Primary acute lymphoblastic leukemia cells are susceptible to microtubule depolymerization in G1 and M phases through distinct cell death pathways.

Microtubule targeting agents (MTAs) are widely used cancer chemotherapeutics which conventionally exert their effects during mitosis, leading to mitotic or postmitotic death. However, accumulating evidence suggests that MTAs can also generate death signals during interphase, which may represent a key mechanism in the clinical setting. We reported previously that vincristine and other microtubule destabilizers induce death not only in M phase but also in G1 phase in primary acute lymphoblastic leukemia cells. Here, we sought to investigate and compare the pathways responsible for phase-specific cell death. Primary acute lymphoblastic leukemia cells were subjected to centrifugal elutriation, and cell populations enriched in G1 phase (97%) or G2/M phases (80%) were obtained and treated with vincristine. We found death of M phase cells was associated with established features of mitochondrial-mediated apoptosis, including Bax activation, loss of mitochondrial transmembrane potential, caspase-3 activation, and nucleosomal DNA fragmentation. In contrast, death of G1 phase cells was not associated with pronounced Bax or caspase-3 activation but was associated with loss of mitochondrial transmembrane potential, parylation, nuclear translocation of apoptosis-inducing factor and endonuclease G, and supra-nucleosomal DNA fragmentation, which was enhanced by inhibition of autophagy. The results indicate that microtubule depolymerization induces distinct cell death pathways depending on during which phase of the cell cycle microtubule perturbation occurs. The observation that a specific type of drug can enter a single cell type and induce two different modes of death is novel and intriguing. These findings provide a basis for advancing knowledge of clinical mechanisms of MTAs.

Crystal structure of the mouse endonuclease G.

Endonuclease G (EndoG) is a mitochondrial enzyme that responds to apoptotic stimuli by translocating to the nucleus and cleaving the chromatin DNA. The molecular mechanism of EndoG still remains unknown in higher organisms. Here, we determined the crystal structure of mouse EndoG at ∼1.96 Å resolution. The EndoG shows an altered dimeric configuration in which N-terminal region of one subunit interact to the other subunit in dimer. The deletion of this region that is highly conserved in mammalian EndoGs resulted in a monomer with significantly reduced activity suggesting the association of the dimeric arrangement into the nuclease activity. Furthermore, we observed a large conformational change in the loop of the active site groove in EndoG, which corresponds to the DNA binding region. Intriguingly, EndoG dimers are linked by oxidation of the reactive cysteine 110 in this flexible loop to form a long oligomeric chain in the crystal lattice. The structural analysis and ensuing biochemical data suggest that this flexible loop region in the active site is important to the regulation of EndoG nuclease function in mouse.

Induction of Alternative Splicing and Inhibition of Activity of Telomerase Catalytic Subunit by Apoptotic Endonuclease EndoG in Human T, B, and NK Cells.

The effect of apoptotic endonuclease EndoG on alternative splicing of mRNA of human telomerase catalytic subunit hTERT (human telomerase reverse transcriptase) and telomerase activity in normal human lymphocytes were studied. Human CD4+, CD8+, B, and NK cells were transfected with a plasmid pEndoG-GFP containing EndoG gene or control plasmid pGFP. The levels of mRNA of EndoG or hTERT splicing variants were analyzed by real-time PCR. Protein content was assessed by Western blotting. Telomerase activity was measured by the telomere repeats amplification protocol. EndoG overexpression reduced the expression of active full-length hTERT and increased the expression of inactive splice variant. Shifted balance of hTERT splice variants in cells led to a significant decrease in telomerase activity within 72 h after transfection.

AKT2 deficiency induces retardation of myocyte development through EndoG-MEF2A signaling in mouse heart.

Protein kinase B2 (AKT2) is implicated in diverse process of cardiomyocyte signaling including survival and metabolism. However, the role of AKT2 in myocardium development and the signaling pathway is rarely understood. Therefore, we sought to determine the effect of AKT2 deletion on heart development and its downstream targets. By using experimental animal models and neonatal rat cardiomyocytes (NRCMs), we observed that AKT2 deficiency induces retardation of heart development and increased systemic blood pressure (BP) without affecting cardiac function. Further investigation suggested that deficiency of AKT2 in myocardium results in diminished MEF2A abundance, which induced decreased size of cardiomyocytes. We additionally confirmed that EndoG, which is also regulated by AKT2, is a suppressor of MEF2A in myocardium. Finally, our results proved that AKT2 deficiency impairs the response to ß-adrenergic stimuli that normally causes hypertrophy in cardiomyocytes by downregulating MEF2A expression. Our data are the first to show the important role of AKT2 in determining the size of myocardium, its deficiency causes retardation of cardiomyocyte development. We also proved a novel pathway of heart development involving EndoG and MEF2A regulated by AKT2.

