Ubiquitinated inclusions and neuronal cell death

Ubiquitinated inclusions and selective neuronal cell death are considered the pathological hallmarks of Parkinson's disease and other neurodegenerative diseases. Recent genetic, pathological and biochemical evidence suggests that dysfunction of ubiquitin-dependent protein degradation by the proteasome might be a contributing, if not initiating factor in the pathogenesis of these diseases. In neuronal cell culture models inhibition of the proteasome leads to cell death and formation of fibrillar ubiquitin and alpha-synuclein-positive inclusions, thus modeling some aspects of Lewy body diseases. The processes of inclusion formation and neuronal cell death share some common mechanisms, but can also be dissociated at a certain level.     

Glioblastoma cell death induced by asiatic acid

Asiatic acid (AA), a triterpene, is known to be cytotoxic to several tumor cell lines. AA induces dose- and time-dependent cell death in U-87 MG human glioblastoma. This cell death occurs via both apoptosis and necrosis. The effect of AA may be cell type-specific as AA-induced cell death was mainly apoptotic in colon cancer RKO cells. AA-induced glioblastoma cell death is associated with decreased mitochondrial membrane potential, activation of caspase-9 and -3, and increased intracellular free Ca²⁺. Although treatment of glioblastoma cells with the caspase inhibitor zVAD-fmk completely abolished AA-induced caspase activation, it did not significantly block AA-induced cell death. AA-induced cell death was significantly prevented by an intracellular Ca²⁺ inhibitor, BAPTA/AM. Taken together, these results indicate that AA induces cell death by both apoptosis and necrosis, with Ca²⁺-mediated necrotic cell death predominating.     

Identification of Mallory bodies with rhodamine B fluorescence and other stains for keratin

Rhodamine B staining in conjunction with fluorescence microscopy is shown to demonstrate Mallory bodies. Mallory body morphology, localization, and distribution in hepatocytes from griseofulvin-fed mice, human hepatoma, and human alcoholics were similar to those observed in the same tissues after conventional staining methods for Mallory bodies. The presence of these inclusions was further confirmed by specific cytochemical localization with indirect immunoperoxidase labeling, horseradish peroxidase labeling, and electron microscopy. Other tinctorial or histochemical procedures previously used for keratin or prekeratin (modified Mallory stain, Kreyberg method, Pauly method for histidine) also stained Mallory bodies for study with white light microscopy but with decreasing sensitivity respectively. Mallory bodies from mouse and human liver both appear to contain a keratin-like moiety. This entity may be simply, rapidly, and permanently stained with rhodamine B, and selectively and reproducibly demonstrated with fluorescence microscopy.     

Tim18, a component of the mitochondrial translocator, mediates yeast cell death induced by arsenic

Evidence is presented that Tim18, a mitochondria translocase, plays a role in the previously described apoptosis induced by arsenite in Saccharomyces cerevisiae. Tim18 deletion mutant exhibited resistance to arsenite. After arsenite treatment, both the wild type and Tim18-deficient cells showed reactive oxygen species (ROS) production. Arsenite induced the higher expression of tim18 in wild type yeast cells. We found that the tim18 deletion mutant also exhibited resistance to other apoptotic stresses such as acetic acid, H2O2, and hyperosmotic stress. These results suggest that Tim18 is important for yeast cell death induced by arsenic, and it may act downstream of ROS production.     

Autophagic-like cell death in neutrophils induced by autoantibodies

Human neutrophils undergo autophagic-like cell death following Sialic acid binding immunoglobulin-like lectin-9 (Siglec-9) ligation and concurrent stimulation with certain, but not all, neutrophil survival cytokines. Caspase inhibition by these cytokines is required, but is not sufficient, to trigger this particular form of cell death. Additional mechanisms may involve reactive oxygen species (ROS), and blocking of ROS or prevention of ROS production prevents autophagic-like neutrophil death. Interestingly, human intravenous immunoglobulin (IVIg) preparations contain natural anti-Siglec-9 autoantibodies, which are able to ligate Siglec-9 on neutrophils and induce autophagic-like cell death in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and some other survival cytokines. Here, we discuss the pathophysiological and therapeutic implications of these recent findings.     

