DNA fragmentation and DNA repair of mammalian cells as an indicator for the complex interactions between carcinogens and modulating factors.
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Animals↗
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Two in situ labeling techniques reveal different patterns of DNA fragmentation during spontaneous apoptosis in vivo and induced apoptosis in vitro.
BACKGROUND: Two new enzymatic reactions were described recently to detect apoptotic cell death in situ viz in situ end labeling (ISEL) and terminal deoxynucleotidyl transferase mediated UTP nick end labeling (TUNEL) of fragmented DNA. A comparative study was conducted to detect in vivo and in vitro apoptotic death using these two techniques. Experimental design; Spontaneous apoptous cell death was detected in plastic embedded tumor biopsies from patients with non-Hodgkin's lymphoma (NHL), head and neck squamous cell carcinomas (HNSCC), and breast cancer using these two in situ methods. Uninvolved normal tissues adjacent to breast tumors and a lymph node metastasis of breast tumor were also studied. Furthermore, apoptotic death induced by different doses of etoposide (VP16) was also studied in HL60 cells by in situ methods and by agarose gel electrophoresis. RESULTS: Interestingly, whereas NH1 and HNSCC biopsies showed comparable levels of detectability with the two techniques, the breast tissues be it neoplastic, normal or metastatic, revealed apoptosis detectable only by TUNEL and not by ISEL. Similarly in HL60 cells, the percentage of apoptotic cells or apoptotic index (AI) determined by TUNEL was significantly higher than that determined by ISEL. A double labelling of these HL60 cells for ISEL and TUNEL also revealed a higher proportion of cells labeled positively for TUNEL as compared to those labeled for ISEL. Agarose gel electrophoresis revealed characteristic DNA laddering only at 35 microM dose of VP 16. No smearing of DNA was found in any group ruling out the necrotic death. In vivo, in one HNSCC specimen apoptosis and necrosis could be differentiated by the difference in staining intensity. Both methods stained necrotic chromatin fragments very lightly. The DNA fragments generated during apoptosis could be of unique lengths (ie 180-200 bp or multiples) but have differently staggered ends. These fragments may be 3' recessed, 5' recessed or blunt ended. While TUNEL can label all three types, ISEL labels only those with 3' recessed ends. CONCLUSIONS: Thus our data show that the DNA fragments formed during spontaneous apoptosis in breast tissues and preferentially during VP16 induced apoptosis in HL60 cells are either 5' recessed or blunt ended, being distinctly different from 3' recessed fragments seen in NHL and HNSCC or with a lesser frequency in VP 16 treated HL60 cells. Specific fragmentation patterns could be a result of activation of different endonucleases which as indicated by our data could be tissue specific and may be differentially activated by different chemotherapeutic agents. Therefore, screening for the presence of specific endonucleases in different tissues and for agents specifically activating them would have major clinical implications.
Apoptosis↗
The irreversible binding of azacytosine-containing DNA fragments to bacterial DNA(cytosine-5)methyltransferases.
DNA containing 5-azacytosine is an irreversible inhibitor of DNA(cytosine-5)methyltransferase. This paper describes the binding of DNA methyltransferase to 32P-labeled fragments of DNA containing 5-azacytosine. The complexes were identified by gel electrophoresis. The EcoRII methyltransferase specified by the R15 plasmid was purified from Escherichia coli B(R15). This enzyme methylates the second C in the sequence CCAGG and has a molecular mass of 60,000 Da. Specific binding of enzyme to DNA fragments could be detected if either excess unlabeled DNA or 0.8% sodium dodecyl sulfate was added to the reaction mixture prior to electrophoresis. Binding was dependent upon the presence of both the CCAGG sequence and azacytosine in the DNA fragment. S-Adenosylmethionine stimulated the formation of the complex. The complex was stable to 6 M urea but could be digested with pronase. These DNA fragments could be used to detect the presence of several different methyltransferases in crude extracts of E. coli. No DNA protein complexes could be detected in E. coli B extracts, a strain that contains no DNA(cytosine-5)methyltransferases. The chromosomally determined methylase with the same specificity as the purified EcoRII methylase could be detected in crude extracts of E. coli K12 strains. The MspI methylase cloned in E. coli HB101 could also be detected in crude extracts. These enzymes are the only proteins that bind azacytosine-containing DNA in crude extracts of E. coli.
Cytosine↗
Biochemical screening of stable dinucleosomes using DNA fragments from a dinucleosome DNA library.
