DNA fragmentation in apoptosis

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

DNA fragmentation in apoptosis and homeostasis

Apoptosis is a highly regulated physiological process critical in development and tissue homeostasis. Abnormal apoptosis can lead to disease conditions including neurodegeneration, autoimmunity and cancer. DNA fragmentation is an integral part of apoptosis and has long been suspected to be of critical importance in cleaning up potentially antigenic DNA and genetic material capable of inducing neoplasmic transformation in neighboring cells. Direct evidence for this function of DNA fragmentation however, is still lacking. The identification of a heterodimeric DNA fragmentation factor 45 and 40 (DFF45 and DFF40, also called ICAD for Inhibitor of Caspase Activated DNase and CAD for Caspase Activated DNase respectively) as well as     

The role of DNA fragmentation in apoptosis

The formation of distinct DNA fragments of oligonucleosomal size (180-200 bp lengths) is a biochemical hallmark of apoptosis in many cells. Recent observations also suggest large DNA fragments and even single-strand cleavage events occur during cell death. These observations have raised many questions. What are the types of DNA cleavage observed during apoptosis? What are the nucleases involved? And what is the role of these nucleolytic events in apoptosis?     

DNA loop organization and DNA fragmentation during radiation-induced apoptosis in human lymphocytes

Apoptotic DNA fragmentation induced by gamma-rays has been compared with the DNA loop sizes in G0-human lymphocytes using pulsed field gel electrophoresis (PFGE). Genomic DNA was cleaved into the DNA loops at the topoisomerase II mediated attachment points using short treatment of cells with etoposide. The apoptotic fragmentation, with a distinct cut-off around 50 kb for a maximum length of fragments, appeared 5 h after irradiation when the most part of radiation-induced DNA double strand breaks (DSBs) have been repaired. The data indicate that apoptotic fragmentation of DNA in the G0-human lymphocytes begins when repair of radiation-induced DSBs has been completed. Similar apoptotic DNA fragmentation was also observed following the treatment of cells with etoposide. All genomic DNA was fragmented into 50-kb fragments during the final stages of apoptosis. Most of the DNA in resting lymphocytes is organized into Mb-size loops but loops of sizes down to 50 kb were also observed. A sharp border between the size distributions of DNA loops and apoptotic fragments was found. The data suggest that 50 kb apoptotic fragmentation is not based on excision of the DNA loops. No apoptotic fragments with the sizes more than 5.7 Mb were seen during the whole course of apoptosis. This observation indicates that despite intensive apoptotic fragmentation into the 50-kb fragments the chromosomes maintain integrity during radiation-induced apoptosis in human lymphocytes. We propose a model for radiation-induced apoptotic fragmentation in human lymphocytes that involves four stages: induction of DNA breaks and relaxation of DNA loops; DNA repair followed by reorganization of the DNA loops into the 50-kb units of condensed chromatin; co-operative fragmentation of the reorganized DNA loops into the distinct 50-kb fragments and resealing of the chromosome ends at the sites of this fragmentation; cleavage of the 50-kb fragments at the internucleosomal spacers.     

Ionophore-induced apoptosis: role of DNA fragmentation and calcium fluxes

Two ionophores specific for K+, valinomycin and beauvericin, induce a type of cell death very similar to apoptosis due to tumor necrosis factor (TNF alpha). Both ionophores cause cytolysis accompanied by internucleosomal DNA fragmentation of the dying cell into units of 200 base pairs. Morphologically, the cell death appears to consist of a mixture of nuclear apoptotic changes and cytoplasmic necrotic changes. As in the case for TNF alpha-mediated death, metabolic inhibitors have no effect on the course of cell death, but DNA fragmentation and cytolysis are decreased by the endonuclease inhibitor, zinc. Beauvericin and valinomycin trigger an increase in the cytoplasmic calcium concentration, most likely due to release of calcium from intracellular stores, and chelation of cytoplasmic calcium with quin-2 inhibits DNA fragmentation. Thus, these ionophores set off apoptosis through a calcium-activatable endonuclease, suggesting that other nonphysiological toxins might also cause apoptosis through their ability to indirectly elevate the cytoplasmic calcium concentration, without the need to invoke specific surface receptors.     

