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PubMed2002

The Poly(ADP-ribose) polymerase-1-regulated endonuclease DNAS1L3 is required for etoposide-induced internucleosomal DNA fragmentation and increases etoposide cytotoxicity in transfected osteosarcoma cells.

The cytotoxic effect of the chemotherapeutic drug etoposide (VP-16) is thought to result from its ability to induce DNA damage and thereby to trigger apoptosis. Internucleosomal DNA fragmentation occurs late during apoptosis in many cell types. However, whereas human osteosarcoma cells undergo internucleosomal DNA fragmentation during staurosporine-induced apoptosis, they fail to do so in response to VP-16. Recently, we showed that these cells also do not express the poly(ADP-ribosyl)ation-regulated Ca(2+)- and Mg(2+)-dependent endonuclease DNAS1L3. The possibility that this deficiency underlies the failure of these cells to undergo internucleosomal DNA fragmentation in response to VP-16 was investigated. The proteolytic processing and consequent activation of procaspase-3, cleavage of the inhibitory subunit of DNA fragmentation factor, and the degradation of DNA into 50-kb fragments occurred similarly in osteosarcoma cells exposed to either staurosporine or VP-16. However, the additional processing of the 50-kb DNA fragments to oligonucleosomal fragments was not apparent in the VP-16-treated cells. Ectopic expression of DNAS1L3 conferred on osteosarcoma cells the ability to undergo VP-16-induced internucleosomal DNA fragmentation. Furthermore, expression of DNAS1L3 markedly potentiated the cytotoxic effect of VP-16 in these cells. Both DNAS1L3-mediated and staurosporine-induced internucleosomal DNA fragmentation were Ca(2+) dependent, but only the DNAS1L3-mediated DNA cleavage was blocked by expression of a caspase-3-resistant mutant of poly(ADP-ribose) polymerase-1. The present work results suggest a direct relation between the activity of a chemotherapeutic drug (VP-16) and a specific endonuclease (DNAS1L3). They also indicate that internucleosomal DNA fragmentation plays an active role in apoptosis and that the failure of cancer cells to undergo such DNA degradation may contribute to the development of resistance to chemotherapeutic drugs.

Antineoplastic Agents, Phytogenic↗
PubMed1985

Selective detection of Mycoplasma hyorhinis using cloned genomic DNA fragments.

A 13-kilobase DNA fragment from a genomic library of Mycoplasma hyorhinis demonstrated specific Southern hybridization and dot hybridization when tested against a group of different mycoplasmas. This probe selectively recognized M. hyorhinis sequences in purified DNA, broth-grown organisms, and infected cell cultures, providing a direct method for differential detection of this species.

Base Sequence↗
PubMed1998

Trinucleotide (CAG) repeat length is positively correlated with the degree of DNA fragmentation in Huntington's disease striatum.

Recent studies using DNA fragmentation assays suggest a role for apoptosis in cell death in Huntington's disease. In this study, we investigated the relationship between the degree of DNA fragmentation and the number of trinucleotide (CAG) repeats of the Huntington's disease gene in striatal tissue from Huntington's disease brains. We used frozen striatal tissue from 27 post mortem Huntington's disease brains (graded 0-4 on the Vonsattel classification, post mortem delay ranging from 4 to 41 h), plus control sections which were age, sex and post mortem delay matched from neurologically normal and Alzheimer's diseased striatal tissue. Our results show a significant positive correlation between the number of CAG repeats in the Huntington's disease gene and the degree of DNA fragmentation in Huntington's disease striatum. These results suggest that expanded CAG repeats in the Huntington's disease gene may lead to neuronal degeneration in Huntington's disease through an apoptotic mechanism.

Adult↗
PubMed1996

Poly(ADP-ribosyl)ation of histone H1 correlates with internucleosomal DNA fragmentation during apoptosis.

