Functional characterization of DNase X, a novel endonuclease expressed in muscle cells

The activation of endonucleases resulting in the degradation of genomic DNA is one of the most characteristic changes in apoptosis. Here, we report the characterization of a novel endonuclease, termed DNase X due to its X-chromosomal localization. The active nuclease is a 35 kDa protein with 39% identity to DNase I. When incubated with isolated nuclei, recombinant DNase X was capable of triggering DNA degradation at internucleosomal sites. Similarly to DNase I, the nuclease activity of DNase X was dependent on Ca(2+) and Mg(2+) and inhibited by Zn(2+) ions or chelators of bivalent cations. Overexpression of DNase X caused internucleosomal DNA degradation and induction of cell death associated with increased caspase activation. Despite the presence of two potential caspase cleavage sites, DNase X was processed neither in vitro nor in vivo by different caspases. Interestingly, after initiation of apoptosis DNase X was translocated from the cytoplasm to the nuclear compartment and aggregated as a detergent-insoluble complex. Abundant expression of DNase X mRNA was detected in heart and skeletal muscle cells, suggesting that DNase X may be involved in apoptotic or other biological events in muscle tissues.     

Granzyme A activates an endoplasmic reticulum-associated caspase-independent nuclease to induce single-stranded DNA nicks

The cytotoxic T lymphocyte protease granzyme A (GzmA) initiates a novel caspase-independent cell death pathway characterized by single-stranded DNA nicking. The previously identified GzmA substrate SET is in a multimeric 270-420-kDa endoplasmic reticulum-associated complex that also contains the tumor suppressor protein pp32. GzmA cleaved the nucleosome assembly protein SET after Lys(176) and disrupted its nucleosome assembly activity. The purified SET complex required only GzmA to reconstitute single-stranded DNA nicking in isolated nuclei. DNA nicking occurred independently of caspase activation. The SET complex contains a 25-kDa Mg(2+)-dependent nuclease that degrades calf thymus DNA and plasmid DNA. Thus, GzmA activates a DNase (GzmA-activated DNase) within the SET complex to produce a novel form of DNA damage during cytotoxic T lymphocyte-mediated death.     

Multiparameter kinetic analysis of killer cell initiation by using immune RNA.

By treating nonsensitized C57BL/6J spleen derived lymphocytes with EL-4 tumor cell directed xenogeneic extracted RNA we were able to monitor early changes in cellular DNA content by flow cytometric (FCM) analysis and 3H-thymidine uptake. These kinetic parameters were correlated with cell mediated cytotoxicity which appeared as early as 8 hr after activation as measured by release of chromium-51 from labeled EL-4 target cells. Flow cytometric analysis and 3H-thymidine uptake data shown peak S phase activity at 72 hr. Maximum cytotoxicity was observed at 48 hr. Cell cycle kinetic parameters were correlated with the appearance of cell mediated cytotoxicity.     

Deoxyribonucleoside-triphosphate imbalance death: deoxyadenosine-induced dNTP imbalance and DNA double strand breaks in mouse FM3A cells and the mechanism of cell death

The mechanism of deoxyadenosine (dAdo)-induced death of mouse mammary tumor FM3A cells was studied. When the cells were exposed to dAdo at 3 mM, an imbalance of intracellular dNTP pool resulted: dATP concentration was elevated 100-fold and the dGTP concentration was reduced to less than 1% of the control values. The imbalance was followed by breakage of mature DNA. DNA double strand breaks were observed in the dAdo treated cells 12 hr after the administration. We assume that the double strand breaks play an important role in the process of the dAdo-mediated cell death, and that the intracellular dNTP imbalance is the trigger of these events.     

