Peripheral Chromatin Condensation Research Articles

Use of Secondary Metabolites Derived from Aspergillus Species as Anticancer Agents and Related Histological and Genetic Alterations: In Vitro Study

The present work aimed to use natural derived fungal secondary metabolites as anticancer agents concerning cytotoxicity, apoptotic, genetic and histopathological profile. It was noticed that Asp. Terrius (Asp. T), Flavous (Asp. Fl) and Fumegatgus (Asp. Fu) induced variable toxic potential that was cell type, secondary metabolite type and concentration dependent. CaCo-2 cells showed less sensitivity than HuH-7 and in turn the IC50 was variable. Also, the apoptotic potential of Asp. species derived fungal secondary metabolites was proven via detection of up regulated pro apoptotic genes and down regulation of anti-apoptotic genes. The expression rate was cell type dependent. Concurrently apoptotic profile was accompanied with cellular DNA accumulation at the G2/M phase, an insignificant accumulation during the G0/G1 phase but there was not during the Pre-G1 and S phases. Also, there were a characteristic apoptotic features of treated cells presented as abnormal intra-nuclear eosinophyllic structures, necrotic cells with mixed euchromatin and heterochromatin, ruptured cell membranes, intranuclear eosinophyllic structures, apoptotic cells with irregular cellular and nuclear membranes, peripheral chromatin condensation and necrotic swollen cells with mixed euchromatin and heterochromatin. It can be concluded that A. secondary metabolites are promising agents can be used as a supplementary agents to current cancer drugs regimen applied.

Stony Coral Tissue Loss Disease in Florida Is Associated With Disruption of Host–Zooxanthellae Physiology

Samples from eight species of corals (Colpophyllia natans, Dendrogyra cylindrus, Diploria labyrinthiformis, Meandrina meandrites, Montastraea cavernosa, Orbicella faveolata, Pseudodiploria strigosa, and Siderastrea siderea) that exhibited gross clinical signs of acute, subacute, or chronic tissue loss attributed to stony coral tissue loss disease (SCTLD) were collected from the Florida Reef Tract during 2016–2018 and examined histopathologically. The hallmark microscopic lesion seen in all eight species was focal to multifocal lytic necrosis (LN) originating in the gastrodermis of the basal body wall (BBW) and extending to the calicodermis, with more advanced lesions involving the surface body wall. This was accompanied by other degenerative changes in host cells such as mucocyte hypertrophy, degradation and fragmentation of gastrodermal architecture, and disintegration of the mesoglea. Zooxanthellae manifested various changes including necrosis (cytoplasmic hypereosinophilia, pyknosis); peripheral nuclear chromatin condensation; cytoplasmic vacuolation accompanied by deformation, swelling, or atrophy; swollen accumulation bodies; prominent pyrenoids; and degraded chloroplasts. Polyhedral intracytoplasmic eosinophilic periodic acid–Schiff-positive crystalline inclusion bodies (∼1–10 μm in length) were seen only in M. cavernosa and P. strigosa BBW gastrodermis in or adjacent to active lesions and some unaffected areas (without surface lesions) of diseased colonies. Coccoidlike or coccobacilloidlike structures (Gram-neutral) reminiscent of microorganisms were occasionally associated with LN lesions or seen in apparently healthy tissue of diseased colonies along with various parasites and other bacteria all considered likely secondary colonizers. Of the 82 samples showing gross lesions of SCTLD, 71 (87%) were confirmed histologically to have LN. Collectively, pathology indicates that SCTLD is the result of a disruption of host–symbiont physiology with lesions originating in the BBW leading to detachment and sloughing of tissues from the skeleton. Future investigations could focus on identifying the cause and pathogenesis of this process.

