Culture filtrates of Aspergillus fumigatus induce different modes of cell death in human cancer cell lines

Aspergillus fumigatus culture filtrate (CF) has a potent cytotoxic effect on three human cancer cell lines (DLKP, A549 and HEp-2) and initiates cell death by apoptosis but the execution of the apoptotic process is incomplete. DLKP cells treated with A. fumigatus CF demonstrate features associated with apoptosis but cytoplasmic and nuclear fragmentation were not observed and cells ultimately underwent necrosis. The apoptotic process commenced in A549 and HEp-2 cells upon exposure to CF, cell shrinkage was observed but membrane blebbing and apoptotic body formation were not detected and detached cells died by necrosis. In contrast, extensive nuclear fragmentation and apoptotic body formation were evident in DLKP and A549 cells treated with anti-neoplastic agents. This work indicates that A. fumigatus CF is cytotoxic to cancer cells and can initiate apoptosis but that the complete apoptotic pathway is not followed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Recombinant Galectins of <Emphasis Type="Italic">Haemonchus contortus</Emphasis> Parasite Induces Apoptosis in the Peripheral Blood Lymphocytes of Goat

The effect of Haemonchus contortus galectin peptides rHco-gal-m/f to induce apoptosis in the peripheral blood lymphocytes (PBLCs) of goats was investigated. Analysis of apoptosis was carried out with agarose gel electrophoresis, flow cytometry and transmission electron microscopy. The results indicated that there were visible apoptosis bodies and typical DNA ladders by genomic DNA fragmentation. The quantitative analysis of apoptosis by flow cytometry indicated that rHco-gal-m/f peptides induced apoptosis was time and dose dependent. Ultrastructural studies of the PBLCs revealed that a large number of apoptotic cells were present in galectin-treated cells, which had the typical morphologic changes of apoptosis such as reduction of the cytoplasmic volume, loss of cell surface microvilli, chromatin condensation and fragmentation of the apoptotic cells into small apoptotic bodies.     

Mitochondrion-mediated apoptosis induced by Rana grylio virus infection in fish cells

A fish cell line, fathead minnow (FHM) cell, was used to investigate the alteration of mitochondrial dynamics and the mechanism of apoptosis under Rana grylio virus (RGV) infection. Microscopy observations, flow-cytometry analysis and molecular marker detection revealed the apoptotic fate of the RGV-infected cells. Some typical apoptotic characteristics, such as chromatin condensation, DNA fragmentation and mitochondrial fragmentation, were observed, and significantly morphological changes of mitochondria, including size, shape, internal structure and distribution, were revealed. The mitochondria in RGV-infected cells were aggregated around the viromatrix, and the aggregation could be blocked by colchicine. Moreover, the Δψm collapse was induced, and caspase-9 and caspase-3 were activated in the RGV-infected cells. In addition, NF-κB activation and intracellular Ca²⁺ increase were also detected at different times after infection. The data revealed the detailed dynamics of mitochondrion-mediated apoptosis induced by an iridovirus, and provided the first report on mitochondrial fragmentation during virus-induced apoptosis in fish cells.     

Thai Acanthamoeba isolate (T4) induced apoptotic death in neuroblastoma cells via the Bax-mediated pathway

A Thai Acanthamoeba isolate named AS recovered from a corneal scraping of a keratitis patient was genotypically determined as T4. AS trophozoites were used for studying Acanthamoeba-induced apoptosis in mouse neuroblastoma NA cells during in vitro co-cultivation. The Acanthamoeba-exposed NA cells showed signs of apoptosis including cell shrinkage, nuclear condensation and DNA fragmentation. The effect was confirmed by DNA laddering electrophoresis. Involvement of caspase enzymes and mitochondrial pro- and anti-apoptotic proteins (Bax and Bcl-2) in AS-induced apoptosis was determined. The use of Z-VAD-FMK, a pan-caspase inhibitor, significantly reduced the apoptotic effect, while Bax/Bcl-2 ratio analysis showed a significant increase in the expression of apoptotic proteins in AS-exposed NA cells. These results strongly indicated that apoptosis induced by AS trophozoites is caspase-dependent and is mediated by over-expression of pro-apoptotic proteins in the mitochondrial pathway. This is the first report on the role of Bax in mediating apoptosis induced by Acanthamoeba.     