Interleukin-6 deficiency attenuates angiotensin II-induced cardiac pathogenesis with increased myocyte hypertrophy.

Interleukin-6 (IL-6) signaling is critical for cardiomyocyte hypertrophy, while the role of IL-6 in the pathogenesis of myocardium hypertrophy remains controversial. To determine the essential role of IL-6 signaling for the cardiac development during AngII-induced hypertension, and to elucidate the mechanisms, wild-type (WT) and IL-6 knockout (IL-6 KO) mice were infused subcutaneously with either vehicle or AngII (1.5 µg/h/mouse) for 1 week. Immunohistological and serum studies revealed that the extents of cardiac fibrosis, inflammation and apoptosis were reduced in IL-6 KO heart during AngII-stimulation, while cardiac hypertrophy was obviously induced. To investigate the underlying mechanisms, by using myocardial tissue and neonatal cardiomyocytes, we observed that IL-6/STAT3 signaling was activated under the stimulation of AngII both in vivo and in vitro. Further investigation suggested that STAT3 activation enhances the inhibitory effect of EndoG on MEF2A and hampers cardiomyocyte hypertrophy. Our study is the first to show the important role of IL-6 in regulating cardiac pathogenesis via inflammation and apoptosis during AngII-induced hypertension. We also provide a novel link between IL-6/STAT3 and EndoG/MEF2A pathway that affects cardiac hypertrophy during AngII stimulation.

Mechanism of graphene-induced cytotoxicity: Role of endonucleases.

Graphene, a crystalline allotrope or carbon, presents numerous useful properties; however, its toxicity is yet to be determined. One of the most dramatic and irreversible toxic abilities of carbon nanomaterials is the induction of DNA fragmentation produced by endogenous cellular endonucleases. This study demonstrated that pristine graphene exposed to cultured kidney tubular epithelial cells is capable of inducing DNA fragmentation measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which is usually associated with cell death. TUNEL (cell death) and endonuclease activity measured using a near infrared fluorescence probe was significantly higher in cells containing graphene aggregates detected by Raman spectroscopy. The elevation of TUNEL coincided with the increased abundance of heme oxygenase 1 (HO-1), heat shock protein 90 (HSP90), active caspase-3 and endonucleases (deoxyribonuclease I [DNase I] and endonuclease G [EndoG]), as measured by quantitative immunocytochemistry. Specific inhibitors for HO-1, HSP90, caspase-3, DNase I and EndoG almost completely blocked the DNA fragmentation induced by graphene exposure. Therefore, graphene induces cell death through oxidative injury, caspase-mediated and caspase-independent pathways; and endonucleases DNase I and EndoG are important for graphene toxicity. Inhibition of these pathways may ameliorate cell injury produced by graphene. Copyright © 2017 John Wiley & Sons, Ltd.

AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF) and Endonuclease G (EndoG) While Promoting Caspase Activation during Cardiac Ischemia.

The AKT (protein kinase B, PKB) family has been shown to participate in diverse cellular processes, including apoptosis. Previous studies demonstrated that protein kinase B2 (AKT2-/-) mice heart was sensitized to apoptosis in response to ischemic injury. However, little is known about the mechanism and apoptotic signaling pathway. Here, we show that AKT2 inhibition does not affect the development of cardiomyocytes but increases cell death during cardiomyocyte ischemia. Caspase-dependent apoptosis of both the extrinsic and intrinsic pathway was inactivated in cardiomyocytes with AKT2 inhibition during ischemia, while significant mitochondrial disruption was observed as well as intracytosolic translocation of cytochrome C (Cyto C) together with apoptosis-inducing factor (AIF) and endonuclease G (EndoG), both of which are proven to conduct DNA degradation in a range of cell death stimuli. Therefore, mitochondria-dependent cell death was investigated and the results suggested that AIF and EndoG nucleus translocation causes cardiomyocyte DNA degradation during ischemia when AKT2 is blocked. These data are the first to show a previous unrecognized function and mechanism of AKT2 in regulating cardiomyocyte survival during ischemia by inducing a unique mitochondrial-dependent DNA degradation pathway when it is inhibited.

