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Senescence is an irreversible permanent cell cycle arrest accompanied by changes in cell morphology and physiology. Bioactive compounds including tocotrienols (vitamin E) can affect important biological functions. The aim of this study was to investigate how γ- and δ-tocotrienols can affect stress-induced premature senescence. We established two different models of premature stress senescence by induction of senescence with either hydrogen peroxide or etoposide in human lung fibroblasts MRC-5 (ECACC, England). We observed increased percentage of cells with increased SA-β-galactosidase activity, decreased cell viability/proliferation and increased level of p21 in both models. In addition, γ-tocotrienol or δ-tocotrienol (both at concentrations of 150, 200 and 300 μM) were added to the cells along with the inductor of senescence (cotreatment). We have found that this cotreatment led to the decrease of cell viability/proliferation in both models of premature stress senescence, but did not change the percentage of senescent cells. Moreover, we detected no expression of caspase-3 or apoptotic DNA fragmentation in any models of premature stress senescence after the cotreatment with γ- as well as δ-tocotrienols. However, an increased level of autophagic protein LC-3 II was detected in cells with hydrogen peroxide-induced senescence after the cotreatment with γ-tocotrienol as well as δ-tocotrienol. In case of etoposide-induced senescence only δ-tocotrienol cotreatment led to an increased level of LC-3 II protein in cells. According to our work δ-tocotrienol is more effective compound than γ-tocotrienol.

Briefly depicted are the publications in CYTOMETRY that received the highest frequency of citations. Among them are seminal papers describing application of metachromatic fluorochrome acridine orange to differentially stain DNA versus RNA or to analyze susceptibility of DNA in situ to denaturation; both features being markers of different sections of the cell cycle including identification of noncycling quiescent cells. The papers reviewing detection of cyclins D1, E, A or B1, each in relation to cell cycle phase, were also among the highly cited ones. The highest citation rates received publications describing development of the TUNEL methodology to detect apoptotic DNA fragmentation, and more recently expression of ϒH2AX to reveal DNA damage. © 2020 International Society for Advancement of Cytometry.

Chalcones found in fruits and vegetables have promising cancer chemopreventive properties. This study attempts to identify the anticancer efficacies of chalcone flavokawain B (FKB) in the rhizomes of Alpinia pricei Hayata by examining key molecular events in non-small-cell lung cancer (A549) cells. Our results indicated that in human A549 cells, FKB (0-15 μg/ml) decreases cell viability and colony formation, dysregulates the Bax:B-cell lymphoma 2 ratio and increases apoptotic DNA fragmentation. Mitochondrial (caspase-9/-3 and poly ADP ribose polymerase [PARP]) signaling was found to be involved in FKB-induced apoptosis. In addition, FKB-induced reactive oxygen species (ROS) generation, and N-acetylcysteine attenuated FKB-induced apoptotic cell death. Moreover, FKB triggered autophagy, as evidenced by the improved acidic vesicular organelle formation, lipidated light chain 3 (microtubule-related light chain 3) accumulation, and ATG7 expression and the decreased mammalian target of rapamycin phosphorylation. Furthermore, FKB suppressed ROS-mediated ATG4B expression. Inhibiting autophagy using 3-methyladenine/chloroquine diminished FKB-induced cell death, indicating that autophagy is triggered as a death mechanism by FKB. In summary, FKB has a crucial role in the execution and propagation of ROS-mediated apoptotic and autophagic cell death of lung adenocarcinoma cells.

DNA double strand breaks (DSB) induced by ionizing radiation (IR) are usually measured using γH2AX/53BP1 DNA repair foci, that is considered to be the most sensitive assay for DSB analysis. While fluorescence microscopy (FM) is the gold standard for this analysis, imaging flow cytometry (IFC) may offer number of advantages such as lack of the fluorescence background, higher number of cells analyzed, and higher sensitivity in detection of DNA damage induced by IR at low doses. Along with appearance of γH2AX foci, the variable fraction of the cells exhibits homogeneously stained γH2AX signal resulting in so-called γH2AX pan-staining, which is believed to appear at early stages of apoptosis. Here, we investigated incidence of γH2AX pan-staining at different time points after irradiation with γ-rays using IFC and compared the obtained data with the data from FM. Appearance of γH2AX pan-staining during the apoptotic process was further analyzed by fluorescence-activated cell sorting (FACS) of cells at different stages of apoptosis and subsequent immunofluorescence analysis. Our results show that IFC was able to reveal dose dependence of pan-staining, while FM failed to detect all pan-staining cells. Moreover, we found that γH2AX pan-staining could be induced by therapeutic, but not low doses of γ-rays and correlate well with percentage of apoptotic cells was analyzed using flow cytometric Annexin-V/7-AAD assay. Further investigations showed that γH2AX pan-staining is formed in the early phases of apoptosis and remains until later stages of apoptotic process. Apoptotic DNA fragmentation as detected with comet assay using FM correlated with the percentage of live and late apoptotic/necrotic cells as analyzed by flow cytometry. Lastly, we successfully tested IFC for detection of γH2AX pan-staining and γH2AX/53BP1 DNA repair foci in lymphocyte of breast cancer patients after radiotherapy, which may be useful for assessing individual radiosensitivity in a clinically relevant cohort of patients.

Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different species1,2. However, their biogenesis and functions are largely unknown. By developing a new circular DNA enrichment method, here we purified and sequenced full-length eccDNAs with Nanopore sequencing. We found that eccDNAs map across the entire genome in a close to random manner, suggesting a biogenesis mechanism of random ligation of genomic DNA fragments. Consistent with this idea, we found that apoptosis inducers can increase eccDNA generation, which is dependent on apoptotic DNA fragmentation followed by ligation by DNA ligase 3. Importantly, we demonstrated that eccDNAs can function as potent innate immunostimulants in a manner that is independent of eccDNA sequence but dependent on eccDNA circularity and the cytosolic DNA sensor Sting. Collectively, our study not only revealed the origin, biogenesis and immunostimulant function of eccDNAs but also uncovered their sensing pathway and potential clinical implications in immune response. By developing a new eccDNA purification and profiling method, the study revealed close-to-random genomic origination, mechanism of biogenesis and function of eccDNAs.

Oligonucleosomal DNA fragmentation and chromatin condensation are two hallmarks of apoptosis. However, their generation mechanisms are not entirely understood. Histone H1, a positively charged nuclear protein located in the linker region of chromatin, is involved in higher-order chromatin structures and tight chromatin packing. On the basis of the physical and biochemical characteristics of histone H1, we hypothesized that histone H1 plays a role in determining the efficiencies of apoptotic DNA fragmentation and chromatin condensation. Therefore, we examined histone H1 quantity in five human leukemia cell lines and compared the efficiencies. The cell lines were categorized into two groups according to their origins: (i) Ramos and Molt-4 cells of lymphoid origin and (ii) U937, ML-1, and HL60 cells of myeloid origin. Compared to the lymphoid-origin group, the myeloid-origin group had lower levels of histone H1 but more open chromatin. Furthermore, the myeloid-origin group showed marked DNA fragmentation but less chromatin condensation during apoptosis. These results suggested that histone H1 determined chromatin structure and that its quantity affected the efficiencies of DNA fragmentation and chromatin condensation in apoptosis.