Directional cell-to-cell communication in theArabidopsis root apical meristem III. Plasmodesmata turnover and apoptosis in meristem and root cap cells during four weeks after germination

Summary Plasmodesmata frequency and distribution in root cap cells ofArabidopsis thaliana root tips were characterized during four weeks after germination to understand the symplasmic control of apoptosis. Apoptotic cells in some of the root apical-meristem cells and in root cap cells were identified by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling reaction and characterized by electron microscopy. Starting at the second week after germination, cells in the outermost layers of the root cap showed typical apoptotic features, including nuclear DNA fragmentation, chromatin condensation, cytoplasmic vacuolation, and organelle destruction. Intercellular connections, indicated by the frequency and number of plasmodesmata per cell length, were significantly reduced in the walls of outer root cap cells. This shows that cells become symplasmically isolated during the apoptosis process. In apoptotic root cap cells, the majority of nonfunctional plasmodesmata were observed to be associated with degenerated endoplasmic reticulum; this state was prior to the detection of any nuclear DNA fragmentation. Other nonfunctional plasmodesmata were sealed by heterogeneous cell wall materials. However, in immature epidermal and cortical cells in 4-week-old arrested roots the endoplasmic reticulum associated with plasmodesmata became disconnected as a result of protoplast condensation and shrinkage. No degenerated endoplasmic reticulum was observed in these cells. These observations suggest that the apoptotic processes in the root body and the root cap are different.     

20-Hydroxyecdysone-induced differentiation and apoptosis in the Drosophila cell line, l(2)mbn

20-Hydroxyecdysone has an inhibitory effect on the proliferation of l(2)mbn cells, causes vacuolization and fragmentation of cells, and promotes a strong phagocytotic activity. From several lines of evidence, it can be concluded that 20-hydroxyecdysone induces apoptosis. Long-term video observations following the fate of individual cells, scanning and transmission electron microscopy reveal the typical characteristics of apoptosis: sequestration of small cellular protuberances or larger parts of the cell with nuclear fragments (apoptotic bodies), chromatin condensation, condensation and vesiculation of cytoplasm, whereas the mitochondria retain their normal appearance. The induction of apoptosis by 20-hydroxyecdysone was confirmed by the TUNEL reaction and quantitatively determined by a method based on this reaction. Onset of apoptosis precedes phagocytotic activity. JH III alone has no clear-cut effect on l(2)mbn cells. In double treatments, the inhibitory effect of 20-hydroxyecdysone on cell proliferation is significantly reduced by the addition of JH III. Whether or not JH III also reduces apoptotic activity is not yet clear. It is shown that the l(2)mbn cell line is an advantageous model system for the exploration of steroid-induced apoptosis.     

Apoptotic cell death in the central nervous system of Bufo arenarum tadpoles induced by cypermethrin

Tadpoles of the toad Bufo arenarum treated with cypermethrin (CY) at concentrations above 39 μg CY/L showed dose-dependent apoptotic cell death in immature cells of the central nervous system as demonstrated by morphometric analysis, the TUNEL method, and DNA fragmentation assay. Light-and electron-microscopic studies showed structural alterations in the intermediate and marginal layers of the brain. Immature cerebral tissue showed cellular shrinkage, nuclear fragmentation and increase of intercellular spaces. In this study we demonstrated high toxicity of CY to larval stages of Bufo arenarum. Our results show that doses lower than those used in routine insecticide applications can cause massive apoptosis in the immature cells of the central nervous system. These results coincide with our previous studies in Physalaemus biligonigerus, confirming the severe toxic effects of CY to the central nervous system of anuran species from Argentina. This may increase the mortality index in wild animals and contribute to the loss of biodiversity in our agroecosystems. We postulate that CY induces apoptosis in central nervous system cells of Bufo arenarum tadpoles by specific neurotoxic mechanisms.     

