Role of β-adrenergic signaling in ovarian cancer cells biology

Tese de mestrado. Biologia (Biologia Evolutiva e do Desenvolvimento). Universidade de Lisboa, Faculdade de Ciências, 2014

Role of the ASPP Family in the Regulation of p53-Mediated Apoptotic Death of Retinal Ganglion Cells after Optic Nerve Injury

Le glaucome est la première cause de cécité irréversible à travers le monde. À présent il n’existe aucun remède au glaucome, et les thérapies adoptées sont souvent inadéquates. La perte de vision causée par le glaucome est due à la mort sélective des cellules rétiniennes ganglionnaires, les neurones qui envoient de l’information visuelle de la rétine au cerveau. Le mécanisme principal menant au dommage des cellules rétiniennes ganglionnaires lors du glaucome n’est pas bien compris, mais quelques responsables putatifs ont été proposés tels que l’excitotoxicité, le manque de neurotrophines, la compression mécanique, l’ischémie, les astrocytes réactifs et le stress oxidatif, parmis d’autres. Indépendamment de la cause, il est bien établi que la perte des cellules rétiniennes ganglionnaires lors du glaucome est causée par la mort cellulaire programmée apoptotique. Cependant, les mécanismes moléculaires précis qui déclenchent l’apoptose dans les cellules rétiniennes ganglionnaires adultes sont mal définis. Pour aborder ce point, j’ai avancé l’hypothèse centrale que l’identification de voies de signalisations moléculaires impliquées dans la mort apoptotique des cellules rétiniennes ganglionnaires offrirait des avenues thérapeutiques pour ralentir ou même prévenir la mort de celles-ci lors de neuropathies oculaires telles que le glaucome. Dans la première partie de ma thèse, j’ai caractérisé le rôle de la famille de protéines stimulatrices d’apoptose de p53 (ASPP), protéines régulatrices de la famille p53, dans la mort apoptotique des cellules rétiniennes ganglionnaires. p53 est un facteur de transcription nucléaire impliqué dans des fonctions cellulaires variant de la transcription à l’apoptose. Les membres de la famille ASPP, soit ASPP1, ASPP2 et iASPP, sont des protéines de liaison de p53 qui régulent l’apoptose. Pourtant, le rôle de la famille des ASPP dans la mort des cellules rétiniennes ganglionnaires est inconnu. ASPP1 et ASPP2 étant pro-apoptotiques, l’hypothèse de cette première étude est que la baisse ciblée de ASPP1 et ASPP2 promouvrait la survie des cellules rétiniennes ganglionnaires après une blessure du nerf optique. Nous avons utilisé un modèle expérimental bien caractérisé de mort apoptotique neuronale induite par axotomie du nerf optique chez le rat de type Sprague Dawley. Les résultats de cette étude (Wilson et al. Journal of Neuroscience, 2013) ont démontré que p53 est impliqué dans la mort apoptotique des cellules rétiniennes ganglionnaires, et qu’une baisse ciblée de ASPP1 et ASPP2 par acide ribonucléique d’interference promeut la survie des cellules rétiniennes ganglionnaires. Dans la deuxième partie de ma thèse, j’ai caractérisé le rôle d’iASPP, le membre anti-apoptotique de la famille des ASPP, dans la mort apoptotique des cellules rétiniennes ganglionnaires. L’hypothèse de cette seconde étude est que la surexpression d’iASPP promouvrait la survie des cellules rétiniennes ganglionnaires après axotomie. Mes résultats (Wilson et al. PLoS ONE, 2014) démontrent que le knockdown ciblé de iASPP exacerbe la mort apoptotique des cellules rétiniennes ganglionnaires, et que la surexpression de iASPP par virus adéno-associé promeut la survie des cellules rétiniennes ganglionnaires. En conclusion, les résultats présentés dans cette thèse contribuent à une meilleure compréhension des mécanismes régulateurs sous-jacents la perte de cellules rétiniennes ganglionnaires par apoptose et pourraient fournir des pistes pour la conception de nouvelles stratégies neuroprotectrices pour le traitement de maladies neurodégénératives telles que le glaucome.