Canine osteosarcoma is an aggressive primary bone tumor that shows metastasis to distal regions and is associated with a high mortality rate. However, the pathophysiological mechanisms of canine osteosarcoma are not well characterized. In addition, development of prognostic factors and novel therapeutic agents is necessary to efficiently treat osteosarcoma. Therefore, we studied the effects of myricetin, an antioxidant found in berries, nuts, teas, wine, and vegetables, on apoptosis and signal transduction in the canine osteosarcoma cell lines, D-17 and DSN. Results of the present study demonstrated that treatment with myricetin decreased cell proliferation and DNA replication, while it increased apoptotic DNA fragmentation in D-17 and DSN cells. In addition, it increased generation of ROS, lipid peroxidation, and depolarization of MMP in both D-17 and DSN cells. Myricetin treatment activated phosphorylation of AKT, p70S6K, ERK1/2, JNK, and p90RSK in canine osteosarcoma cells. Moreover, inhibition of PI3K and MAPK using LY294002, U0126, or SP600125, in addition to myricetin treatment, effectively suppressed cell proliferation compared to treatment with myricetin or each inhibitor alone. Therefore, we concluded that myricetin may be a potentially effective and less toxic therapeutic agent to prevent and control progression of canine osteosarcoma.

Significance Cell death-inducing DFF45-like effector (CIDE) domains, initially identified in apoptotic nucleases, form a highly conserved family with diverse functions ranging from cell death to lipid homeostasis and synaptic regulation. Through structural determination of two CIDE family proteins, Drep2 and Drep4, we found that CIDE domains can form helical oligomers. Our results reveal that such higher-order structures not only are conserved in the CIDE family, but also are critically important for both DNA fragmentation and lipid droplet fusion. Therefore, our findings identify the CIDE domain as a scaffolding component for higher-order structure assembly. Our results expand the importance of higher-order structures from the established field of immune signaling to broader biological functions.

Background: Apoptosis results in specific and stage-dependent morphological alterations of the cell nucleus, including pyknosis and cell shrinking. The experimental investigation of apoptotic processes is still challenging and routinely based on the assessment of molecular events like chromatin fragmentation and caspase enzyme activity. Alternatively, the establishment of a fluorescence microscopy nuclear morphology assay would provide a simple and robust low-cost method for detection and quantification of apoptotic cascades. Materials and Methods: Model cell lines LNCaP and MDA-MB-231 were incubated in the presence of the apoptosis-inducer cycloheximide (CHX). After evaluation of apoptotic cascades by terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assay, stained cell nuclei were analyzed regarding area, perimeter, major and minor axis, as well as brightness of nuclear fluorescence signal. Results: When compared to vehicle-treated control cells, administration of CHX led to significantly reduced cell growth and elevated rates of chromatin fragmentation of both cell lines as shown by cell counting and TUNEL assay, respectively. These apoptotic effects were accompanied by apoptosis-specific modulations of the nuclei demonstrated by diminished nuclear morphology parameters, such as area, perimeter, major and minor axis, as well as elevated levels of nuclear staining intensity. Conclusion: We present a computerized method for apoptosis detection and quantification using images of fluorescent dye-stained cell nuclei. The advantages of this nuclear morphology assay include the (i) ability to routinely assess apoptosis by a fast, highly reproducible low-cost technique, (ii) applicability of an experimental approach analyzing high numbers of single nuclei and (iii) detection of apoptosis in early, as well as late, stages of the apoptotic cascade.

Type I/II interferons (IFNα,β/IFNɣ) are cytokines that activate signal-transducer-and-activator-of-transcription-1 (STAT1). The STAT1 N-terminal domain (NTD) mediates dimerization and cooperative DNA-binding. The STAT1 DNA-binding domain (DBD) confers sequence-specific DNA-recognition. STAT1 has been connected to growth inhibition, replication stress and DNA-damage. We investigated how STAT1 and NTD/DBD mutants thereof affect fibrosarcoma cells. STAT1 and indicated mutants do not affect proliferation of resting and IFNα-treated cells as well as checkpoint kinase signaling, and phosphorylation of the tumor-suppressive transcription factor p53 ensuing ɣ-irradiation. Of the STAT1 reconstituted U3A cells those with STAT1 NTD mutants accumulate the highest levels of the replication stress/DNA-damage marker S139-phosphorylated histone H2AX (ɣH2AX). This is similarly seen with a STAT1 NTD/DBD double mutant, indicating transcription-independent effects. Furthermore, U3A cells with STAT1 NTD mutants are most susceptible to apoptotic DNA fragmentation and cleavage of the DNA repair protein PARP1. These data provide novel insights into the relevance of the STAT1 NTD.

Lectins are proteins that show a variety of biological activities. Nevertheless, information on lectin from Gluttonous beauts and their anticancer activities are very limited. In this study, we purified a lectin from hemolymph of G. beauts and identified its molecular weight to be 66 kDa. The effect of lectin at different concentrations (μg/mL) on the cell growth and apoptosis were evaluated against MCF-7 and MCF-10A cells, whereas cytotoxicity to the MCF-7 cells mediated by lectin was observed and the mechanism of action of the lectin in including apoptosis in cancer cells via the intrinsic pathway was also proposed. The MCF-7 cells were employed for in vitro studies on cytotoxicity, induction of apoptosis and apoptotic DNA fragmentation. In MCF-10A cells lectin did not show any adverse effect even at higher concentration. Cell cycle analysis also showed a significant cell cycle arrest on selected cells after lectin treatment. Western blotting suggested that lectin up regulates the apoptotic protein expression in MCF-7 cells while it down regulates the level of Bcl-2 expression.

Treatment of Jurkat T cells with the dynamin inhibitor, myristyl trimethyl ammonium bromides (MiTMAB) caused cytokinesis impairment and apoptotic DNA fragmentation along with down-regulation of anti-apoptotic BAG3 and Mcl-1 levels, Bak activation, mitochondrial membrane potential (Δψm) loss, activation of caspase-9 and -3, and PARP cleavage, without accompanying necrosis. Bcl-xL overexpression completely abrogated these MiTMAB-induced mitochondrial damage and resultant caspase cascade activation, except for impaired cytokinesis and down-regulated BAG3 and Mcl-1 levels. Additionally, autophagic responses including Akt-mTOR pathway inhibition, formation of acridine orange-stainable acidic vesicular organelles, LC3-I/II conversion, and p62/SQSTM1 down-regulation were detected regardless of Bcl-xL overexpression. The autophagy inhibitors 3-methyladenine and LY294002 enhanced MiTMAB-induced apoptotic sub-G1 peak, BAG3 and Mcl-1 down-regulation, Bak activation, Δψm loss, and caspase activation. These results indicate that MiTMAB-caused cytokinesis failure leads to concomitant induction of apoptosis and cytoprotective autophagy, and suggest that inhibition of autophagy is a promising strategy to augment antitumor activity of MiTMAB.

Cell death-inducing DFF45-like effect (CIDE) domain-containing proteins, DFF40, DFF45, CIDE-A, CIDE-B, and FSP27, play important roles in apoptotic DNA fragmentation and lipid homeostasis. The function of DFF40/45 in apoptotic DNA fragmentation is mediated by CIDE domain filament formation. Although our recent structural study of DREP4 CIDE revealed the first filament-like structure of the CIDE domain and its functional importance, the filament structure of DREP2 CIDE is unclear because this structure was not helical in the asymmetric unit. In this study, we present the crystal structure and mutagenesis analysis of the DREP2 CIDE mutant, which confirmed that DREP2 CIDE also forms a filament-like structure with features differing from those of DREP4 CIDE.

Cells contain several apoptotic endonucleases, which appear to act simultaneously before and after cell death by destroying the host cell DNA. It is largely unknown how the endonucleases are being induced and whether they can regulate each other. This study was performed to determine whether apoptotic mitochondrial endonuclease G (EndoG) can regulate expression of other apoptotic endonucleases. The study showed that overexpression of mature EndoG in kidney tubular epithelial NRK-52E cells can increase expression of caspase-activated DNase (CAD) and four endonucleases that belong to DNase I group including DNase I, DNase X, DNase IL2, and DNase γ, but not endonucleases of the DNase 2 group. The induction of DNase I-type endonucleases was associated with DNA degradation in promoter/exon 1 regions of the endonuclease genes. These results together with findings on colocalization of immunostained endonucleases and TUNEL suggest that DNA fragmentation after EndoG overexpression was caused by DNase I endonucleases and CAD in addition to EndoG itself. Overall, these data provide first evidence for the existence of the integral network of apoptotic endonucleases regulated by EndoG.

