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PubMed1998

Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa.

The objective of this study was to evaluate the effect of the generation of reactive oxygen species (ROS) on the integrity of the DNA of human spermatozoa, and to determine if pretreatment with antioxidants can reduce DNA damage. Samples were obtained from 47 men undergoing infertility investigation. ROS were generated in the samples by the addition of xanthine/xanthine oxidase (X/XO) with or without antioxidants. After incubation at timed intervals (0-2 h) with X/XO, the percentage of spermatozoa with DNA fragmentation was determined using the method of TdT-mediated DNA end-labelling (TUNEL). Time intervals were selected to mimic the clinical situation in which spermatozoa are held for a period of time after swim-up while the oocytes are prepared for ICSI. A significant increase in sperm DNA damage was evident when samples were incubated in the presence of ROS for intervals of 1 and 2 h, but not when incubated with ROS for <1 h (P = 0.0001). The addition of antioxidants significantly decreased the amount of DNA damage induced by ROS generation (P < 0.04). ROS can cause an increase in DNA fragmentation and pretreatment with antioxidants can reduce DNA damage.

Antioxidants↗
PubMed2003

Activation of caspases-3/7 is dispensable for idarubicin-induced apoptotic DNA fragmentation in human leukemia cells.

Idarubicin (IDA) is a 4-demethoxy-anthracycline analogue of daunorubicin (DNR). IDA has been recognized as a potent anti-leukemic agent. However, the molecular mechanism of IDA-induced cell death remains unclear. In the present study, we investigated the activity of IDA to induce apoptosis in human leukemia HL-60 and Jurkat cells, and studied its relationship with activation of caspases-3/7. IDA induced apoptotic DNA fragmentation in a time- and dose-dependent manner. The kinetics of apoptotic DNA fragmentation induced by IDA was well correlated with that of caspase-3/7 activation. We examined the effect of caspase-3/7 inhibitor Ac-DEVD-CHO on IDA-induced apoptosis in HL-60 and Jurkat cells. Ac-DEVD-CHO abolished IDA-induced caspases-3/7 activation in both cell lines. We have also found that L-carnitine can inhibit recombinant caspase-3 activity in vitro. L-carnitine treatment prevented IDA-induced caspases-3/7 activation in both cell lines in a dose-dependent manner. However, neither Ac-DEVD-CHO nor L-carnitine inhibited IDA-induced apoptotic internucleosomal DNA fragmentation in HL-60 or Jurkat cells. These data suggest that caspase-3/7 may be dispensable for idarubicin-induced internucleosomal DNA cleavage during apoptosis in human leukemia cells.

Antineoplastic Agents↗
PubMed2005

[Impact of sperm DNA fragmentation on ART outcome].

We have used the sperm chromatin structure assay (SCSA) test in order to determine if correlations can be found between sperm DNA fragmentation and spermogram parameters. Only necrospermia and DNA fragmentation index are strongly correlated (P<0.0001). Neither fertilization rates for ICSI and IVF, nor blastocyst formation rates are impaired by a high DFI. However when the critical DFI>30% is reached, the chances of having ongoing pregnancies after blastocyst transfer are reduced by three. Treatments with antioxidants are of limited efficacy even though we obtained 2 deliveries after DFI treatments with such treatments. New strategies in order to improve the pregnancy rates for these peculiar cases of reduced fertility are discussed.

Adult↗
PubMed1995

Cell death in Alzheimer's disease evaluated by DNA fragmentation in situ.

Loss of nerve cells is a hallmark of the pathology of Alzheimer's disease (AD), yet the patterns of cell death are unknown. By analyzing DNA fragmentation in situ we found evidence for cell death not only of nerve cells but also of oligodendrocytes and microglia in AD brains. In average, 30 times more brain cells showed DNA fragmentation in AD as compared to age-matched controls. Nuclear alterations suggestive of apoptosis were rare in degenerating cells. Even though the majority of degenerating cells were not located within amyloid deposits and did not contain neurofibrillary tangles, neurons situated within areas of amyloid deposits or affected by neurofibrillary degeneration revealed a higher risk of DNA fragmentation and death than cells not exposed to these AD changes.