Apoptotic cell death induced by serum and its prevention by thiols

We recently reported that serum contains low molecular weight factors that inhibit growth and cause cell death in vitro. The present study focused on identifying components of basal media that counteract the toxic effects of serum. Amino acids L-cyst(e)ine and L-tryptophan were found to prevent serum-induced cell death of TIG-1 human fetal lung fibroblasts and other cell types. In addition to L-cysteine, other thiol-bearing and dithiol-cleaving compounds showed a similar ability to rescue the cells. Various inhibitors of protein or RNA synthesis also prevented the cell death. By contrast, nonthiol-containing reducing agents and super oxide dismutase (SOD), an active oxygen-eliminating enzyme, were ineffective. Thiol compounds appeared to exert a supportive level in TIG-1 cells cultured in FBS, whereas protein synthesis inhibitors did not alter the reduced intracellular thiol content. Fragmentation of DNA occurred prior to the plasma membrane breakdown of dying cells. Taken together, these data suggest that serum-induced cell death represents a form of apoptosis in which molecules containing thiol groups are active participants. © 1994 Wiley-Liss, Inc.     

Mitochondrial control of cell death induced by hyperosmotic stress

HeLa and HCT116 cells respond differentially to sorbitol, an osmolyte able to induce hypertonic stress. In these models, sorbitol promoted the phenotypic manifestations of early apoptosis followed by complete loss of viability in a time-, dose-, and cell type-specific fashion, by eliciting distinct yet partially overlapping molecular pathways. In HCT116 but not in HeLa cells, sorbitol caused the mitochondrial release of the caspase-independent death effector AIF, whereas in both cell lines cytochrome c was retained in mitochondria. Despite cytochrome c retention, HeLa cells exhibited the progressive activation of caspase-3, presumably due to the prior activation of caspase-8. Accordingly, caspase inhibition prevented sorbitol-induced killing in HeLa, but only partially in HCT116 cells. Both the knock-out of Bax in HCT116 cells and the knock-down of Bax in A549 cells by RNA interference reduced the AIF release and/or the mitochondrial alterations. While the knock-down of Bcl-2/Bcl-X_L sensitized to sorbitol-induced killing, overexpression of a Bcl-2 variant that specifically localizes to mitochondria (but not of the wild-type nor of a endoplasmic reticulum-targeted form) strongly inhibited sorbitol effects. Thus, hyperosmotic stress kills cells by triggering different molecular pathways, which converge at mitochondria where pro- and anti-apoptotic members of the Bcl-2 family exert their control. A. Criollo and L. Galluzzi contributed equally to this work.     

Oxidative DNA damage and glioma cell death induced by tetrahydropapaveroline

A series of naturally occurring isoquinoline alkaloids, besides their distribution in the environment and presence in certain food stuffs, have been detected in human tissues including particular regions of brain. An example is salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) that not only induces neuronal cell death, but also causes DNA damage and genotoxicity. Tetrahydropapaveroline [THP; 6,7-dihydroxy-1-(3',4'-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline], a dopamine-derived tetrahydroisoquinoline alkaloid, has been reported to inhibit mitochondrial respiration and is considered to contribute to neurodegeneration implicated in Parkinson's disease. Since THP bears two catechol moieties, the compound may readily undergo redox cycling to produce reactive oxygen species (ROS) as well as toxic quinoids. In the present study, we have examined the capability of THP to cause oxidative DNA damage and cell death. Incubation of THP with phiX174 supercoiled DNA or calf thymus DNA in the presence of cupric ion caused substantial DNA damage as determined by strand scission or formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. THP plus copper-induced DNA damage was ameliorated by some ROS scavengers/antioxidants and catalase. Treatment of C6 glioma cells with THP led to a concentration-dependent reduction in cell viability, which was prevented by the antioxidant N-acetyl-L-cysteine. When these cells were treated with 10microM THP, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were rapidly activated via phosphorylation, whereas activation of extracellular signal-regulated protein kinase (ERK) was inhibited. Furthermore, pretreatment with inhibitors of JNK and p38 MAPK rescued the glioma cells from THP-induced cytotoxicity, suggestive of the involvement of these kinases in THP-induced C6 glioma cell damage.     