The biochemical role of poly(ADP-ribosyl)ation on internucleosomal DNA fragmentation associated with apoptosis was investigated in HL 60 human premyelocytic leukemia cells. It was found that UV light and chemotherapeutic drugs including adriamycin, mitomycin C, and cisplatin increased poly(ADP-ribosyl)ation of nuclear proteins, particularly histone H1. A poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide, prevented both internucleosomal DNA fragmentation and histone H1 poly(ADP-ribosyl)ation in cells treated with the apoptosis inducers. When nuclear chromatin was made accessible to the exogenous nuclease in a permeabilized cell system, chromatin of UV-treated cells was more susceptible to micrococcal nuclease than the chromatin of control cells. Suppression of histone H1 poly(ADP-ribosyl)ation by 3-aminobenzamide reduced the micrococcal nuclease digestibility of internucleosomal chromatin in UV-treated cells. These results suggest that the poly(ADP-ribosyl)ation of histone H1 correlates with the internucleosomal DNA fragmentation during apoptosis mediated by DNA damaging agents. This suggestion is supported by the finding that xeroderma pigmentosum cells which are defective in introducing incision at the site of DNA damage, failed to induce DNA fragmentation as well as histone H1 poly(ADP-ribosyl)ation after UV irradiation. We propose that poly(ADP-ribosyl)ation of histone H1 protein in the early stage of apoptosis facilitates internucleosomal DNA fragmentation by increasing the susceptibility of chromatin to cellular endonuclease.

Adenosine Diphosphate Ribose↗
PubMed1992

Ultraviolet B-induced DNA fragmentation (apoptosis) in activated T-lymphocytes and Jurkat cells is augmented by inhibition of RNA and protein synthesis.

UVB (280-320 nm) light profoundly affects cellular immunity and immunogenicity. To further characterize the interaction of UVB light with T cells, we examined UVB-induced DNA fragmentation patterns in normal T cells and the T-cell line Jurkat. Resting or preactivated peripheral blood T cells from normal donors or Jurkat cells were exposed to various doses of UVB or gamma irradiation. Cells were sampled at 0-48 h after exposure, and DNA fragmentation was analyzed by electrophoresis in agarose gels. UVB and gamma irradiation, in a dose- and time-dependent manner, induced DNA fragmentation in Jurkat cells and in T cells preactivated with phytohemagglutinin (PHA; 10 micrograms/ml) and phorbol myristate acetate (PMA; 10 ng/ml), but not in resting T cells. The presence of RNA or protein synthesis inhibitors such as actinomycin D or cycloheximide neither inhibited nor delayed DNA fragmentation; in fact, DNA fragmentation was augmented above control values. Similarly, DNA fragmentation increased in the presence of the calcium-chelating agent ethylene-glycol-bis-tetraacetic acid (EGTA) and decreased in the presence of the calcium ionophore A23187. In the presence of ethylenediaminetetraacetic acid (EDTA), DNA fragmentation decreased. In summary, these data show that UVB-induced DNA fragmentation strongly depends upon the cell activation status, and upon the presence of divalent cations other than calcium, possibly magnesium. The data indicate furthermore that in this model, inhibition of RNA or protein synthesis can induce rather than inhibit apoptosis, suggesting that the synthesis inhibitors disrupted primarily the synthesis or action of enzymes ordinarily aimed at repairing DNA fragmentation.

Calcium↗
PubMed1999

Cerebral ischemia produces laddered DNA fragments distinct from cardiac ischemia and archetypal apoptosis.

The electrophoretic pattern of laddered DNA fragments which has been observed after cerebral ischemia is considered to indicate that neurons are dying by apoptosis. Herein the authors directly demonstrate using ligation-mediated polymerase chain reaction methods that 99% of the DNA fragments produced after either global or focal ischemia in adult rats, or produced after hypoxia-ischemia in neonatal rats, have staggered ends with a 3' recess of approximately 8 to 10 nucleotides. This is in contrast to archetypal apoptosis in which the DNA fragments are blunt ended as seen during developmental programmed cell death in dying cortical neurons, neuroblastoma, or thymic lymphocytes. It is not simply ischemia that results in staggered ends in DNA fragments because ischemic myocardium is similar to archetypal apoptosis with a vast majority of blunt-ended fragments. It is concluded that the endonucleases that produce this staggered fragmentation of the DNA backbone in ischemic brain must be different than those of classic or type I apoptosis.

