Attenuation of apoptotic DNA fragmentation by amiloride

Amiloride is a K⁺-sparing diuretic that effectively inhibits the Na⁺/H⁺ transporter in the plasma membrane of most mammalian cells. We have examined the effects of amiloride on the progression of apoptosis in HL-60 cells induced by camptothecin (CAM), cycloheximide (CHX), and 20 Gy gamma irradiation. Spectrofluorometric measurements on cell populations showed an inhibition of Na⁺/H⁺ transporter activity and a corresponding decrease in intracellular pH following treatment with amiloride alone, or in combination with the apoptosis-inducing agents. Flow cytometric cell cycle analysis, in combination with DNA strand break analysis, indicated that amiloride diminished endonuclease-mediated degradation of nuclear chromatin 3 h following treatment with CAM or CHX, and prevented degradation for 3 h following gamma radiation treatment. Apoptosis-associated DNA degradation was significantly greater for all three agents in the absence of amiloride. Protection from radiation-induced apoptosis was transient, since apoptotic subpopulations were observed, but still at a decreased level, 5 h following irradiation. Amiloride was as effective as zinc, an inhibitor of Ca²⁺/Mg²⁺-dependent endonucleases, in reducing or delaying the onset of endonuclease activity. Data presented show that effects of amiloride on membrane Na⁺/H⁺ transporter activity and intracellular pH can potentially affect apoptotic signaling cascades, leading to a retardation in the rate of progression to an apoptotic cell death. Results also point to the involvement of intracellular pH and Ca²⁺ in the regulation of apoptotic endonuclease activity, and the need for a functional Na⁺/H⁺ exchanger for the induction of apoptosis. J. Cell. Physiol. 175:59–67, 1998. © 1998 Wiley-Liss, Inc.     

Expression of DNase gamma during Fas-independent apoptotic DNA fragmentation in rodent hepatocytes

Endonuclease-induced DNA fragmentation is a hallmark of apoptosis. DNase gamma (DNase ) was recently identified as one of the endonucleases responsible for apoptotic DNA fragmentation. In this study, immunohistochemistry for DNase was performed on paraffin sections of rodent liver in well-defined models of hepatocyte apoptosis induced by Fas antibody (Fas) or cycloheximide (CHX), and necrosis induced by lipopolysaccharide (LPS) or carbon tetrachloride (CCl₄). DNase immunoreactivity was compared with TdT-mediated dUTP nick-end labeling (TUNEL) reactivity. Our results showed TUNEL reactivity in both apoptotic and necrotic hepatocytes. DNase immunoreactivity was not detected during LPS-induced or CCl₄-induced hepatocyte necrosis. In contrast, it was evident during CHX-induced, but not Fas-induced, apoptotic DNA fragmentation. These findings suggest that DNase plays an important role in Fas-independent apoptotic DNA fragmentation in hepatocytes.     

Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation.

Caspase-3 initiates apoptotic DNA fragmentation by proteolytically inactivating DFF45 (DNA fragmentation factor-45)/ICAD (inhibitor of caspase-activated DNase), which releases active DFF40/CAD (caspase-activated DNase), the inhibitor's associated endonuclease. Here, we examined whether other apoptotic proteinases initiated DNA fragmentation via DFF45/ICAD inactivation. In a cell-free assay, caspases-3, -6, -7, -8, and granzyme B initiated benzoyloxycarbonyl-Asp-Glu-Val-Asp (DEVD) cleaving caspase activity, DFF45/ICAD inactivation, and DNA fragmentation, but calpain and cathepsin D failed to initiate these events. Strikingly, only the DEVD cleaving caspases, caspase-3 and caspase-7, inactivated DFF45/ICAD and promoted DNA fragmentation in an in vitro DFF40/CAD assay, suggesting that granzyme B, caspase-6, and caspase-8 promote DFF45/ICAD inactivation and DNA fragmentation indirectly by activating caspase-3 and/or caspase-7. In vitro, however, caspase-3 inactivated DFF45/ICAD and promoted DNA fragmentation more effectively than caspase-7 and endogenous levels of caspase-7 failed to inactivate DFF45/ICAD in caspase-3 null MCF7 cells and extracts. Together, these data suggest that caspase-3 is the primary inactivator of DFF45/ICAD and therefore the primary activator of apoptotic DNA fragmentation.     