bFGF expression mediated by a hypoxia-regulated adenoviral vector protects PC12 cell death induced by serum deprivation

Basic fibroblast growth factor (bFGF) is a known neuroprotectant against a number of brain injury conditions such as cerebral ischemia. However, bFGF also regulates a plethora of brain developmental processes and functions as a strong mitogen. Therefore, unregulated long-term expression of bFGF in brain may potentially be tumorigenic, limiting its utility in brain therapy. Here, we report the successful construction of an adenoviral vector (Ad-5HRE-bFGF) expressing bFGF under the regulation of five hypoxia-responsive elements (5HRE) and a minimal cytomegalovirus promoter (CMVmp). Following hypoxia treatment in a hypoxic chamber with less than 1% of oxygen, Ad-5HRE-bFGF induced a significant and time-dependent expression of bFGF protein and the fluorescent tag, humanized GFP (hrGFP) protein, in infected PC12 cells. In contrast, normoxia treatment evoked extremely low level of bFGF and hrGFP expression, demonstrating that the 5HRE-CMVmp cassette was effective in regulating the expression of bFGF gene in response to hypoxia. More importantly, bFGF expressed by the Ad-5HRE-bFGF viral vector under the regulation of hypoxia was significantly neuroprotective against PC12 cell death evoked by serum deprivation. Taken together, these studies demonstrated the feasibility to express bFGF in a hypoxia-regulated fashion to provide neuroprotection. The Ad-5HRE-bFGF can be further developed as an effective tool to provide neuroprotection against hypoxia-induced brain diseases, such as cerebral ischemia.     

Necrotic cell death by hydrogen peroxide in immortal DF-1 chicken embryo fibroblast cells expressing deregulated MnSOD and catalase

The reactive oxygen species are known as endogenous toxic oxidant damaging factors in a variety of cell types, and in response, the antioxidant genes have been implicated in cell proliferation, senescence, immortalization, and tumorigenesis. The expression of manganese superoxide dismutase mRNA was shown to increase in most of the immortal chicken embryo fibroblast (CEF) cells tested, while expression of catalase mRNA appeared to be dramatically decreased in all immortal CEF cells compared to their primary counterparts. The expression of copper-zinc superoxide dismutase mRNA was shown to increase slightly in some immortal CEF cells. The glutathione peroxidase expressed relatively similar levels in both primary and immortal CEF cells. As primary and immortal DF-1 CEF cells were treated with 10-100 microM of hydrogen peroxide (concentrations known to be sublethal in human diploid fibroblasts), immortal DF-1 CEF cells were shown to be more sensitive to hydrogen peroxide, and total cell numbers were dramatically reduced when compared with primary cell counterparts. This increased sensitivity to hydrogen peroxide in immortal DF-1 cells occurred without evident changes in either antioxidant gene expression, mitochondrial membrane potential, cell cycle distribution or chromatin condensation. However, the total number of dead cells without chromatin condensation was dramatically elevated in immortal DF-1 CEFs treated with hydrogen peroxide, indicating that the inhibition of immortal DF-1 cell growth by low concentrations of hydrogen peroxide is due to increased necrotic cell death, but not apoptosis. Taken together, our observation suggests that the balanced antioxidant function might be important for cell proliferation in response to toxic oxidative damage by hydrogen peroxide.     

Ectopic coexpression of keratin 8 and 18 promotes invasion of transformed keratinocytes and is induced in patients with cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) results from transformation of epidermal keratinocytes. Invasion of transformed keratinocytes through the basement membrane into the dermis results in invasive cSCC with substantial metastatic potential. To better understand the mechanisms for invasion and metastasis, we compared the protein expression profiles of a non-metastatic transformed mouse keratinocyte line and its metastatic derivative. Keratin 8 (Krt8) and Krt18, not seen in normal keratinocytes, were coexpressed and formed Krt8/18 filaments in the metastatic line. The metastatic line efficiently invaded an artificial basement membrane in vitro owing to the Krt8/18-coexpression, since coexpression of exogenous Krt8/18 in the non-invasive parental line conferred invasiveness. To test whether the Krt8/18-coexpression is induced and is involved in cSCC invasion, we examined specimens from 21 pre-invasive and 24 invasive cSCC patients by immunohistochemistry, and the ectopic Krt8/18-coexpression was almost exclusively found in invasive cSCC. Further studies are needed to examine the clinical significance of ectopic Krt8/18-coexpression in cSCC.     