Animals↗
PubMed1983

An agarose gel resolving a wide range of DNA fragment lengths.

To resolve DNA fragments ranging from several kilobases to some tens of base pairs in length, an agarose slab gel of steadily increasing thickness has been designed. During electrophoresis a gradient of decreasing electric-field strength is generated throughout the gel from the cathode end to the anode end. Shorter fragments which migrate further are decelerated, resulting in an increased linearity of the relationship between mobility and molecular weight.

DNA↗
PubMed2005

Chromatin supraorganization, DNA fragmentation, and cell death in snake erythrocytes.

In nucleate erythrocytes of several vertebrate groups, the frequency and intensity of DNA fragmentation associated with programmed cell death vary considerably. Although hemoglobin efficiency may be related to erythrocyte life span, and hemoglobin types and erythrocyte life spans are assumed to vary in reptiles, no data on DNA fragmentation and chromatin organization as related to cell death exist for snakes. In the present study, chromatin supraorganization, DNA fragmentation, and cell death were investigated in four snake species (Crotalus durissus terrificus, Bothrops jararaca, Bothrops alternatus, and Bothrops neuwiedii), which differ in their geographical distribution and habitats, by using image analysis of Feulgen hydrolysis kinetics, the TUNEL assay, single-cell gel electrophoresis, and transmission electron microscopy. Relatively few circulating erythrocytes were found to be simultaneously committed to cell death, although there was some variation among the snake species. Conspicuous nuclear and cytoplasmic organelles suggestive of metabolic activity were seen ultrastructurally in most snake erythrocytes. The DNA of the snake erythrocyte chromatin was much more resistant to Feulgen acid hydrolysis (DNA depurination and breakdown) than that of young adult bullfrog erythrocytes, which had a high frequency and intensity of DNA fragmentation. Of the species studied, B. neuwiedii and C. d. terrificus showed the greatest resistance to Feulgen acid hydrolysis and to the DNA fragmentation, revealed by the TUNEL assay. Although B. neuwiedii also showed the lowest frequency of cells with more damaged DNA in the single-cell gel electrophoresis assay, C. d. terrificus had the highest frequency of damaged cells, possibly because of the abundance of alkaline-sensitive DNA sites. The results for DNA fragmentation and cell death in erythrocytes of B. jararaca and B. alternatus generally differed from those for C. d. terrificus and B. neuwiedii and may reflect differences in the biology of these species selected under different geographical habitats. The differences in erythrocyte cell biology reported here may be related to hemoglobin variants selected in the mentioned snake species and that would lead the cells to different resistances to unfavorable environmental conditions.

Animals↗
PubMed2004

GP7 can induce apoptotic DNA fragmentation of human leukemia cells through caspase-3-dependent and -independent pathways.

GP7 (4-[4"-(2",2",6",6"-tetramethyl-l"-piperidinyloxy)amino]-4'-demethyl epipodophyllotoxin), a new spin-labeled derivative of podophyllotoxin, is a promising anticancer drug of podophyllotoxin class. The primary effect of GP7 is the anticancer activity on transplanted mouse tumors and cultured tumor cells. However, its molecular mechanism of action is still obscure. In this study, we investigated the activity of GP7 to induce apoptosis in human leukemia HL-60 and Jurkat cells. Apoptosis was determined by detection of DNA fragmentation in agarose gel electrophoresis. GP7 induced apoptotic DNA fragmentation of HL-60 and Jurkat cells in time- and dose-dependent manner. We further investigated the activity of caspase-3 in GP7-induced apoptotic DNA fragmentation of HL-60 and Jurkat cells. GP7 also induced time- and dose-dependent caspase-3 activation in both cell lines, and the kinetics of caspase-3 activation induced by GP7 was well correlated with that of apoptotic DNA fragmentation. To determine the role of caspase-3 in GP7-induced apoptotic DNA fragmentation, we examined the effect of specific caspase-3 inhibitor, Ac-DEVD-CHO, on GP7-induced apoptotic DNA fragmentation. Ac-DEVD-CHO prevented GP7-induced caspase-3 activation in both HL-60 and Jurkat cells, however, it only inhibited GP7-induced apoptotic internucleosomal DNA fragmentation in HL-60 cells. We then employed L-carnitine to investigate the role of caspase-3 in GP7-induced apoptotic DNA fragmentation. L-carnitine treatment prevented GP7-induced caspase-3 activation in both cell lines in a dose-dependent manner. Similar to Ac-DEVD-CHO, L-carnitine only inhibited GP7-induced apoptotic internucleosomal DNA fragmentation in HL-60 cells. These findings suggest that GP7 exerts an anti-leukemic effect by both caspase-3-dependent and -independent apoptotic signaling pathways.

