RESUMEN

Vittorio Gremigni is a scientific leader in the field of planarian biology with a very long historical perspective. By using electron microscopy, he contributed to the reconstruction of the phylogenesis of free living "Turbellaria", and he pioneered the study of the origin of blastema cells by using chromosomal markers. In this interview, Professor Gremigni describes the steps that moved his career towards the planarian field, the main scientific achievements he obtained and the changes that are taking place in the field. He also discusses recent progress and unanswered questions on planarian neoblasts and regeneration.

Asunto(s)

Planarias/citología , Planarias/fisiología , Regeneración/fisiología , Animales

RESUMEN

Adenine nucleotide translocases (ANTs) are multitask proteins involved in several aspects of cell metabolism, as well as in the regulation of cell death/survival processes. We investigated the role played by ANT isoforms 1 and 2 in the growth of a human glioblastoma cell line (ADF cells). The silencing of ANT2 isoform, by small interfering RNA, did not produce significant changes in ADF cell viability. By contrast, the silencing of ANT1 isoform strongly reduced ADF cell viability by inducing a non-apoptotic cell death process resembling paraptosis. We demonstrated that cell death induced by ANT1 depletion cannot be ascribed to the loss of the ATP/ADP exchange function of this protein. By contrast, our findings indicate that ANT1-silenced cells experience oxidative stress, thus allowing us to hypothesize that the effect of ANT1-silencing on ADF is mediated by the loss of the ANT1 uncoupling function. Several studies ascribe a pro-apoptotic role to ANT1 as a result of the observation that ANT1 overexpression sensitizes cells to mitochondrial depolarization or to apoptotic stimuli. In the present study, we demonstrate that, despite its pro-apoptotic function at a high expression level, the reduction of ANT1 density below a physiological baseline impairs fundamental functions of this protein in ADF cells, leading them to undertake a cell death process.

Asunto(s)

Translocador 1 del Nucleótido Adenina/genética , Apoptosis , Silenciador del Gen , Glioblastoma/metabolismo , Glioblastoma/patología , Estrés Oxidativo , Translocador 1 del Nucleótido Adenina/metabolismo , Glioblastoma/enzimología , Humanos , Células Tumorales Cultivadas

RESUMEN

Retinoblastoma-associated proteins 46 and 48 (RbAp46 and RbAp48) are factors that are components of different chromatin-modelling complexes, such as polycomb repressive complex 2, the activity of which is related to epigenetic gene regulation in stem cells. To date, no direct findings are available on the in vivo role of RbAp48 in stem-cell biology. We recently identified DjRbAp48 - a planarian (Dugesia japonica) homologue of human RBAP48 - expression of which is restricted to the neoblasts, the adult stem cells of planarians. In vivo silencing of DjRbAp48 induces lethality and inability to regenerate, even though neoblasts proliferate and accumulate after wounding. Despite a partial reduction in neoblast number, we were always able to detect a significant number of these cells in DjRbAp48 RNAi animals. Parallel to the decrease in neoblasts, a reduction in the number of differentiated cells and the presence of apoptotic-like neoblasts were detectable in RNAi animals. These findings suggest that DjRbAp48 is not involved in neoblast maintenance, but rather in the regulation of differentiation of stem-cell progeny. We discuss our data, taking into account the possibility that DjRbAp48 might control the expression of genes necessary for cell differentiation by influencing chromatin architecture.

Asunto(s)

Proteínas del Helminto/metabolismo , Planarias/citología , Planarias/metabolismo , Proteína 4 de Unión a Retinoblastoma/metabolismo , Células Madre/citología , Células Madre/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Cromatina/genética , Cromatina/metabolismo , Cromatina/ultraestructura , Citometría de Flujo , Proteínas del Helminto/genética , Humanos , Inmunohistoquímica , Hibridación in Situ , Microscopía Electrónica de Transmisión , Planarias/genética , Planarias/ultraestructura , Interferencia de ARN , Proteína 4 de Unión a Retinoblastoma/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/ultraestructura