Growth Kinetics of Yaba Tumor Poxvirus After In Vitro Adaptation to Cercopithecus Kidney Cells

Yaba tumor poxvirus has been adapted to continuous in vitro cultivation in monolayers of cercopithecus kidney cells. At 35 C, the minimum replicative cycle, after synchronous infection of CV-1 cells with multiplicity of infection of 135 focusforming units per cell, was 35 hr; however, maximum virus yields were not obtained until 75 hr postinfection (PI). Cytoplasmic incorporation of (3)H-thymidine [viral deoxyribonucleic acid (DNA) synthesis] was detected 3 hr PI and was preceded by synthesis of nonstructural associated antigens (YS). Synthesis of YS antigens was not inhibited by the DNA inhibitor, arabinofuranosyl cytosine (ARA-C). Synthesis of at least two virion structural antigens, although not detected by immunofluorescence until 2 hr after the onset of DNA synthesis, occurred in the presence of ARA-C, indicating potential translation of these structural antigens from parental DNA. The first progeny DNA was completed by 20 hr PI but was not detected in infectious form until 35 hr PI. The maximum rate of progeny DNA completion occurred between 20 and 30 hr PI. DNA synthesis continued 45 to 50 hr PI. The adapted virus retained its oncogenicity and, like the wild type, replicated better at 35 C than at 37 C. A synthetic step associated with viral DNA synthesis appears to be temperature-sensitive.     

Cytoplasmic DNA in cancer cells: Several pathways that potentially limit DNase2 and TREX1 activities

The presence of DNA in the cytoplasm of tumor cells induces the dendritic cell to produce type-I IFNs. Classically, the presence of foreign DNA in host cells' cytoplasm during viral infection elicits cGAS-STING mediated type-I IFN signaling and cytokine production. It is likely that cytosolic DNA leads to senescence and immune surveillance in transformed cells during the early stages of carcinogenesis. However, multiple factors, such as loss of cell-cycle checkpoint, mitochondrial damage and chromosomal instability, can lead to persistent accumulation of DNA in the cytoplasm of metastatic tumor cells. That is why aberrant activation of the type I IFN pathway is frequently associated with highly aggressive tumors. Intriguingly, two powerful intracellular deoxyribonucleases, DNase2 and TREX1, can target the cytoplasmic DNA for degradation. Yet the tumor cells consistently accumulate cytoplasmic DNA. This review highlights recent work connecting the lack of DNase2 and TREX1 function to innate immune signaling. It also summarizes the possible mechanisms that limit the activity of DNase2 and TREX1 in tumor cells and contributes to chronic inflammation.     

Study of cell death in Friend leukaemia.

Cell death of splenic Friend leukaemic cells has been studied in vivo, using 125I-UdR and 3H-TdR pulse labelling. The evolution of the splenic specific activity has been measured by autoradiography and external counting during 40 hr after injection of the labelled precursor. These two techniques show the existence of a large reutilization of 3H-TdR (50%), which is measurable as soon as 7 hr after the injection. The DNA turnover rate is rapid, 83-8% of the splenic cellular DNA being renewed per day. Those results confirm that most of the cells produced in the Friend leukaemic spleen are rapidly lost; they also demonstrate that this cell loss is mainly due to a massive death, which occurs in proerythroblastic and erythroblastic compartments after one or two cell divisions. Friend leukaemic cells, which are characterized by a limited capacity of proliferation and a short lifespan, do not appear to be malignant.     

Assessment of cell viability by flow cytometric analysis using DNase exclusion

A new method was developed for selective measurement of DNA distributions in viable cell populations. The method is based on the fact that non-viable cells lose membrane integrity and treatment of such cells with DNase should remove their DNA. The DNase-treated cells were stained with DNA fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) in the presence of Triton X-100. DNA distribution was measured by flow cytometry prior to and after treatment with DNase. Percentage of cells stained after DNase treatment was considered as an index of cell viability. Optimal conditions for DNase treatment and application of DNase exclusion test for the analysis of spontaneous cell death, selective death of cells arrested in S/G2 phases, instant cell disintegration induced by cytotoxic compounds and cell death induced by hyperthermia are described.     