The dinucleosome is an informative unit for analysis of the higher-order chromatin structure. DNA fragments forming stable dinucleosomes were screened from a dinucleosome DNA library after the reconstitution of nucleosomes in vitro and digestion with micrococcal nuclease. Reconstituted dinucleosomes showed a diversity of sensitivity to micrococcal nuclease, suggesting that the biochemical stability of a dinucleosome depends, in part, on the DNA fragments. The DNA fragments after the screening were classified into three groups represented by clones bf10, af14 and af32 according to the sensitivity to micrococcal nuclease. Mapping of the nucleosome boundaries by Southern blotting of the DNA after restriction digestion and by primer extension analysis showed that each nucleosome position of clone af32 was fixed. Analysis of reconstituted dinucleosomes using mutant DNA fragments of clone af32 revealed a unique property characteristic of a key nucleosome, given that the replacement of a DNA fragment corresponding to the right nucleosome position resulted in marked sensitivity to micrococcal nuclease, whereas the replacement of the other nucleosome fragment had almost no effect on sensitivity as compared to the original af32 construct. The mutant construct in which the right nucleosome was removed showed multiple nucleosome phases, suggesting that the right nucleosome stabilized first each mononucleosome and then the dinucleosome. An oligonucleotide bending assay revealed that the DNA fragment in the right nucleosome included curved DNA, suggesting that the positioning activity of the nucleosome was attributed to its DNA structure. These results suggest that information for forming stable dinucleosome is embedded in the genomic DNA and that a further characterization of the key nucleosome is useful for understanding the building up of the chromatin structure.
Animals↗
Doxorubicin induces apoptosis with profile of large-scale DNA fragmentation and without DNA ladder in anaplastic thyroid carcinoma cells via histone hyperacetylation.
Doxorubicin is known to be the most effective single cytotoxic drug against anaplastic thyroid carcinoma (ATC). Although doxorubicin has been shown to cause cell death, at least partly, by inducing apoptosis in ATC cells, the mechanism underlying its pharmacological efficacy has not been fully delineated. We, in this study, revealed that doxorubicin induced apoptosis in ATC cells by altering the acetylation state of histone. Doxorubicin reduced histone deacetylase activity and induced hyperacetylation of histone 3. Noticeably, ladder-like DNA fragments from their genomic DNA on agarose gel were not detected irrespective of several lines of evidence supporting the induction of apoptosis. Pulse field electrophoresis showed disintegration of nuclear DNA into giant fragments of 1-2 Mbp and high molecular-weight fragments of 100-1000 kbp. We next examined whether a histone deacetylase inhibitor trichostatin A (TsA) augmented doxorubicin-induced apoptosis in ATC cells. TSA potentiated doxorubicin-induced stage I apoptosis in ATC cells. Our study sheds light on the development of a new combination therapy strategy for more effective responses for ATC treatment.
Acetylation↗
Association of double-stranded DNA fragments into multistranded DNA structures.
We have previously observed that double-stranded DNA fragments containing a tract of the tandemly repeated sequence poly(CA). poly(TG) can associate in vitro to form stable complexes of low electrophoretic mobility, which are recognized with high specificity by proteins HMG1 and HMG2. The formation of such complexes has since been observed to depend on interactions of DNA with polypropylene surfaces, with the suggestion that the formation of low mobility complexes might be the result of strand dissociation followed by misaligned reassociation of the repetitive sequences. The data presented here show that at high ionic strength the interactions of DNA with polypropylene are sufficiently strong for DNA to remain bound to the polypropylene surface, which suggests that DNA might also be involved in interactions with hydrophobic molecules in vivo. Under such conditions, low-mobility complexes are found only in the material adsorbed to the polypropylene surface, and all DNA fragments are able to form low-mobility structures, whether or not they contain repetitive sequences. Preventing the separation of strands by ligating hairpin loop oligonucleotides at both ends of the fragments does not prevent the formation of low-mobility complexes. Our results suggest two different pathways for the formation of complexes. In the first, dissociation is followed by misaligned reassociation of repetitive sequences, yielding duplexes with single-stranded end regions that associate to form multimeric complexes. In the second, repetitive as well as nonrepetitive DNA molecules bound to polypropylene adopt a conformation with locally unwound regions, which allows interactions between neighboring duplexes adsorbed on the surface, resulting in the formation of low-mobility complexes.
DNA↗
Induction of differentiation by tumour necrosis factor in HL-60 cells is associated with the formation of large DNA fragments.