RESUMEN

Planarians are a model system for studying adult stem cells, as they possess the neoblasts, a population of pluripotent adult stem cells able to give rise to both somatic and germ cells. Although over the last years several efforts have been made to shed light on neoblast biology, only recent evidence indicate that this population of cells is heterogeneous. In this study we irradiated planarians with different non-lethal X-ray doses (1-5 Gy) and we identified subpopulations of neoblasts with diverse levels of tolerance to X-rays. We demonstrated that a dramatic reduction of neoblasts occurred soon after non-lethal irradiations and that de-novo proliferation of some radioresistant cells re-established the primary neoblast number. In particular, a strong proliferation activity occurred at the ventral side of irradiated animals close to the nervous system. The produced cells migrated towards the dorsal parenchyma and, together with some dorsal radioresistant cells, reconstituted the entire neoblast population demonstrating the extreme plasticity of this adult stem cell system.

Asunto(s)

Células Madre Adultas/citología , Proliferación Celular/efectos de la radiación , Planarias/citología , Células Madre Adultas/fisiología , Animales , Diferenciación Celular/fisiología , Diferenciación Celular/efectos de la radiación , Movimiento Celular/fisiología , Movimiento Celular/efectos de la radiación , Planarias/efectos de la radiación

RESUMEN

BACKGROUND: High grade gliomas are one of the most difficult cancers to treat and despite surgery, radiotherapy and temozolomide-based chemotherapy, the prognosis of glioma patients is poor. Resistance to temozolomide is the major barrier to effective therapy. Alternative therapeutic approaches have been shown to be ineffective for the treatment of genetically unselected glioma patients. Thus, novel therapies are needed. Mitochondria-directed chemotherapy is an emerging tool to combat cancer, and inner mitochondrial permeability transition (MPT) represents a target for the development of cytotoxic drugs. A number of agents are able to induce MPT and some of them target MPT-pore (MPTP) components that are selectively up-regulated in cancer, making these agents putative cancer cell-specific drugs. OBJECTIVE: The aim of this paper is to report a comprehensive analysis of the effects produced by selected MPT-inducing drugs (Betulinic Acid, Lonidamine, CD437) in a temozolomide-resistant glioblastoma cell line (ADF cells). METHODS: EGFRvIII expression has been assayed by RT-PCR. EGFR amplification and PTEN deletion have been assayed by differential-PCR. Drugs effect on cell viability has been tested by crystal violet assay. MPT has been tested by JC1 staining. Drug cytostatic effect has been tested by mitotic index analysis. Drug cytotoxic effect has been tested by calcein AM staining. Apoptosis has been assayed by Hoechst incorporation and Annexine V binding assay. Authophagy has been tested by acridine orange staining. RESULTS: We performed a molecular and genetic characterization of ADF cells and demonstrated that this line does not express the EGFRvIII and does not show EGFR amplification. ADF cells do not show PTEN mutation but differential PCR data indicate a hemizygous deletion of PTEN gene. We analyzed the response of ADF cells to Betulinic Acid, Lonidamine, and CD437. Our data demonstrate that MPT-inducing agents produce concentration-dependent cytostatic and cytotoxic effects in parallel with MPT induction triggered through MPTP. CD437, Lonidamine and Betulinic acid trigger apoptosis as principal death modality. CONCLUSION: The obtained data suggest that these pharmacological agents could be selected as adjuvant drugs for the treatment of high grade astrocytomas that resist conventional therapies or that do not show any peculiar genetic alteration that can be targeted by specific drugs.

