposide, and is completely reversible under conditions that favor topoisomerase II-dependent rejoining reaction. Also, we demonstrate that the formation of HMW-DNA fragments in continuously growing cell lines caused either by serum deprivation or monolayer establishment is of a transient nature and rapidly reverses to the control level following serum addition or dilution of monolayer. The results suggest that the cleavage of nuclear DNA into HMW-DNA fragments is associated not only with apoptosis but also accompanies changes in functional activity of nonapoptotic cells.

Animals↗
PubMed2004

Caspase-dependent and -independent DNA fragmentation in Sertoli and germ cells from men with primary testicular failure: relationship with histological diagnosis.

BACKGROUND: Germ cell elimination and sperm DNA fragmentation in men with primary testiculopathies involve apoptosis-related processes whose mechanisms are poorly understood. This study examines the participation of typical (caspase-dependent) and atypical (caspase-independent) pathways in these processes. METHODS: Caspase activity and DNA fragmentation were evaluated in Sertoli and germ cells from 63 men with non-obstructive azoospermia and with different histological diagnoses who were undergoing testicular biopsy for an assisted reproduction attempt. In eight of these men, phosphatidylserine externalization was also examined. RESULTS: The percentage of Sertoli cells showing caspase activity and DNA fragmentation was low and uniform in all diagnoses. In germ cells that remained tightly associated with Sertoli cells despite vigorous mechanical treatment, the incidence of both caspase activity and DNA fragmentation was high, particularly in men with maturation arrest. In Sertoli cell-free germ cells, high incidence of DNA fragmentation contrasted with low incidence of caspase activity and phosphatidylserine externalization. CONCLUSIONS: In men with primary testicular failure, apoptosis of Sertoli cells is insignificant. Some germ cells undergo caspase-dependent apoptosis, show phosphatidylserine externalization and are tightly associated with Sertoli cells. Other germ cells show caspase-independent DNA fragmentation, do not externalize phosphatidylserine and lack a tight association with Sertoli cells.

Apoptosis↗
PubMed1989

A vector for sequencing long (40-kb) DNA fragments.

An improved vector (pAA113M) has been constructed for sequencing long (40-kb) DNA fragments. The DNA fragment is cloned in the tet gene of the cosmid and subdivided into numerous overlapping segments by IS1-promoted deletions. Plasmids bearing these deletions are fractionated by gel electrophoresis, and shortened further from the opposite end by treatment with specific restriction endonucleases. Thus, a series of short overlapping segments, spread across the entire length of the fragment, become fused to IS1. Each segment can then be sequenced by the dideoxy method using an IS1 primer. The plasmids can replicate either from their normal origin or, in the presence of a filamentous helper phage, from the M13 origin. Hence, each segment can be sequenced as either double-stranded DNA or single-stranded DNA. Sequences of IS1-promoted and restriction enzyme-generated deletions contain overlaps that can be used to assemble the complete 40-kb sequence.

Base Sequence↗
PubMed2001

Early decrease in dna repair proteins, Ku70 and Ku86, and subsequent DNA fragmentation after transient focal cerebral ischemia in mice.

BACKGROUND AND PURPOSE: Ku70 and Ku86, multifunctional DNA repair proteins, bind to broken DNA ends, including double-strand breaks, and trigger a DNA repair pathway. To investigate the involvement of these proteins in DNA fragmentation after ischemia/reperfusion, Ku protein expression was examined before and after transient focal cerebral ischemia (FCI) in mice. METHODS: Adult male CD-1 mice were subjected to 60 minutes of FCI by intraluminal suture blockade of the middle cerebral artery. Ku protein expression was studied by immunohistochemistry and Western blot analysis. DNA fragmentation was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). The spatial relationship between Ku expression and DNA fragmentation was examined by double labeling with Ku and TUNEL after reperfusion. RESULTS: Immunohistochemistry showed constitutive expression of Ku proteins in control brains. The number of Ku-expressing cells was decreased in the entire middle cerebral artery territory as early as 4 hours after reperfusion and remained reduced until 24 hours. Western blot analyses confirmed the significant reduction of these proteins (59.4% and 57.7% reduction in optical density at 4 hours of reperfusion from the normal level of Ku70 and Ku86 bands, respectively; P<0.001). DNA gel electrophoresis demonstrated DNA laddering 24 hours after reperfusion, but not at 4 hours. Double staining with Ku and TUNEL showed a concomitant loss of Ku immunoreactivity and TUNEL-positive staining. CONCLUSIONS: These results suggest that the early reduction of Ku proteins and the loss of defense against DNA damage may underlie the mechanism of DNA fragmentation after FCI.