The DNA fragmentation induced by tumor necrosis factor (TNF) of differentiable human myeloid leukemic HL-60 cells has been further characterized. TNF increased the appearance of very high molecular weight DNA fragments detected by agarose gel electrophoresis. The use of pulsed-field gel electrophoresis (PFGE) revealed these fragments to be as high as 200-400 kilobase pairs. The pattern of HL-60 DNA fragmentation contrasted with that of U937 cells, which exhibited lower molecular weight, nucleosome multiple sized fragments, and greater cytotoxicity in response to TNF. The peak increase of fragments from HL-60 occurred between one and two hours of incubation, with TNF concentrations of 10 U/ml or higher, and was inhibitable by 1 mM Zn2+. Southern blotting of these fragments disclosed enrichment for c-myc related sequences compared with control probes including beta-actin and kappa and lambda light chains. Treatment of DNA with NotI or gamma-irradiation, followed by PFGE, disclosed a class of still higher molecular weight DNA, which decreased following TNF treatment, and which was apparently the precursor of the TNF-induced fragments. TNF thus rapidly increases a class of high molecular weight DNA fragments which are enriched for c-myc related sequences and may arise preferentially from higher molecular weight structures which are detectable following linearization by NotI or gamma-irradiation. Such major but non-random alterations in chromatin structure may contribute to TNF-induced monocytoid differentiation of HL-60.
Blotting, Southern↗
Central nervous system DNA fragmentation induced by the inhibition of nuclear factor kappa B.
Ageing of the central nervous system (CNS) is characterized by a progressive apoptotic loss of neurons that may be in part due to impaired neurotrophin signaling mediated by such elements as the transcription factor nuclear factor kappa B (NFkappaB). To support this hypothesis, we inhibited nuclear translocation of NFkappaB in vivo by injecting a proteasome inhibitor (PSI) directly in the CNS lateral ventricle of rats and then measured fragmented DNA in various CNS areas as an index of ongoing apoptosis. Our results show that after PSI injection there was a significant inhibition of NFkappaB activity in vivo that resulted in the appearance of fragmented (apoptotic) DNA in the CNS of rats. These results suggest that alteration of NFkappaB may cause apoptotic cell death in the rat CNS during ageing.
Animals↗
Renal carcinoma cells undergo apoptosis without oligonucleosomal DNA fragmentation.
Apoptotic DNA fragmentation minimizes the risk of transferring genetic information from apoptotic cancer cells to the neighboring cells. We have reported previously that caspase-deficient human renal cell carcinoma (RCC) lines were almost completely resistant to apoptosis in response to cytotoxic agents. In the present report we examined apoptotic process in caspase competent RCC-91 cells. Apoptosis in RCC-91 cells was accompanied by activation of caspases-3 and -9; cleavage of PARP and DFF45 proteins; typical apoptotic nuclei fragmentation and mitochondrial collapse. Nevertheless, DNA in these cells was not degraded into oligonucleosomal fragments compared to control Jurkat cells. Expression of caspase-activated DNase, DFF40 accountable for characteristic ladder pattern was easily detectable in Jurkat but not renal cancer cells, providing one possible explanation for the lack of oligonucleosomal DNA fragmentation in apoptotic RCC cells. Lack of typical DNA fragmentation indicates a potential threat of transferring genetic information from one tumor cell to another or to the neighboring healthy cells.
Amino Acid Chloromethyl Ketones↗
The CrmA- and TPCK-sensitive pathways that trigger oligonucleosome-sized DNA fragmentation in camptothecin-induced apoptosis: relation to caspase activation and high molecular weight DNA fragmentation.
Abstract: In human B lymphoma Namalwa variant cells expressing the serpin-like CrmA protein, the kinetics of oligonucleosome-sized DNA fragmentation was retarded compared with that of control Namalwa cells following camptothecin treatment. However, no difference in the kinetics of high molecular weight DNA fragmentation was observed between the two lines after camptothecin treatment. Similar delay and inhibition of the oligonucleosome-sized DNA fragmentation was observed in human B lymphoma Namalwa and monocytic-like leukemia U-937 cells coincubated in the presence of various concentrations of N-tosyl-L-phenylalanyl chloromethylketone and camptothecin. The effect of N-tosyl-L-phenylalanyl chloromethylketone was similar to that of CrmA and did not prevent the appearance of high molecular weight DNA fragments. Similar suppression of camptothecin-induced internucleosomal DNA fragmentation was also observed in a cell-free system when cytosolic extracts obtained from camptothecin-treated Namalwa and U-937 cells were coincubated with untreated nuclei in the presence of N-tosyl-L-phenylalanyl chloromethylketone. Furthermore, N-tosyl-L-phenylalanyl chloromethylketone had no significant effects on caspase-3-like activities in camptothecin-treated Namalwa and U-937 cells. Hydrolysis of Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin, a fluorogenic substrate with caspase-3-like activities, was detected in extracts prepared from camptothecin-treated Namalwa and U-937 cells with no apparent difference in the time courses of caspase-3-like activation in the absence or presence of N-tosyl-L-phenylalanyl chloromethylketone. Similarly, N-tosyl-L-phenylalanyl chloromethylketone was a weak inhibitor of caspase-3-like activities in vitro. Taken together, these observations suggest that the pathway sensitive to N-tosyl-L-phenylalanyl chloromethylketone is involved in camptothecin-induced oligonucleosome-sized DNA fragmentation. Furthermore, inhibition of this pathway had no effect on caspase-3-like activation and on the occurrence of high molecular weight DNA fragmentation.