Antineoplastic Agents↗
PubMed1999

The time-course of DNA fragmentation in the choroid plexus and the CA1 region following transient global ischemia in the rat brain. The effect of intra-ischemic hypothermia.

The time-course of DNA fragmentation in the CA1 region of the hippocampus and the choroid plexus was studied following induction of transient forebrain ischemia under lethal normothermic (37 degrees C), or sublethal hypothermic (33 degrees C) conditions. Oligonucleosomal- and high-molecular-weight DNA fragmentation were analysed by conventional agarose gel electrophoresis and pulsed-field gel electrophoresis, respectively. DNA breaks were visualized by the terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridinetriphosphate nick-end labeling method. At 48 h of recovery following normothermic ischemia, in situ labeling of DNA breaks were widespread in medial CA1 and high-molecular-weight DNA cleavage was seen. In contrast, at the same time-point in lateral CA1, many pyknotic but few cells displaying in situ labeling of DNA breaks were observed. Major oligonucleosomal DNA fragmentation was not seen until 72 h of recovery. Following hypothermic ischemia, DNA fragmentation was absent in CA1. DNA fragmentation was seen in the choroid plexus at 24 h of recovery following normothermic ischemia, which was diminished by 48 h of recovery. In conclusion, oligonucleosomal and high-molecular-weight DNA fragmentation at 10-50 kilobase pairs, occur in CA1 after morphological signs, and acidophilia signifying neurodegeneration appear. DNA fragmentation and cell death in the choroid plexus precede neuronal death in CA1 and may play a causative role.

Animals↗
PubMed1988

3'-end labeling of DNA fragments by AMV-reverse transcriptase.

The efficiency on 3'-end labeling of DNA fragments was compared between Klenow fragment of DNA polymerase I from E. coli and AMV-reverse transcriptase. In the case of Klenow fragment, the exonuclease activity of this enzyme rapidly removed the labeled dNMP incorporated in 3'-end of DNA fragment by the polymerase activity of it. AMV-reverse transcriptase caused no decrease of the labeled dNMP incorporated by the polymerase activity because it has no exonuclease activity. Therefore, AMV-reverse transcriptase is easier to use than Klenow fragment for labeling the 3'-end of DNA fragment.

Avian Myeloblastosis Virus↗
PubMed1999

Inhibition of trifluoperazine-induced DNA fragmentation by cyclic AMP mediated signaling.

Trifluoperazine (TFP), a phenothiazine antipsychotic agent with calmodulin antagonist property, induces DNA fragmentation in a dose- and time-dependent manner in PC12 cells. Various agents affecting calcium mediated intracellular signal transduction such as calcium chelators, calcium ionopores, inhibitors of phospholipase C, and activators/inhibitors of protein kinase C did not block TFP-induced DNA fragmentation. Some of these agents themselves induced DNA fragmentation in the conditions under which they were examined. However, cholera toxin (selective Gs activator), forskolin (adenylate cyclase activator) or dibutyryl cyclic AMP (cyclic AMP analogue) inhibited TFP-induced DNA fragmentation in a dose-dependent manner. These results suggest that it is not the calcium but the Gs and adenylate cyclase pathways that play an important role in TFP-induced DNA fragmentation in PC12 cells.

Adenylate Cyclase Toxin↗
PubMed1995

Effect of nuclear Ca2+ uptake inhibitors on Ca(2+)-activated DNA fragmentation in rat liver nuclei.

