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Genomic structure of DNase II and relationship to surrounding genes. A, DNase II is located in 100 kbp of contiguous sequence deposited into the GenBank TM data base by Lawrence Livermore National Laboratories. The following genes are identified on this stretch of chromosome 19p13.2: a, Rad 23a; b, calreticulin; c, phenylalanine tRNA synthetase ; d, glutaryl-CoA dehydrogenase; e, erythroid Krü ppel-like factor; f, deoxyribonuclease II; g, microtubule-associated serine threonine protein kinase. B, DNase II contains six exons (boxes) and introns (straight line). The filled boxes represent the coding sequence and open boxes the untranslated sequences.
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We have previously implicated deoxyribonuclease II (DNase II) as an endonuclease responsible for DNA digestion during apoptosis. The full-length human cDNA has now been cloned. The cDNA contains an open reading frame of 1078 bases coding for a 40-kDa protein. This protein is 10 kDa larger than commercially supplied enzyme, which has been proteolyti...
Citations
... The result suggested that caspases blocked the introduction of exogenous plasmid DNA into cells or gene expression of the introduced plasmid DNA. Activated caspases can activate deoxyribonuclease (DNase) such as DNase II 38 and caspase activated DNase (CAD) 39 . Theses DNases may degrade the transfected plasmid DNA. ...
High transfection efficiency is the most important point for experiments of DNA and RNA introduction into cells. Decrease of cell viability during the transfection procedure is a crucial issue, resulting in transfection failure. However, the mechanism underlying cell growth inhibition has not been fully elucidated. Lipofection is frequently used for transfection experiments, whereases, depending on cell type, it causes a decrease in cell viability. The present study demonstrates here that a potent pan-caspase inhibitor Q-VD-OPh blocked cell death during the lipofection, indicating apoptosis was induced in lipofection. Moreover, Q-VD-OPh drastically increased transfected cells. This method provides easier and more effective transfection system of lipofection and may be useful for transfection of not only cell lines but also clinical uses such as gene therapy and nucleic acids vaccine.
... These two variants differ from each other in a substitution of guanine to adenine in the sequence of the promoter, resulting in reduced expression [52]. This polypeptide consists of 360 amino acids, but just a few of them are part of putative functional sites of an enzyme [53]. The hydrophobic signal peptide is located at the N-end of the enzyme, but its cleavage during posttranslational modifications is still not clarified. ...
Extracellular DNA, also called cell-free DNA, released from dying cells or activated immune cells can be recognized by the immune system as a danger signal causing or enhancing inflammation. The cleavage of extracellular DNA is crucial for limiting the inflammatory response and maintaining homeostasis. Deoxyribonucleases (DNases) as enzymes that degrade DNA are hypothesized to play a key role in this process as a determinant of the variable concentration of extracellular DNA. DNases are divided into two families—DNase I and DNase II, according to their biochemical and biological properties as well as the tissue-specific production. Studies have shown that low DNase activity is both, a biomarker and a pathogenic factor in systemic lupus erythematosus. Interventional experiments proved that administration of exogenous DNase has beneficial effects in inflammatory diseases. Recombinant human DNase reduces mucus viscosity in lungs and is used for the treatment of patients with cystic fibrosis. This review summarizes the currently available published data about DNases, their activity as a potential biomarker and methods used for their assessment. An overview of the experiments with systemic administration of DNase is also included. Whether low-plasma DNase activity is involved in the etiopathogenesis of diseases remains unknown and needs to be elucidated.
... Although these observations do not eliminate DFF40/CAD participation in later stages of the process, they do suggest that other DNases are involved. DNase I and DNase II are of particular interest due to their broad tissue distribution and because they have been implicated as possible effectors of apoptosis [383,384]. ...
... Subcellular fractionation studies have shown that DNase I is localized primarily in the mitochondria, with activity also being detected in the nucleus [216]. A more recent immunolocalization study indicates that substantial amounts of DNase I can be located in the ER/nuclear envelope [384]. ...