Aged↗
PubMed1984

Directional cloning of DNA fragments at a large distance from an initial probe: a circularization method.

The principle of a DNA cloning procedure that directionally generates genomic DNA fragments 50-2000 kilobases away from an initial probe is presented. The method depends on partial digestion of high molecular weight genomic DNA and subsequent ligation at very low concentration to generate covalent DNA circles. A library of the junction fragments from these circles can then be constructed. Biological or physical selection of the junction pieces can be achieved by incorporating a marker DNA fragment into the covalent circles. A 45-kilobase cosmid fragment has been successfully used to test the procedure. At appropriately low ligation concentrations (0.8 micrograms/ml), approximately equal to 90% of the ligated DNA is present as monomeric circles. Larger DNA fragments will require reducing the DNA concentration as the inverse square root of the DNA length. A suppressor tRNA gene has been tested as the selectable marker gene. Ligation of the digested circles into an amber-mutated lambda phage and propagation in a sup- host allows only the phage that contain junction fragments to produce plaques. Potential applications of this approach, such as mapping of complex genetic loci or moving from a linked gene toward a gene of interest, are presented and discussed.

Bacteriophage lambda↗
PubMed1993

A combined molecular and cytogenetic approach to genome evolution in Drosophila using large-fragment DNA cloning.

Methods of genome analysis, including the cloning and manipulation of large fragments of DNA, have opened new strategies for uniting molecular evolutionary genetics with chromosome evolution. We have begun the development of a physical map of the genome of Drosophila virilis based on large DNA fragments cloned in bacteriophage P1. A library of 10,080 P1 clones with average insert sizes of 65.8 kb, containing approximately 3.7 copies of the haploid genome of D. virilis, has been constructed and characterized. Approximately 75% of the clones have inserts exceeding 50 kb, and approximately 25% have inserts exceeding 80 kb. A sample of 186 randomly selected clones was mapped by in situ hybridization with the salivary gland chromosomes. A method for identifying D. virilis clones containing homologs of D. melanogaster genes has also been developed using hybridization with specific probes obtained from D. melanogaster by means of the polymerase chain reaction. This method proved successful for nine of ten genes and resulted in the recovery of 14 clones. The hybridization patterns of a sample of P1 clones containing repetitive DNA were also determined. A significant fraction of these clones hybridizes to multiple euchromatic sites but not to the chromocenter, which is a pattern of hybridization that is very rare among clones derived from D. melanogaster. The materials and methods described will make it possible to carry out a direct study of molecular evolution at the level of chromosome structure and organization as well as at the level of individual genes.

Animals↗
PubMed2002

T and B leukemic cell lines exhibit different requirements for cell death: correlation between caspase activation, DFF40/DFF45 expression, DNA fragmentation and apoptosis in T cell lines but not in Burkitt's lymphoma.