The present study sought to determine the correlation between 2-methoxyestradiol (2-MeO-E2)-induced cell cycle arrest and 2-MeO-E2-induced apoptosis. Exposure of Jurkat T cell clone (JT/Neo) to 2-MeO-E2 (0.5-1.0 μM) caused G2/M arrest, Bak activation, Δψm loss, caspase-9 and -3 activation, PARP cleavage, intracellular ROS accumulation, and apoptotic DNA fragmentation, whereas none of these events except for G2/M arrest were induced in Jurkat T cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, Cdk1 phosphorylation at Thr-161 and dephosphorylation at Tyr-15, up-regulation of cyclin B1 expression, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation at Ser-159/Thr-163, and Bim phosphorylation were detected irrespective of Bcl-2 overexpression. Concomitant treatment of JT/Neo cells with 2-MeO-E2 and the G1/S blocking agent aphidicolin resulted in G1/S arrest and abrogation of all apoptotic events, including Cdk1 activation, phosphorylation of Bcl-2, Mcl-1 and Bim, and ROS accumulation. The 2-MeO-E2-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor, but not by an Aurora A kinase (AURKA), Aurora B kinase (AURKB), JNK, or p38 MAPK inhibitor. Immunofluorescence microscopic analysis revealed that 2-MeO-E2-induced mitotic arrest was caused by mitotic spindle network impairment and prometaphase arrest. Whereas 10-20 μM 2-MeO-E2 reduced the proportion of intracellular polymeric tubulin to monomeric tubulin, 0.5-5.0 μM 2-MeO-E2 increased it. These results demonstrate that the apoptogenic effect of 2-MeO-E2 (0.5-1.0 μM) was attributable to mitotic spindle defect-mediated prometaphase arrest, Cdk1 activation, phosphorylation of Bcl-2, Mcl-1, and Bim, and activation of Bak and mitochondria-dependent caspase cascade.

Exposure of human Jurkat T cells to JNK inhibitor IX (JNKi), targeting JNK2 and JNK3, caused apoptotic DNA fragmentation along with G2/M arrest, phosphorylation of Bcl-2, Mcl-1, and Bim, Δψm loss, and activation of Bak and caspase cascade. These JNKi-induced apoptotic events were abrogated by Bcl-2 overexpression, whereas G2/M arrest, cyclin B1 up-regulation, Cdk1 activation, and phosphorylation of Bcl-2 family proteins were sustained. In the concomitant presence of the G1/S blocking agent aphidicolin and JNKi, the cells underwent G1/S arrest and failed to induce all apoptotic events. The JNKi-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by the Cdk1 inhibitor. Immunofluorescence microscopic analysis revealed that mitotic spindle defect and prometaphase arrest were the underlying factors for the G2/M arrest. These results demonstrate that JNKi-induced mitochondrial apoptosis was caused by microtubule damage-mediated prometaphase arrest, prolonged Cdk1 activation, and phosphorylation of Bcl-2 family proteins in Jurkat T cells.

In this chapter we discuss methods that can be used to study apoptotic cell death in the Drosophila embryo, ovary, as well as in cultured cell lines. These methods assay various aspects of the cell death process, from mitochondrial changes to caspase activation and DNA cleavage. The assays are useful for examining apoptosis in normal development and in response to developmental perturbations and external stresses. These techniques include Acridine Orange staining, TUNEL, cleaved caspase staining, caspase activity assays and assays for mitochondrial fission and permeabilization.

The goal of this study was to determine the short-term effects of the quality (UV-A/UV-B ratio) and quantity (irradiance) of natural ultraviolet radiation (UVR) on the apoptosis levels in Yellow perch (Perca flavescens) larvae. Apoptosis, or programmed cell death, is an essential event in many physiological processes as well as in pathological conditions. Western blots were used to measure the expression of several key proteins of the apoptotic cascade, such as p53, Bax, Bcl-2, and PARP-1, whereas specific apoptotic DNA fragmentation was measured by an ELISA assay. We predicted that higher UVR exposure would be related to higher levels of apoptosis. Our results showed that specific apoptotic DNA fragmentation was reduced by visible light + UV-A as well as by visible light + UV-A and UV-B treatments although it was not significantly affected by light quantity. However, the expression of p53, Bax/Bcl-2 ratio and PARP-1 were not significantly affected in larvae by the quantity or the quality of the light after two days of exposure. Altogether our results suggest that UVR may modulate the apoptotic process in Yellow perch larvae proposing an interesting role for this stressor on the early development of living organism under natural exposure condition.

To determine whether microwave (MW) radiation induces neural cell apoptosis, differentiated PC12 cells and Wistar rats were exposed to 2.856 GHz for 5 min and 15 min, respectively, at an average power density of 30 mW/cm². JC-1 and TUNEL staining detected significant apoptotic events, such as the loss of mitochondria membrane potential and DNA fragmentation, respectively. Transmission electron microscopy and Hoechst staining were used to observe chromatin ultrastructure and apoptotic body formation. Annexin V-FITC/PI double staining was used to quantify the level of apoptosis. The expressions of Bax, Bcl-2, cytochrome c, cleaved caspase-3 and PARP were examined by immunoblotting or immunocytochemistry. Caspase-3 activity was measured using an enzyme-linked immunosorbent assay. The results showed chromatin condensation and apoptotic body formation in neural cells 6h after microwave exposure. Moreover, the mitochondria membrane potential decreased, DNA fragmentation increased, leading to an increase in the apoptotic cell percentage. Furthermore, the ratio of Bax/Bcl-2, expression of cytochrome c, cleaved caspase-3 and PARP all increased. In conclusion, microwave radiation induced neural cell apoptosis via the classical mitochondria-dependent caspase-3 pathway. This study may provide the experimental basis for further investigation of the mechanism of the neurological effects induced by microwave radiation.

Programmed nuclear death (PND) in the ciliate protozoan Tetrahymena thermophila is a novel type of autophagy that occurs during conjugation, in which only the parental somatic macronucleus is destined to die and is then eliminated from the progeny cytoplasm. Other coexisting nuclei, however, such as new micro- and macronuclei are unaffected. PND starts with condensation in the nucleus followed by apoptotic DNA fragmentation, lysosomal acidification, and final resorption. Because of the peculiarity in the process and the absence of some ATG genes in this organism, the mechanism of PND has remained unclear. In this study, we focus on the role of class III phosphatidylinositol 3-kinase (PtdIns3K, corresponding to yeast Vps34) in order to identify central regulators of PND. We identified the sole Tetrahymena thermophila ortholog (TtVPS34) to yeast Vps34 and human PIK3C3 (the catalytic subunit of PtdIns3K), through phylogenetic analysis, and generated the gene knockdown mutant for functional analysis. Loss of TtVPS34 activity prevents autophagosome formation on the parental macronucleus, and this nucleus escapes from the lysosomal pathway. In turn, DNA fragmentation and final resorption of the nucleus are drastically impaired. These phenotypes are similar to the situation in the ATG8Δ mutants of Tetrahymena, implying an inextricable link between TtVPS34 and TtATG8s in controlling PND as well as general macroautophagy. On the other hand, TtVPS34 does not appear responsible for the nuclear condensation and does not affect the progeny nuclear development. These results demonstrate that TtVPS34 is critically involved in the nuclear degradation events of PND in autophagosome formation rather than with an involvement in commitment to the death program.