A pro‐oxidant treatment encompassing a combined administration of ascorbate (VC) and menadione (VK3) on a series of carcinoma cell lines and xenotransplants resulted in specific cytotoxic activities that allowed us to describe a new mode of cell death named autoschizis. Observed in human prostate, bladder, and ovarian carcinoma cells as well as in a transgenic murine prostate cancer model, as well as in vivo, this mode of cell death process differs from apoptosis or necrosis according to the morphological and biochemical investigations collected, using morphological and biochemical techniques.
Comparing the untreated tumor cells with injured ones it was found that, in vitro, this autoschizis cell death is initiated by pro‐oxidant stress in which tumor cells deteriorate through irreversible cell damages caused by reactivation of nucleases. The initial damages consist in superficial and deep cytoplasmic changes involving membranes and the cytoskeleton resulting in dramatic cell size reduction through auto‐ or self‐excisions. Followed by a series of sequential injuries caused by hydrogen peroxide and reactive oxidative species on endomembranes,mitochondria and lysosomes and other organelle's injuries contribute to autophagic activities that also involve sequential nucleus defects. These injuries include karyolysis with nucleollus degradation ruled by reactivated nucleases and cathepsins K and L leaking from lysosomes verified by immunocytochemistry, DNA gel electrophoretic smear pattern, similar to Necrosis. Additionally, flow cytometry disclosed cell cycle blocks in G1/S and G2/M phases in all the treated tumor cells. Biochemical data from in vitro and in vivo tests showed that this mode of cell death is independent from caspase‐3 activation, while inhibiting the repair mechanisms of the cell through depletion of ATP, thiols, impeding protein, nucleic acid synthesis or repairs while starving energetically the tumor cells, i.e. trying to survive through autophagocytosis of reserves. Based on some data from biochemistry, this new mode of cell death verified by morphology and described in 1998 would encompass necroptosis, pathanos, pyroptosis and oxytosis.
In xenotransplanted carcinomas, this pro‐oxidant treatment induces major cell demise by autoschizis with significant reduction of the tumors' size and high survival rate of the host animals. Because this mode of killing specifically targets cancer cells and, based on preliminary clinical data, it has been suggested that this treatment would be a useful, safe and inexpensive strategy to be implemented synergistically with radiation and/or chemotherapy as an adjuvant or treatments in oncology.
... The copy number of DNase II genes varies in different animals; for example, one in Drosophila, two (DNase IIα and DNase IIβ) in mice and humans, and three (nuc-1, crn-6, and crn- 7) in Caenorhabditis elegans [3][4][5]. Several reports have indicated that DNase II exerts its enzymatic activity in the form of a homodimer or heterodimer [6,7], and its biological significance reportedly lies in the degradation of DNA in apoptotic bodies in nematodes, mice and humans [8][9][10]. The loss of DNase II results in embryonic lethality in mice and developmental defects in fruitflies, but no discernible phenotypes in DNase II-deficient nematodes [11,12]. ...
Three waves of apoptosis shape the development of Caenorhabditis elegans. Although the exact roles of the three DNase II genes (nuc-1, crn-6 and crn-7), which are known to mediate degradation of apoptotic DNA, in the embryonic and larval phases of apoptosis have been characterized, the DNase II acting in the third wave of germ cell apoptosis remains undetermined. In the present study, we performed in vitro and in vivo assays on various mutant nematodes to demonstrate that NUC-1 and CRN-7, but not CRN-6, function in germ cell apoptosis. In addition, in situ DNA-break detection and anti-phosphorylated ERK (extracellular-signal-regulated kinase) staining illustrated the sequential and spatially regulated actions of NUC-1 and CRN-7, at the pachytene zone of the gonad and at the loop respectively. In line with the notion that UV-induced DNA fragment accumulation in the gonad activates innate immunity responses, we also found that loss of NUC-1 and CRN-7 lead to up-regulation of antimicrobial genes (abf-2, spp-1, nlp-29, cnc-2, and lys-7). Our observations suggest that an incomplete digestion of DNA fragments resulting from the absence of NUC-1 or CRN-7 in the gonad could induce the ERK signalling, consequently activating antimicrobial gene expression. Taken together, the results of the present study demonstrate for the first time that nuc-1 and crn-7 play a role in degrading apoptotic DNA in distinct sites of the gonad, and act as negative regulators of innate immunity in C. elegans.