The execution phase of apoptosis occurs through the activation and function of caspases which cleave key substrates that orchestrate the death process. Here, we have compared the sensitivity of various T and B cell lines to death receptor or staurosporine-induced apoptosis. First, we found a lack of correlation between death receptor expression and sensitivity to Fas or Trail. By contrast, a correlation between caspase activation, DNA fragmentation and cell death in T cell lines was evidenced. Among T cells, CEM underwent apoptosis in response to CH11 but were resistant to Trail in agreement with the absence of Trail receptors (DR4 and DR5) on their surface. The B cell line SKW 6.4 was sensitive to CH11 and staurosporine but resistant to Trail. As B cell lines expressed significant levels of DR4 and DR5, resistance to Trail in SKW 6.4 is likely due to the expression of the decoy receptor DcR1. Burkitt's lymphoma such as RPMI 8866 and Raji did not exhibit DNA fragmentation in response to CH11, Trail or staurosporine but showed long-term caspase-dependent loss of viability upon effector treatment. The B cell lines used in this study express very weak or undetectable levels of DFF40 and relatively high levels of DFF45. Interestingly, cytosolic extracts from RPMI 88.66 but not other B lymphoma exhibit altered levels of cytochrome c-dependent caspase activation. Taken together, our results show that: (1) death receptor expression does not correlate with sensitivity to apoptosis; (2) the very low ratio of DFF40 vs. DFF45 is unlikely to explain by itself the lack of DNA fragmentation observed in certain B cell lines; and (3) a defective cytochrome c-dependent caspase activation might account at least in part for the insensitivity of certain Burkitt's lymphoma (RPMI 88.66) to apoptosis. Thus it seems that resistance of Burkitt's lymphoma to apoptosis is not governed by a general mechanism, but is rather multifactorial and differs from one cell line to another.

Antineoplastic Agents↗
PubMed2002

In-vitro effects of FSH and testosterone withdrawal on caspase activation and DNA fragmentation in different cell types of human seminiferous epithelium.

BACKGROUND: Caspases are downstream elements of apoptosis-mediating pathways initiated by the Fas ligand/Fas receptor system which is supposed to play a central role in the regulation of apoptosis in the human seminiferous epithelium. However, caspase activity in different cell types of this epithelium has never been addressed. METHODS AND RESULTS: We evaluated caspase activity and DNA integrity in Sertoli and germ cells within in-vitro cultured segments of human seminiferous tubules after induction of apoptosis by FSH or testosterone withdrawal. FSH withdrawal increased the incidence of DNA fragmentation in meiotic (primary spermatocytes) and post-meiotic (spermatids) germ cells without producing any detectable effect on caspase activity in these cells and without affecting DNA integrity or caspase activity in Sertoli cells. Testosterone withdrawal stimulated caspase activity and produced DNA fragmentation in Sertoli cells, but showed only a weak effect on DNA fragmentation in germ cells and did not alter germ cell caspase activity. CONCLUSIONS: These findings confirm the central role of caspases in apoptosis of Sertoli cells. However, they also suggest that acute apoptosis of germ cells in the adult human testis occurs in a caspase-independent way and is controlled by Sertoli cells via an as yet undetermined mechanism.

Apoptosis↗
PubMed1994

Identification and chromosomal localization of a DNA fragment implicated in the partial correction of the Fanconi anemia group D cellular defect.

Fanconi anemia (FA) cells, complementation group D, which had been transfected with mouse genomic DNA were partially corrected for their mitomycin C (MMC) hypersensitivity. A genomic DNA fragment which complements the resistance of FA(D) cells to MMC close to normal level has been cloned; it has no correcting activity in FA group A cells. It contains two highly conserved regions between the mouse and human genome, which flank mouse repeated DNA. This DNA fragment detects a 3.6-4-kb mRNA transcript in human cells. Moreover this fragment maps to chromosome 11q23, a region of particular interest since several genes involved in the control of major cellular functions are located in this area. This DNA fragment may belong to a gene directly or indirectly involved in FA(D) function.

Animals↗
PubMed2000

DNA fragmentation occurs in skeletal muscle during tumor growth: A link with cancer cachexia?

In two different experimental models of cancer cachexia, the rat Yoshida AH-130 ascites hepatoma and the mouse Lewis lung carcinoma, the implantation of the tumor caused a loss of body weight which was associated with a reduction in the weight of different skeletal muscles, as well as with their protein content. The decrease in protein content was accompanied by a reduction in DNA content. Interestingly, the protein/DNA ratio was unchanged in the skeletal muscle of the tumor-bearing animals as compared with the non-tumor-bearing controls. Analysis of DNA fragmentation in skeletal muscle clearly showed enhanced laddering in the skeletal muscle of tumor-bearing animals, suggesting an apoptotic phenomenon. Interestingly, the degree of laddering (total DNA fragmented) increased with tumor burden. These results suggest that DNA fragmentation may be a primary event in cancer-associated cachexia.