Cellular inhibitor of apoptosis protein 1 ubiquitinates endonuclease G but does not affect endonuclease G-mediated cell death.

Inhibitors of Apoptosis Proteins (IAPs) are evolutionarily well conserved and have been recognized as the key negative regulators of apoptosis. Recently, the role of IAPs as E3 ligases through the Ring domain was revealed. Using proteomic analysis to explore potential target proteins of DIAP1, we identified Drosophila Endonuclease G (dEndoG), which is known as an effector of caspase-independent cell death. In this study, we demonstrate that human EndoG interacts with IAPs, including human cellular Inhibitor of Apoptosis Protein 1 (cIAP1). EndoG was ubiquitinated by IAPs in vitro and in human cell lines. Interestingly, cIAP1 was capable of ubiquitinating EndoG in the presence of wild-type and mutant Ubiquitin, in which all lysines except K63 were mutated to arginine. cIAP1 expression did not change the half-life of EndoG and cIAP1 depletion did not alter its levels. Expression of dEndoG 54310, in which the mitochondrial localization sequence was deleted, led to cell death that could not be suppressed by DIAP1 in S2 cells. Moreover, EndoG-mediated cell death induced by oxidative stress in HeLa cells was not affected by cIAP1. Therefore, these results indicate that IAPs interact and ubiquitinate EndoG via K63-mediated isopeptide linkages without affecting EndoG levels and EndoG-mediated cell death, suggesting that EndoG ubiquitination by IAPs may serve as a regulatory signal independent of proteasomal degradation.

The biological function of cytoplasm-translocated ENDOG (endonuclease G).

ENDOG, a mitochondrial intermembrane space located endonuclease, participates in DNA fragmentation and apoptosis by translocating to the nucleus. ENDOG can also relocate to the mitochondrial matrix, where it regulates mitochondrial genome cleavage. However, the biological function of cytoplasm-translocated ENDOG remains unclear. Our previous study reported that starvation induces the release of ENDOG from mitochondria to the cytoplasm, promoting macroautophagy/autophagy in a process conserved across species. We demonstrate that ENDOG can be phosphorylated by GSK3B, which enhances ENDOG binding to YWHAG/14-3-3γ, and leads to the release of TSC2 and PIK3C3/VPS34 from YWHAG/14-3-3γ, followed by MTORC1 pathway suppression and autophagy initiation. Additionally, we recently reported that ENDOG can also activate the MTORC2-AKT-ACLY signaling axis by promoting the release of RICTOR and TSC2 from YWHAG/14-3-3γ, resulting in acetyl-CoA production. Furthermore, cytoplasmic ENDOG can translocate to the endoplasmic reticulum, where it binds with HSPA5/BIP to release ERN1/IRE1a-EIF2AK3/PERK to activate the endoplasmic reticulum stress response, eventually promoting lipid synthesis. Collectively, ENDOG will be released from the mitochondrial intermembrane space, and translocated to the mitochondrial matrix, cytoplasm, and nucleus during different stress stimulation, where it digests DNA or interacts with crucial proteins to regulate different biological functions, including apoptosis, autophagy, mitophagy, and lipid synthesis.

Nuclear endonuclease G controls cell proliferation in ovarian cancer.

Ovarian cancer is characterized by a high degree of genetic heterogeneity. Platinum-based chemotherapy and some gene-targeted therapies have shown limited treatment efficacy due to toxicity and recurrence, and thus, it is essential to identify additional therapeutic targets based on an understanding of the pathological mechanism. Here, we report that endonuclease G, which exhibits altered expression in ovarian cancer, does not function as a cell death effector that digests chromosomal DNA in ovarian cancer. Endonuclease G is modulated by intracellular reactive oxygen species dynamics and plays a role in cell proliferation in ovarian cancer, suggesting that targeting endonuclease G alone or in combination with other antitumor agents may have the potential for development into a treatment for endonuclease G-overexpressing cancers, including ovarian cancer.

ATX-LPA-Dependent Nuclear Translocation of Endonuclease G in Respiratory Epithelial Cells: A New Mode Action for DNA Damage Induced by Crystalline Silica Particles.