DNA-PK phosphorylates histone H2AX during apoptotic DNA fragmentation in mammalian cells

The phosphorylation of histone H2AX at serine 139 is one of the earliest responses of mammalian cells to ionizing radiation-induced DNA breaks. DNA breaks are also generated during the terminal stages of apoptosis when chromosomal DNA is cleaved into oligonucleosomal pieces. Apoptotic DNA fragmentation and the consequent chromatin condensation are important for efficient clearing of genomic DNA and nucleosomes and for protecting the organism from auto-immmunization and oncogenic transformation. In this study, we demonstrate that H2AX is phosphorylated during apoptotic DNA fragmentation in mouse, Chinese hamster ovary, and human cells. We have previously shown that ataxia telangiectasia mutated kinase (ATM) is primarily responsible for H2AX phosphorylation in murine cells in response to ionizing radiation. Interestingly, we find here that DNA-dependent protein kinase (DNA-PK) is solely responsible for H2AX phosphorylation during apoptosis while ATM is dispensable for the process. Moreover, the kinase activity of DNA-PKcs (catalytic subunit of DNA-PK) is specifically required for the induction of gammaH2AX. We further show that DNA-PKcs is robustly activated in apoptotic cells, as evidenced by autophosphorylation at serine 2056, before it is inactivated by cleavage. In contrast, ATM is degraded well before DNA fragmentation and gammaH2AX induction resulting in the predominance of DNA-PK during the later stages of apoptosis. Finally, we show that DNA-PKcs autophosphorylation and gammaH2AX induction occur only in apoptotic nuclei with characteristic chromatin condensation but not in non-apoptotic nuclei from the same culture establishing the most direct link between DNA fragmentation, DNA-PKcs activation, and H2AX phosphorylation. It is well established that DNA-PK is inactivated by cleavage late in apoptosis in order to forestall DNA repair. Our results demonstrate, for the first time, that DNA-PK is actually activated in late apoptotic cells and is able to initiate an early step in the DNA-damage response, namely H2AX phosphorylation, before it is inactivated by proteolysis.     

Crucial role of apopain in the peroxynitrite-induced apoptotic DNA fragmentation

Peroxynitrite, a cytotoxic oxidant formed in the reaction of superoxide and nitric oxide is known to cause programmed cell death. However, the mechanisms of peroxynitrite-induced apoptosis are poorly defined. The present study was designed to characterize the molecular mechanisms by which peroxynitrite induces apoptosis in HL-60 cells, with special emphasis on the role of caspases. Peroxynitrite induced the activation of apopain/caspase-3, but not ICE/caspase-1 as measured by the cleavage of fluorogenic peptides. Considering the short half-life of peroxynitrite and the kinetics of caspase-3 activation (starting 3–4 h after peroxynitrite treatment), the enzyme is not likely to become activated directly by the oxidant. Caspase-3 activation proved to be essential for DNA fragmentation, because pretreatment of the cells with the specific tetrapeptide inhibitor DEVD-fmk completely blocked peroxynitrite-induced DNA fragmentation. Peroxynitrite-induced cytotoxicity was also significantly altered by the inhibition of caspase-3, whereas phosphatidylserine exposure was unaffected by DEVD-fmk treatment. Because many of the effects of peroxynitrite are mediated by poly(ADP-ribose) synthetase (PARS) activation, we have also investigated the effect of PARS-inhibition on peroxynitrite-induced apoptosis. We have found that PARS-inhibition modulates peroxynitrite-induced apoptotic DNA fragmentation in the HL-60 cells. The effect of the PARS inhibitors, 3-aminobenzamide and 5-iodo-6-amino-1,2-benzopyrone were dependent on the concentration of peroxynitrite used. While PARS-inhibition resulted in increased DNA-fragmentation at low doses (15 μM) of peroxynitrite, a decreased DNA-fragmentation was found at high doses (60 μM) of peroxynitrite. PARS inhibition negatively affected viability as determined by flow cytometry. These data demonstrate the crucial role of caspase-3 in mediating apoptotic DNA fragmentation in HL-60 cells exposed to peroxynitrite.     

Evolutionary stability of the R1 retrotransposable element in the genus Drosophila

R1 is a non-long terminal repeat (non-LTR) retrotransposable element that inserts into a specific sequence of insect 28S ribosomal RNA genes. We have previously shown that this element has been maintained through vertical transmission in the melanogaster species subgroup of Drosophila. To address whether R1 elements have been vertically transmitted for longer periods of evolutionary time, the analysis has been extended to 11 other species from four species groups of the genus Drosophila (melanogaster, obscura, testecea, and repleta). All sequenced elements appeared functional on the basis of the preservation of their open-reading frames and consistently higher rate of substitution at synonymous sites relative to replacement sites. The phylogenetic relationships of the R1 elements from all species analyzed were congruent with the species phylogenies, suggesting that the R1 elements have been vertically transmitted since the inception of the Drosophila genus, an estimated 50-70 Mya. The stable maintenance of R1 through the germ line appears to be the major mechanism for the widespread distribution of these elements in Drosophila. In two species, D. neotestecea of the testecea group and D. takahashii of the melanogaster group, a second family of R1 elements was also present that differed in sequence by 46% and 31%, respectively, from the family that was congruent with the species phylogeny. These second families may represent occasional horizontal transfers or, alternatively, they could reflect the ability of R1 elements to diverge into new families within a species and evolve independently.      