FSP27 (CIDE-3 in humans) plays critical roles in lipid metabolism and apoptosis and is known to be involved in regulation of lipid droplet (LD) size and lipid storage and apoptotic DNA fragmentation. Given that CIDE-containing proteins including FSP27 are associated with many human diseases including cancer, aging, diabetes, and obesity, studies of FSP27 and other CIDE-containing proteins are of great biological importance. As a first step toward elucidating the molecular mechanisms of FSP27-mediated lipid droplet growth and apoptosis, we report the crystal structure of the CIDE-N domain of FSP27 at a resolution of 2.0 Å. The structure revealed a possible biologically important homo-dimeric interface similar to that formed by the hetero-dimeric complex, CAD/ICAD. Comparison with other structural homologues revealed that the PB1 domain of BEM1P, ubiquitin-like domain of BAG6 and ubiquitin are structurally similar proteins. Our homo-dimeric structure of the CIDE-N domain of FSP27 will provide important information that will enable better understanding of the function of FSP27.

Midazolam is a widely used anesthetic of the benzodiazepine class that has shown cytotoxicity and apoptosis-inducing activity in neuronal cells and lymphocytes. This study aims to evaluate the effect of midazolam on growth of K562 human leukemia cells and HT29 colon cancer cells. The in vivo effect of midazolam was investigated in BALB/c-nu mice bearing K562 and HT29 cells human tumor xenografts. The results show that midazolam decreased the viability of K562 and HT29 cells by inducing apoptosis and S phase cell-cycle arrest in a concentration-dependent manner. Midazolam activated caspase-9, capspase-3 and PARP indicating induction of the mitochondrial intrinsic pathway of apoptosis. Midazolam lowered mitochondrial membrane potential and increased apoptotic DNA fragmentation. Midazolam showed reactive oxygen species (ROS) scavenging activity through inhibition of NADPH oxidase 2 (Nox2) enzyme activity in K562 cells. Midazolam caused inhibition of pERK1/2 signaling which led to inhibition of the anti-apoptotic proteins Bcl-XL and XIAP and phosphorylation activation of the pro-apoptotic protein Bid. Midazolam inhibited growth of HT29 tumors in xenograft mice. Collectively our results demonstrate that midazolam caused growth inhibition of cancer cells via activation of the mitochondrial intrinsic pathway of apoptosis and inhibited HT29 tumor growth in xenograft mice. The mechanism underlying these effects of midazolam might be suppression of ROS production leading to modulation of apoptosis and growth regulatory proteins. These findings present possible clinical implications of midazolam as an anesthetic to relieve pain during in vivo anticancer drug delivery and to enhance anticancer efficacy through its ROS-scavenging and pro-apoptotic properties.

Apoptosis is programmed cell death that normally occurs during development and aging in multicellular animals. Apoptosis also occurs as a defense mechanism against disease or harmful external agents. It can be initiated by a variety of stimuli including viruses and viral proteins. Canine parvovirus type 2 (CPV-2) that causes acute disease in dogs has been found to induce cell cycle arrest and DNA damage leading to cellular lysis. Though non structural protein 1 (NS1) of many parvoviruses has been found to be apoptotic, no report on the apoptotic potential of NS1 of CPV-2 (CPV-2.NS1) exists. In this study, we evaluated the apoptotic potential of CPV-2.NS1 in HeLa cells. CPV-2.NS1 has been found to induce apoptosis which was evident through characteristic DNA fragmentation, increase in hypodiploid cell count, phosphatidyl serine translocation and activation of caspase-3. Increase in caspase-3 activity and no change in p53 activity with time in CPV-2.NS1 expressing HeLa cells showed the induction of apoptosis to be caspase dependent and p53 independent.

Cancer stem cells (CSCs) are believed to be responsible for tumor metastasis, recurrence, and high mortality of cancer patients due to their high tumorigenicity resistance to chemo-radiotherapy. Morusin possesses anti-cancer activity through attenuation of NF-κB activity, which is up-regulated in cancer stem cells. The purpose of this study is to confirm the growth and migration inhibition effect of morusin on human cervical CSCs, and to clarify its partial mechanism of activity. Human cervical CSCs were enriched using non-adhesive culture system. Their stemness characteristics were identified with tumor sphere formation, self-renewal, toluidine blue staining, migration assays, RT-PCR analysis, and immunofluorescence staining of putative stem cell markers, Oct4, SOX2, and ALDH1; the epithelial-to-mesenchymal (EMT) transition markers and relevant transcription factors were evaluated with Western blotting. The growth and migration inhibition effects of morusin on human cervical CSCs were tested by cell proliferation, tumor sphere formation, and transwell assay; apoptotic death of human cervical CSCs in response to morusin was measured with DAPI staining, apoptotic DNA fragmentation; NF-κBp65, Bcl-2, Bax, and caspase-3 protein expressions were detected through Western blotting. Under this non-adhesive culture system, typical tumor spheres appeared within 5-7 days, the tumor sphere formation, self-renewal, and cell migration, expressions of putative stem cell markers, EMT markers, and relevant transcription factors of the tumor sphere cells were increased significantly. After morusin treatment, the proliferation, tumor sphere formation, and migration of human cervical CSCs were decreased significantly, DAPI-stained apoptotic cells increased, apoptotic DNA fragmentations formed evidently; the expression levels of NF-κBp65 and Bcl-2 decreased significantly, Bax, and caspase-3 increased significantly in a dose-dependent manner. Using the non-adhesive culture system, human cervical CSCs were enriched and expanded. Morusin has the potential to target and kill CSCs, and can inhibit human cervical growth and migration through NF-κB attenuation mediated apoptosis induction.

Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD(-/-) cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3'-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3'-OH ends in single-strand rather than double-strand DNA nicks/breaks.

Objective The present study aimed to elucidate the cell death mechanism in Leishmania donovani upon treatment with KalsomeTM10, a new liposomal amphotericin B. Methodology/Principal findings We studied morphological alterations in promastigotes through phase contrast and scanning electron microscopy. Phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and disruption of mitochondrial integrity was determined by flow cytometry using annexinV-FITC, JC-1 and mitotraker, respectively. For analysing oxidative stress, generation of H2O2 (bioluminescence kit) and mitochondrial superoxide O2− (mitosox) were measured. DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) and DNA laddering assay. We found that KalsomeTM10 is more effective then Ambisome against the promastigote as well as intracellular amastigote forms. The mechanistic study showed that KalsomeTM10 induced several morphological alterations in promastigotes typical of apoptosis. KalsomeTM10 treatment showed a dose- and time-dependent exposure of PS in promastigotes. Further, study on mitochondrial pathway revealed loss of mitochondrial membrane potential as well as disruption in mitochondrial integrity with depletion of intracellular pool of ATP. KalsomeTM10 treated promastigotes showed increased ROS production, diminished GSH levels and increased caspase-like activity. DNA fragmentation and cell cycle arrest was observed in KalsomeTM10 treated promastigotes. Apoptotic DNA fragmentation was also observed in KalsomeTM10 treated intracellular amastigotes. KalsomeTM10 induced generation of ROS and nitric oxide leads to the killing of the intracellular parasites. Moreover, endocytosis is indispensable for KalsomeTM10 mediated anti-leishmanial effect in host macrophage. Conclusions KalsomeTM10 induces apoptotic-like cell death in L. donovani parasites to exhibit its anti-leishmanial function.

DNA damage is one of the common consequences of exposure to various stress conditions. Different methods have been developed to accurately assess DNA damage and fragmentation in cells and tissues exposed to different stress agents. However, owing to the presence of firm cellulosic cell wall and phenolics, plant cells and tissues are not easily amenable to be subjected to these assays. Here, we describe an optimized TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay-based protocol to determine the extent of DNA fragmentation and programmed cell death in plant root cells subjected to various stress conditions. The method described here has the advantages of simplicity, reliability and reproducibility.