Crystalline silica particles (CSi) are an established human carcinogen, but it is not clear how these particles cause necessary mutations. A well-established scenario includes inflammation caused by retained particles in the bronchioles, activated macrophages, and reactive oxygen species (ROS) that cause DNA damage. In previous studies, we showed that CSi in contact with the plasma membrane of human bronchial epithelium induced double strand breaks within minutes. A signaling pathway implicating the ATX-LPA axis, Rac1, NLRP3, and mitochondrial depolarization upstream of DSB formation was delineated. In this paper, we provide in vitro and in vivo evidence that this signaling pathway triggers endonuclease G (EndoG) translocation from the mitochondria to the nucleus. The DNA damage is documented as γH2AX and p53BP1 nuclear foci, strand breaks in the Comet assay, and as micronuclei. In addition, the DNA damage is induced by low doses of CSi that do not induce apoptosis. By inhibiting the ATX-LPA axis or by EndoG knockdown, we prevent EndoG translocation and DSB formation. Our data indicate that CSi in low doses induces DSBs by sub-apoptotic activation of EndoG, adding CSi to a list of carcinogens that may induce mutations via sub-apoptotic and "minority MOMP" effects. This is the first report linking the ATX-LPA axis to this type of carcinogenic effect.

Biallelic Variants in ENDOG Associated with Mitochondrial Myopathy and Multiple mtDNA Deletions.

Endonuclease G (ENDOG) is a nuclear-encoded mitochondrial-localized nuclease. Although its precise biological function remains unclear, its proximity to mitochondrial DNA (mtDNA) makes it an excellent candidate to participate in mtDNA replication, metabolism and maintenance. Indeed, several roles for ENDOG have been hypothesized, including maturation of RNA primers during mtDNA replication, splicing of polycistronic transcripts and mtDNA repair. To date, ENDOG has been deemed as a determinant of cardiac hypertrophy, but no pathogenic variants or genetically defined patients linked to this gene have been described. Here, we report biallelic ENDOG variants identified by NGS in a patient with progressive external ophthalmoplegia, mitochondrial myopathy and multiple mtDNA deletions in muscle. The absence of the ENDOG protein in the patient's muscle and fibroblasts indicates that the identified variants are pathogenic. The presence of multiple mtDNA deletions supports the role of ENDOG in mtDNA maintenance; moreover, the patient's clinical presentation is very similar to mitochondrial diseases caused by mutations in other genes involved in mtDNA homeostasis. Although the patient's fibroblasts did not present multiple mtDNA deletions or delay in the replication process, interestingly, we detected an accumulation of low-level heteroplasmy mtDNA point mutations compared with age-matched controls. This may indicate a possible role of ENDOG in mtDNA replication or repair. Our report provides evidence of the association of ENDOG variants with mitochondrial myopathy.

Assessment of the Role of Nuclear ENDOG Gene and mtDNA Variations on Paternal Mitochondrial Elimination (PME) in Infertile Men: An Experimental Study.

In humans and most animals, maternal inheritance of mitochondria and mitochondrial DNA (mtDNA) is considered as an universal assumption. Recently, several lines of evidence suggest that different species seem to employ distinct mechanisms to prevent the inheritance of paternal mtDNA. There are few studies in the literature on the molecular basis of sperm mtDNA elimination in mammals and paternal mtDNA transmission in humans. Endonuclease G (ENDOG) is a mitochondrial nuclease encoded by nuclear ENDOG gene. The critical importance of ENDOG gene on paternal mitochondrial elimination (PME) has been previously demonstrated in model organisms such as C. elegans and D. melanogaster. However, its mechanism in human is still unclear. Therefore, we aimed to evaluate whether nuclear ENDOG gene copy number could be a potential marker of paternal mtDNA transmission or not.Male factor infertility patients diagnosed with different infertility subgroups such as azoospermia, oligoteratozoospermia, astheno-teratozoospermia were included in this study: 13 infertile men and 25 healthy men as control group. Quantitative real-time polymerase chain reaction (qPCR) analysis and dual-color Fluorescence in situ hybridization (FISH) method were used to compare the groups. FISH method was applied to verify qPCR results and two signals were observed in nearly all patients. ENDOG gene copy number data were evaluated by comparing them with entire human mtDNA next-generation sequencing (NGS) analysis results obtained through bioinformatics and proteomics tools. Mitochondrial whole genome sequencing (WGS) data allowed determination of novel and reported variations such as single nucleotide polymorphisms (SNPs), multiple nucleotide polymorphism (MNP), insertion/deletion (INDEL). Missense variants causing amino acid substitution were filtered out from patients' mtDNA WGS data.Relative copy number of target ENDOG gene in male infertility patients [0.49 (0.31 - 0.77)] was lower than healthy controls [1.00 (0.66 - 1.51)], and statistical results showed significant differences between the groups (p < 0.01). A total of 38 missense variants were detected in the genes encoding the proteins involved in the respiratory chain complex. Moreover, we detected paternal mtDNA transmissions in the children of these patients who applied to assisted reproductive techniques.In conclusion, this study reveals that ENDOG gene may be an important factor for the PME mechanism in humans. To the best of our knowledge, this is the first study in humans about this topic and assessment of ENDOG gene sequencing and gene expression studies in a larger sample size including patients with male factor infertility would be our future project.