APOPTOTIC AND NECROTIC EFFECTS OF CHITOSAN NANOPARTICLES LOADED WITH THE HONEYBEE, APIS MELLIFERA VENOM ON DIFFERENT CANCER CELL LINES

Nanoparticles encapsulated with natural venoms have a considerable attention to improve illnesses therapies by overcoming toxic effects secreted by different pathogens. The present study explored conjugation of chitosan extracted from cuticle of naturally died honeybees nanoparticles (B-Cs-NPs) with the honeybee, Apis mellifera venom (BV). Detected related toxicity and anticancer potential of BV and BV loaded NPs- against Colon (CaCO-2), Larynx (HEp-2) and breast (MCF-7) cancer cell lines. The Inotropic gelation of chitosan (CS) with sodium tripolyphosphate (TPP) was the method used to prepare nanoparticles. Cytotoxic effects of BV and BV loaded NPs nanoparticles against the three cancer cell lines studied, then they were assessed using MTT stain assay. Data recorded revealed that combination of bee venom with bee chitosan nanoparticles enhanced the inhibitory effect against cancer cells studied when compared with bee venom alone. Also, it was cleared that by treating CaCO-2, HEp-2 & MCF-7 cell lines with IC50 concentrations of BV alone and BV loaded chitosan nanoparticles, this induced apoptotic features as cell shrinking, peripheral condensation of chromatin, apoptoticbodies in late stage, nucleoli fragmentation, membrane blebbing, shrunken nucleus with some necrotic features. Control cells showed normal pathway of mitotic divisions of obvious cell membrane and nucleus when stained by (H&E) and by visualization using inverted microscope.

Nuclear apoptotic volume decrease in individual cells: Confocal microscopy imaging and kinetic modeling

The dynamics of nuclear morphology changes during apoptosis remains poorly investigated and understood. Using 3D time-lapse confocal microscopy we performed a study of early-stage apoptotic nuclear morphological changes induced by etoposide in single living HepG2 cells. These observations provide a definitive evidence that nuclear apoptotic volume decrease (AVD) is occurring simultaneously with peripheral chromatin condensation (so called “apoptotic ring”). In order to describe quantitatively the dynamics of nuclear morphological changes in the early stage of apoptosis we suggest a general molecular kinetic model, which fits well the obtained experimental data in our study. Results of this work may clarify molecular mechanisms of nuclear morphology changes during apoptosis.

The Possible Protective Role of Melatonin on Doxorubicin Induced Cardiomyopathy of Adult Male Albino Rats

Introduction: Doxorubicin is one of the major antitumor treatment. The essential limiting factor of using this drug is the production of cardiotoxicity. However, melatonin is a powerful antioxidant that may protect the heart.Aim: This study was aimed to study possible protective role of melatonin in adult male albino rats following doxorubicin administrationMaterial and Method: In this study, 40 adult male albino rats were used. They were divided into four groups (10 rats for each): control group, Melatonin group, Doxorubicin group and Doxorubicin & Melatonin group. Heart specimens were obtained at the end and processed.Results: Light studies showed degenerative changes. Some fibers showed dark acidophilic cytoplasm and pyknotic nuclei. Apoptosis were detected where the nuclei varying from peripheral condensation of chromatin up to pyknosis, confirmed with positively caspase-3 activity. Dilatation of the vessels with mononuclear cellular infiltrations and deposited collagen fibers were seen. Ultrastructural examination showed disarrangement of the sarcomeres, disruption of microfilaments and Z- line, the number and size of mitochondria apparently increased, dilated SER and T tubules were also noticed. The presence of oval shaped cells (Telocyte) with thin long processes (Telopodes) were detected.Conclusion: From this study, it was concluded that, melatonin markedly suppressed cardiomyopathy induced by doxorubicin.