... AAW25360.1 (Sjda), with a sequence characteristic of DNase II, was the only DNase that showed significantly increased expression in the adult parasites ( Figure 3A). Since the identification of human DNase II in 1998, murine [16], porcine [17] and bovine [18] DNase II genes have been sequentially identified. Other putative genes encoding proteins homologous to DNase II have recently been observed in the genomes of many species, including Gallus gallus (chicken), Fugu rubripes (puffer fish), Xenopus laevis (frog), Anopheles (See figure on previous page.) ...
Recent advances in studies of the Schistosoma japonicum genome have opened new avenues for the elucidation of parasite biology and the identification of novel targets for vaccines, drug development and early diagnostic tools.
In this study, we surveyed the S. japonicum genome database for genes encoding nucleases. A total of 130 nucleases of 3 classes were found. Transcriptional analysis of these genes using a genomic DNA microarray revealed that the majority of the nucleases were differentially expressed in parasites of different developmental stages or different genders, whereas no obvious transcriptional variation was detected in parasites from different hosts. Further analysis of the putative DNases of S. japonicum revealed a novel DNase II homologue (Sjda) that contained a highly conserved catalytic domain. A recombinant Sjda-GST protein efficiently hydrolysed genomic DNA in the absence of divalent iron. Western-blot and immunofluorescence assays showed that Sjda was mainly expressed on the teguments of female adult parasites and induced early humoral immune responses in infected mice.
A novel DNase II homologue, Sjda, was identified in S. japonicum. Sjda was mainly distributed on the teguments of adult female parasites and possessed a typical divalent iron-independent DNA catalytic activity. This protein may play an important role in the host-parasite interaction.
... The type-II DNAse has its pH optimum at pH 5.0 (17, 18, 115, 228, 362). The cytosolic acidification fosters activation of DNA degradating enzymes (17,18,115,228,362). As a result, cytosolic acidification accelerates apoptosis of a variety of cells including BAF-3 cells (139), HL-60 cells (18, 351), CTLL-2 cells (371), MCF-7 cells (421), and MOE-7 cells (370). ...
Cell shrinkage is a hallmark and contributes to signaling of apoptosis. Apoptotic cell shrinkage requires ion transport across the cell membrane involving K(+) channels, Cl(-) or anion channels, Na(+)/H(+) exchange, Na(+),K(+),Cl(-) cotransport, and Na(+)/K(+)ATPase. Activation of K(+) channels fosters K(+) exit with decrease of cytosolic K(+) concentration, activation of anion channels triggers exit of Cl(-), organic osmolytes, and HCO3(-). Cellular loss of K(+) and organic osmolytes as well as cytosolic acidification favor apoptosis. Ca(2+) entry through Ca(2+)-permeable cation channels may result in apoptosis by affecting mitochondrial integrity, stimulating proteinases, inducing cell shrinkage due to activation of Ca(2+)-sensitive K(+) channels, and triggering cell-membrane scrambling. Signaling involved in the modification of cell-volume regulatory ion transport during apoptosis include mitogen-activated kinases p38, JNK, ERK1/2, MEKK1, MKK4, the small G proteins Cdc42, and/or Rac and the transcription factor p53. Osmosensing involves integrin receptors, focal adhesion kinases, and tyrosine kinase receptors. Hyperosmotic shock leads to vesicular acidification followed by activation of acid sphingomyelinase, ceramide formation, release of reactive oxygen species, activation of the tyrosine kinase Yes with subsequent stimulation of CD95 trafficking to the cell membrane. Apoptosis is counteracted by mechanisms involved in regulatory volume increase (RVI), by organic osmolytes, by focal adhesion kinase, and by heat-shock proteins. Clearly, our knowledge on the interplay between cell-volume regulatory mechanisms and suicidal cell death is still far from complete and substantial additional experimental effort is needed to elucidate the role of cell-volume regulatory mechanisms in suicidal cell death. © 2012 American Physiological Society. Compr Physiol 2:2037-2061, 2012.