Animals↗
PubMed1986

The lac operator as a phenotypic label for DNA fragments cloned in Escherichia coli.

To confer a detectable phenotype on any DNA fragment cloned in Escherichia coli, one can label the fragment by ligating it to the lac operator so that host cells can be identified as blue colonies on agar plates. This screening strategy is similar to that used for the pUC and M13 series of vectors, but does not require the vector to contain the lacZ gene. Instead, the presence of the lac operator on the multicopy vector results in the induction of the host cell beta-galactosidase by titrating out the repressor. This paper describes how pUC/M13 vectors or synthetic oligodeoxynucleotides can be used to supply the operator label, and shows how this method has been used to position unique restriction sites for initiating BAL 31 deletions. This approach may be particularly helpful when a given DNA fragment is to be cloned in many different constructs or is to pass through many sequential cloning steps.

Base Sequence↗
PubMed2003

Subunit structures and stoichiometries of human DNA fragmentation factor proteins before and after induction of apoptosis.

DNA fragmentation factor (DFF) is one of the major endonucleases responsible for internucleosomal DNA cleavage during apoptosis. Understanding the regulatory checkpoints involved in safeguarding non-apoptotic cells against accidental activation of this nuclease is as important as elucidating its activation mechanisms during apoptosis. Here we address these issues by determining DFF native subunit structures and stoichiometries in human cells before and after induction of apoptosis using the technique of native pore-exclusion limit electrophoresis in combination with Western analyses. For comparison, we employed similar techniques with recombinant proteins in conjunction with atomic force microscopy. Before induction of apoptosis, the expression of DFF subunits varied widely among the cell types studied, and the chaperone/inhibitor subunits DFF45 and DFF35 unexpectedly existed primarily as monomers in vast excess of the latent nuclease subunit, DFF40, which was stoichiometrically associated with DFF45 to form heterodimers. DFF35 was exclusively cytoplasmic as a monomer. Nuclease activation upon caspase-3 cleavage of DFF45/DFF35 was accompanied by DFF40 homo-oligomer formation, with a tetramer being the smallest unit. Interestingly, intact DFF45 can inhibit nuclease activity by associating with these homo-oligomers without mediating their disassembly. We conclude that DFF nuclease is regulated by multiple pre- and post-activation fail-safe steps.

Apoptosis↗
PubMed1999

Changes in the protein pattern of H1 histones associated with apoptotic DNA fragmentation.

Characteristic steps in the course of cellular apoptosis are the induction of chromatin condensation and cleavage of the DNA, leading to the formation of oligomers of nucleosomes. Since the H1 histones represent functional elements that are essential for the generation of highly condensed chromatin structures, we analysed the total cellular H1 histones of five leukaemic and three solid human tumour cell lines, comparing the H1 pattern of exponentially growing cells with that of apoptotic cells. For the induction of apoptosis, cell lines were treated with the water-soluble camptothecin derivative, topotecan (a topoisomerase I inhibitor), or with an apoptosis-inducing monoclonal anti-CD95 (Fas/APO-1) antibody. Total histone H1 proteins were isolated by extraction with 5% perchloric acid and were analysed by means of capillary zone electrophoresis (CZE) separation. The identities of the peaks representing different histone H1 subtypes on CZE electropherograms were confirmed by analysis of preparations (recombinant proteins purified from transformed yeast used as internal standards) mixed with each of the subtypes respectively. The progress of topotecan- or anti-CD95-induced cell death was monitored by flow cytometry analysis and also by agarose electrophoresis of fragmented DNA. During early apoptosis of three of these cell lines, we observed the induction of internucleosomal DNA cleavage and, simultaneously, a typical change in the histone H1 protein pattern, leading to an increase in the relative amounts of histone subtypes H1.4 and H1.5. Upon apoptosis induction, these changes were only observed in correlation with the occurrence of DNA fragmentation, thus possibly reflecting a prerequisite for DNA accessibility and/or endonuclease activity.