Influence of one bout of vigorous exercise on ascorbic acid in plasma and oxidative damage to DNA in blood cells and muscle in untrained rats

We investigated the influence of a single exhaustive bout of downhill running on oxidative damage to DNA and changes of antioxidant vitamin concentrations in rats. Plasma vitamin E levels were unchanged up to 48 hr postexercise. However, plasma ascorbic acid (AA) levels increased after the exercise, then decreased thereafter. This increase corresponded to a marked decrease in AA concentration in the adrenal glands. The activity of hepatic l-gulono-gamma-lactone oxidase, which catalyzes AA synthesis, was unaltered after the exercise. The weight of the adrenal glands was significantly increased 24 hr postexercise. These results indicate that the change in the plasma AA concentration after vigorous exercise was due mainly to the release of AA from the adrenal glands. The plasma creatine phosphokinase (CPK) activity and white blood cell (WBC) count increased 3 to 6 hr postexercise. Over this same period, a marker of oxidative DNA damage, 8-hydroxydeoxyguanosine in DNA, increased in the WBC, but not in the foreleg muscle. Lipid peroxide and vitamin E levels were also unchanged in the foreleg muscle. There was a positive correlation between CPK activity in the plasma and DNA damage in the WBC, suggesting that the DNA damage in the WBC was closely related with muscle damage due to exercise.     

Caspase-2 activation is redundant during seizure-induced neuronal death

Seizure-induced neuronal death may be under the control of the caspase family of cell death proteases. We examined the role of caspase-2 in a model of focally evoked limbic seizures with continuous EEG recording. Seizures were elicited by microinjection of kainic acid into the amygdala of the rat and terminated after 40 min by diazepam. Caspase-2 was constitutively present in brain, mostly within neurons, and was detected in both cytoplasm and nucleus. Cleaved caspase-2 (12 kDa) was detected immediately following seizure termination within injured ipsilateral hippocampus, contiguous with increased Val-Asp-Val-Ala-Asp (VDVADase) activity, a putative measure of activated caspase-2. Expression of receptor interacting protein (RIP)-associated Ich-1-homologous protein with death domain (RAIDD) was increased following seizures, whereas expression of RIP and tumor necrosis factor receptor associated protein with death domain (TRADD), other components thought to be linked to the caspase-2 activation and signaling mechanism, were unchanged. Intracerebroventricular administration of z-VDVAD-fluoromethyl ketone blocked seizure-induced caspase-2 activity but did not alter caspase-8 activity and failed to affect DNA fragmentation or neuronal death. These data support activation of caspase-2 following seizures but suggest that parallel caspase pathways may circumvent deficits in caspase-2 function to complete the cell death process.     

Direct involvement of p53 in programmed cell death of oligodendrocytes.

A covalent dimer of interleukin (IL)-2, produced in vitro by the action of a nerve-derived transglutaminase, has been shown previously to be cytotoxic to mature rat brain oligodendrocytes. Here we report that this cytotoxic effect operates via programmed cell death (apoptosis) and that the p53 tumor suppressor gene is involved directly in the process. The apoptotic death of mature rat brain oligodendrocytes in culture following treatment with dimeric IL-2 was demonstrated by chromatin condensation and internucleosomal DNA fragmentation. The peak of apoptosis was observed 16-24 h after treatment, while the commitment to death was already observed after 3-4 h. An involvement of p53 in this process was indicated by the shift in location of constitutively expressed endogenous p53 from the cytoplasm to the nucleus, as early as 15 min after exposure to dimeric IL-2. Moreover, infection with a recombinant retrovirus encoding a C-terminal p53 miniprotein, shown previously to act as a dominant negative inhibitor of endogenous wild-type p53 activity, protected these cells from apoptosis.     

Plasma membrane biogenesis in the avian salt gland: a biochemical and quantitative electron microscopic autoradiographic study