Characterisation of cell death in bagged baby salad leaves

Baby leaf salads are gaining in popularity over traditional whole head lettuce salads in response to consumer demand for greater variety and convenience in their diet. Baby lettuce leaves are mixed, washed and packaged as whole leaves, with a shelf-life of approximately 10 days post-processing. End of shelf-life, as determined by the consumer, is typified by bruising, water-logging and blackening of the leaves, but the biological events causing this phenotype have not been studied to date. We investigated the physiological and ultrastructural characteristics during postharvest shelf-life of two lettuce varieties with very different leaf morphologies. Membrane disruption was an important determinant of cell death in both varieties. although the timing and characteristics of breakdown was different in each with Lollo rossa showing signs of aging such as thylakoid disruption and plastoglobuli accumulation earlier than Cos. Membranes in Lollo rossa showed a later, but more distinct increase in permeability than in Cos. as indicated by electrolyte leakage and the presence of cytoplasmic fragments in the vacuole, but Cos membranes show distinct fractures towards the end of shelf-life. The tissue lost less than 25% fresh weight during shelf-life and there was little protein loss compared to developmentally aging leaves in an ambient environment. Biophysical measurements showed that breakstrength was significantly reduced in Lollo rossa, whereas irreversible leaf plasticity was significantly reduced in Cos leaves. The reversible elastic properties of both varieties changed throughout shelf-life. We compared the characteristics of shelf-life in both varieties of bagged lettuce leaves with other leafy salad crops and discuss the potential targets for future work to improve postharvest quality of baby leaf lettuce. (C) 2007 Elsevier B.V. All rights reserved.

The pgip family in soybean and three other legume species: evidence for a birth-and-death model of evolution

Background Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) plant cell wall glycoproteins involved in plant immunity. They are typically encoded by gene families with a small number of gene copies whose evolutionary origin has been poorly investigated. Here we report the complete characterization of the full complement of the pgip family in soybean (Glycine max [L.] Merr.) and the characterization of the genomic region surrounding the pgip family in four legume species. Results BAC clone and genome sequence analyses showed that the soybean genome contains two pgip loci. Each locus is composed of three clustered genes that are induced following infection with the fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary, and remnant sequences of pgip genes. The analyzed homeologous soybean genomic regions (about 126 Kb) that include the pgip loci are strongly conserved and this conservation extends also to the genomes of the legume species Phaseolus vulgaris L., Medicago truncatula Gaertn. and Cicer arietinum L., each containing a single pgip locus. Maximum likelihood-based gene trees suggest that the genes within the pgip clusters have independently undergone tandem duplication in each species. Conclusions The paleopolyploid soybean genome contains two pgip loci comprised in large and highly conserved duplicated regions, which are also conserved in bean, M. truncatula and C. arietinum. The genomic features of these legume pgip families suggest that the forces driving the evolution of pgip genes follow the birth-and-death model, similar to that proposed for the evolution of resistance (R) genes of NBS-LRR-type.

CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice

Purkinje cell degeneration (pcd) mice have a mutation within the gene encoding cytosolic carboxypeptidase 1 (CCP1/Nna1), which has homology to metallocarboxypeptidases. To assess the function of CCP1/Nna1, quantitative proteomics and peptidomics approaches were used to compare proteins and peptides in mutant and wild-type mice. Hundreds of peptides derived from cytosolic and mitochondrial proteins are greatly elevated in pcd mouse hypothalamus, amygdala, cortex, prefrontal cortex, and striatum. However, the major proteins detected on 2-D gel electrophoresis were present in mutant and wild-type mouse cortex and hypothalamus at comparable levels, and proteasome activity is normal in these brain regions of pcd mice, suggesting that the increase in cellular peptide levels in the pcd mice is due to reduced degradation of the peptides downstream of the proteasome. Both nondegenerating and degenerating regions of pcd mouse brain, but not wild-type mouse brain, show elevated autophagy, which can be triggered by a decrease in amino acid levels. Taken together with previous studies on CCP1/Nna1, these data suggest that CCP1/Nna1 plays a role in protein turnover by cleaving proteasome-generated peptides into amino acids and that decreased peptide turnover in the pcd mice leads to cell death.-Berezniuk, I., Sironi, J., Callaway, M. B., Castro, L. M., Hirata, I. Y., Ferro, E. S., Fricker, L. D. CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice. FASEB J. 24, 1813-1823 (2010). www.fasebj.org

Cell death and lumen formation in spheroids of MCF-7 cells

3D (three-dimensional) cell culture permits a more integrated analysis of the relationship between cells, inserting them into a structure more closely resembling the cellular microenvironment in vivo. The development of in vitro parameters to approximate in vivo 3D cellular environments makes a less reductionist interpretation of cell biology possible. For breast cells, in vitro 3D culture has proven to be an important tool for the analysis of luminal morphogenesis. A greater understanding of this process is necessary because alterations in the lumen arrangement are associated with carcinogenesis. Following lumen formation in 3D cell culture using laser scanning confocal microscopy, we observed alterations in the arrangement of cytoskeletal components (F-actin and microtubules) and increasing levels of cell death associated with lumen formation. The formation of a polarized monolayer facing the lumen was characterized through 3D reconstructions and the use of TEM (transmission electron microscopy), and this process was found to occur through the gradual clearing of cells from the medullary region of the spheroids. This process was associated with different types of cell death, such as apoptosis, autophagy and entosis. The present study showed that changes in the extracellular matrix associated with long periods of time in 3D cell culture lead to the formation of a lumen in MCF-7 cell spheroids and that features of differentiation such as lumen and budding formation occur after long periods in 3D culture, even in the absence of exogenous extracellular compounds.