Infectious Bovine Rhinotracheitis in a Cattle Farm at Sharkia Governorate with Special Reference to its Effect on T lymphocytes

This study was applied on a cattle farm of Holstein cows at Sharkia Governorate for the isolation and identification of Bovine herpesvirus type-1 (BoHV-1) and to examine its effect on T lymphocytes. The results of clinical examination revealed that there were respiratory disorders in 30 out of 150 (20%) of cattle including elevated body temperature (40 to 42°C), nasal and ocular discharges, some animals developed severe rhinitis, conjunctivitis, corneal opacity, cough and diarrhea. Out of 30 nasal swabs, 15 swabs were positive for virus isolation as indicated by cytopothic effect (CPE) on MDBK cells. Only 11 of the 15 isolates were confirmed by virus neutralization test (VNT) as BoHV-1 isolates. In addition, only 3 out of 4 BoHV-1 isolates were detected by PCR. Peripheral blood T lymphocytes (PBTL) were analyzed using electron microscopy and comet assay to examine the effect of BoHV-1 on lymphocytes. Electron micrographs of T lymphocytes revealed peripheral condensation of chromatin and fragmentation of the nucleus end of the cell leading to the formation of apoptoic bodies. Comet assay denoted fragmentation of cellular DNA. It could be concluded that BoHV-1 can infect T lymphocytes of cattle, causing directly and indirectly apoptosis which subsequently lead to suppression of cell-mediated immunity, enhancing establishment of latency and increasing the probability for secondary bacterial infection.

Cyclosporine A prevents ex vivo PCO formation through induction of autophagy-mediated cell death

The purpose of this study was to determine the Cyclosporine A (CsA) dose and minimum drug delivery time needed to prevent posterior capsule opacification (PCO) in an ex vivo canine model and evaluate the mechanism of CsA-induced cell death. Canine lens epithelial cells (LEC) were treated with CsA and changes in cell migration, proliferation, and density were monitored over time. CsA-treated LEC underwent transmission electron microscopy (TEM), immunofluorescence, and immunoblotting in the presence or absence of autophagy inhibitors to evaluate the mechanism of cell death. Lens capsules were harvested from canine cadaver eyes for an ex vivo model of PCO. Lens capsules were treated with CsA for 1, 2, 3, 4, 5, 6, or 7 days, and subsequently maintained in culture for a total of 28 days in the absence of drug. CsA reduced LEC viability in a dose dependent manner. Morphologically, CsA-treated LEC were swollen, had intact nuclei, lacked peripheral chromatin condensation, and demonstrated prominent vacuolization; TEM revealed autophagosomes. LC3-II protein expression and acridine orange fluorescence increased in CsA-treated cells. A small non-significant induction of cleaved caspase-3 was observed in CsA-treated LEC. Lens capsules treated with 5, 6, or 7 days of 10 μg/mL CsA showed a significant decrease in ex vivo PCO formation; 6 days of drug delivery prevented PCO. This study finds that morphologic changes, formation of acidic vesicles, and increased expression of LC3-II supports the hypothesis that CsA mediates LEC death via autophagy; this is a novel finding in the lens. Induction of CsA-induced apoptosis was minimal. Six days of intracapsular CsA drug delivery prevented ex vivo PCO formation.

Differential Modulation of the Oligodendrocyte Transcriptome by Sonic Hedgehog and Bone Morphogenetic Protein 4 via Opposing Effects on Histone Acetylation

Differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes is regulated by the interplay between extrinsic signals and intrinsic epigenetic determinants. In this study, we analyze the effect that the extracellular ligands sonic hedgehog (Shh) and bone morphogenetic protein 4 (BMP4), have on histone acetylation and gene expression in cultured OPCs. Shh treatment favored the progression toward oligodendrocytes by decreasing histone acetylation and inducing peripheral chromatin condensation. BMP4 treatment, in contrast, inhibited the progression toward oligodendrocytes and favored astrogliogenesis by favoring global histone acetylation and retaining euchromatin. Pharmacological treatment or silencing of histone deacetylase 1 (Hdac1) or histone deacetylase 2 (Hdac2) in OPCs did not affect BMP4-dependent astrogliogenesis, while it prevented Shh-induced oligodendrocyte differentiation and favored the expression of astrocytic genes. Transcriptional profiling of treated OPCs, revealed that BMP4-inhibition of oligodendrocyte differentiation was accompanied by increased levels of Wnt (Tbx3) and Notch-target genes (Jag1, Hes1, Hes5, Hey1, and Hey2), decreased recruitment of Hdac and increased histone acetylation at these loci. Similar upregulation of Notch-target genes and increased histone acetylation were observed in the corpus callosum of mice infused with BMP4 during cuprizone-induced demyelination. We conclude that Shh and Bmp4 differentially regulate histone acetylation and chromatin structure in OPCs and that BMP4 acts as a potent inducer of gene expression, including Notch and Wnt target genes, thereby enhancing the crosstalk among signaling pathways that are known to inhibit myelination and repair.