The effect of nuclear Ca2+ uptake inhibitors on the Ca(2+)-activated DNA fragmentation in rat liver nuclei was investigated. The addition of Ca2+ (40 microM) into the reaction mixture containing liver nuclei in the presence of 2.0 mM ATP caused a remarkable increase in nuclear DNA fragmentation. This Ca(2+)-activated DNA fragmentation was not seen in the absence of ATP, because nuclear Ca2+ uptake is not initiated without ATP addition. Moreover, the presence of various reagents (10 microM arachidonic acid, 2.0 mM NAD+, 10 microM zinc sulfate and 0.2 mM N-ethylmaleimide), which could inhibit Ca(2+)-ATPase activity and Ca2+ uptake in the nuclei, produced a significant inhibition of the Ca(2+)-activated DNA fragmentation in the nuclei. The results show that the Ca(2+)-activated DNA fragmentation is involved in the uptake of Ca2+ by the nuclei, suggesting a role of Ca2+ transport system in the regulation of liver nuclear functions.

Animals↗
PubMed2000

Analysis of an SMN gene-like DNA fragment.

Part of a survival motor neuron (SMN) gene-like DNA fragment has been identified. This DNA fragment was accidentally isolated from cDNA by RT-PCR using primers specific for the region between exon 3 and 6 of the SMN gene. This fragment was used as a probe to hybridize the mRNA from several tissues, but we have been unable to detect any transcript of this SMN-like gene in these tissues. Thus, we have inferred this SMN gene-like fragment was a genomic product contaminant that was amplified in the reaction. Sequencing analysis of this fragment, which contains several stop codons, revealed a 74.6% nucleotide homology with the SMN gene. From these results, we believe that this DNA fragment is not a mutated form of SMN gene. Rather, it is an SMN-like pseudogene, which is variably present even in normal individuals.

Base Sequence↗
PubMed1997

DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis.

We have identified and purified from HeLa cytosol a protein that induces DNA fragmentation in coincubated nuclei after it is activated by caspase-3. This protein, designated DNA Fragmentation Factor (DFF), is a heterodimer of 40 kDa and 45 kDa subunits. The amino acid sequence of the 45 kDa subunit, determined from its cDNA sequence, reveals it to be a novel protein. Caspase-3 cleaves the 45 kDa subunit at two sites to generate an active factor that produces DNA fragmentation without further requirement for caspase-3 or other cytosolic proteins. In cells undergoing apoptosis, the 45 kDa subunit is cleaved in the same pattern as it is cleaved by caspase-3 in vitro. These data delineate a direct signal transduction pathway during apoptosis: caspase-3 to DFF to DNA fragmentation.

Amino Acid Sequence↗
PubMed1994

Structural organization and differential expression of three stilbene synthase genes located on a 13 kb grapevine DNA fragment.

A 13 kb DNA fragment was isolated from a grapevine (Vitis var. Optima) genomic library by hybridizing with elicitor-induced stilbene synthase cDNA as a probe. After fragmentation with Eco RI, subcloning and sequencing, two full-size stilbene synthase genes (Vst1 and Vst2) and the 3' end of a third stilbene synthase gene (Vst3) were located within the 13 kb fragment. Vst1 and Vst2, differing only slightly in the coding region, are distinguished in the intron size and in the structure of the promoter region. The 5' flanking region of gene Vst1 contains a TATAA box at nucleotide -48. The substantial structural differences found for the promoters of the two genes are paralleled by a striking difference in the expression of the two genes in elicitor-treated cells. Moreover, the accumulation upon elicitation of six different stilbene synthase mRNAs was studied and found to differ by two orders of magnitude.

Acyltransferases↗
PubMedDate not supplied

Comparison of chromatin assays for DNA fragmentation evaluation in human sperm.