Molecular Characterization of Propolis-Induced Cell Death in Saccharomyces cerevisiae

Propolis, a natural product of plant resins, is used by the bees to seal holes in their honeycombs and protect the hive entrance. However, propolis has also been used in folk medicine for centuries. Here, we apply the power of Saccharomyces cerevisiae as a model organism for studies of genetics, cell biology, and genomics to determine how propolis affects fungi at the cellular level. Propolis is able to induce an apoptosis cell death response. However, increased exposure to propolis provides a corresponding increase in the necrosis response. We showed that cytochrome c but not endonuclease G (Nuc1p) is involved in propolis-mediated cell death in S. cerevisiae. We also observed that the metacaspase YCA1 gene is important for propolis-mediated cell death. To elucidate the gene functions that may be required for propolis sensitivity in eukaryotes, the full collection of about 4,800 haploid S. cerevisiae deletion strains was screened for propolis sensitivity. We were able to identify 138 deletion strains that have different degrees of propolis sensitivity compared to the corresponding wild-type strains. Systems biology revealed enrichment for genes involved in the mitochondrial electron transport chain, vacuolar acidification, negative regulation of transcription from RNA polymerase II promoter, regulation of macroautophagy associated with protein targeting to vacuoles, and cellular response to starvation. Validation studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis.

Peptide gomesin triggers cell death through L-type channel calcium influx, MAPK/ERK, PKC and PI3K signaling and generation of reactive oxygen species

Gomesin is an antimicrobial peptide isolated from hemocytes of a common Brazilian tarantula spider named Acanthoscurriagomesiana. This peptide exerts antitumor activity in vitro and in vivo by an unknown mechanism. In this study, the cytotoxic mechanism of gomesin in human neuroblastoma SH-SY5Y and rat pheochromocytoma PC12 cells was investigated. Gomesin induced necrotic cell death and was cytotoxic to SH-SY5Y and PC12 cells. The peptide evoked a rapid and transient elevation of intracellular calcium levels in Fluo-4-AM loaded PC12 cells, which was inhibited by nimodipine, an L-type calcium channel blocker. Preincubation with nimodipine also inhibited cell death induced by gomesin in SH-SY5Y and PC12 cells. Gomesin-induced cell death was prevented by the pretreatment with MAPK/ERK, PKC or PI3K inhibitors, but not with PKA inhibitor. In addition, gomesin generated reactive oxygen species (ROS) in SH-SY5Y cells, which were blocked with nimodipine and MAPK/ERK, PKC or PI3K inhibitors. Taken together, these results suggest that gomesin could be a useful anticancer agent, which mechanism of cytotoxicity implicates calcium entry through L-type calcium channels, activation of MAPK/ERK, PKC and PI3K signaling as well as the generation of reactive oxygen species. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Major determinants of photoinduced cell death: Subcellular localization versus photosensitization efficiency

We present a study on whether and to what extent subcellular localization may compete favorably with photosensitization efficiency with respect to the overall efficiency of photoinduced cell death. We have compared the efficiency with which two cationic photosensitizers, namely methylene blue (MB) and crystal violet (CV), induce the photoinduced death of human cervical adenocarcinoma (HeLa) cells. Whereas MB is well known to generate singlet oxygen and related triplet excited species with high quantum yields in a variety of biological and chemical environments (i.e., acting as a typical type II photosensitizer), the highly mitochondria-specific CV produces triplet species and singlet oxygen with low yields, acting mostly via the classical type I mechanism (e.g., via free radicals). The findings described here indicate that the presumably more phototoxic type II photosensitizer (MB) does not lead to higher degrees of cell death compared to the type I (CV) photosensitizer. In fact, CV kills cells with the same efficiency as MB, generating at least 10 times fewer photoinduced reactive species. Therefore, subcellular localization is indeed more important than photochemical reactivity in terms of overall cell killing, with mitochondrial localization representing a highly desirable property for the development of more specific/efficient photosensitizers for photodynamic therapy applications. (C) 2011 Elsevier Inc. All rights reserved.