Delta- and Gamma-Tocotrienols Induce Classical Ultrastructural Apoptotic Changes in Human T Lymphoblastic Leukemic Cells

Tocotrienols are isomers of the vitamin E family, which have been reported to exert cytotoxic effects in various cancer cells. Although there have been some reports on the effects of tocotrienols in leukemic cells, ultrastructural evidence of tocotrienol-induced apoptotic cell death in leukemic cells is lacking. The present study investigated the effects of three isomers of tocotrienols (alpha, delta, and gamma) on a human T lymphoblastic leukemic cell line (CEM-SS). Cell viability assays showed that all three isomers had cytotoxic effects (p < 0.05) on CEM-SS cells with delta-tocotrienol being the most potent. Transmission electron microscopy showed that the cytotoxic effects by delta- and gamma-tocotrienols were through the induction of an apoptotic pathway as demonstrated by the classical ultrastructural apoptotic changes characterized by peripheral nuclear chromatin condensation and nuclear fragmentation. These findings were confirmed biochemically by the demonstration of phosphatidylserine externalization via flow cytometry analysis. This is the first study showing classical ultrastructural apoptotic changes induced by delta- and gamma-tocotrienols in human T lymphoblastic leukemic cells.

β‐Cyclodextrin/Glycyrrhizic Acid Functionalised Quantum Dots Selectively Enter Hepatic Cells and Induce Apoptosis

The use of active components from important medical herbs has proved effective in treating various cancers. Glycyrrhizic acid (GA) is one of the many interesting triterpenoic acids with anticancerogenic potential, and is known to trigger apoptosis in hepatocarcinoma cells. In this study we combined quantum dots (QDs) with GA in the presence of β-cyclodextrin (β-CD), and prepared β-CD/GA-functionalised QDs, which led to improved antitumor activity and induced apoptosis in hepatocarcinoma cells. These compounds showed a better selectivity for hepatic cells compared to HeLa and ECV-304 cells. Hoechst and annexin V-FITC staining and mitochondrial membrane potential (MMP) experiments proved an apoptotic effect of these compounds on HepG2 cells. At the same time, transmission electron microscopy (TEM) showed obvious features of apoptosis, for example, irregularities of nuclear shapes, mitochondria swelling, clumping and peripheral chromatin condensation, zeiosis or blebbing of the plasma membrane and formation of apoptotic bodies. It is notable that β-CD/GA-functionalised QDs showed effective cell growth inhibition by triggering G0/G1 phase arrest and inducing apoptosis through an reactive oxygen species mediated mitochondrial dysfunction pathway. β-CD/GA-functionalised QDs primarily induced apoptotic response in a time- and dose-dependent manner, but little apoptosis appeared with L-Cys-β-CD-functionalised QDs or GA alone. These studies suggest that β-CD/GA-functionalised QDs have therapeutic potential against cancer.