Apoptosis↗
PubMed1989

Two methods to detect DNA fragments produced by restriction enzymes.

This report summarizes two methods for detecting limited amounts of DNA from restriction endonuclease digests. The first is a photographic system for visualizing ethidium bromide-stained DNA fragments in agarose gels which can detect as little as 50-100 pg DNA per band. The second technique is direct sulfonation of DNA fragments bound to nylon membranes followed by visualization of the fragments by nonradioactive immunoblot methods. The immunohistochemical staining can detect 10 pg DNA per band. The direct sulfonation technique is not intended to identify specific DNA sequences; DNA-DNA hybridization with sulfonated probes has previously been described (P. Lebacq, D. Squalli, M. Duchenne, P. Poulety, and M. Johannes (1988) J. Biochem. Biophys. Methods 15, 255-266). Direct sulfonation can be used when samples are relatively free of contaminating nucleic acids and is a useful alternative to end-labeling. These highly sensitive techniques may be suitable when the DNA source is of limited quantity or in instances where radiolabeling is not permitted.

Animals↗
PubMed2006

Relationships between sperm DNA fragmentation, sperm apoptotic markers and serum levels of CB-153 and p,p'-DDE in European and Inuit populations.

Persistent organochlorine pollutants (POPs) are suspected to interfere with hormone activity and the normal homeostasis of spermatogenesis. We investigated the relationships between sperm DNA fragmentation, apoptotic markers identified on ejaculated spermatozoa and POP levels in the blood of 652 adult males (200 Inuits from Greenland, 166 Swedish, 134 Polish and 152 Ukrainian). Serum levels of 2, 2', 4, 4', 5, 5'-hexachlorobiphenyl (CB-153), as a proxy of the total POP burden, and of 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p'-DDE), as a proxy of the total DDT exposure were determined. Sperm DNA fragmentation was measured by using the TUNEL assay, whereas immunofluorescence methods were utilized for detecting pro-apoptotic (Fas) and anti-apoptotic (Bcl-xL) markers. Both TUNEL assay and apoptotic markers were statistically differed across the four populations. No correlation between neither sperm DNA fragmentation nor apoptotic sperm parameters and the large variations in POPs exposure was observed for the separate study groups. However, considering the European populations taken together, we showed that both %TUNEL positivity and Bcl-xL were related to CB-153 serum levels, whereas our study failed to demonstrate any relations between DDE and %TUNEL positivity and apoptotic sperm biomarkers (Fas and Bcl-xL) in any region or overall regions. These results suggest that CB-153 and related chemicals might alter sperm DNA integrity and Bcl-xL levels in European adult males, but not in the highly exposed Inuit men. Additional issues (genetic background, lifestyle habits and characterization of total xeno-hormonal activities) need to be investigated in order to fully assess the population variations observed.

Adult↗
PubMed1999

Imbalance between cell proliferation and cellular DNA fragmentation in hepatocellular carcinoma.