The avian salt gland provides an ideal system for the study of plasma membrane (PM) biogenesis. Feeding ducklings 1% sodium chloride (salt stress) induces the secretory cells of the gland to synthesize large amounts of PM, which forms an extensive basolateral PM domain after 7-9 days of treatment. In the present study, the initial biosynthetic events following salt stress were investigated. In vivo studies using 3H-uridine indicated that increased rates of RNA synthesis could be detected by 2 hr after the beginning of salt stress and continued through at least 12 hr. Under in vitro conditions, increased rates of protein and glycoprotein synthesis (as monitored by 3H-leucine and 3H-fucose incorporation, respectively) were also detected after 2 hr and continued through 7-9 days. Increased levels of Na,K-ATPase, a specific secretory cell PM marker, were detected after 8 hr of treatment as monitored by specific activity and 3H-ouabain binding. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis coupled with fluorography indicated that both 3H-leucine and 3H-fucose were incorporated into partially purified preparations of Na,K-ATPase isolated after 12 hr. Light microscopic autoradiographic analysis of pulse-chase experiments indicated that in secretory cells of 12-hr salt-stressed glands, 3H-leucine- and 3H-fucose-labelled products reached the cell periphery by 1-2 hr after the initial pulse. The incorporation of both tritiated precursors was predominantly associated with the secretory cells. Quantitative electron microscopic autoradiography indicated that 3H-leucine is initially taken up by elements of the rough endoplasmic reticulum (RER) and cytoplasm (5 min postpulse), subsequently transported to and concentrated within components of the Golgi apparatus (10 min of chase), and ultimately incorporated into all domains of the plasma membrane of secretory cells by 1-2 hr of chase. The data is consistent with a flow of newly synthesized membrane components from RER to Golgi to plasma membrane and is analogous to the pattern previously found for the synthesis and processing of PM proteins in a wide variety of cell types.     

Inhibition of growth of ASL-1w murine leukemia cells by anti-thymus leukemia antigen (TL) serum in the absence of complement.

Previous studies have shown that the cell surface expression of thymus leukemia antigen (TL) on ASL-1w leukemia cells varies with the progression of the cells through the growth cycle. Expression of TL is maximal in S phase, and its quantitative expression varies directly with DNA synthesis. In the present study, the effect of anti-TL serum on the growth of ASL-1w cells was examined. The antiserum, tested in the absence of complement, affected the growth of these cells in biphasic manner. When the antiserum concentration was 0.1% or greater, there was a rapid decline in the rate of DNA synthesis, and after 5 to 7 hr, cell death. When the antiserum concentration was 0.067% or less, the decline in the rate of synthesis of DNA did not become apparent until 5 to 6 hr after treatment. Under these conditions, there was approximately a 20% increase in cell number after 24 hr of culture. The hypothesis that treatment of ASL-1w cells with the lesser concentration of anti-TL serum blocks the cells in G2 phase of the cell cycle is discussed.     

Prostaglandin F2alpha-mediated activation of apoptotic signaling cascades in the corpus luteum during apoptosis: involvement of caspase-activated DNase

Prostaglandin F(2alpha) (PGF(2alpha)) acting via a G protein-coupled receptor has been shown to induce apoptosis in the corpus luteum of many species. Studies were carried out to characterize changes in the apoptotic signaling cascade(s) culminating in luteal tissue apoptosis during PGF(2alpha)-induced luteolysis in the bovine species in which initiation of apoptosis was demonstrable at 18 h after exogenous PGF(2alpha) treatment. An analysis of intrinsic arm of apoptotic signaling cascade elements revealed that PGF(2alpha) injection triggered increased ratio of Bax to Bcl-2 in the luteal tissue as early as 4 h posttreatment that remained elevated until 18 h. This increase was associated with the elevation in the active caspase-9 and -3 protein levels and activity (p < 0.05) at 4-12 h, but a spurt in the activity was seen only at 18 h posttreatment that could not be accounted for by the changes in the Bax/Bcl-2 ratio or changes in translocation of Bax to mitochondria. Examination of luteal tissue for FasL/Fas death receptor cascade revealed increased expression of FasL and Fas at 18 h accompanied by a significant (p < 0.05) induction in the caspase-8 activity and truncated Bid levels. Furthermore, intrabursal administration of specific caspase inhibitors, downstream to the extrinsic and intrinsic apoptotic signaling cascades, in a pseudopregnant rat model revealed a greater importance of extrinsic apoptotic signaling cascade in mediating luteal tissue apoptosis during PGF(2alpha) treatment. The DNase responsible for PGF(2alpha)-induced apoptotic DNA fragmentation was found to be Ca(2+)/Mg(2+)-dependent, temperature-sensitive DNase, and optimally active at neutral pH conditions. This putative DNase was inhibited by the recombinant inhibitor of caspase-activated DNase, and immunodepletion of caspase-activated DNase from luteal lysates abolished the observed DNA fragmentation activity. Together, these data demonstrate for the first time temporal and spatial changes in the apoptotic signaling cascades during PGF(2alpha)-in-duced apoptosis in the corpus luteum.     