Animals↗
PubMed1997

Nuclear DNA fragmentation and expression of Bcl-2 in primary biliary cirrhosis.

It is uncertain whether or not apoptosis is involved in the pathogenesis of primary biliary cirrhosis (PBC). The aims of this study were to assess the nuclear DNA fragmentation and expression of Bcl-2 in the biliary epithelial cells (BECs) and in the hepatocytes of PBC. Additionally, the effects of ursodeoxycholic acid (UDCA) on DNA fragmentation and Bcl-2 expression in PBC were evaluated. Liver tissue specimens from 35 PBC patients were examined by in situ nick-end labeling to detect any nuclear DNA fragments, and by immunohistochemistry for Bcl-2. Ten of these patients underwent a second liver biopsy after the treatment with UDCA. Sixteen histologically normal liver tissues and 17 chronic viral hepatitis C (CVHC) samples were chosen as controls. DNA fragmentation in BECs was more frequently found in PBC than in CVHC and more frequently than in the normal controls (both P < .05), and fragmentation in the hepatocytes of PBC more frequently than in normal controls (P < .01). Bcl-2 expression was more frequently found in both the BECs and hepatocytes of PBC than in the controls. UDCA significantly decreased the DNA fragmentation in BECs (P < .05) and the positivity for Bcl-2 in BECs (P < .01), although no significant decrease was found in hepatocytes. In conclusion, de novo Bcl-2 expression in hepatocytes of PBC was shown, and the increased nuclear DNA fragmentation and the Bcl-2 expression both in BECs and in hepatocytes may reflect apoptotic stress, although nuclear DNA fragmentation in BECs did not necessarily represent apoptosis. UDCA showed a potential effect of reducing nuclear DNA fragmentation in BECs.

Apoptosis↗
PubMed1976

A DNA fragment from the origin of single-strand to double-strand DNA replication of bacteriophage fd.

A specific complex is formed between fd DNA, Escherichia coli DNA unwinding protein, and RNA polymerase (EC 2.7.7.6; nucleosidetriphosphate:RNA nucleotidyltransferase) during the first steps in the conversion of the single-stranded viral DNA to the double-stranded replicative form. In this complex a unique DNA fragment of about 120 nucleotides is protected against nuclease digestion. Both the requirements for its isolation and its position on the map of the phage genome indicate that the fragment contains the origin of single-strand to double-strand DNA replication. The isolated DNA fragment possesses double-strand-like characteristics, which protect it from being covered by the DNA unwinding protein and thus indirectly positions the RNA polymerase to the origin of replication.

Chromosome Mapping↗
PubMed2003

BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium.

Development of efficient methods to transfer large DNA fragments into plants will greatly facilitate the map-based cloning of genes. The recently developed BIBAC and TAC vectors have shown potential to deliver large DNA fragments into plants via Agrobacterium-mediated transformation. Here we report that BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium. We tested the possible factors that may cause instability, including the insert sizes of the BIBAC and TAC constructs, potato DNA fragments consisting of highly repetitive or largely single-copy DNA sequences, different Agrobacterium transformation methods and different Agrobacterium strains. The insert sizes of the potato BIBAC and TAC constructs were found to be critical to their stability in Agrobacterium. All constructs containing a potato DNA fragment larger than 100 kb were not stable in any of the four tested Agrobacterium strains, including two recA deficient strains. We developed a transposon-based technique that can be used to efficiently subclone a BAC insert into two to three BIBAC/TAC constructs to circumvent the instability problem.

Chromosomes, Artificial, Bacterial↗
PubMed2004

Cisplatin-evoked DNA fragmentation in normal and cancer cells and its modulation by free radical scavengers and the tyrosine kinase inhibitor STI571.