Glutathione depletion-induced chromosomal DNA fragmentation associated with apoptosis and necrosis

Chromosomal DNA and mitochondrial dysfunctions play a role on mammalian cell death induced by oxidative stress. The major biochemical dysfunction of chromosome is the presence of an ordered cleavage of the DNA backborn, which is separated and visualized as an electrophoretic pattern of fragments. Oxidative stress provides chromatin dysfunction such as single strand and double strand DNA fragmentation leading to cell death. More than 1 Mb of giant DNA, 200-800 kb or 50-300 kb high molecular weight (HMW) DNA and internucleosomal DNA fragments are produced during apoptosis or necrosis induced by oxidative stress such as glutathione (GSH) depletion in several types of mammalian cells. Reactive oxygen species (ROS)-mediated DNA fragmentation is enhanced by polyunsaturated fatty acids including arachidonic acid or their hydroperoxides, leading to necrosis. Mitochondrial dysfunction on decrease of trans membrane potential, accumulation of ROS, membrane permeability transition and release of apoptotic factors during apoptosis or necrosis has been implicated. This review refers to the correlation of chromosomal DNA fragmentation and apoptosis or necrosis induced by GSH depletion, and the possible mechanisms of oxidative stress-induced cell death.     

Cell nucleus and DNA fragmentation are not required for apoptosis

Apoptosis is the predominant form of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Activation of an endonuclease which cleaves genomic DNA into internucleosomal DNA fragments is considered to be the hallmark of apoptosis. However, no clear evidence exists that DNA degradation plays a primary and causative role in apoptotic cell death. Here we show that cells enucleated with cytochalasin B still undergo apoptosis induced either by treatment with menadione, an oxidant quinone compound, or by triggering APO-1/Fas, a cell surface molecule involved in physiological cell death. Incubation of enucleated cells with the agonistic monoclonal anti-APO-1 antibody revealed the key morphological features of apoptosis. Moreover, in non-enucleated cells inhibitors of endonuclease blocked DNA fragmentation, but not cell death induced by anti-APO-1. These data suggest that DNA degradation and nuclear signaling are not required for induction of apoptotic cell death.     

Detection of methylated sequences in eukaryotic DNA with the restriction endonucleases Smai and Xmai

araC protein was identified on two-dimensional O'Farrell gels (O'Farrell, 1975) as a protein electrophoresing as two spots, both of molecular weight 30,500 and pI near 7.1, but differing by about 0.1 pH unit. The two spots were seen in crude extracts from cells overproducing C protein as specified by a plasmid, by a phage, and were also seen in C protein purified to about 20% purity on the basis of biological activity. A label-chase experiment indicated that both species of araC are unstable in vivo and possess half-lives of about 60 minutes. The normal intracellular level of C protein is about 40 monomers per cell.     

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.     

Detection of measles virus-induced apoptosis of human monocytic cell line (THP-1) by DNA fragmentation ELISA

Abstract Measles virus (wild strain, Toyoshima strain)-induced cell death is characterized by cell shrinkage, chromatin condensation, and nuclear fragmentation in a human monocytic cell line (THP-1). DNA fragmentation of measles virus-infected THP-1 cells was demonstrated by DNA agarose gel electrophoresis as well as by DNA fragmentation ELISA. When measles virus-infected THP-1 cells were cultured on monolayers of fibroblasts or human umbilical vein endothelial cells (HUVEC), the percentage of measles virus antigen-positive THP-1 cells and DNA fragmentation were significantly decreased. Addition of anti-intercellular adhesion molecule (ICAM)-1 (CD54) monoclonal antibody to culture of measles virus-infected THP-1 cells reduced significantly DNA fragmentation induced by measles virus. These findings suggest that inhibition of virus spread by fibroblasts and HUVEC reduces apoptosis, and ICAM-1 (CD54) may participate in the DNA fragmentation pathway.     

Mitochondrial fragmentation in apoptosis

Mitochondrial outer membrane permeabilization (MOMP) is associated with most pro-apoptotic stimuli. MOMP results in the release of cytochrome c from the mitochondria into the cytosol, triggering caspase activation and subsequent apoptosis. Several theories explaining the mechanisms of MOMP have been proposed, one of which suggests that MOMP relies on the activation of the molecular machinery involved in fission, resulting in mitochondrial fragmentation. By contrast, several recent studies suggest that mitochondrial fragmentation occurs following MOMP. Moreover, under some conditions. MOMP occurs without mitochondrial fragmentation and, in fact, fragmentation even inhibits MOMP. Here, I discuss the apparently conflicting data and conclude that mitochondrial fragmentation is probably not a prerequisite for MOMP and cytochrome c release.     