Inhibition of Deoxyribonuclease Activity in Synchronized Tetrahymena

SYNOPSIS. Deoxyribonuclease (DNase) activity in crude homogenates of synchronized cultures of Tetrahymena pyriformis GL was inhibited by added acidic polymers (which inactivated the enzyme) and polyamines (which blocked enzyme sites on the substrate). The inhibition appeared to be a nonspecific reaction between basic groups on the enzyme and acidic groups on the substrate. The relation between variation in DNase activity and amount of DNA was analyzed by means of puromycin and mitomycin C. DNase activity was lower after addition of each antibiotic, without significant change in amount of DNA. The results suggest that induction of DNase synthesis is governed by the amount of DNA.     

Temporal variation in the effects of warfarin on the vitamin K cycle

In this in vivo study, the time-dependent effect of oral sodium warfarin was studied in male rats synchronized under a 12-hr light-dark cycle (light 0600-1800). Groups of 5 animals received an oral dose of 500 micrograms/kg of warfarin or saline at 0600 or 1800 and 1 mg/kg of vitamin K 8 hr later and the rats were sacrificed 240 min after vitamin K administration. The activities of the vitamin K reductase and vitamin K epoxide reductase were measured indirectly by determining the content of vitamin K1 and vitamin K epoxide reductase in the plasma and liver. The data obtained in control rats indicated that vitamin K and vitamin K 2,3 epoxide concentrations in plasma and liver were higher (P less than 0.05) at 1800 than at 0600. Warfarin had a greater (P less than 0.05) inhibitory effect on the vitamin K and vitamin K-epoxide reductases at 0600 compared to 1800; plasma levels of S- and R-warfarin did not vary with time of administration. The findings suggest that the activity of both reductases under control conditions, and the warfarin-induced inhibition of these enzymes varied depending on the time of drug administration.     

Spontaneous release of nucleosome cores from embryoblast chromatin

During in vitro incubation of the rabbit blastocyst, chromatin fragments of the nucleosome series are released spontaneously by cells of the pluripotent embryoblast but not by the developmentally restricted trophoblast. The DNA of these fragments is double stranded and linear, and represents total genomic DNA. The fragments are stable for at least 24 hr and are initially found in the cytoplasm of intact cells. The kinetics of radiolabeling of the DNA component of these fragments suggest that they originate from actively replicating cells, possibly only at the time of mitosis. DNase II-like activity, one of the endonucleases known preferentially to attack spacer DNA between nucleosome cores, is readily detectable in the blastocyst, although the evidence for its involvement in the observed chromatin fragmentation remains circumstantial. These nonrandom chromatin breaks, presumably representing an early event in cell death, occur more frequently under suboptimal conditions of incubation but are still detectable in embryos developing normally. There is no evidence for lysosomal activation in these dying cells. These findings, together with previous reports of identical chromatin fragmentation in other cell systems, suggest that this form of cell death in the embryoblast may be only one possible result of an active process, and that the mechanisms responsible for generating double-strand breaks in internucleosomal DNA may be of more biological significance than DNA degradation alone.     

Activation of intrinsic and extrinsic proapoptotic signaling pathways in interleukin-18-mediated human cardiac endothelial cell death