Lack of chromatin and nuclear fragmentation in vivo impairs the production of lupus anti-nuclear antibodies

Nuclear autoantigens in systemic lupus erythematosus are thought to derive primarily from apoptotic cells, yet there is no direct evidence that interfering with apoptosis impairs the generation of lupus autoantibodies. Here we use a mouse model that lacks the endonuclease caspase-activated DNase (CAD), resulting in an absence of chromatin and nuclear fragmentation during apoptotic cell death. We show that in this mouse, production and release into circulation of chromatin is impaired after exposure to several apoptotic triggers, but that the absence of CAD does not interfere with upstream steps of apoptosis or immune system function. Finally we show that in CAD-mutant mice, impaired lupus autoimmunity is skewed toward known cytoplasmic components, and autoimmunity toward membrane autoantigens is preserved, while autoimmunity toward chromatin and other lupus nuclear targets is severely impaired or absent. We also show, as control, that the induction of experimental autoimmune encephalomyelitis is not affected by the absence of CAD. Thus, our work in vivo strongly suggests that apoptotic molecular steps during cell death generate nuclear autoantigens to sustain the specific autoimmune response in systemic lupus erythematosus.     

Rapid cold-hardening protects <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> from cold-induced apoptosis

The rapid cold-hardening (RCH) response increases the cold tolerance of insects by protecting against non-freezing, cold-shock injury. Apoptosis, or programmed cell death, plays important roles in development and the elimination of sub-lethally damaged cells. Our objectives were to determine whether apoptosis plays a role in cold-shock injury and, if so, whether the RCH response protects against cold-induced apoptosis in Drosophila melanogaster. The present study confirmed that RCH increased the cold tolerance of the adults at the organismal level. No flies in the cold-shocked group survived direct exposure to ‒7°C for 2 h, whereas significantly more flies in the RCH group survived exposure to ‒7°C for 2 h after a 2-h exposure to 5°C. We used a TUNEL assay to detect and quantify apoptotic cell death in five groups of flies including control, cold-shocked, RCH, heat-shocked (37.5°C, 30 min), and frozen (‒20°C, 24 h) and found that apoptosis was induced by cold shock, heat shock, and freezing. The RCH treatment significantly improved cell viability by 38% compared to the cold-shocked group. Cold shock-induced DNA fragmentation shown by electrophoresis provided further evidence for apoptosis. SDS-PAGE analysis revealed an RCH-specific protein band with molecular mass of ∼150 kDa. Western-blotting revealed three proteins that play key roles in the apoptotic pathway: caspase-9-like (apoptotic initiator), caspase-3-like (apoptotic executioner) and Bcl-2 (anti-apoptotic protein). Consequently, the results of this study support the hypothesis that the RCH response protects against cold-shock-induced apoptosis.     

Apoptosis is not the cause of seasonal testicular involution in roe deer

Apoptosis is involved in the regulation of spermatogenesis. The involution of testes in seasonal breeders might be expected to involve enhanced apoptotic cell elimination. We have compared seasonally changing testicular apoptosis in roe deer with that in non-seasonally breeding cattle. Apoptotic cells were detected as TUNEL-positive cells by both flow-cytometric analysis and in situ localisation of fragmented DNA in tissue sections. Apoptosis-induced DNA fragments were also assessed by enzyme-linked immunosorbent assay (ELISA) in homogenised testicular parenchyma. As expected, the testis mass and the percentage of haploid cells in roe deer showed a seasonal pattern with a significant maximum during the rut (August), whereas no annual variation of these parameters was found in bulls. All three methods for determining apoptosis showed similar findings. Roe deer exhibited significant seasonal fluctuation of total apoptotic activity (ELISA, apoptotic cells per tubule cross section) with a maximum during the breeding season. However, the seasonal differences in the number of apoptotic cells corresponded to the variable total numbers of spermatogonia and spermatocytes per tubule cross section. Thus, the percentages of TUNEL-positive cells related to the combined number of both germ cell types showed no seasonal variance, as confirmed by percentages of apoptotic cells analysed flow-cytometrically. The maximum level of apoptosis during the rut in roe deer was similar to the values obtained during the invariably high spermatogenic activity in cattle. These results suggest that, in roe deer, apoptosis is not the cause of the seasonal involution of testes. This study was partially supported by grant Bl 319/6-1 from the Deutsche Forschungsgemeinschaft.     