Human fibroblasts undergo oxidative stress-induced apoptosis without internucleosomal DNA fragmentation

In order to evaluate the reliability of fibroblasts as a cell model for studying apoptosis, we tested the response of normal human fibroblasts to the oxidative stress inducers H(2)O(2) and 2-deoxy-D-ribose (dRib). Our results showed that fibroblasts treated with dRib and H(2)O(2) are induced to undergo apoptosis as demonstrated by reduction in total cell number, chromatin condensation, phosphatidylserine (PS) exposure, activation of caspase-3 and 7, changes in mitochondrial membrane potential and increase in the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive nuclei. However we only found a slight increase in the percentage of cells in the sub-G1 region evaluated by flow cytometry, and we did not observe DNA fragmentation by agarose gel electrophoresis. Early in apoptosis, DNA cleavage generates high molecular weight (HMW) fragments which can be detected by TUNEL assay; successively followed by a pronounced DNA brake down into low molecular weight (LMW) fragments, detected as a "DNA ladder" by conventional agarose gel electrophoresis and as an hypodiploid peak by propidium iodide (PI) flow cytometry assay. Our results thus suggest that only HMW fragmentation occurs in fibroblasts exposed to dRib or H(2)O(2) and the lack of internucleosomal DNA fragmentation may depend on the peculiar characteristics of human fibroblasts themselves, irrespective of the apoptotic stimulus used. The existence of distinct events leading to cell death in different cell types makes it necessary to use a combination of strategies and techniques to evaluate the occurrence of apoptosis.     

Evidence that DNA fragmentation in apoptosis is initiated and propagated by single-strand breaks

Apoptosis is characterised by the degradation of DNA into a specific pattern of high and low molecular weight fragments seen on agarose gels as a distribution of sizes between 50-300 kb and sometimes, but not always, a ladder of smaller oligonucleosomal fragments. Using a 2D pulsed field-conventional agarose gel electrophoresis technique, where the second dimension is run under either normal or denaturing conditions, we show that single-strand breaks are introduced into DNA at the initial stages of fragmentation. Using single-strand specific nuclease probes we further show that the complete fragmentation pattern, including release of small oligonucleosomal fragments can also be generated by a single-strand endonuclease. Three classes of sites where single-strand breaks accumulate were identified. The initial breaks produce a distribution of fragment sizes (50 kb to >1 Mb) similar to those generated by Topoisomerase II inhibitors suggesting that cleavage may commence at sites of attachment of DNA to the nuclear matrix. A second class of rare sites is also cut further reducing the size distribution of the fragments to 50-300 kb. Thirdly, single-strand breaks accumulate at the linker region between nucleosomes eventually causing double-strand scissions which release oligonucleosomes. These observations further define the properties of the endonuclease responsible for DNA fragmentation in apoptosis.     

Apparent inhibition of apoptosis by polyamines and aminothiols in DNA fragmentation assays is artifactual

We report that, in commonly used DNA fragmentation assays, polyamines and the radioprotective aminothiol WR1065 artifactually depress the degree of spontaneous or induced cellular apoptosis in two distinct ways. Firstly, in assays utilizing Hoechst 33258 dye to measure apoptotic DNA, both amines quench the fluorescence of low affinity dye/DNA binding resulting in preferential underestimation of DNA in the apoptotic DNA fraction and a resultant underestimation of the extent of DNA fragmentation. Secondly, these amines can cause aggregation and condensation of apoptotic DNA, causing anomalous sedimentation under conditions universally employed to separate apoptotic from intact DNA in cell lysates. This anomalous sedimentation of apoptotic DNA leads to underestimation of fragmentation in fluorescence assays as well as in agarose gel assays. We demonstrate that manipulation of the ionic strength of the lysis buffer or lowering the dye concentration ameliorates the effects of dye quenching in the Hoechst assay. Alternatively, this effect is alleviated by substituting DAPI for Hoechst in this assay. Inclusion of a polyanion to the lysis buffer antagonizes the condensation and anomalous sedimentation of apoptotic DNA observed regardless of which dye is used in the assay. These studies call into question the validity of previously reported studies suggesting that polyamines and the radioprotective aminothiol, WR1065, inherently suppress the apoptotic process and underline the need to consider alternative endpoints of apoptosis such as morphology in order to assess effects on cellular apoptosis of exogenously added agents, particularly di- or polycations.     