Lysosomal DNase II in phagocytic digestion produces DNA ends with 3'PO4/5'OH, which differ from those created in apoptotic DNA fragmentation, and can be used to label phagocytic clearance of cell death. Here, we describe the use of these specific DNA ends as selective markers of phagocytic reaction in cell suspensions. The approach does not require cell fixation. It selectively labels blunt-ended DNA breaks with terminal 5'OH. The detection is performed by ultra-fast FRET probes in a single step, closed-tube procedure. It takes 3 min and is signaled by fluorescence. The full step-by-step protocol is presented as well as instructions on analysis and representation of the results.The described DNA-end-based phagocytosis marker and the new rapid FRET assay can be useful in studies of phagocytosis, apoptosis and in immune system assessments.

Previous study has found that a new nitroxyl spin-labeled derivative of podophyllotoxin, 4-[4"-(2",2",6",6"-tetramethyl-1"-piperidinyloxy)amino]-4'-demethyl-epipodophyllotoxin (GP7), can induce apoptosis in human leukemia cells. However, there have been no studies about the effects of GP7 on osteosarcoma (OS) cells. Here, we observed the anti-OS effects of GP7 in mouse and human OS cells with the comparison of etoposide. GP7 and etoposide inhibited the proliferation of a panel of mouse and human OS cells in a concentration- or time-dependent manner, and the inhibitory effect of GP7 on the proliferation of mouse LM8 or human U2OS cells was 1.28- or 1.35-fold higher than that of etoposide. GP7 or etoposide augmented the anti-OS effects of methotrexate, adriamycin, cisplatin, or their combination, and the combined inhibitory effects of GP7 with MTX on the proliferation of LM8 cells was higher than those of etoposide with MTX. GP7 arrested the cell cycle in S phase but etoposide in G(2)/M phase. GP7 or etoposide induced sub-G(1) peak, apoptotic DNA fragmentation, activations of caspase-3, -8, -9, and DNA fragmentation factor, downregulation of Bcl-2 and Bcl-xL, upregulation of Bax and Bak, and cytochrome-c release from mitochondria in both mouse and human OS cells. GP7 or etoposide also induced endonuclease G translocation from mitochondria into cytosol in mouse cells. GP7- or etoposide-induced apoptotic DNA fragmentation of human OS cells was inhibited by the pan caspase inhibitor and caspase-9 inhibitor, not by caspase-8 inhibitor whereas it was not inhibited by the pan caspase inhibitor in mouse OS cells. Our findings indicate that GP7 is effective against mouse and human OS cells in vitro. The apoptotic DNA fragmentation in mouse OS cells may be mediated by caspase-independent pathway with the involvement of endonuclease G whereas in human OS cells by caspase-9-dependent pathway downstream of the cytochrome-c-initiated caspase cascade.

DNA fragmentation is common phenomenon for apoptotic cell death. DNA fragmentation factor, called DFF40 (CAD: mouse homologue), is a main nuclease for apoptotic DNA fragmentation. Nuclease activity of DFF40 is normally inhibited by DFF45 by tight interaction via CIDE domain without apoptotic stimuli. Once effector caspase is activated during apoptosis signaling, it cleave DFF45, allowing DFF40 to enter the nucleus and cleave chromosomal DNA. Unlike mammalian system, apoptotic DNA fragmentation in the fly might be controlled by four DFF-related proteins, known as Drep1, Drep2, Drep3 and Drep4. Although the function of Drep1 and Drep4 is well known as DFF45 and DFF40 homologues, respectively, the function of Drep2 and Drep3 is still unclear. DFF-related proteins contain a conserved CIDE domain of ~90 amino acid residues that is involved in protein-protein interaction. Here, we showed that Drep1 directly bind to Drep2 as well as Drep4 via CIDE domain. In addition, we found that the interaction of Drep2 and Drep4 to Drep1 was not competitive indicating that Drep2 and Drep4 bind different place of Drep1. All together, we suggest that Drep1 might be involved in apoptotic DNA fragmentation of fly system by direct interaction with Drep2 as well as Drep4.

Caspase-activated DNase (CAD) is the most favorable candidate for chromatin degradation during apoptosis. Ca2+-dependent endonucleases are equally important in internucleosomal DNA fragmentation (INDF), including the PARP-1-regulated DNAS1L3. Despite the elaborate work on these endonucleases, the question of whether these enzymes cooperate during INDF was not addressed. Here, we show a lack of correlation between INDF and CAD expression levels and inactivation by cleavage of its inhibitor (ICAD) during apoptosis. The cells that failed to induce INDF accumulated large amounts of 50-kb breaks, which is suggestive of incomplete chromatin processing. Similarly, INDF was blocked by Ca2+ chelation without a block in ICAD cleavage or caspase-3 activation, which is consistent with the involvement of CAD in 50-kb DNA fragmentation and its Ca2+ independence. However, DNAS1L3 expression in INDF-deficient cells promoted INDF during apoptosis and was blocked by Ca2+ chelation. Interestingly, expression of DNAS1L3 in ICAD-deficient cells failed to promote tumor necrosis factor α-induced INDF but required the coexpression of ICAD. These results suggest a cooperative activity between CAD and DNAS1L3 to accomplish INDF. In HT-29 cells, endogenous DNAS1L3 localized to the endoplasmic reticulum (ER) and translocated to the nucleus upon apoptosis induction but prior to INDF manifestation, making it the first reported Ca2+-dependent endonuclease to migrate from the ER to the nucleus. The nuclear accumulation of DNAS1L3, but not its exit out of the ER, required the activity of cysteine and serine proteases. Interestingly, the endonuclease accumulated in the cytosol upon inhibition of serine, but not cysteine, proteases. These results exemplify the complexity of chromatin degradation during apoptosis. Background: The mechanism by which apoptotic internucleosomal DNA fragmentation occurs remains unclear. Results: CAD and DNAS1L3 cooperate to process chromatin degradation during apoptosis. DNAS1L3 achieves such function by translocating from the ER to the nucleus. Conclusion: The results provide new insight on the mechanism by which chromatin degradation takes place during apoptosis. Significance: Our results exemplify the complexity of chromatin degradation during apoptosis.

The in vitro anticancer activity of the dinuclear trithiolato-bridged arene ruthenium complex diruthenium-1 (DiRu-1) was evaluated against a panel of human cancer cell lines used as in vitro models for hepatocellular carcinoma (HepG2 cells), estrogen-responsive breast adenocarcinoma (MCF-7 cells), and triple-negative breast adenocarcinoma (MDA-MB-231 cells). DiRu-1 is highly cytotoxic to these cell lines, demonstrating half-maximal inhibitory concentrations (IC50) in the low-nanomolar range (77±1.4 to 268.2±4.4 nm). The main molecular mechanisms responsible for the high cytotoxicity of DiRu-1 against the most responsive MCF-7 cell line (IC50=77±1.4 nm) were investigated on the basis of the capacity of DiRu-1 to induce oxidative stress, apoptosis, and DNA damage, and to inhibit the cell cycle and proliferation. The results show that DiRu-1 triggers caspase-dependent apoptosis in MCF-7 cells on both the intrinsic and extrinsic pathways. Moreover, the Ru complex also causes necrosis, mitotic catastrophe, and autophagy. DiRu-1 increases the intracellular levels of reactive oxygen species (ROS), which play a significant role in its cytotoxicity and pro-apoptotic activity. An important mechanism of the anticancer activity of DiRu-1 appears to be the induction of DNA lesions, mainly due to apoptotic DNA fragmentation and cell-cycle arrest at the G2/M checkpoint. These changes are correlated with the concentration of DiRu-1, the duration of the cell treatment, and the post-treatment time.