Spermatogenesis in the marine snail Adelomelon ancilla (Volutidae) from Patagonia

This study describes euspermatogenesis and paraspermatogenesis in Adelomelon ancilla (Volutidae) and compares them with the same processes in other caenogastropods. Spermatogenesis in A. ancilla followed the general developmental pattern of caenogastropods. The pattern of nuclear condensation involves granular, fibrillar, lamellar and homogenous electron-dense phases. Acrosome development begins with the posteriorly located proacrosomal vesicle arising from the Golgi complex in euspermatids. The proacrosomal vesicle develops into a preattachment acrosome which, together with the Golgi body, later moves towards the apex of the nucleus. In the early stage of paraspermatogenesis, the pattern of chromatin distribution in dense patches, together with the cytoplasmatic characteristics of paraspermatogonia, allows the recognition of the apyrene line from the euspermatogonia. Later features, including the peripheral condensation of nuclear chromatin followed by nuclear invagination, the posterior breakdown into nuclear vesicles (‘caryomerites’), centriole multiplication and the synthesis of secretory products, are the most conspicuous changes in the process of paraspermatogenesis.

NAD+ depletion or PAR polymer formation: which plays the role of executioner in ischaemic cell death?

Multiple cell death pathways are activated in cerebral ischaemia. Much of the initial injury, especially in the core of the infarct where cerebral blood flow is severely reduced, is necrotic and secondary to severe energy failure. However, there is considerable evidence that delayed cell death continues for several days, primarily in the penumbral region. As reperfusion therapies grow in number and effectiveness, restoration of blood flow early after injury may lead to a shift towards apoptosis. It is important to elucidate what are the key mediators of apoptotic cell death after stroke, as inhibition of apoptosis may have therapeutic implications. There are two well described pathways that lead to apoptotic cell death; the caspase pathway and the more recently described caspase-independent pathway triggered by poly-ADP-ribose polymers (PARP) activation. Caspase-induced cell death is initiated by release of mitochondrial cytochrome c, formation of the cytosolic apoptosome, and activation of endonucleases leading to a multitude of small randomly cleaved DNA fragments. In contrast caspase-independent cell death is secondary to activation of apoptosis inducing factor (AIF). Mitochondrial AIF translocates to the nucleus, where it induces peripheral chromatin condensation, as well as characteristic high-molecular-weight (50 kbp) DNA fragmentation. Although caspase-independent cell death has been recognized for some time and is known to contribute to ischaemic injury, the upstream triggering events leading to activation of this pathway remain unclear. The two major theories are that ischaemia leads to nicotinamide adenine dinucleotide (NAD+) depletion and subsequent energy failure, or alternatively that cell death is directly triggered by a pro-apoptotic factor produced by activation of the DNA repair enzyme PARP. PARP activation is robust in the ischaemic brain producing variable lengths of poly-ADP-ribose (PAR) polymers as byproducts of PARP activation. PAR polymers may be directly toxic by triggering mitochondrial AIF release independently of NAD+ depletion. Recently, sex differences have been discovered that illustrate the importance of understanding these molecular pathways, especially as new therapeutics targeting apoptotic cell death are developed. Cell death in females proceeds primarily via caspase activation whereas caspase-independent mechanisms triggered by the activation of PARP predominate in the male brain. This review summarizes the current literature in an attempt to clarify the roles of NAD+ and PAR polymers in caspase-independent cell death, and discuss sex specific cell death to provide an example of the possible importance of these downstream mediators.

Carbon black and titanium dioxide nanoparticles elicit distinct apoptotic pathways in bronchial epithelial cells