Cis-diamminedichloroplatinum(II) (cisplatin, cis-DDP) is well studied anticancer drug, whose activity can be attributed to its ability to form adducts with DNA, but this drug can also form DNA-damaging free radicals, however this mechanism of cisplatin action is far less explored. Using the comet assay we studied cisplatin-induced DNA damage in the presence of spin traps: DMPO and PBN, Vitamins A, C and E as well as the tyrosine kinases inhibitor STI571 in normal human lymphocytes and leukemic K562 cells. The latter cells express the BCR/ABL fusion protein, which can be a target of the tyrosine kinase inhibitor STI571. A 20 h incubation with cisplatin at 1-10 microM induced DNA cross-links and DNA fragmentation in normal and cancer cells. Cisplatin could induce intra- and interstrand DNA-DNA cross-links as well as DNA-protein cross-links. DNA damage in K562 cells was more pronounced than in normal lymphocytes. In the presence of spin traps and vitamins we noticed a decrease in the DNA fragmentation in both cell types. Co-treatment of the lymphocytes with cisplatin at 10 microM and STI571 at 0.25 microg/ml caused an increase of DNA fragmentation in comparison with DNA fragmentation induced by cisplatin alone. In the case of K562 cells, an increase of DNA fragmentation was observed after treatment with cisplatin at 1 microM. Our results indicate that the free radicals scavengers could decrease DNA fragmentation induced by cisplatin in the normal and cancer cells, but probably they have no effect on DNA cross-linking induced by the drug. The results obtained with the BCR/ABL inhibitor suggest that K562 cells could be more sensitive towards co-treatment of cisplatin and STI571. Our results suggest also that aside from the BCR/ABL other factors such as p53 level, signal transduction pathways and DNA repair processes can be responsible for the increased sensitivity of K562 cells to cisplatin compared with normal lymphocytes.

Antineoplastic Agents↗
PubMed2004

Chemosensitivity assay in mice prostate tumor: Preliminary report of flow cytometry, DNA fragmentation, ion ratiometric methods of anti-neoplastic drug monitoring.

Flow cytometry, DNA fragmentation, ion ratiomateric analysis and NMR peaks characterized drug chemosensitivity of antineoplastic drugs. Hypotheses were: 1. The chemosensitive effect of different cancer cell lines is characteristic; 2. DNA fragmentation, ion ratiometric analysis suggest apoptosis status of tumor cells. METHODS: PC-3 cell lines were compared with DU-145, LNCaP cell lines in culture for the [Na]i and [Ca]i ion sensing dyes, cell death, NMR peaks and apoptosis staining for chemotherapeutic action of different drugs. RESULTS: DNA fragmentation, ratiometric ions and fluorescence endlabelling plots were characteristic for cell lines and drug response. 31P-23Na NMR spectra showed characteristic high phospho-choline and sodium peaks. CONCLUSION: Flow cytometry, DNA fragmentation, ion ratiometric methods and NMR peaks indicated apoptosis and offered in vivo drug monitoring method.

Journal Article↗
PubMed1994

Cell death due to ACNU-induced DNA fragmentation: inhibition by cycloheximide.

Several anticancer drugs have recently been shown to induce cell death in a manner similar to programmed cell death or apoptosis. The purpose of this study is to explore the mode of cell death caused by ACNU, a water-soluble nitrosourea. Exposure of rat glioma cell line KEG-1 to ACNU for 2 hours resulted in oligonucleosomal DNA fragmentation, creating a 'ladder' on agarose gel electrophoresis. DNA fragmentation began 18 hours after ACNU treatment, and preceded loss of membrane integrity as evaluated by the trypan blue exclusion test. The extent of DNA fragmentation increased in a dose-dependent manner, and the cell survival rate decreased reciprocally. A translational inhibitor, cycloheximide, suppressed this DNA fragmentation and enhanced cell survival rate with partial inhibition of protein synthesis. However, a transcriptional inhibitor, actinomycin D, failed to inhibit DNA fragmentation or enhance cell survival. Cycloheximide-inhibitable DNA fragmentation was also found in the KEG-1 implanted in vivo rat model following the administration of ACNU. These findings suggest that ACNU induces cell death associates with DNA fragmentation and partially with protein synthesis.

Animals↗
PubMed2001

Presence of DNA fragmentation and lack of neuroprotective effect in DFF45 knockout mice subjected to traumatic brain injury.