Involvement of both caspase-dependent and -independent pathways in apoptotic induction by hexaminolevulinate-mediated photodynamic therapy in human lymphoma cells

Photodynamic therapy (PDT) is a cancer treatment based on the interaction of a photosensitizer, light and oxygen. PDT with the endogenous photosensitizer, protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) or its derivatives is a modification of this treatment modality with successful application in dermatology. However, the mechanism of cell destruction by ALA-PDT has not been elucidated. In this study a human T-cell lymphoma Jurkat cell line was treated with PDT using hexaminolevulinate (HAL, hexylester of ALA). Four hours following treatment nearly 80% of the cells exhibited typical apoptotic features. Mitochondrial pro-apoptotic proteins were evaluated by Western blots in subcellular fractionated samples. PDT caused cytosolic translocation of cytochrome c and nuclear redistribution of apoptosis-inducing factor (AIF), but the release of mitochondrial Smac/DIABLO, Omi/HtrA2 and EndoG was not observed. The release of cytochrome c was followed by the cleavage of caspase-9 and caspase-3 as well as its downstream substrates, together with oligonucleosomal DNA fragmentation. The pan-caspases inhibitor, z-VAD.fmk, prevented oligonucleosomal DNA fragmentation, but failed to inhibit PDT-mediated apoptosis. The apoptotic induction by AIF-mediated caspase-independent pathway was also found after HAL-PDT with large-scale DNA fragmentation in the presence of z-VAD.fmk. These results demonstrate that cytochrome c-mediated caspase-dependent pathway and AIF-induced caspase-independent pathway are simultaneously involved in the apoptotic induction by PDT. When the cytochrome c-induced caspase-dependent pathway is blocked, the cells go into apoptosis via AIF-mediated pathway, clearly demonstrating that the cytochrome c-mediated caspase-dependent pathway is not required for such apoptotic induction. This finding may have an impact on improved PDT effectiveness.     

Betanin a betacyanin pigment purified from fruits of Opuntia ficus-indica induces apoptosis in human chronic myeloid leukemia Cell line-K562

Betalains are water-soluble nitrogenous vacuolar pigments present in flowers and fruits of many caryophyllales with potent antioxidant properties. In the present study the antiproliferative effects of betanin, a principle betacyanin pigment, isolated from the fruits of Opuntia ficus-indica, was evaluated on human chronic myeloid leukemia cell line (K562). The results show dose and time dependent decrease in the proliferation of K562 cells treated with betanin with an IC(50) of 40 microM. Further studies involving scanning and transmission electron microscopy revealed the apoptotic characteristics such as chromatin condensation, cell shrinkage and membrane blebbing. Agarose electrophoresis of genomic DNA of cells treated with betanin showed fragmentation pattern typical for apoptotic cells. Flow cytometric analysis of cells treated with 40 microM betanin showed 28.4% of cells in sub G0/G1 phase. Betanin treatment to the cells also induced the release of cytochrome c into the cytosol, poly (ADP) ribose polymerase (PARP) cleavage, down regulation Bcl-2, and reduction in the membrane potentials. Confocal microscopic studies on the cells treated with betanin suggest the entry of betanin into the cells. These studies thus demonstrate that betanin induces apoptosis in K562 cells through the intrinsic pathway and is mediated by the release of cytochrome c from mitochondria into the cytosol, and PARP cleavage. The antiproliferative effects of betanin add further value to the nutritional characteristics of the fruits of O. ficus-indica.     