BackgroundIncreasing environmental and occupational exposures to nanoparticles (NPs) warrant deeper insight into the toxicological mechanisms induced by these materials. The present study was designed to characterize the cell death induced by carbon black (CB) and titanium dioxide (TiO2) NPs in bronchial epithelial cells (16HBE14o- cell line and primary cells) and to investigate the implicated molecular pathways.ResultsDetailed time course studies revealed that both CB (13 nm) and TiO2(15 nm) NP exposed cells exhibit typical morphological (decreased cell size, membrane blebbing, peripheral chromatin condensation, apoptotic body formation) and biochemical (caspase activation and DNA fragmentation) features of apoptotic cell death. A decrease in mitochondrial membrane potential, activation of Bax and release of cytochrome c from mitochondria were only observed in case of CB NPs whereas lipid peroxidation, lysosomal membrane destabilization and cathepsin B release were observed during the apoptotic process induced by TiO2 NPs. Furthermore, ROS production was observed after exposure to CB and TiO2 but hydrogen peroxide (H2O2) production was only involved in apoptosis induction by CB NPs.ConclusionsBoth CB and TiO2 NPs induce apoptotic cell death in bronchial epithelial cells. CB NPs induce apoptosis by a ROS dependent mitochondrial pathway whereas TiO2 NPs induce cell death through lysosomal membrane destabilization and lipid peroxidation. Although the final outcome is similar (apoptosis), the molecular pathways activated by NPs differ depending upon the chemical nature of the NPs.

Effect of Amikacin on the Postnatal Development of the Cochlea in Albino Rats

Background: The cochlea is the anterior part of the labyrinth and its development is sensitive to large variety of noxious influences. Aim of the Work: This work was conducted to study the effect of amikacin on the postnatal development of cochlea in albino rats. Material and Methods: A total number of 30 albino rats were divided into two groups, a control group and an experimental group. The ages studied in this research were newborn, 7 days, 15 days, 1 month and 3 months postnatally in both control and experimental groups. The pregnant mothers of the experimental group received 250 mg/kg body weight of amikacin intraperitoneally daily from day 10 of gestation until the end of pregnancy. The same dose was given to the offsprings from day 1 until day 16 postnatally. The specimens were processed for histological study and stained with H&E. Ultrastructural study of the inner and outer hair cells and the large spiral ganglion cells was done through preparation of semithin and ultrathin sections. Morphometric measurements were performed to study the number of spiral ganglion cells in all the studied groups and were statistically analyzed. Results: This study revealed that the cochlea was still immature at birth and reached the adult appearance at the age of 15 days postnatally. The cochlear structures including the organ of Corti were found to be mature from the base to the apex of the cochlea. We observed that amikacin has no apparent effect on development of the cochlea in the newborn rats. In the 7-day-old rats, some degenerative changes appeared in the outer and inner hair cells. This effect became manifested in 15-day-old animals. Ultrastructural study of the outer hair cells showed peripheral condensation of nuclear chromatin with presence of many cytoplasmic vacuoles. At 1-month-age, there was disorganization of the organ of Corti, where atypical cells found in the region of the outer hair cells while the inner hair cells showed signs of degeneration. At the age of three months, there was complete destruction of the organ of Corti along the whole cochlea. Morphometric study for the number of the spiral ganglion cells showed highly significant decrease in the amikacin treated animals at the age of 15- day-old rats. This decrease became very highly significant at the age of one month and three months old amikacin treated rats Conclusions: Amikacin has a damaging effect on the organ of Corti and so considered as one of the causes of sensory deafness. The effect of amikacin was manifested during the second postnatal week is corresponding to the period of maturity of the cochlea.

Inhibition of Nuclear Translocation of Apoptosis-Inducing Factor Is an Essential Mechanism of the Neuroprotective Activity of Pigment Epithelium-Derived Factor in a Rat Model of Retinal Degeneration