Apoptotic cell death of oestrogen activated lactotrophs induced by tamoxifen

Stimulation and inhibition of lactotroph cells cause remarkable morphological and functional changes. In keeping with these changes, the size of the lactotroph cell population undergoes striking alterations due to proliferation or cell death. Factors involved in the induction of apoptosis of pituitary cells are not well established. We demonstrated earlier that oestrogens prevent lactotroph cells of female rats to die by apoptosis induced by bromocryptine treatment, a fact that can be reversed in ovariectomised rats. In this study, we developed experimental models for in vivo and in vitro studies to gain further insight on the survival effect of oestrogens on lactotrophs. In rats pretreated with oestrogens, tamoxifen generates a massive cell death by apoptosis as validated by the TUNEL technique and DNA electrophoresis of pituitary gland. On electron microscope observations, numerous lactotrophs exhibited progressive morphological changes in the nuclei compatible with the apoptotic process. The cells remaining intact also exhibit signs of inhibition due to a significant transformation of regular lactotrophs in atypical subtypes. In pituitary cell cultures exposed to tamoxifen and oestrogen simultaneously, most of the lactotrophs displayed features of apoptosis in the nucleus. The present reports gathered new evidences on the apoptogenic potential of tamoxifen on lactotroph cells, and corroborates the contribution of oestrogens to sustain both a balanced population of lactotrophs and a competent secretory activity. The concept that opposed activities, such as inhibition and stimulation, can activate apoptosis is also strengthen by these observations.     

Kinetics of apparent cell death in yeasts induced by ethanol

Summary Evidence is presented that adaptation of yeast cells to ethanol results in a reduced loss of cell viability induced by exposure to that agent. In line with earlier work, an exponential model is shown to apply when the concentration of ethanol exceeds a critical value, beyond which cell growth cannot occur. Such an exponential model is consistent with the absolute theory of reaction rates. Adaptation of yeast cells to 7% w/v ethanol lowers the specific rate of cell death at various ethanol concentrations by a factor of some 40 fold compared to a non-adapted culture.     

Metaphase arrest and cell death induced by etoposide on HeLa cells

DNA damage, cell cycle and apoptosis form a network with important implications for cancer chemotherapy. Dysfunctions of the cycle checkpoints can allow cancer cells to acquire drug resistance. Etoposide is a well-known inducer of apoptosis, which is widely used in cell biology and in clinical practice. In this work we report that a pulse of 50 μM etoposide (incubation for only 3 h) on HeLa cells causes a sequence of events that leads to abnormal mitotic figures that could be followed either by cell death or, more commonly, by interphase restitution and endocycle. The endocycling polyploid cells enter immediately into mitosis and suffer metaphase blockage with multiple spindle poles, which were generally followed by a direct triggering of apoptosis from metaphase (mitotic catastrophe), or by a new process of endocycling, until surviving cells finally became apoptotic (96 h after the treatment).     