H2AX is a target of the JNK signaling pathway that is required for apoptotic DNA fragmentation

The mechanism of apoptotic signaling by JNK is incompletely understood. In the July 7 issue of Molecular Cell, Lu et al. (2006) report that JNK phosphorylation of H2AX at a noncanonical site is required for caspase-induced DNA fragmentation.     

Molecular basis of apoptotic DNA fragmentation by DFF40

Although the functions of CIDE domain-containing proteins, including DFF40, DFF45, CIDE-A, CIDE-B, and FSP27, in apoptotic DNA fragmentation and lipid homeostasis have been studied extensively in mammals, the functions of four CIDE domain-containing proteins identified in the fly, namely DREP1, 2, 3, and 4, have not been explored much. Recent structural study of DREP4, a fly orthologue of mammalian DFF40 (an endonuclease involved in apoptotic DNA fragmentation), showed that the CIDE domain of DREP4 (and DFF40) forms filament-like assembly, which is critical for the corresponding function. The current study aimed to investigate the mechanism of filament formation of DREP4 CIDE and to characterize the same. DREP4 CIDE was shown to specifically bind to histones H1 and H2, an event important for the nuclease activity of DREP4. Based on the current experimental results, we proposed the mechanism underlying the process of apoptotic DNA fragmentation.Subject terms: Proteins, Enzymes, X-ray crystallography     

Suppression of apoptotic DNA fragmentation in herpes simplex virus type 1-infected cells.

HEp-2 cells underwent apoptosis when the cells were incubated in medium containing sorbitol. Infection with herpes simplex virus type 1 (HSV-1) prior to the sorbitol treatment suppressed this apoptosis completely, indicating that HSV-1 carries an antiapoptosis gene. In addition, HSV-1 multiplication was restricted in these apoptotic HEp-2 cells.     

Oligomerization state of the DNA fragmentation factor in normal and apoptotic cells

The caspase-activated DNase (CAD) is the primary nuclease responsible for oligonucleosomal DNA fragmentation during apoptosis. The DNA fragmentation factor (DFF) is composed of the 40-kDa CAD (DFF40) in complex with its cognate 45-kDa inhibitor (inhibitor of CAD: ICAD or DFF45). The association of ICAD with CAD not only inhibits the DNase activity but is also essential for the co-translational folding of CAD. Activation of CAD requires caspase-3-dependent proteolysis of ICAD. The tertiary structures of neither the inactive nor the activated DFF have been conclusively established. Whereas the inactive DFF is thought to consist of the CAD/ICAD heterodimer, activated CAD has been isolated as a large (>MDa) multimer, as well as a monomer. To establish the subunit stoichiometry of DFF and some of its structural determinants in normal and apoptotic cells, we utilized size-exclusion chromatography in combination with co-immunoprecipitation and mutagenesis techniques. Both endogenous and heterologously expressed DFF have an apparent molecular mass of 160-190 kDa and contain 2 CAD and 2 ICAD molecules (CAD/ICAD)2 in HeLa cells. Although the N-terminal (CIDE-N) domain of CAD is not required for ICAD binding, it is necessary but not sufficient for ICAD homodimerization in the DFF. In contrast, the CIDE-N domain of ICAD is required for CAD/ICAD association. Using bioluminescence resonance energy transfer (BRET), dimerization of ICAD in DFF was confirmed in live cells. In apoptotic cells, endogenous and exogenous CAD forms limited oligomers, representing the active nuclease. A model is proposed for the rearrangement of the DFF subunit stoichiometry in cells undergoing programmed cell death.     

Distribution of the retrotransposable element 412 in Drosophila species

Copy numbers of sequences homologous to the Drosophila melanogaster retrotransposable element 412, their distribution between the chromosome arms and the chromocenter, and whether they contain full- size copies were analyzed for 55 species of the Drosophila genus. Element 412 insertion sites were detected on the chromosome arms of D. melanogaster, Drosophila simulans, and a few species of the obscura group, but the chromocenter was labeled in almost all species. The presence of element 412 sequences in the majority of species shows that this element has a long evolutionary history in Drosophilidae, although it may have recently invaded the chromosomes in some species, such as D. simulans. Differences in copy number between species may be due to population size or specific endogenous or environmental factors and may follow the worldwide invasion of the species. Putative full-length copies were detected in the chromocenters of some species with no copies on the chromosome arms, suggesting that the chromocenter may be a shelter for such copies and not only for deleted ones.      