Conserved role of ENDOG in promoting autophagy.

ENDOG (endonuclease G), a mitochondrial endonuclease, is known to participate in apoptosis and paternal mitochondria elimination. However, the role and underlying mechanism of ENDOG in regulating macroautophagy remain unclear. We recently reported that ENDOG released from mitochondria promotes autophagy during starvation, which we demonstrated is evolutionarily conserved across species by performing experiments in human cell lines, mice, Drosophila, and C. elegans. This study demonstrates that ENDOG can be phosphorylated by GSK3B, which enhances the interaction between ENDOG with YWHAG and leads to the release of TSC2 and PIK3C3 from YWHAG, followed by MTOR pathway suppression and autophagy initiation. Additionally, the endonuclease activity of ENDOG is essential for activating the DNA damage response and thus inducing autophagy. Consequently, this study uncovered an exciting new role for ENDOG as a crucial regulator of autophagy.

ENDOG Impacts on Tumor Cell Proliferation and Tumor Prognosis in the Context of PI3K/PTEN Pathway Status.

EndoG influences mitochondrial DNA replication and is involved in somatic cell proliferation. Here, we investigated the effect of ENDOG/Endog expression on proliferation in different tumor models. Noteworthy, ENDOG deficiency reduced proliferation of endometrial tumor cells expressing low PTEN/high p-AKT levels, and Endog deletion blunted the growth of PTEN-deficient 3D endometrial cultures. Furthermore, ENDOG silencing reduced proliferation of follicular thyroid carcinoma and glioblastoma cell lines with high p-AKT expression. High ENDOG expression was associated with a short time to treatment in a cohort of patients with chronic lymphocytic leukemia (CLL), a B-cell lymphoid neoplasm with activation of PI3K/AKT. This clinical impact was observed in the less aggressive CLL subtype with mutated IGHV in which high ENDOG and low PTEN levels were associated with worse outcome. In summary, our results show that reducing ENDOG expression hinders growth of some tumors characterized by low PTEN activity and high p-AKT expression and that ENDOG has prognostic value for some cancer types.

The telomere complex and the origin of the cancer stem cell.

Exquisite regulation of telomere length is essential for the preservation of the lifetime function and self-renewal of stem cells. However, multiple oncogenic pathways converge on induction of telomere attrition or telomerase overexpression and these events can by themselves trigger malignant transformation. Activation of NFκB, the outcome of telomere complex damage, is present in leukemia stem cells but absent in normal stem cells and can activate DOT1L which has been linked to MLL-fusion leukemias. Tumors that arise from cells of early and late developmental stages appear to follow two different oncogenic routes in which the role of telomere and telomerase signaling might be differentially involved. In contrast, direct malignant transformation of stem cells appears to be extremely rare. This suggests an inherent resistance of stem cells to cancer transformation which could be linked to a stem cell'specific mechanism of telomere maintenance. However, tumor protection of normal stem cells could also be conferred by cell extrinsic mechanisms.

Recommendations for the use of the acetaminophen hepatotoxicity model for mechanistic studies and how to avoid common pitfalls.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, which is safe at therapeutic doses but can cause severe liver injury and even liver failure after overdoses. The mouse model of APAP hepatotoxicity recapitulates closely the human pathophysiology. As a result, this clinically relevant model is frequently used to study mechanisms of drug-induced liver injury and even more so to test potential therapeutic interventions. However, the complexity of the model requires a thorough understanding of the pathophysiology to obtain valid results and mechanistic information that is translatable to the clinic. However, many studies using this model are flawed, which jeopardizes the scientific and clinical relevance. The purpose of this review is to provide a framework of the model where mechanistically sound and clinically relevant data can be obtained. The discussion provides insight into the injury mechanisms and how to study it including the critical roles of drug metabolism, mitochondrial dysfunction, necrotic cell death, autophagy and the sterile inflammatory response. In addition, the most frequently made mistakes when using this model are discussed. Thus, considering these recommendations when studying APAP hepatotoxicity will facilitate the discovery of more clinically relevant interventions.