Apoptosis↗
Evidence for DNA fragmentation in the CNS of aged Fischer-344 rats.
The aged central nervous system (CNS) is characterized by a loss of neurons. Apoptosis has been reported to be responsible for neuronal death during development and may also be involved in some age-related neurodegenerative diseases of the CNS. No evidence is currently available as to whether apoptosis is also responsible for the loss of neurons associated with physiological aging. In this study, we have found fragmented DNA, a characteristic trait of cells undergoing apoptosis, in the hippocampus and, to a minor extent, in the frontal cortex and basal forebrain of aged (24-month-old) Fisher-344 rats, but not in the cerebellum. These data represent important evidence to support the idea that apoptosis may be associated with the neuronal death observed during normal aging.
Aging↗
Enzymatic amplification and characterization of large DNA fragments from genomic DNA.
Conditions for DNA amplification in vitro using modified T7 DNA polymerase have been devised to obtain 2000-bp DNA fragments of the HGPRT gene directly from human genomic DNA. The DNA obtained from a 1.2 x 10(5)-fold amplification has been used for direct sequencing.
Base Sequence↗
[Use PCR synthesis large fragment DNA].
Synthesis Large Fragment DNA using PCR (SLFD PCR) is a useful method to synthesize large fragment DNA. Use a known 500 approximately 600 basepair DNA fragment as PCR template, a series of 5' terminal primers are designed, and these primers are overlap one by one from 5' terminal to 3' terminal, the net DNA is just what you want to synthesize. The last 3' terminal primer of these primers has a BamH I site, and behind the BamH I site there are 15 bp overlap the 5' terminal of the template. Another downstream primer complement the 3' terminal of the template, and has a BamH I site too. PCR begin using the innerest 5' terminal primer and the downstream primer. After 10 cycles of PCR, use the product of the PCR as the template of next round PCR, but this time the upstream primer change to the 5' terminal outer primer . So do the PCRs, till all the 5' terminal primers take part in the PCR. Clone the final PCR product and BamH I cut the original DNA template, The DNA synthesis complete. It's a useful method to synthesize 100 approximately 200 bp DNA fragment, even more long DNA fragment.
Antimicrobial Cationic Peptides↗
Homologous recombination of exogenous DNA fragments with genomic DNA in somatic cells of mice.
We compared liposomes and empty viral capsids for their use as vehicles for DNA transfer into cells and animals. DNA binding capacity was high for liposomes, but DNase I protection of DNA bound to liposomes was only moderate in comparison to DNA incorporated into viral capsids. Cellular uptake of radiolabeled and physiologically active DNA was also compared. For animal studies we chose an endogenous retroposon as target gene. To identify recombinational events we replaced a part of this gene with an artificial sequence not present in the mouse genome. The recombination rate for DNA fragments transfected in Polyoma capsids in live mice was higher than for liposome mediated transfection. Homologous recombination could be observed for both DNA transfer methods, mediated by positively charged liposomes (DOTMA) and by empty Polyoma viral capsids.
Animals↗
High resolution preparative gel electrophoresis of DNA fragments and plasmid DNA using a continuous elution apparatus.
An apparatus designed for preparative gel electrophoretic separation of proteins (M. A. Hediger, (1984) Anal. Biochem. 142, 445-454) has been used successfully for separating DNA restriction fragments. The apparatus displayed yields and resolutions that are higher than those obtainable with commercially available devices. The amounts of DNA applied to the column range from a few micrograms to milligram quantities. Restriction DNA fragments very similar in size were isolated in pure form with the apparatus. After ethanol precipitation, these fragments were successfully used for restriction enzyme cleavages, ligation, or chemical sequencing. Furthermore the apparatus provides a convenient method for the large-scale isolation of plasmid DNA. The method requires only 4 h of electrophoresis and therefore greatly reduces the preparation time compared with the conventional equilibrium centrifugation method which requires centrifugation times of up to 60 h. In contrast to the centrifugation method, contaminants such as RNA, proteins, and chromosomal DNA are efficiently removed by this technique.