Photoreceptor apoptosis is a critical process of retinal degeneration in retinitis pigmentosa (RP), a group of retinal degenerative diseases that result from rod and cone photoreceptor cell death and represent a major cause of adult blindness. We previously demonstrated the efficient prevention of photoreceptor apoptosis by intraocular gene transfer of pigment epithelium-derived factor (PEDF) in animal models of RP; however, the underlying mechanism of the neuroprotective activity of PEDF remains elusive. In this study, we show that an apoptosis-inducing factor (AIF)-related pathway is an essential target of PEDF-mediated neuroprotection. PEDF rescued serum starvation-induced apoptosis, which is mediated by AIF but not by caspases, of R28 cells derived from the rat retina by preventing translocation of AIF into the nucleus. Nuclear translocation of AIF was also observed in the apoptotic photoreceptors of Royal College of Surgeons rats, a well-known animal model of RP that carries a mutation of the Mertk gene. Lentivirus-mediated retinal gene transfer of PEDF prevented the nuclear translocation of AIF in vivo, resulting in the inhibition of the apoptotic loss of their photoreceptors in association with up-regulated Bcl-2 expression, which mediates the mitochondrial release of AIF. These findings clearly demonstrate that AIF is an essential executioner of photoreceptor apoptosis in inherited retinal degeneration and provide a therapeutic rationale for PEDF-mediated neuroprotective gene therapy for individuals with RP.

Early RB94-produced cytotoxicity in cancer cells is independent of caspase activation or 50 kb DNA fragmentation

RB94, which lacks the N-terminal 112 amino-acid residues of the full-length retinoblastoma protein (RB110) is a more potent inhibitor of cancer cell growth than RB110, being cytotoxic to all cancer cell lines studied, independent of their genetic abnormalities. Although we initially thought RB94-induced cell death was caspase-dependent, such caspase activation now appears to be a late event. Cells that remained attached 48 h after transduction with Ad-RB94 showed, among other changes, nuclear enlargement, peripheral nuclear chromatin condensation and often micronucleation. In addition, the cells were TdT-mediated dUTP nick end labeling (TUNEL) positive but showed no cleavage of caspase 3 or 9. Only after the cells detached was cleavage of both caspase 3 and 9 observed. These TUNEL-positive cells showed neither cytochrome c mitochondrial translocation usually found in typical apoptotic cells nor DNA laddering indicative of oligonucleosomal DNA fragmentation. In addition, although 50 kb DNA fragmentation was produced in these TUNEL-positive cells, which was dependent on apoptosis-inducing factor (AIF), inhibiting this fragmentation by siAIF did not inhibit TUNEL formation or cytotoxicity. As RB94 will soon be used for gene therapy further understanding the molecular basis of these early changes in killing cancer cells is one of our particularly important present goals.

Mechanism of 4-HPR-induced apoptosis in glioma cells: evidences suggesting role of mitochondrial-mediated pathway and endoplasmic reticulum stress

N-(4-hydroxyphenyl)retinamide (4-HPR), a synthetic retinoid is under clinical evaluation as a therapeutic agent in a variety of cancers. Its mechanism(s) of action involves multiple overlapping pathways that still remain unclear. In glioma cells its mechanism of action is not well elucidated. Here, we show that 4-HPR and not all-trans retinoic acid and 9-cis retinoic acid effectively induce apoptosis in glioma cells. 4-HPR-induced apoptosis is associated with hydroperoxide production and loss of mitochondrial membrane potential (Delta Psi(m)). Ultrastructural changes further indicate 4-HPR-induced mitochondrial swelling, endoplasmic reticulum (ER) dilation as well as close proximity of mitochondria and ER. As suggested by dilated ER, 4-HPR treatment increased the free cytosolic Ca(2+) as well as mitochondrial Ca(2+). Chelation of extracellular Ca(2+) by EGTA did not prevent Ca(2+) elevation, thus suggesting involvement of intracellular calcium stores in the release. Buffering of intracellular calcium by BAPTA-AM did not prevent 4-HPR-induced apoptosis; however, blocking the release of Ca(2+) from ER by heparin inhibited apoptosis, indicating the role of depletion of Ca(2+) from ER stores in apoptosis. 4-HPR treatment also resulted in an increase in Bax levels along with its translocation to mitochondria that promote mitochondrial membrane permeabilization. 4-HPR-induced apoptosis was further associated with the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria to cytosol and nucleus, respectively, along with caspase-3 and caspase-7 activation. However, AIF nuclear translocation, peripheral chromatin condensation and apoptosis were not completely prevented by general caspase inhibitors, thus suggesting involvement of a caspase-dependent and caspase-independent pathway in 4-HPR-induced apoptosis. Taken together, these results suggest the role of mitochondrial-mediated pathway and ER stress as a key event in 4-HPR-induced apoptosis in glioma cells.