Sperm chromatin integrity is vital for successful pregnancy and transmission of genetic material to the offspring. We evaluated chromatin integrity in sperm from 60 infertile men and 7 fertile donors comparing the sperm chromatin structure assay (SCSA), TdT-mediated-dUTP nick end labeling (TUNEL), the sperm chromatin dispersion (SCD) test, and acridine orange staining technique (AOT). The TUNEL and SCD assays showed a strong relationship with the SCSA (r > .866; P < .001) for sperm DNA fragmentation, both in infertile men and donors of known fertility. AOT did not show any relationship with SCSA. The breakdown of the DNA fragmentation index (DFI) into 3 categories (< or =15%, >15%-<30%, and > or =30%) showed that the SCSA, TUNEL, and SCD test predict the same levels of DNA fragmentation. AOT consistently showed higher levels of DNA fragmentation for each DFI category. DNA fragmentation in sperm between infertile men and donor sperm was significantly different (P < .05) under SCSA (22.0 +/- 1.6 vs 11.8 +/- 1.4), TUNEL (19.5 +/- 1.3 vs 11.1 +/- 0.9) and SCD (20.4 +/- 1.3 vs 10.8 +/- 1.1), respectively. DNA fragmentation in sperm evaluated by AOT did not differ (P > .05) between infertile men (31.3 +/- 2.4) and donors (32.7 +/- 4.8). AOT showed extreme variations for sperm DNA fragmentation in semen from both infertile men and donors. The problems of indistinct colors, rapid fading, and the heterogeneous staining were also faced. In conclusion, SCSA, TUNEL, and SCD show similar predictive values for DNA fragmentation, and AOT shows variable and increased levels of DNA fragmentation, which makes it of questionable value in clinical practice.

Chromatin↗
PubMed1994

Induction of apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C.

The present studies were undertaken to characterize further the potential role of protein kinase C (PKC) in the regulation of apoptosis in HL-60 promyelocytic leukemia cells. The capacity of acute exposure to specific and nonspecific pharmacological inhibitors of PKC to promote apoptotic DNA fragmentation was examined both quantitatively and qualitatively and correlated with effects on cellular differentiation and proliferation. Incubation of HL-60 cells for 6 h with chelerythrine and calphostin C (highly specific inhibitors that act at the regulatory domain) or H7 and gossypol (nonspecific inhibitors that act at the PKC catalytic domain) produced concentration-dependent increases in DNA fragmentation. Induction of DNA fragmentation by chelerythrine, calphostin C, and gossypol was biphasic, resulting in a sharp decline in effect at concentrations above 5 microM, 0.1 microM, and 100 microM, respectively, whereas maximal and more stable effects were observed in response to H7 (100 microM). A 6-h exposure to staurosporine, a nonspecific but potent PKC inhibitor, failed to induce DNA fragmentation at concentrations generally used to achieve maximal inhibition of enzyme activity (e.g., 50 nM) but promoted fragmentation at considerably higher concentrations (e.g., > or = 200 nM). In contrast, 6-h exposures to the nonspecific protein kinase inhibitor hypericin (0.1 to 100 microM) or to the nonspecific inhibitor of protein kinase A, HA1004 (50 microM), were without effect on DNA fragmentation. DNA obtained from cells exposed to chelerythrine (5 microM), calphostin C (100 nM), H7 (50 microM), gossypol (50 microM), and staurosporine (200 nM)--but not hypericin (25 microM)--exhibited clear evidence of internucleosomal DNA cleavage on agarose gel electrophoresis; moreover, these cells exhibited the classical morphological features of apoptosis (cell shrinkage, nuclear condensation, and the formation of apoptotic bodies). All of the PKC inhibitors that induced apoptosis, and one of the inhibitors that did not (hypericin), substantially inhibited HL-60 cell clonogenicity at the concentrations evaluated. None of the agents tested induced cellular maturation as assessed by nonspecific esterase and nitro-blue tetrazolium positivity. DNA fragments obtained from cells exposed to specific and nonspecific PKC inhibitors possessed predominantly 5'-phosphate termini, consistent with the action of a Ca(2+)-/Mg(2+)-dependent endonuclease. Finally, Northern blot analysis revealed that exposure to calphostin C at a concentration that induced apoptosis (100 nM) failed to alter expression of bcl-2, an oncogene known to block apoptosis in both lymphoid and myeloid leukemia cells.(ABSTRACT TRUNCATED AT 400 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