Adsorption behavior of two glucanases on three lignins and the effect by adding sulfonated lignin.

The adsorption behaviors of two glucanases, TvEG and TrCel7A, on three lignins were investigated. Three lignins were isolated from raw aspen and its pretreated solid residue. The isolated lignins were labeled as Asp-MWL, DA-MWL (pretreated by dilute acid), and GL-MWL (pretreated by green liquor), respectively. The surface properties of lignins and spin-coated lignin films were characterized by zeta potential, atomic force microscope (AFM) and contact angle. The enzyme adsorption behavior was monitored by quartz crystal microbalance (QCM) and fluorescence spectrometer. TlCel7A had similar adsorption capacities on the three lignin films but were higher than those of TvEG. The TrCel7A adsorptions on the three lignin films were affected by synergistic effect of electrostatic and hydrophobic interaction while the TvEG adsorptions on the three lignin films were mainly dominated by hydrophobic action. The adsorption capacities of TlCel7A and TvEG on the three lignin films were decreased by adding SL. Plausible explanation was that the SL and glucanase formed a complex with more negative charges, which suppressed the adsorption of glucannase on lignin through electrostatic repulsion. It also explained the improved enzymatic hydrolysis efficiency of lignocellulose upon adding SL.

Involvement of the mitochondrial nuclease EndoG in the regulation of cell proliferation through the control of reactive oxygen species.

The apoptotic nuclease EndoG is involved in mitochondrial DNA replication. Previous results suggested that, in addition to regulate cardiomyocyte hypertrophy, EndoG could be involved in cell proliferation. Here, by using in vivo and cell culture models, we investigated the role of EndoG in cell proliferation. Genetic deletion of Endog both in vivo and in cultured cells or Endog silencing in vitro induced a defect in rodent and human cell proliferation with a tendency of cells to accumulate in the G1 phase of cell cycle and increased reactive oxygen species (ROS) production. The defect in cell proliferation occurred with a decrease in the activity of the AKT/PKB-GSK-3ß-Cyclin D axis and was reversed by addition of ROS scavengers. EndoG deficiency did not affect the expression of ROS detoxifying enzymes, nor the expression of the electron transport chain complexes and oxygen consumption rate. Addition of the micropeptide Humanin to EndoG-deficient cells restored AKT phosphorylation and proliferation without lowering ROS levels. Thus, our results show that EndoG is important for cell proliferation through the control of ROS and that Humanin can restore cell division in EndoG-deficient cells and counteracts the effects of ROS on AKT phosphorylation.

Endonuclease G modulates the alternative splicing of deoxyribonuclease 1 mRNA in human CD4+ T lymphocytes and prevents the progression of apoptosis.

Apoptotic endonucleases act cooperatively to fragment DNA and ensure the irreversibility of apoptosis. However, very little is known regarding the potential regulatory links between endonucleases. Deoxyribonuclease 1 (DNase I) inactivation is caused by alternative splicing (AS) of DNase I pre-mRNA skipping exon 4, which occurs in response to EndoG overexpression in cells. The current study aimed to determine the role of EndoG in the regulation of DNase I mRNA AS and the modulation of its enzymatic activity. A strong correlation was identified between the EndoG expression levels and DNase I splice variants in human lymphocytes. EndoG overexpression in CD4+ T cells down-regulated the mRNA levels of the active full-length DNase I variant and up-regulated the levels of the non-active spliced variant, which acts in a dominant-negative fashion. DNase I AS was induced by the translocation of EndoG from mitochondria into nuclei during the development of apoptosis. The DNase I spliced variant was induced by recombinant EndoG or by incubation with EndoG-digested cellular RNA in an in vitro system with isolated cell nuclei. Using antisense DNA oligonucleotides, we identified a 72-base segment that spans the adjacent segments of exon 4 and intron 4 and appears to be responsible for the AS. DNase I-positive CD4+ T cells overexpressing EndoG demonstrated decreased progression towards bleomycin-induced apoptosis. Therefore, EndoG is an endonuclease with the unique ability to inactivate another endonuclease, DNase I, and to modulate the development of apoptosis.