Germ cells with nuclear DNA fragmentation related to apoptotic cells in rat testis in experimental hyperprolactinemia induced by metoclopramide

The cells with nuclear DNA fragmentation related to apoptosis were detected by TUNEL technique in the seminiferous epithelium of control rats and of rats with experimental hyperprolactinemia induced by metoclopramide. The percentage of convoluted tubules with apoptotic cells and the number of apoptotic cells (predominantly spermatogonia and spermatocytes) was increased in the experimental group. The results indicated stage-specific germ cell apoptosis. In the experimental group, apoptotic cells were most evident at early (I-IV), middle (VII-VIII) and late (XII-XIV) stages of the seminiferous epithelium cycle, as revealed by light and electron microscopy. We suggest that a decreased concentration of testosterone and an increased concentration of prolactin could disturb spermatogenesis and contribute to the intensive apoptosis of germ cells in rats with hyperprolactinemia. Sertoli cells which have receptors for testosterone and prolactin and play an important role in spermatogenesis and in the initiation of apoptosis in seminiferous epithelium, could mediate such an influence of both hormones.     

cDNA cloning and functional characterization of Xenopus laevis DNase γ

We report here the cDNA cloning and functional analysis of Xenopus DNase γ (xDNase γ). Two forms of cDNAs are isolated from adult spleen: one composing a 933 bp open reading frame for the enzymatically active xDNase γ protein, and the other encoding an inactive short alternative form. Northern blot analysis revealed that the xDNase γ mRNA is expressed in spleen, liver, testis, and ovary. xDNase γ expression is scarcely detected in the tail muscle of tadpoles; however, it increases during metamorphosis and reaches a maximum during the late metamorphic climax. The ectopic expression of xDNase γ results in the appearance of extensive DNA fragmentation in C2C12 myoblasts after the induction of apoptosis. In contrast, Xenopus DNase I fails to induce apoptotic DNA ladder formation under the same conditions. Our results suggest a possible involvement of xDNase γ in apoptosis during amphibian metamorphosis. The nucleotide sequence of Xenopus DNase γ has been submitted to DDBJ/EMBL/GenBank database under the accession number AF059612     

Chronic heat-shock treatment driven differentiation induces apoptosis in Leishmania donovani

The present study investigates the role of apoptosis in the regulation of cell numbers of Leishmania donovani during the in vitro differentiation of promastigote stage to amastigote stage in axenic conditions. We report that apoptosis is induced in Leishmania donovani due to chronic heat-shock treatment of 37 ^°C that also mediates the differentiation of promastigotes to amastigotes. This is characterized by the fragmentation of DNA, blebbing in the parasite cell membrane, nuclear condensation, formation of preapoptotic bodies and involvement of Ca⁺⁺ in the apoptotic process. The flowcytometric analysis shows an early and steep rise in percentage apoptotic nuclei till 48-hour stage of differentiation and then a gradual decline, suggesting synergistic action of Ca⁺⁺ ATPase and probably Hsp70. Hsp70 might be rescuing cells from apoptosis in the death signaling pathway. Incubation of the culture with Ca⁺⁺ chelator EGTA (1 mM) brings down the percentage of apoptotic nuclei considerably showing thereby that calcium is needed for the process of cell death here that occurs by apoptosis. The survival of the infective individuals appears to be decided by the parasite in the early stages of its differentiation. Our studies show the potential of the physiological temperature of 37 ^°C in inducing apoptosis in Leishmania donovani and the therapeutic use it can be put to.     