Serum endonucleases are essential for degrading the chromatin released from dead cells and preventing autoimmune diseases such as systemic lupus erythematosus. Serum DNase I is known as the major endonuclease, but recently, another endonuclease, DNase γ/DNase I-like 3, gained attention. However, the precise role of each endonuclease, especially that of DNase γ, remains unclear. In this study, we distinguished the activities of DNase γ from those of DNase I in mouse serum and concluded that both cooperated in degrading DNA during necrosis: DNase γ functions as the primary chromatolytic activity, causing internucleosomal DNA fragmentation, and DNase I as the secondary one, causing random DNA digestion for its complete degradation. These results were confirmed by two in vivo experimental mouse models, in which necrosis was induced, acetaminophen-induced hepatic injury and streptozotocin-induced β-cell necrosis models. We also determined that DNase γ functions as a backup endonuclease for caspase-activated DNase (CAD) in the secondary necrosis phase after γ-ray-induced apoptosis in vivo.

The DNase domain-containing protein TATDN1 is a conserved nuclease in both prokaryotes and eukaryotes. It was previously implicated to play a role in apoptotic DNA fragmentation in yeast and C. elegans. However, its biological function in higher organisms, such as vertebrates, is unknown. Here, we report that zebrafish TATDN1 (zTATDN1) possesses a novel endonuclease activity, which first makes a nick at the DNA duplex and subsequently converts the nick into a DNA double-strand break in vitro. This biochemical property allows zTATDN1 to catalyze decatenation of catenated kinetoplast DNA to produce separated linear DNA in vitro. We further determine that zTATDN1 is predominantly expressed in eye cells during embryonic development. Knockdown of TATDN1 in zebrafish embryos results in an abnormal cell cycle progression, formation of polyploidy and aberrant chromatin structures. Consequently, the TATDN1-deficient morphants have disordered eye cell layers and significantly smaller eyes compared with the WT control. Altogether, our current studies suggest that zTATDN1 plays an important role in chromosome segregation and eye development in zebrafish.

Treatment of human promyelocytic leukemia HL-60 cells with apigeninidin could induce cytotoxicity (IC 50 = ∼80 μM), along with apoptotic sub-G 1 cells, TUNEL-positive apoptotic DNA fragmentation, activation of the multidomain pro-apoptotic Bcl-2 proteins (Bak and Bax), mitochondrial membrane potential (Δ ψ m ) loss, release of mitochondrial cytochrome c and AIF into the cytoplasm, activation of caspase-9, -3, -8, and -7, and cleavage of PARP and lamin B. These induced apoptotic events were accompanied by decrease of Bcl-2 level and increase of Bak and Bax levels. Apigeninidin-induced sub-G 1 cells and activation of Bak and Bax were also detected in human acute leukemia Jurkat T cells, but not in Jurkat T cells overexpressing Bcl-2. Pretreatment of HL-60 cells with the pan-caspase inhibitor z-VAD-fmk reduced significantly apigeninidin-induced sub-G 1 cells and caspase cascade activation, whereas it failed to suppress Bak and Bax activations, Δ ψ m loss, and release of mitochondrial cytochrome c and AIF. None of FADD and caspase-8 deficiencies affected the sensitivity of Jurkat T cells to apigeninidin-induced cytotoxicity. These results demonstrated that apigeninidin-induced apoptosis was mediated by activation of Bak and Bax, mitochondrial damage and resultant release of not only cytochrome c , causing caspase cascade activation, but also caspase-independent death effector AIF in HL-60 cells.

In plants, different forms of programmed cell death (PCD) have been identified, but they only partially correspond to those described for animals, which is most probably due to structural differences between animal and plant cells. Here, the results show that in tobacco BY-2 cells, bleomycin (BLM), an inducer of double-strand breaks (DSBs), triggers a novel type of non-apoptotic PCD with paraptotic-like features. Analysis of numerous PCD markers revealed an extensive vacuolization, vacuolar rupture, and chromatin condensation, but no apoptotic DNA fragmentation, fragmentation of the nuclei, or sensitivity to caspase inhibitors. BLM-induced PCD was cell cycle regulated, occurring predominantly upon G(2)/M cell cycle checkpoint activation. In addition, this paraptotic-like PCD was at least partially inhibited by caffeine, a known inhibitor of DNA damage sensor kinases ATM and ATR. Interestingly, overexpression of one NtE2F transcriptional factor, whose homologues play a dual role in animal apoptosis and DNA repair, reduced PCD induction and modulated G(2)/M checkpoint activation in BY-2 cells. These observations provide a solid ground for further investigations into the paraptotic-like PCD in plants, which might represent an ancestral non-apoptotic form of PCD conserved among animals, protists, and plants.

The apoptogenic effect of p-coumaric acid, a phenolic acid found in various edible plants, on human acute leukemia Jurkat T cells was investigated. Exposure of Jurkat T cells to p-coumaric acid (50-150 μM) caused cytotoxicity and TdT-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic DNA fragmentation along with Bak activation, Δψm loss, activation of caspase-9, -3, -7, and -8, and PARP degradation in a dose-dependent manner. However,these apoptotic events were completely abrogated in Jurkat T cells overexpressing Bcl-2.Under these conditions, necrosis was not accompanied. Pretreatment of the cells with the pan-caspase inhibitor (z-VAD-fmk) could prevent p-coumaric acid-induced sub-G1 peak representing apoptotic cells, whereas it failed to block Δψm loss, indicating that the activation of caspase cascade was prerequisite for p-coumaric acid-induced apoptosis as a downstream event of Δψm loss. FADD- and caspase-8-positive wild-type Jurkat T cell clone A3, FADD-deficient Jurkat T cell clone I2.1, and caspase-8-deficient Jurkat T cell clone I9.2 exhibited similar susceptibilities to the cytotoxicity of p-coumaric acid, excluding an involvement of Fas/FasL system in triggering the apoptosis. The apoptogenic activity of p-coumaric acid is more potent in malignant Jurkat T cells than in normal human peripheral T cells. Together, these results demonstrated that p-coumaric acid-induced apoptogenic activity in Jurkat T cellswas mediated by Bak activation, Δψm loss, and subsequent activation of multiple caspases such as caspase-9, -3, -7, and-8, and PARP degradation, which could be regulated by anti-apoptotic protein Bcl-2.

DNA double strand breaks (DSBs) induced by etoposide, an inhibitor of DNA topoisomerase II, are repaired mainly by non-homologous end joining (NHEJ). Unexpectedly, it was found that at high doses of etoposide, proteins involved in NHEJ, such as KU70/80, DNA-PKcs and ARTEMIS/SNM1C, trigger apoptosis rather than repair of DSBs. Because ARTEMIS is a member of the SNM1 protein family that includes SNM1A and APOLLO/SNM1B, this study examined whether SNM1A and/or APOLLO are also involved in etoposide-induced apoptosis. Using SNM1A(-/-) and APOLLO(-/-) cells, it was found that both SNM1A and APOLLO participate in etoposide-induced apoptosis. Although cell viability monitored by MTT assay did not differ between SNM1A(-/-)/APOLLO(-/-)/ARTEMIS(-/-), SNM1A(-/-)/APOLLO(-/-), and single gene knockout cells, DNA fragmentation monitored by TUNEL assay differed between these cells, suggesting that the three SNM1 family nucleases function independently, at least during the induction of apoptotic DNA fragmentation.