DNA↗
Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation.
The protease granzyme B (GrB) plays a key role in the cytocidal activity during cytotoxic T lymphocyte (CTL)-mediated programmed cell death. Multiple caspases have been identified as direct substrates for GrB, suggesting that the activation of caspases constitutes an important event during CTL-induced cell death. However, recent studies have provided evidence for caspase-independent pathway(s) during CTL-mediated apoptosis. In this study, we demonstrate caspase-independent and direct cleavage of the 45 kDa unit of DNA fragmentation factor (DFF45) by GrB both in vitro and in vivo. Using a novel and selective caspase-3 inhibitor, we show the ability of GrB to process DFF45 directly and mediate DNA fragmentation in the absence of caspase-3 activity. Furthermore, studies with DFF45 mutants reveal that both caspase-3 and GrB share a common cleavage site, which is necessary and sufficient to induce DNA fragmentation in target cells during apoptosis. Together, our data suggest that CTLs possess alternative mechanism(s) for inducing DNA fragmentation without the requirement for caspases.
Apoptosis↗
Characterization of the rat DNA fragmentation factor 35/Inhibitor of caspase-activated DNase (Short form). The endogenous inhibitor of caspase-dependent DNA fragmentation in neuronal apoptosis.
Nuclear changes, including internucleosomal DNA fragmentation, are classical manifestations of apoptosis for which the biochemical mechanisms have not been fully elucidated, particularly in neuronal cells. We have cloned the rat DNA fragmentation factor 35/inhibitor of caspase-activated DNase (short form) (DFF35/ICAD(S)) and found it to be the predominant form of ICAD present in rodent brain cells as well as in many other types of cells. DFF35/ICAD(S) forms a functional complex with DFF40/caspase-activated DNase (CAD) in the nucleus, and when its caspase-resistant mutant is over-expressed, it inhibits the nuclease activity, internucleosomal DNA fragmentation, and nuclear fragmentation but not the shrinkage and condensation of the nucleus, in neuron-differentiated PC12 cells in response to apoptosis inducers. DFF40/CAD is found to be localized mainly in the nucleus, and during neuronal apoptosis, there is no evidence of further nuclear translocation of this molecule. It is further suggested that inactivation of DFF40/CAD-bound DFF35 and subsequent activation of DFF40/CAD during apoptosis of neuronal cells may not occur in the cytosol but rather in the nucleus through a novel mechanism that requires nuclear translocation of caspases. These results establish that DFF35/ICAD(S) is the endogenous inhibitor of DFF40/CAD and caspase-dependent apoptotic DNA fragmentation in neurons.
Amino Acid Sequence↗
Telomere dysfunction triggers extensive DNA fragmentation and evolution of complex chromosome abnormalities in human malignant tumors.
Although mechanisms for chromosomal instability in tumors have been described in animal and in vitro models, little is known about these processes in man. To explore cytogenetic evolution in human tumors, chromosomal breakpoint profiles were constructed for 102 pancreatic carcinomas and 140 osteosarcomas, two tumor types characterized by extensive genomic instability. Cases with few chromosomal alterations showed a preferential clustering of breakpoints to the terminal bands, whereas tumors with many changes showed primarily interstitial and centromeric breakpoints. The terminal breakpoint frequency was negatively correlated to telomeric TTAGGG repeat length, and fluorescence in situ hybridization with telomeric TTAGGG probes consistently indicated shortened telomeres and >10% of chromosome ends lacking telomeric signals. Because telomeric dysfunction may lead to formation of unstable ring and dicentric chromosomes, mitotic figures were also evaluated. Anaphase bridges were found in all cases, and fluorescence in situ hybridization demonstrated extensive structural rearrangements of chromosomes, with terminal transferase detection showing fragmented DNA in 5-20% of interphase cells. Less than 2% of cells showed evidence of necrosis or apoptosis, and telomerase was expressed in the majority of cases. Telomeric dysfunction may thus trigger chromosomal fragmentation through persistent bridge-breakage events in pancreatic carcinomas and osteosarcomas, leading to a continuous reorganization of the tumor genome. Telomerase expression is not sufficient for completely stabilizing the chromosome complement but may be crucial for preventing complete genomic deterioration and maintaining cellular survival.
Adolescent↗