Preapoptotic Chromatin Condensation Upstream of the Mitochondrial Checkpoint

When added for a short period (2-4 h) to cells, the kinase inhibitor staurosporine (STS), can trigger double strand breaks, the formation of nuclear foci containing phosphorylated H2AX, Chk2, and p53, a decrease in transcription, and a minor degree of peripheral chromatin condensation. This "preapoptotic chromatin condensation" (PACC) occurs before mitochondrial membrane permeabilization (MMP) and caspase activation become detectable and is not inhibited by Z-VAD-fmk or Bcl-2. PACC is followed by classical apoptosis, when cells are cultured overnight, even when STS is removed from the system. After overnight incubation, STS-pretreated cells manifest mitochondrial cytochrome c release, caspase activation, phosphatidylserine exposure, and apoptotic DNA fragmentation. Caspase or MMP inhibitors did not influence the advent of PACC yet did suppress the evolution of PACC toward apoptosis. Importantly, two unrelated MMP inhibitors (viral mitochondrial inhibitor of apoptosis (vMIA) from cytomegalovirus and mitochondrion-targeted Bcl-2) had a larger range of effects than the pan-caspase inhibitor Z-VAD-fmk. Caspase inhibition simply prevented the transition from PACC to apoptosis yet did not reverse PACC and did not restore transcription. In contrast, Bcl-2 and vMIA allowed for the repair of the DNA lesions, correlating with the reestablishment of active transcription. PACC could also be induced by a gross perturbation of RNA synthesis or primary DNA damage. Again, inhibition of MMP (but not that of caspases) reversed PACC induced by these stimuli. In synthesis, our data reveal the unexpected capacity of STS to induce DNA lesions and suggest qualitative differences in the cytoprotective and DNA repair-inducing potential of different apoptosis inhibitors.

Cross-linking of P-selectin glycoprotein ligand-1 induces death of activated T cells

AbstractIncreasing evidence has shown that death signaling in T cells is regulated in a complicated way. Molecules other than death receptors can also trigger T-cell death. Here, we demonstrate for the first time that P-selectin glycoprotein ligand-1 (PSGL-1) or CD162 molecules cross-linked by an anti–PSGL-1 monoclonal antibody, TAB4, can trigger a death signal in activated T cells. In contrast to classic cell death, PSGL-1–mediated T-cell death is caspase independent. It involves translocation of apoptosis-inducing factor from mitochondria to nucleus and mitochondrial cytochrome c release. Ultrastructurally, both peripheral condensation of chromatin and apoptotic body were observed in PSGL-1–mediated T-cell death. Collectively, this study demonstrates a novel role for PSGL-1 in controlling activated T-cell death and, thus, advances our understanding of immune regulation.

Down-regulation of apoptosis-inducing factor protein by RNA interference inhibits UVA-induced cell death

Apoptosis-inducing factor (AIF) is a caspase-independent apoptosis effector. UVA-induced Raji cell death was not completely inhibited by pan-caspase inhibitor zVAD.fmk. Moreover, AIF translocated from its normal location, the mitochondrial intermembrane space, into the nucleus, and induced peripheral chromatin condensation during the early stage of UVA-inducing cell death. Enforced expression of AIF can induce Raji cell death in a caspase-independent manner. Down-regulation of AIF protein level by RNA interference (RNAi) can reduce UVA-induced Raji cell death, but the combination of down-regulation of AIF and zVAD.fmk almost completely inhibited UVA-induced Raji cell death. All these suggest that caspase and AIF are two independent pathways and that UVA-induced Raji cell death is dependent on caspase and AIF.