The inhibitory mechanism of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (CMEP-NQ) against apoptosis induced by the microtubule-damaging agents (MDAs), nocodazole (NOC) and 2-methoxyestradiol (2-MeO-E2), or a DNA-damaging agent (DDA), camptothecin (CPT) were investigated in human Jurkat T cell clones (J/Neo and J/BCL-XL cells). Treatment of J/Neo cells with NOC, 2-MeO-E2, or CPT caused cytotoxicity and apoptotic DNA fragmentation but these events were significantly attenuated in the presence of CMEP-NQ. Although not only MDA (NOC or 2-MeO-E2)-induced mitotic arrest, CDK1 activation, and BCL-2, BCL-XL and BIM phosphorylation, but also DDA (CPT)-induced S-phase arrest and ATM-CHK1/CHK2-p53 pathway activation were not or were barely affected in the presence of CMEP-NQ, the levels of anti-apoptotic BAG3 and MCL-1, which were markedly downregulated after MDA- or DDA-treatment, were rather elevated by CMEP-NQ. Under the same conditions, MDA- or DDA-induced mitochondrial apoptotic events including BAK activation, mitochondrial membrane potential (Δψm) loss, caspase-9 activation, and PARP cleavage were significantly inhibited by CMEP-NQ. While MDA- or DDA-induced sub-G1 peak and Δψm loss were abrogated in J/BCL-XL cells, MDA-induced mitotic arrest and DDA-induced S-arrest were more apparent in J/BCL-XL cells than in J/Neo cells. Simultaneously, the induced cell cycle arrest in J/BCL-XL cells was not significantly disturbed by CMEP-NQ. MDA- or DDA-treatment caused intracellular reactive oxygen species (ROS) production; however, MDA- or DDA-induced ROS production was almost completely abrogated in J/BCL-XL cells. MDA- or DDA-induced ROS production in J/Neo cells was significantly suppressed by CMEP-NQ, but the suppressive effect was hardly observed in J/BCL-XL cells. Together, these results show that CMEP-NQ efficiently protects Jurkat T cells from apoptotic cell death via the elevation of BAG3 and MCL-1 levels, which results in the inhibition of intrinsic BAK-dependent mitochondrial apoptosis pathway, as does the overexpression of BCL-XL.

Phenethyl isothiocyanate (PEITC), a constituent of edible cruciferous vegetables such as watercress, not only affords significant protection against chemically induced cancer in experimental rodents but also inhibits growth of human cancer cells by causing apoptotic and autophagic cell death. However, the underlying mechanism of PEITC-induced cell death is not fully understood. Using LNCaP and PC-3 human prostate cancer cells as a model, we demonstrate that the PEITC-induced cell death is initiated by production of reactive oxygen species (ROS) resulting from inhibition of oxidative phosphorylation (OXPHOS). Exposure of LNCaP and PC-3 cells to pharmacologic concentrations of PEITC resulted in ROS production, which correlated with inhibition of complex III activity, suppression of OXPHOS, and ATP depletion. These effects were not observed in a representative normal human prostate epithelial cell line (PrEC). The ROS production by PEITC treatment was not influenced by cyclosporin A. The Rho-0 variants of LNCaP and PC-3 cells were more resistant to PEITC-mediated ROS generation, apoptotic DNA fragmentation, and collapse of mitochondrial membrane potential compared with respective wild-type cells. The PEITC treatment resulted in activation of Bax in wild-type LNCaP and PC-3 cells, but not in their respective Rho-0 variants. Furthermore, RNA interference of Bax and Bak conferred significant protection against PEITC-induced apoptosis. The Rho-0 variants of LNCaP and PC-3 cells also resisted PEITC-mediated autophagy. In conclusion, the present study provides novel insight into the molecular circuitry of PEITC-induced cell death involving ROS production due to inhibition of complex III and OXPHOS.

Leukemia inhibitory factor (LIF) is an important regulator of skeletal muscle regeneration and has been suggested to be mitogenic for myogenic cells because it has been shown to increase the quantity of myoblast cells grown in culture over extended periods of time. Using the established C2C12 murine myoblast cell line, we observed that LIF treatment did not significantly increase the rate at which myoblasts synthesise DNA under conditions which increased cell quantity by 73% above control, whilst the known mitogen fibroblast growth factor-2 significantly increased DNA synthesis under these conditions. Consequently, we examined the capacity of LIF to prevent apoptotic cell death. LIF treatment significantly reduced staurosporine-induced apoptotic DNA fragmentation by 37% compared to control and also reduced the proteolytic activation of caspase-3 by 40% compared to control. This effect of LIF was completely abolished by addition of the phosphatidylinositol 3-kinase inhibitor wortmannin, indicating that the phosphatidylinositol 3-kinase signalling pathway, previously shown to be linked to LIF-dependent increases in cell number, is necessary in mediating the anti-apoptotic effects of LIF. LIF treatment was also associated with increased levels of Bcl-xL and XIAP transcripts compared to control. Therefore, we suggest that the role of LIF in skeletal muscle regeneration and myogenesis is that of a survival factor rather than a mitogen.

Histone H2B phosphorylation at Serine 14 (phosS14) has been proposed as an epigenetic marker of apoptotic cells, whereas acetylation at the adjacent Lysine 15 (acK15) is a property of non-dying cells. We investigated the relationship and the potential regulatory mechanisms between these two epigenetic histone modifications and internucleosomal DNA degradation during apoptosis. Using rat primary thymocytes induced to undergo apoptosis with glucocorticoids we found that H2B phosphorylated at Ser14 was associated with soluble, cleaved DNA in apoptotic nuclei. In contrast acK15 was prevalent in non-apoptotic nuclei and scarce in apoptotic nuclei. This switch between K15 acetylation and S14 phosphorylation on H2B was also observed in apoptotic thymocytes from animals treated in vivo with glucocorticoids and in a rat hepatoma cell line (HTC) induced to die by UV-C or Fas ligand. It is interesting to note that the combined use of a histone deacetylase inhibitor and glucocorticoid suppressed both S14 phosphorylation and internucleosomal DNA degradation without inhibiting apoptosis in thymocytes. Using synthetic peptides and a PKC phosphorylation assay system, we show that the deacetylation of K15 was necessary to allow the S14 phosphorylation. These findings suggest that selective chromatin post-translational modifications are associated with DNA degradation during apoptosis.

In the present study, we investigated the effect of 3alpha,23-isopropylidenedioxyolean-12-en-27-oic acid (IPA), an active compound isolated from Aceriphyllum rossii, on the apoptotic activity and the molecular mechanism of the action in human cervical cancer HeLa cells. Treatment with IPA significantly increased externalization of phosphatidylserine residues and apoptotic DNA fragmentation as shown by Annexin V staining and 4',6-diamidino-2-phenylindole-dihydrochloride (DAPI) staining, respectively. In addition, IPA induced the activations of caspase-8, -9, -3, and cleavage of poly(ADP ribose) polymerase (PARP-1) in HeLa cells. Pretreatment with a specific caspase-8, -9, or -3 inhibitor neutralized the pro-apoptotic activity of IPA in HeLa cells. Furthermore, IPA was found to induce the loss of mitochondrial membrane potential, the release of cytochrome c to the cytosol, and the increased ratio of mitochondrial Bax/Bcl-2. Moreover, we demonstrated that IPA triggered endoplasmic reticulum (ER) stress, as shown by changes in cytosol-calcium level, activation of mu-calpain and caspase-12, and up-regulation of glucose-regulated protein 78 (GRP78) and growth arrest DNA damage-inducible gene 153 (GADD153). IPA-induced apoptosis was substantially reduced in the presence of an intracellular calcium chelator BAPTA/AM. Taken together, these results suggest that both mitochondrial dysfunction and ER stress contribute to IPA-induced apoptosis of human cervical cancer HeLa cells.