... These findings demonstrated that SO4-TAM is biologically active within the tumor cell, mediating an apoptotic response that is dependent on SULT1A1 expression, and is associated with increased expression of EndoG, which to our knowledge has not been reported previously in 4-OHT-mediated apoptosis. EndoG belongs to a family of cell death endonucleases normally described as downstream effectors of apoptotic cascades, though capable of acting alone following activation or overexpression in cell lines [34,35]. In several cancers, EndoG has been identified as a key enzyme in caspaseindependent apoptosis following treatment with different chemotherapeutic drugs [14][15][16]. ...
Previously, we reported a strong association of the high activity SULT1A1*1 allele and overall survival of patients receiving tamoxifen therapy, indicating that sulfation of 4-hydroxytamoxifen (4-OHT) via SULT1A1 may contribute to the therapeutic efficacy of tamoxifen treatment. In most, but not all cases, sulfation is considered to be an elimination pathway; therefore we sought to define the biological mechanism by which increased sulfation of tamoxifen could provide a therapeutic benefit. We compared the antiproliferative and apoptotic responses between MCF7-SULT1A1 expressing cells and control MCF7 pcDNA3 cells when treated with 4-OHT. We observed a greater than 30% decrease in cell proliferation in MCF7-SULT1A1 expressing cells at physiological concentrations of 4-OHT, and significant cell death in SULT1A1-expressing cells treated with 2µM 4-OHT for 48 hours compared to control cells (p<0.05). Within 24 hours of drug treatment, an 80% increase in apoptosis in SULT1A1-expressing cells was apparent when compared to similarly treated cells that did not express SULT1A1. We also observed an increase in endonuclease G, the primary endonuclease expressed in ER-dependent breast cancer cells, which participates in caspaseindependent apoptosis. These data confirm that SULT1A1-mediated biotransformation of 4-OHT is important in the efficacy of 4-OHT cytotoxicity in breast tumors, and reveals a potential role for sulfated metabolites in the efficacy of tamoxifen therapy.
... Cellular events paralleling apoptosis include inhibition of the Na + /H + exchanger [20,21] leading to profound cytosolic acidosis and participating in apoptotic cell shrinkage [21][22][23]. The cytosolic acidification may support the activation of enzymes catalyzing DNA degradation [24][25][26][27][28]. Accordingly, cytosolic acidification has been shown to accelerate apoptosis of a variety of cells including BAF-3 cells [29], HL-60 cells [24,30], CTLL-2 cells [31], MCF-7 cells [32] and MOE-7 cells [33]. ...
... Caspase activity and DNA fragmentation may in turn be enhanced by cytosolic acidification. Previous observations disclosed that the type-II DNAse has a pH optimum at pH 5.0 [24][25][26][27][28]. Another type of endonuclease has a pH optimum at 7.5 [70], which may provide an explanation for the paradoxically enhanced apoptosis in some cells following cytosolic alkalinization [71,72]. ...
Infection with parvovirus B19 (B19) may induce apoptosis resulting in anemia, acute fulminant liver failure, placental insufficiency and myocarditis. Apoptosis has been attributed to proapoptotic activity of the non-structural viral protein NS1, which is known to trigger a signaling cascade eventually leading to activation of caspases. In several cell types apoptosis was found to be paralleled by profound cytosolic acidification, which may be secondary to inhibition of the Na+/H+ exchanger. The acidification has been considered to support the activation of pH sensitive caspases and endonucleases. However, nothing is known about the effect of NS1 on Na+/H+ exchanger activity and cytosolic pH. The present study thus explored whether NS1 expression affects cytosolic pH (pHi) and Na+-dependent realkalinization (DeltapHi) following acidification by an ammonium pulse. According to FACS analysis, overexpression of NS1 in RXR-10SW cells led within 72 hours to activation of caspase 3 and DNA fragmentation. NS1 overexpression resulted within 24 hours in a significant decline of pHi from 6.93 +/- 0.03 (n = 6) to 6.78 +/- 0.04 (n = 7), and to a significant decrease of DeltapHi from 0.159 +/- 0.017 (n = 6) to 0.039 +/- 0.004, (n = 7). The decrease of pHi and of DeltapHi following NS1 expression could be significantly blunted by inhibition of caspase 3 with zVAD. Western blot analysis revealed degradation of NHE1 following NS1 expression. In vitro, caspase 3, but not caspase 6, caspase 7 and caspase 8 degraded NHE1 protein of cell lysates. In conclusion, overexpression of NS1 triggers a signaling cascade eventually leading to activation of caspase 3 and subsequent degradation of NHE1. The effect contributes to cytosolic acidification which may in turn favor activation of caspases and endonucleases and thus participate in the pathophysiology of B19-infection.