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.     

Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation

A method combining the advantages of electrophoretic DNA fractionation and autoradiography is described for the qualitative and quantitative analysis of internucleosomal DNA fragmentation that occurs during apoptosis, or “programmed cell death”. This procedure utilizes terminal transferase enzyme to uniformly add one molecule of [α ³²P]-to the 3′-of DNA fragments. Following gel electrophoresis and autoradiographic analysis, the total amount of radiolabel incorporated into the low molecular weight DNA fraction can be quantitated and used to estimate the degree of apoptotic DNA fragmentation in any given sample. This method requires as little as 15 ng of total cellular DNA and increases the sensitivity of apoptotic DNA detection by at least 100-fold over the widely used ethidium bromide staining method. The procedure should prove valuable for the analysis of apoptosis in minute quantities of tissues and cultured cells. © 1993 Wiley-Liss, Inc.     

DNA fragmentation is regulated by ethylene during carpel senescence in Pisum sativum

Treatment of the pea carpel with the ethylene action inhibitors, silver thiosulphate and 2,5-norbornadiene, retarded its senescence and extended the growth time in which the unpollinated carpel was able to respond to gibberellins. Cells of the senescent carpel have characteristics of apoptotic cells, such as nuclei condensation and internucleosomal DNA fragmentation. Both DNA laddering and condensed nuclei can be prevented by the use of the ethylene action inhibitors, while ethylene treatment accelerates DNA fragmentation. It is postulated that ethylene produced by the flower after anthesis determines the fate of the ovary/ovule; in other words, if the ovary receives no additional stimulus (pollination or gibberellin treatment), it undergoes a senescence process, with some cells showing characteristics similar to those of programmed cell death already identified in animal systems.     

Dual apoptotic DNA fragmentation system in the fly: Drep2 is a novel nuclease of which activity is inhibited by Drep3

DNA fragmentation is the hallmark of apoptotic cells and mainly mediated by the DNA fragmentation factor DFF40(CAD)/DFF45(ICAD). DFF40 is a novel nuclease, whereas DFF45 is an inhibitor that can suppress the nuclease activity. Apoptotic DNA fragmentation in the fly is controlled by four DFF-related proteins, known as Drep1, 2, 3 and 4. However, the functions of Drep2 and Drep3 are totally unknown. Here, we found that Drep2 is a novel nuclease whose activity is inhibited by Drep3 through a tight interaction with the CIDE domain. Our results suggest that the fly has dual apoptotic DNA fragmentation systems: Drep1: Drep4 and Drep2: Drep3 complexes.Structured summary of protein interactionsDrep2 CIDE and Drep-3 CIDE bind by blue native page (View interaction)Drep2 CIDE and Drep-3 CIDE bind by molecular sieving (View interaction)     

The apoptotic suppressor P35 is required early during baculovirus replication and is targeted to the cytosol of infected cells.

The p35 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) is required to block virus-induced apoptosis. The trans-dominant activity of p35 suppresses premature cell death and facilitates AcMNPV replication in a cell line- and host-specific manner. To characterize the p35 gene product (P35), a specific polyclonal antiserum was raised. As revealed by immunoblot analyses of wild-type AcMNPV-infected cells, P35 appeared early (8 to 12 h) and accumulated through the late stages of infection (24 to 36 h). Biochemical fractionation of cells both early and late in infection and indirect immunochemical staining demonstrated that P35 localized predominantly to the cytosol (150,000 x g supernatant); comparatively minor quantities of P35 were associated with intracellular membranes. The cytoplasmic localization of P35 was independent of virus infection. The functional significance of the early and late synthesis of P35 was examined by constructing recombinant viruses in which the timing and level of p35 expression were altered. Delaying P35 synthesis by placing p35 under exclusive control of a strong, very late promoter failed to suppress intracellular DNA fragmentation and apoptotic blebbing in most cells. Thus, earlier expression of p35 was required to block virus-induced apoptosis. Site-specific mutagenesis of the p35 promoter demonstrated that low levels of P35 were sufficient to block apoptosis, whereas higher levels were required to maintain wild-type virus gene expression. Consistent with an early role in infection, P35 was also detected in the budded form of AcMNPV. Because of the lack of sequence similarity and its cytosolic targeting, P35 may function in a manner that is mechanistically distinct from other apoptotic regulators, including Bcl-2 and the adenovirus E1B 19-kDa protein.