Endothelial cells are the primary targets of circulating immune and inflammatory mediators. We hypothesize that interleukin-18, a proinflammatory cytokine, induces endothelial cell apoptosis. Human cardiac microvascular endothelial cells (HCMEC) were treated with interleukin (IL) 18. mRNA expression was analyzed by ribonuclease protection assay, protein levels by immunoblotting, and cell death by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis. We also investigated the signal transduction pathways involved in IL-18-mediated cell death. Treatment of HCMEC with IL-18 increases 1) NF-kappaB DNA binding activity; 2) induces kappaB-driven luciferase activity; 3) induces IL-1beta and TNF-alpha expression via NF-kappaB activation; 4) inhibits antiapoptotic Bcl-2 and Bcl-X(L); 5) up-regulates proapoptotic Fas, Fas-L, and Bcl-X(S) expression; 6) induces fas and Fas-L promoter activities via NF-kappaB activation; 7) activates caspases-8, -3, -9, and BID; 8) induces cytochrome c release into the cytoplasm; 9) inhibits FLIP; and 10) induces HCME cell death by apoptosis as seen by increased annexin V staining and increased levels of mono- and oligonucleosomal fragmented DNA. Whereas overexpression of Bcl-2 significantly attenuated IL-18-induced endothelial cell apoptosis, Bcl-2/Bcl-X(L) chimeric phosphorothioated 2'-MOE-modified antisense oligonucleotides potentiated the proapoptotic effects of IL-18. Furthermore, caspase-8, IKK-alpha, and NF-kappaB p65 knockdown or dominant negative IkappaB-alpha and dominant negative IkappaB-beta or kinase dead IKK-beta significantly attenuated IL-18-induced HCME cell death. Effects of IL-18 on cell death are direct and are not mediated by intermediaries such as IL-1beta, tumor necrosis factor-alpha, or interferon-gamma. Taken together, our results indicate that IL-18 activates both intrinsic and extrinsic proapoptotic signaling pathways, induces endothelial cell death, and thereby may play a role in myocardial inflammation and injury.     

Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death).

Cell death by apoptosis occurs in a wide range of physiological events including repertoire selection of lymphocytes and during immune responses in vivo. A hallmark of apoptosis is the internucleosomal DNA degradation for which a Ca2+,Mg(2+)-dependent endonuclease has been postulated. This nuclease activity was extracted from both rat thymocyte and lymph node cell nuclei. When incubated with nuclei harbouring only limited amounts of endogenous nuclease activity, the ladder pattern of DNA fragments characteristic of apoptosis was induced. This extractable nucleolytic activity was immunoprecipitated with antibodies specific for rat deoxyribonuclease I (DNase I) and was inhibited by actin in complex with gelsolin segment 1, strongly pointing to the presence of a DNase I-type enzyme in the nuclear extracts. COS cells transiently transfected with the cDNA of rat parotid DNase I expressed the enzyme, and their nuclei were able to degrade their DNA into oligosome-sized fragments. PCR analysis of mRNA isolated from thymus, lymph node cells and kidney yielded a product identical in size to that from rat parotid DNase I. Immunohistochemical staining with antibodies to rat DNase I confirmed the presence of DNase I antigen in thymocytes and lymph node cells. The tissue distribution of DNase I is thus extended to tissues with no digestive function and to cells which are known to be susceptible to apoptosis. We propose that during apoptosis, an endonuclease indistinguishable from DNase I gains access to the nucleus due to the breakdown of the ER and the nuclear membrane.     

Cytoplasmic poly(ADP-ribose) polymerase during the HeLa cell cycle.

Poly(ADP-ribose) polymerase, an enzyme that has reportedly been confined to the nucleus of eukaryotic cells, has been found in the cytoplasm of HeLa cells. The enzyme activity is stimulated more than 30-fold by the addition of both DNA and histones. These two macromolecules are absolutely necessary for maximal activity and they act in a synergistic manner. The product of the reaction was characterized as poly(ADP-ribose) by its acid insolubility, its insensitivity to hydrolysis by DNase, RNase, spleen phosphodiesterase or Pronase and by release of 5′-AMP and 2′-(5″-phosphoribosyl)-5′-AMP by incubation with snake venom phosphodiesterase. A covalent attachment between histone F1 and poly(ADP-ribose) has been established by using the cytoplasmic enzyme. The enzyme is primarily associated with ribosomes, both free ribosomes and those found in polysomes. Inhibition of protein synthesis in the intact cell reduced the level of activity in the cytoplasm. The enzyme can be removed from the ribosomes by centrifugation through sucrose gradients containing 0.6 m ammonium chloride. A relationship between this enzyme and DNA replication is suggested by the fact that the specific activity in the cytoplasm parallels the rate of DNA synthesis during the HeLa cell cycle.