AIM/BACKGROUND: Hepatocellular carcinoma (HCC) is known for its rapid growth. This study was undertaken to determine the expression of proliferative markers, apoptosis (DNA fragmentation) and oncogene products known to regulate apoptosis (p53, bcl-2) in HCC. METHODS: 150 Chinese patients with HCC were studied (M:F 128:22, age 14-88 years). Immunohistochemistry was employed to detect cell proliferative markers (PCNA, Ki67), and oncogene products known to regulate apoptosis (p53, bcl-2). DNA fragmentation was determined by terminal dUTP nick end labeling (TUNEL). RESULTS: 98% and 95% of HCC had PCNA (median 2+) and Ki67 (median 2+) detected respectively. TUNEL labeling was detected in only a small number of tumor cells (no labeling in 11%, median 1/1000 cell labeled, range: 0-70/1000 cells). There was no correlation between TUNEL labeling and the clinical parameters (sex, age, cirrhosis, and survival) and the expression of cell proliferative markers. p53 was detected in 53% of the patients (median 1+, range: 0-4+) and bcl-2 was detected in a small proportion of tumor cells in only 13% of the HCCs (range: 0-1 +). The expression of p53 and Bcl-2 did not correlate with TUNEL labeling or the natural survival. CONCLUSIONS: Cell proliferation in HCC is unmatched by apoptosis, accounting for the rapid growth of this tumor. This lack of apoptosis in HCC is unrelated to the expression of p53 or bcl-2 over-expression.

Adolescent↗
PubMed2006

Increase in DNA fragmentation and apoptosis-related gene expression in frozen-thawed bovine blastocysts.

Evaluation of apoptosis and expression level of apoptosis-related genes is useful for examining the variation in embryo quality according to environmental change. The objective of this study was to investigate DNA fragmentation and apoptosis-related gene expression patterns in frozen-thawed bovine blastocysts. In vitro produced day 7 blastocysts were frozen by two different vitrification methods (conventional 0.25 ml straw or MVC straw). After thawing, DNA fragmentation of surviving embryos was examined by TUNEL assay, and the expression patterns of their apoptotic genes (survivin, Fas, Hsp 70 and caspase-3) were evaluated using real-time quantitative reverse transcriptase polymerase chain reaction. In vitro survival rates of frozen-thawed embryos were higher following the MVC vitrification method (88.2% re-expanded at 24 h, 77.1% hatching at 48 h) than the conventional (C) vitrification method (77.0% re-expanded at 24 h, 66.7% hatching at 48 h). However, both vitrified methods resulted in a significantly higher apoptotic index (C vitrification method 11.9%, MVC vitrification method 11.0%) than in non-frozen embryos (3.0%). Expression levels of survivin, Fas, caspase-3, and Hsp 70 were also increased in the frozen-thawed embryos compared with non-frozen embryos. These results indicate that the cryopreservation procedure might cause damage that results in an increase in DNA fragmentation and apoptosis-related gene transcription, reducing developmental capacity of frozen-thawed embryos.

Animals↗
PubMed1993

Effect of Bcl-2 on ionizing radiation and 1-beta-D-arabinofuranosylcytosine-induced internucleosomal DNA fragmentation and cell survival in human myeloid leukemia cells.

1-beta-D-Arabinofuranosylcytosine (ara-C) is an anti-leukemic agent that incorporates into cellular DNA leading to inhibition of DNA synthesis and loss of clonogenic survival. In contrast, ionizing radiation induces DNA damage through the generation of reactive oxygen intermediates. Although little is known of the specific determinants of ara-C and ionizing radiation-induced cytotoxicity, recent work has shown that both are capable of inducing internucleosomal DNA fragmentation in a pattern consistent with programmed cell death (apoptosis). In order to assess the importance of apoptosis in drug and ionizing radiation-induced cytotoxicity in the U-937 myelomonocytic cell line, we created cell lines that constitutively express a transfected bcl-2 gene. Bcl-2 was capable of inhibiting 40-50% of the ara-C and ionizing radiation-induced internucleosomal DNA fragmentation at all tested concentrations. However, cell survival following exposure to these agents was only increased in the bcl-2 transfectants at relatively low doses of ara-C and ionizing radiation. These data demonstrate that although bcl-2 is capable of inhibiting ara-C and ionizing radiation-induced DNA fragmentation in myeloid cells, it increases cell survival only at low doses of these agents. This suggests that apoptosis may be a less important mechanism of cytotoxicity at higher doses of ara-C and ionizing radiation than it is at lower doses.

Cell Survival↗