Cell shape and organelle modification in apoptotic U937 cells

U937 cells induced to apoptosis, progressively and dramatically modified their cell shape by intense blebbing formation, leading to the production of apoptotic bodies. The blebs evolved with time; milder forms of blebbing involving only a region or just the cortical part of the cytoplasm were observed within the first hour of incubation with puromycin; blebbing involving the whole cell body with very deep constrictions is the most frequent event observed during late times of incubation. The ultrastructural analysis of apoptotic cells revealed characteristic features of nuclear fragmentation (budding and cleavage mode) and cytoplasmatic modifications. The cytoplasm of blebs does not contain organelles, such as ribosomes or mitochondria. Scarce presence of endoplasmic reticulum can be observed at the site of bleb detachment. However, blebbing is a dispensable event as evaluated by using inhibitor of actin polymerization. In the present study, the progressive modifications of the nucleus, mitochondria, nuclear fragmentation, cytoplasmic blebs formation and production of apoptotic bodies in U937 monocytic cells induced to apoptosis by puromycin (an inhibitor of protein synthesis) were simultaneously analyzed.     

Relationships between DNA Fragmentation, Chromatin Condensation, and Changes in Flow Cytometry Profiles Detected during Apoptosis

The determination of whether a cell dies by apoptosis as opposed to necrosis is usually best made on the basis of distinct structural changes in the chromatin. These changes include extensive condensation of the chromatin and DNA fragmentation. We have shown that the cytotoxic drug bleomycin (BLM) is able to cleave the DNA between the nucleosomes when it enters into the cell. If sufficient amounts of BLM are internalized, the nuclear morphological changes characteristic of apoptosis are detected. In this work, we describe the nuclear changes that occurred after DNA fragmentation as a function of the number of DNA double-strand breaks generated per cell and of the time after their generation. Our results show that DNA fragmentation and degradation of higher-order DNA structure were directly responsible for the nuclear morphological changes associated with apoptosis. During apoptosis reduced fluorescence with respect to the G₀/G₁ cell cycle region (the sub-G₁ region) is often detected if fixed cells from cultures undergoing apoptosis are analyzed by flow cytometry. We demonstrate here that, depending on the extent of the DNA fragmentation and on ulterior changes in chromatin structure, the content of the fluorescent sub-G₁ region can be either soluble pieces of DNA or apoptotic bodies or cells depleted in the DNA content by partial loss of fragmented DNA dissolved in the washing media and/or by the release of apoptotic bodies.     

Human mature erythroblasts are resistant to apoptosis

Apoptosis plays an important role in red blood cell development, notably by regulating the fate of early erythroid progenitors. We show here that, by contrast, mature erythroblasts are resistant to apoptosis. Treatment of these cells with several apoptosis-inducing agents failed to trigger caspase activation and oligonucleosomal DNA fragmentation. Interestingly, we find that cytochrome c levels are dramatically reduced even though the cells contain mitochondria. Supplementation of cytosolic extracts from mature erythroblasts with cytochrome c, however, did not rescue caspase activation. This was not due to the presence of inhibitors of apoptosis, as these proteins were also missing in these cells. We also show that cytochrome c depletion is a normal event during erythroblast differentiation, which follows transient, developmentally induced caspase activation and correlates with the loss of response to cytokine withdrawal or drug-induced apoptosis. Our data therefore suggest that erythroblasts acquire resistance to apoptosis during maturation through the developmentally induced depletion of cytochrome c and other crucial regulators of the apoptotic machinery.     

Ultraviolet B radiation-induced cell death: critical role of ultraviolet dose in inflammation and lupus autoantigen redistribution

The nuclear self-Ags targeted in systemic lupus erythematosus translocate to the cell membrane of UV-irradiated apoptotic keratinocytes and may represent an important source of self-immunization. It is hard to understand how the noninflammatory milieu accompanying most apoptosis might provoke an immunogenic response leading to autoantibodies. We have found that the precise amount of keratinocyte UV exposure is crucial in determining the rate of apoptosis, the amount of inflammatory cytokine production, and the degree of autoantigen translocation. Low doses of UVB (相似文献