Oligonucleosome DNA fragmentation of caspase 3 deficient MCF-7 cells in palmitate-induced apoptosis

Oligonucleosomal fragmentation of nuclear DNA is the late stage hallmark of the apoptotic process. In mammalian apoptotic cells fragmentation is catalyzed by DFF40/ CAD DNase. DFF40/CAD primary activated through site-specific proteolytic cleavage by caspase 3. The absence of caspase 3 in MCF-7 leads to lack of oligonucleosomal DNA fragmentation under numerous apoptotic stimuli. In this study it was shown that palmitate induces apoptotic changes of nuclei and oligonucleosomal DNA fragmentation in casp3 deficient MCF-7. Activation and accumulation of 40-50 kDa DFF40 like DNases in nuclei and cytoplasm of palmitate-treated MCF-7 were detected by SDS-DNA-PAGE assay. Microsomes of apoptotic MCF-7 activate 40-50 kDa nucleases when incubated with human placental chromatin and induce oligonucleosomal fragmentation of chromatin in cell free system. Both DNases activation and chromatin fragmentation are suppressed in presence of caspase 3/7 inhibitor Ac-DEVD-CHO. Microsome associated caspase 7 is suggested to play the principal role in induction of oligonucleosomal DNA fragmentation of casp3 defitient MCF-7.     

DNA fragmentation during apoptosis is caused by frequent single-strand cuts.

One of the hallmarks of apoptosis is the digestion of genomic DNA by an endonuclease, generating a ladder of small fragments of double-stranded DNA. We have examined the nature of the DNA breaks produced in mouse thymocytes triggered to undergo apoptosis by steroids or by stimulation of the T cell receptor. Whereas the typical ladder pattern of oligonucleosomal fragments was observed after agarose gel electrophoresis, numerous single-strand cuts were detected after electrophoresis under denaturing conditions. Single-strand nicks were found to be very frequent in the internucleosomal regions, but also to occur in the core particle-associated DNA. An identical pattern of single-strand nicks was obtained when chromatin DNA was exposed to the single-strand cleaving deoxyribonuclease I. The nicked DNA fragments, extracted from apoptotic thymocytes, were sensitive to the action of S1-nuclease. We propose that DNA fragmentation induced during apoptosis is not due to a double-strand cutting enzyme as previously postulated, but rather is the result of single-strand breaks. This ensures the dissociation of the DNA molecule at sites where cuts are found within close proximity.     

Prothymosin alpha fragmentation in apoptosis

We observed fragmentation of an essential proliferation-related human nuclear protein prothymosin alpha in the course of apoptosis induced by various stimuli. Prothymosin alpha cleavage occurred at the DDVD(99) motif. In vitro, prothymosin alpha could be cleaved at D(99) by caspase-3 and -7. Caspase hydrolysis disrupted the nuclear localization signal of prothymosin alpha and abrogated the ability of the truncated protein to accumulate inside the nucleus. Prothymosin alpha fragmentation may therefore be proposed to disable intranuclear proliferation-related function of prothymosin alpha in two ways: by cleaving off a short peptide containing important determinants, and by preventing active nuclear uptake of the truncated protein.     

Differentiation and apoptosis without DNA fragmentation in cultured Schwann cells derived from wallerian-degenerated nerve

The Schwann cell cables provide particularly favorable sites for the growth of regenerating axonal sprouts. However, if they remain denervated, endoneurial fibrosis takes place with the Schwann cells atrophying and total Schwann cell number gradually decrease with time. Even when regenerating axonal sprouts invade into the cables, Schwann cells do not survive for long periods if they fail to make axonal contact. These observations strongly suggest the involvement of apoptosis in peripheral nerve degeneration and regeneration. So, we investigated the behavior of Schwann cells prepared from walleriandegenerated adult rat sciatic nerve in vitro. The secondary cultured Schwann cells showed serial changes in morphology, mitotic activity and migratory activity as they do during Schwann cell cable formation in vivo. At the final stage of differentiation, the Schwann cells became rounded and detached from the flask with extensive blebbing. Electron micrographs clearly demonstrated typical cytoplasmic changes of apoptosis, but, nuclei of most of the cells retained their size and morphology with residual nucleolar structures. An agarose gel electrophoresis of DNA clearly demonstrated that there was not any DNA fragmentation up to 120 h after detachment. Results by in situ apoptosis detection assay did not show any DNA degradation despite the substantial decrease in Schwann cell number. In conclusion, during peripheral nerve degeneration and regeneration, supernumerary Schwann cells are removed by apoptosis, however, it lacks most of the nuclear events of usual apoptosis.