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) which is being introduced into clinic for the treatment of hematological diseases. We studied the effect of this compound on six human hematopoietic cell lines (JURL-MK1, K562, CML-T1, Karpas-299, HL-60, and ML-2) as well as on normal human lymphocytes and on leukemic primary cells. SAHA induced dose-dependent and cell type-dependent cell death which displayed apoptotic features (caspase-3 activation and apoptotic DNA fragmentation) in most cell types including the normal lymphocytes. At subtoxic concentrations (0.5–1 µM), SAHA increased the cell adhesivity to fibronectin (FN) in all leukemia/lymphoma-derived cell lines but not in normal lymphocytes. This increase was accompanied by an enhanced expression of integrin β1 and paxillin, an essential constituent of focal adhesion complexes, both at the protein and mRNA level. On the other hand, the inhibition of ROCK protein, an important regulator of cytoskeleton structure, had no consistent effect on SAHA-induced increase in the cell adhesivity. The promotion of cell adhesivity to FN seems to be specific for SAHA as we observed no such effects with other HDAC inhibitors (trichostatin A and sodium butyrate). J. Cell. Biochem. 109: 184–195, 2010. © 2009 Wiley-Liss, Inc.

A major hallmark of the terminal stages of apoptosis is the internucleosomal DNA fragmentation. The endonuclease responsible for this type of DNA degradation is the DNA fragmentation factor (DFF). DFF is a complex of the endonuclease DFF40 and its chaperone/inhibitor, DFF45. In vitro work has shown that histone H1 and HMGB1/2 recruit/target DFF40 to the internucleosomal linker regions of chromatin and that histone H1 directly interacts with DFF40 conferring DNA binding ability and enhancing its nuclease activity. The histone H1 family is comprised of many subtypes, which recent work has shown may have distinct roles in chromatin function. Thus we studied the binding association of DFF40 with specific H1 subtypes and whether these binding associations are altered after the induction of apoptosis in an in vivo cellular context. The apoptotic agent used in this study is the histone deacetylase inhibitor, trichostatin A (TSA). We separated the insoluble chromatin-enriched fraction from the soluble nuclear fraction of the NB4 leukemic cell line. Using MNase digestion, we provide evidence which strongly suggests that the heterodimer, DFF40-DFF45, is localized to the chromatin fraction under apoptotic as well as non-apoptotic conditions. Moreover, we present results that show that DFF40 interacts with the all H1 subtypes used in this study, but preferentially interacts with specific H1 subtypes after the induction of apoptosis by TSA. These results illustrate for the first time the association of DFF40 with individual H1 subtypes, under a specific apoptotic stimulus in an in vivo cellular context.

Daily injection of parathyroid hormone (PTH) is a clinically approved treatment for osteoporosis. It suppresses apoptosis of bone-forming osteoblasts although its exact anti-apoptotic mechanism(s) is incompletely understood. In this study, PTH treatment of cultured osteoblasts blocked the pro-apoptotic effects of serum withdrawal and nutrient deprivation; hydrogen peroxide induced oxidative stress, and UV irradiation. We hypothesized that PTH might suppress osteoblast apoptosis by enhancing DNA repair. Evidence is provided showing that post-confluent, non-proliferating osteoblasts treated with PTH exhibited a protein kinase A-mediated activation of two proteins that regulate DNA repair processes (proliferating cell nuclear antigen and forkhead box transcription factor 3a) as well as a suppression of the pro-apoptotic growth arrest and DNA damage protein 153. Additional proof of a connection between DNA damage and osteoblast apoptosis came from an unexpected finding whereby a majority of fixed PTH-treated osteoblasts scored weakly positive for Terminal Deoxynucleotidyl dUTP Nick-End Labeling (TUNEL), even though similar cultures were determined to be viable via a trypsin replating strategy. TUNEL identifies DNA excision repair, not just apoptotic DNA fragmentation, and the most likely explanation of these TUNEL results is that PTH's activation of DNA repair processes would permit nucleotide incorporation as a result of enhanced excision repair. This explanation was confirmed by an enhanced incorporation of bromodeoxyuridine in PTH-treated cells even though a majority of the cell population was determined to be non-replicating. An augmentation of DNA repair by PTH is an unreported finding, and provides an additional explanation for its anti-apoptotic mechanism(s).

The time-course of programmed cell death (apoptosis) during reorganization of gill epithelium in salinity-stressed tilapia was analyzed using a recently developed method based on laser scanning cytometry (LSC) of dissociated gill cells. Apoptosis in mitochondria-rich cells (MRC) was distinguished from that in other cell types using Na(+)/K(+) ATPase (NKA) as a cell-specific marker. Caspase 3/7 activity in MRC, assessed using LSC and microplate assays, increased significantly starting at 6 h of salinity stress and remained elevated for at least 5 days. This time-course of apoptosis in MRC during acute salinity stress was reflected in elevated apoptotic DNA fragmentation. In parallel to induction of apoptosis, MRC showed a pronounced shift to G2 phase of the cell cycle, which is indicative of G2/M cell cycle arrest, and an increase in NKA abundance per MRC. Unlike in MRC, apoptosis was not significantly increased in other gill cell types, although there was a small transient increase in DNA fragmentation at 6 h. G2 arrest was also observed. Overall, we interpret our data as evidence for a significant role of apoptosis in the extensive reorganization of MRC populations that takes place during salinity acclimation, perhaps similar to its well-established role during organismal development.

In the present study, we investigated the effect of KPS-A on the apoptotic activity and the molecular mechanism of the action in human leukemia. Treatment with KPS-A significantly increased apoptotic DNA fragmentation in human histiocytic lymphoma U937 cells as shown by DAPI staining, flow cytometry, and agarose gel electrophoresis. In addition, stimulation of U937 cell with KPS-A induced a series of intracellular events: (1) the activations of caspase-8, caspase-9, and caspase-3; (2) the translocations of Bid and Bax proteins to mitochondria; (3) the loss of mitochondrial membrane potential; and (4) the increased release of cytochrome c to the cytosol. Pretreatment with a specific caspases-8, -9 or -3 inhibitor, neutralized the pro-apoptotic activity of KPS-A in U937 cells. We further demonstrated that KPS-A markedly induced an increase in intracellular Ca2+ level, which was reversed by EGTA, a general calcium chelator, but not by TMB-8 and dantrolene, intracellular Ca2+ release blockers. Moreover, KPS-A-induced DNA fragmentation and caspase activation were substantially reduced in the presence of EGTA. Taken together, these results suggest that KPS-A may play therapeutic role for leukemia via the potent apoptotic activity through Ca2+/caspases-8/MPT/caspases-9/caspases-3 signaling pathway.

Specific DNA fragmentation into oligonucleosomal units occurs during programmed cell death (PCD) in both animal and plant cells, usually being regarded as an indicator of its apoptotic character. This internucleosomal DNA fragmentation is demonstrated in tobacco suspension and leaf cells, which were killed immediately by freezing in liquid nitrogen, and homogenization or treatment with Triton X-100. Although these cells could not activate and realize the respective enzymatic processes in a programmed manner, the character of DNA fragmentation was similar to that in the cells undergoing typical gradual PCD induced by 50 microM CdSO4. This internucleosomal DNA fragmentation was connected with the action of cysteine proteases and the loss of membrane, in particular tonoplast, integrity. The mechanisms of DNase activation in the rapidly killed cells, hypothetical biological relevance, and implications for the classification of cell death are discussed.

Chromatin condensation and degradation of DNA into internucleosomal DNA fragments are key hallmarks of apoptosis. The phosphorylation of protein kinase ataxia telangiectasia mutated (ATM) and histone H2A.X was recently shown to occur concurrently with apoptotic DNA fragmentation. We have used immunofluorescence microscopy, Western blot analysis and alkali comet assays to show that phosphorylation of ATM in NIH3T3 fibroblasts occurs prior to apoptotic DNA fragmentation, nuclease degradation and phosphorylation of histone H2A.X in cells treated with low levels of either staurosporine (STS) or tumor necrosis factor-alpha mixed with cycloheximide (TNF-alpha/CHX). In extension to previous findings, ATM phosphorylation was associated with chromatin decondensation, i.e., by loss of dense foci of constitutive heterochromatin. These results suggest that chromatin is decondensed and that ATM is activated independently of DNA damage signaling pathways during the very early stages of apoptosis.