Mechanism of Trypanosoma cruzi death induced by Cratylia mollis seed lectin

Incubation of T. cruzi epimastigotes with the lectin Cramoll 1,4 in Ca(2+) containing medium led to agglutination and inhibition of cell proliferation. The lectin (50 A mu g/ml) induced plasma membrane permeabilization followed by Ca(2+) influx and mitochondrial Ca(2+) accumulation, a result that resembles the classical effect of digitonin. Cramoll 1,4 stimulated (five-fold) mitochondrial reactive oxygen species (ROS) production, significantly decreased the electrical mitochondrial membrane potential (Delta I(m)) and impaired ADP phosphorylation. The rate of uncoupled respiration in epimastigotes was not affected by Cramoll 1,4 plus Ca(2+) treatment, but oligomycin-induced resting respiration was 65% higher in treated cells than in controls. Experiments using T. cruzi mitochondrial fractions showed that, in contrast to digitonin, the lectin significantly decreased Delta I(m) by a mechanism sensitive to EGTA. In agreement with the results showing plasma membrane permeabilization and impairment of oxidative phosphorylation by the lectin, fluorescence microscopy experiments using propidium iodide revealed that Cramoll 1,4 induced epimastigotes death by necrosis.

A truncated fragment of Src protein kinase generated by calpain-mediated cleavage is a mediator of neuronal death in excitotoxicity

Excitotoxicity resulting from overstimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke. The overstimulated ionotropic glutamate receptors exert their neurotoxic effects in part by overactivation of calpains, which induce neuronal death by catalyzing limited proteolysis of specific cellular proteins. Here, we report that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpains at a site in the N-terminal unique domain. This generates a truncated Src fragment of ?52 kDa, which we localized predominantly to the cytosol. A cell membrane-permeable fusion peptide derived from the unique domain of Src prevents calpain from cleaving Src in neurons and protects against excitotoxic neuronal death. To explore the role of the truncated Src fragment in neuronal death, we expressed a recombinant truncated Src fragment in cultured neurons and examined how it affects neuronal survival. Expression of this fragment, which lacks the myristoylation motif and unique domain, was sufficient to induce neuronal death. Furthermore, inactivation of the prosurvival kinase Akt is a key step in its neurotoxic signaling pathway. Because Src maintains neuronal survival, our results implicate calpain cleavage as a molecular switch converting Src from a promoter of cell survival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new pathological action of Src, our discovery of the neurotoxic action of the truncated Src fragment suggests new therapeutic strategies with the potential to minimize brain damage in ischemic stroke.

The 5S rDNA family evolves through concerted and birth-and-death evolution in fish genomes: an example from freshwater stingrays

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Modes of cell death in the hypopharyngeal gland of the honey bee (Apis mellifera L)

Different modes of cell death have been revealed in the regressing hypopharyngeal glands of worker honey bees. The hypopharyngeal gland, which is well developed in young nursing bees to produce protein for larval food, was seen to regress naturally in foraging adult worker bees. A range of techniques including histology, cytochemistry, in situ TUNEL, Annexin V and Comet assays indicated that cells within the gland demonstrate progressive symptoms of apoptosis, necrosis and a vacuolar form of programmed cell death. The latter mode of cell death did not display chromatin margination, but was accompanied by an enhanced level of autophagic and hydrolytic activity in which a cytosolic source of acid phosphatase became manifest in the extra-cisternal spaces. Normal and annexin-positive cells were found to occur in the younger nursing bees, whilst necrosis and an aberrant vacuolar type of apoptosis predominated in the older foraging bees. The relevance of these results to the classification of programmed cell death is discussed. (C) 2000 Academic Press.

Morphological characterization of cell death during the ovary differentiation in worker honey bee

Cell death that occurs during ovary differentiation in the honeybee worker's larval development accounts for ovariole reabsorption. From a morphological standpoint, three modes of death were detected. Germinative cells in the ovarioles die by an apoptotic-like process, whereas the somatic cells die by an autophagic process, type 11 cell death; and during pupation, stromatic and ovarian capsular cells die through cytoplasmic disintegration, releasing their components into the hemolymph. These modes of cell death are in part determined by the pattern of tissue organization within which the cell occurs. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Canine transmissible venereal tumors: Aspects related to programmed cell death

Canine transmissible venereal tumor (CTVT) is a neoplasm transmitted by the physical transfer of viable tumor cells by direct contact with injured skin and/or mucous tissue. These cells can transpose across histocompatibility barriers into unrelated hosts. This review focuses on the biology of apoptosis and the interaction of proteins involved in this process, as well as p53, p63 and the antiapoptotic protein Bcl-2. As such, this disease offer unique opportunity to study the biology of transplantable tumours and the interaction of proteins involved in apoptosis process and the prognosis of CTVT.