BACKGROUND: Apoptosis plays an important pathophysiologic role in neuronal cell loss and associated neurologic deficits following traumatic brain injury (TBI). DNA fragmentation represents one of the characteristic biochemical features of neuronal apoptosis and is observed after experimental TBI. DFF45 and DFF40 are essential for DNA fragmentation in various models of apoptosis. MATERIALS AND METHODS: We used mice deficient in DFF45 and wild-type controls. Oligonucleosomal DNA fragmentation induced by TBI was analyzed using in vivo and in vitro assays. Expression and integrity of DFF45 and DFF40 proteins was assessed by Western analysis. Other outcome measurements included neurologic scoring, learning/memory tests, lesion volume measurements (MRI), and assessment of cell viability in vitro among others. RESULTS: We compared the effects of controlled cortical impact (CCI) trauma in DFF45 knockout mice and wild-type controls. Analysis of TBI-induced DNA fragmentation in brain cortex from wild-type and DFF45 knockout mice indicates that, although somewhat delayed, oligonucleosomal cleavage of DNA occurs after TBI in DFF45 knockout mice. DFF45 knockouts showed no significant differences in behavioral outcomes or lesion volumes after TBI as compared to wild-type controls. Using an in vitro reconstitution system, we also demonstrated that cleavage of DFF45 by caspase-3 is not sufficient for DNA fragmentation induced by protein extracts from rat brain cortex. We found that endonuclease activity induced in rat brain cortex following TBI depends on the presence of Mg2+ and Ca2+, but is not inhibited by Zn2+. Primary neuronal cultures from DFF45 knockouts failed to show DNA laddering in response to staurosporine, but did show prominent, albeit delayed, DNA fragmentation following treatment with etoposide. In contrast, primary neurons from wild-type animals demonstrated marked DNA fragmentation following treatment with staurosporine or etoposide. CONCLUSIONS: The results of this study suggest that, in addition to DFF45/40, other endonucleases may be essential for chromatin degradation during neuronal apoptosis in adult brain after TBI.

Animals↗
PubMed1998

Level of HgCl2-mediated phosphorylation of intracellular proteins determines death of thymic T-lymphocytes with or without DNA fragmentation.

Exposure to Hg2+ at a wide range of concentrations (approximately 1-100 microM) more or less caused the death of murine thymic T-lymphocytes, and exposure to 1 microM but not 10 microM (or more) of Hg2- induced DNA fragmentation. Exposure of cells to Hg2+ caused phosphorylation of multiple cellular proteins at the tyrosine residue in a concentration-dependent manner. We found that not only the DNA fragmentation induced by 1 microM Hg2+ but also the cell death bypassing DNA fragmentation caused by 10 microM or more Hg2+ was partly inhibited by protein kinase inhibitors such as staurosporine and herbimycin A. This result suggested the involvement of a protein phosphorylation-linked signal in the mechanism of the Hg2+-mediated cell death with or without DNA fragmentation. Analysis of proteins by both one- and two-dimensional electrophoresis and immunoblot showed that a 52-kDa Shc protein was heavily phosphorylated by an early signal delivered by a high concentration of Hg2+, which also phosphorylated extracellular signal-regulated kinase 1 (ERK1; p44) and ERK2 (p42) of the mitogen-activated protein kinase (MAPK) family in a concentration- and time-dependent manner. The c-Jun amino terminal kinase (p54), which is a distant relative of the MAPK family, was also phosphorylated by the treatment with Hg2+. This eventually formed the signaling cascade that ended with a nuclear target by phosphorylating c-jun at the serine 73. This phosphorylation of c-jun was inhibited by staurosporine. These results suggest that a high level of Hg2+-mediated protein phosphorylation-linked signal induces rapid cell death bypassing DNA fragmentation, whereas a lower level induces cell death accompanying DNA fragmentation. This conclusion in turn implies that DNA fragmentation is not always a prerequisite for the signal transduction-dependent cell death of T-lymphocytes.

Animals↗
PubMed1999

[pCSE4 plasmid for cloning of promoter-containing DNA fragments in francisella tularensis].

Recombinant pCSE4 plasmid has been constructed, which contains the cat gene of the Tn9 without its own promoter and with the restriction BamH1 site in front of the Shine-Delgarno region of this gene. pCSE4 is consistently inherited in the cells of F. tularensis and E. coli and makes the cells of both microorganisms to be resistant to tetracycline. By cloning the Sau3A fragments of F. tularensis chromosomal DNA at the BamH1 site of pCSE4. Promoter-containing DNA fragments were selected. The plasmids with such chromosomal DNA fragments retained their structural integrity and functional activity in F. tularensis.

Chromatin↗
PubMed1986

Flying squirrel-associated Rickettsia prowazekii (epidemic typhus rickettsiae) characterized by a specific DNA fragment produced by restriction endonuclease digestion.

The DNA from flying squirrel-associated Rickettsia prowazekii was characterized by using a specific DNA fragment produced by digestion with the enzyme BamHI. The DNA fragment was cloned into a plasmid vector and used to readily distinguish between available human- and flying squirrel-associated R. prowazekii DNAs derived from crude cytoplasmic extracts.

Animals↗