... Cytotoxic endonucleases, also called "cell death endonucleases," are the recently recognized group of enzymes responsible for premortem and postmortem DNA fragmentation associated with cell death by apoptosis or necrosis [19,20]. Major representatives of this group of enzymes include: deoxyribonuclease I (DNase I) [21], deoxyribonuclease II (DNase II) [22], EndoG [23], caspase-activated DNase (CAD) [24], and DNase gamma [25]. Cell death endonucleases were found in all studied cells and tissues, including the prostate [26,27]. ...
... Endonuclease-generated breaks have been shown to strongly interfere with DNA synthesis in both normal and cancer cells [28]. While often considered downstream effectors of apoptotic cascades, endonucleases can cause DNA fragmentation and imminent, irreversible cell death when acting alone after overexpression or introduction into the cell [21,22,24]. Some cell death endonucleases seem to be dispensable in normal apoptosis [29][30][31]. ...
Analysis of promoter sequences of all known human cytotoxic endonucleases showed that endonuclease G (EndoG) is the only endonuclease that contains a CpG island, a segment of DNA with high G+C content and a site for methylation, in the promoter region. A comparison of three human prostate cancer cell lines showed that EndoG is highly expressed in 22Rv1 and LNCaP cells. In PC3 cells, EndoG was not expressed and the EndoG CpG island was hypermethylated. The expression of EndoG correlated positively with sensitivity to cisplatin and etoposide, and the silencing of EndoG by siRNA decreased the sensitivity of the cells to the chemotherapeutic agents in the two EndoG-expressing cell lines. 5-aza-2'-deoxycytidine caused hypomethylation of the EndoG promoter in PC3 cells, induced EndoG mRNA and protein expression, and made the cells sensitive to both cisplatin and etoposide. The acetylation of histones by trichostatin A, the histone deacetylase inhibitor, induced EndoG expression in 22Rv1 cells, while it had no such effect in PC3 cells. These data are the first indication that EndoG may be regulated by methylation of its gene promoter, and partially by histone acetylation, and that EndoG is essential for prostate cancer cell death in the used models.
... It may be that DNase II is activated in lysosomes and transferred to nuclei to degrade DNA. In humans and rodents, three acidic DNase II have been identified: DNase IIa (Baker et al., 1998; Krieser et al., 1998), DNase IIb (Shiokawa et al., 1999), and L-DNase II (Torriglia et al., 1998). Among the three acidic DNase II, DNase IIa and DNase IIb belong to a DNase II family; DNase IIb shares 34.4% identity with DNase IIa (Shiokawa et al., 1999); and L-DNase II has some similar features when compared with DNase IIa or DNase IIb. ...
Apoptosis, which is usually accompanied by DNA degradation, is important not only for the homeostasis of metazoans but also for mammalian development. If DNA is not properly degraded in these processes, it can cause diverse diseases, such as anemia, cataracts, and some autoimmune diseases. A large effort has been made to identify these nucleases that are responsible for these effects. In contrast to Deoxyribonuclease I (DNase I), Deoxyribonuclease II (DNase II) has been less well characterized in these processes. Additionally, enzymes of DNase II family in Trichinella spiralis, which is an intracellular parasitic nematode, are also considered involved in the development of the nematode. We have compiled information from studies on DNase II from various organisms and found some nonclassic features in these enzymes of T. spiralis. Here we have reviewed the characterization and functions of DNase II in these processes and predicted the functions of these enzymes in T. spiralis during host invasion and development.