Proteolysis and cell death in clover leaves is induced by grazing

Summary.  Programmed plant cell death is a widespread phenomenon resulting in the formation of xylem vessels, dissected leaf forms, and aerenchyma. We demonstrate here that some characteristics of programmed cell death can also be observed during the cellular response to biotic and abiotic stress when plant tissue is ingested by grazing ruminants. Furthermore, the onset and progression of plant cell death processes may influence the proteolytic rate in the rumen. This is important because rapid proteolysis of plant proteins in ruminants is a major cause of the inefficient conversion of plant to animal protein resulting in the release of environmental N pollutants. Although rumen proteolysis is widely believed to be mediated by proteases from rumen microorganisms, proteolysis and cell death occurred concurrently in clover leaves incubated in vitro under rumenlike conditions (maintained anaerobically at 39 °C) but in the absence of a rumen microbial population. Under rumenlike conditions, both red and white clover cells showed progressive loss of DNA, but this was only associated with fragmentation in white clover. Cell death was indicated by increased ionic leakage and the appearance of terminal deoxynucleotidyl transferase-mediated dUTP-nick-end-labelled nuclei. Foliar protein decreased to 50% of the initial values after 3 h incubation in white clover and after 4 h in red clover, while no decrease was observed in ambient (25 °C, aerobic) incubations. In white clover, decreased foliar protein coincided with an increased number of protease isoforms. Received June 24, 2002; accepted August 15, 2002; published online March 11, 2003     

Aberrant in vitro expression of keratin K13 induced by Ca2+ and vitamin A acid in mouse epidermal cell lines.

Normally the expression of the murine type I keratin K13 is restricted to differentiating cells of internal squamous epithelia which line the oral cavity and the upper digestive tract. Recently, however, we were able to show that K13 is aberrantly but constitutively expressed without its normal type II partner K4 also in differentiating parts of 7,12-dimethylbenz(a)anthracene (DMBA/TPA) 12-O-tetradecanoylphorbol-13-acetate-induced squamous cell carcinomas of mouse back skin, whereas its likewise suprabasal expression in papillomas is variable (Nischt et al., Mol. Carcinogenesis 1, 96-108, 1988). In an attempt to reproduce the aberrant expression of K13 in a mouse in vitro system, we have investigated eight established murine epidermal cell lines for their putative ability to express K13. The cell lines differed distinctly in their derivation and comprised cell lines originating from DMBA/TPA induced papillomas (line SP1) or DMBA-treated adult mouse epidermis (line 308) as well as cell lines derived from DMBA or DMBA/TPA-treated primary epidermal keratinocytes (lines PDV and MCA 3D) and cell lines which arose spontaneously by long-term culture of normal epidermal keratinocytes (lines HEL 30 degrees HEL 37 degrees, HELP I and HELP III). We show that, independent of their derivation, all cell lines possess the intrinsic property to aberrantly express K13. Invariably the K13 gene is not expressed when the lines are cultured under low Ca2+ conditions (0.05 mM) and thus prevented from differentiation. Its expression can, however, be induced either by increasing the extracellular Ca2+ concentration or by the addition of physiological concentrations of vitamin A acid to low Ca2+ medium. Whereas in the latter case, K13 expression occurs without concomitant induction of morphological differentiation of the cells, Ca2+ elevation in the culture medium induces squamous differentiation and K13 expression occurs only in differentiating cells, thus reflecting the situation observed in in vivo tumors. All cell lines exhibit a concentration optimum for the stimulatory agents; however, the degree of maximal K13 expression varies considerably among the individual cell lines and shows a striking correlation with the reported tumorigenicity of the lines after transplantation to animals. In contrast, a tentatively suggested correlation between the activation of the Ha-ras gene and the aberrant expression of K13 (Nischt et al., Mol. Carcinogenesis 1, 96-108, 1988) could not definitely be confirmed since we observed K13 expression also in three cell lines which did not carry a mutation in codon 61 of the Ha-ras gene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mitochondrial apoptotic pathways induced by Drosophila programmed cell death regulators

Multicellular organisms eliminate unwanted or damaged cells by cell death, a process essential to the maintenance of tissue homeostasis. Cell death is a tightly regulated event, whose alteration by excess or defect is involved in the pathogenesis of many diseases such as cancer, autoimmune syndromes, and neurodegenerative processes. Studies in model organisms, especially in the nematode Caenorhabditis elegans, have been crucial in identifying the key molecules implicated in the regulation and execution of programmed cell death. In contrast, the study of cell death in Drosophila melanogaster, often an excellent model organism, has identified regulators and mechanisms not obviously conserved in other metazoans. Recent molecular and cellular analyses suggest, however, that the mechanisms of action of the main programmed cell death regulators in Drosophila include a canonical mitochondrial pathway.