Asunto(s)

Citostáticos/farmacología , Dacarbazina/análogos & derivados , Resistencia a Antineoplásicos/efectos de los fármacos , Glioma/patología , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Naranja de Acridina , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ciclosporina/farmacología , Dacarbazina/farmacología , Receptores ErbB/metabolismo , Glioma/genética , Humanos , Indazoles/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Poro de Transición de la Permeabilidad Mitocondrial , Triterpenos Pentacíclicos , Reacción en Cadena de la Polimerasa , Retinoides/farmacología , Temozolomida , Triterpenos/farmacología , Ácido Betulínico

RESUMEN

Regeneration in planarians is an intriguing phenomenon, based on the presence of pluripotent stem cells, known as neoblasts. Following amputation, these cells activate mitotic divisions, migrate distally and undergo differentiation, giving rise to the regeneration blastema. We have identified two msh/msx-related genes, Djmsh1 and Djmsh2, which are expressed in distinct cell populations of the planarian Dugesia japonica and activated, with different patterns, during head regeneration. We demonstrate that RNA interference of Djmsh1 or Djmsh2 generates a delay in the growth of cephalic blastema, interfering with the dynamics of mitoses during its initial formation. Our data also reveal that the activity of the two planarian msh genes is required to regulate Djbmp expression during head regeneration. This study identifies, for the first time, a functional association between muscle segment homeobox (MSH) homeoproteins and BMP signaling during stem cell-based regeneration of the planarian head and provides a functional analysis of how msh genes may regulate in vivo the regenerative response of planarian stem cells.

Asunto(s)

Genes de Helminto , Crecimiento/genética , Proteínas del Helminto/genética , Planarias/genética , Regeneración/genética , Animales , Proteínas del Helminto/fisiología , Planarias/fisiología , Interferencia de ARN , Regeneración/fisiología

RESUMEN

Gliomas are the most common brain tumours with a poor prognosis due to their aggressiveness and propensity for recurrence. The 18 kDa translocator protein (TSPO) has been demonstrated to be greatly expressed in glioma cells and its over-expression has been correlated with glioma malignance grades. Due to both its high density in tumours and the pro-apoptotic activity of its ligands, TSPO has been suggested as a promising target in gliomas. With the aim to evidence if the TSPO expression level alters glioma cell susceptibility to undergo to cell death, we analysed the effects of the specific TSPO ligand, PK 11195, in human astrocytoma wild-type and TSPO-silenced cell lines. As first step, TSPO was characterised in human astrocytoma cell line (ADF). Our data demonstrated the presence of a single class of TSPO binding sites highly expressed in mitochondria. PK 11195 cell treatment activated an autophagic pathway followed by apoptosis mediated by the modulation of the mitochondrial permeability transition. In TSPO-silenced cells, produced by siRNA technique, a reduced cell proliferation rate and a decreased cell susceptibility to the PK 11195-induced anti-proliferative effect and mitochondrial potential dissipation were demonstrated respect to control cells. In conclusion, for the first time, PK 11195 was demonstrated to differentially affect glioma cell survival in relation to TSPO expression levels. These results encourage the development of specific-cell strategies for the treatment of gliomas, in which TSPO is highly expressed respect to normal cells.

Asunto(s)

Antineoplásicos/farmacología , Astrocitoma/metabolismo , Isoquinolinas/farmacología , Receptores de GABA/genética , Receptores de GABA/metabolismo , Astrocitoma/genética , Muerte Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Humanos , Microscopía Electrónica de Transmisión , Membranas Mitocondriales/metabolismo , Interferencia de ARN , Receptores de GABA/análisis , Transfección

RESUMEN

Gliomas are one of the most malignant cancers. The molecular bases regulating the onset of such tumors are still poorly understood. The translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, is a mitochondrial permeability transition (MPT)-pore protein robustly expressed in gliomas and involved in the regulation of apoptosis and cell proliferation. TSPO expression levels have been correlated with tumor malignancy. Here we describe the production of C6 rat glioma cells engineered to over-express the TSPO protein with the aim of providing the first direct evidence of a correlation between TSPO expression level and glioma cell aggressiveness. We observed that TSPO potentiates proliferation, motility and transmigration capabilities as well as the ability to overcome contact-induced cell growth inhibition of glioma cells. On the whole, these data demonstrate that TSPO density influences metastatic potential of glioma cells. Since several data suggest that TSPO ligands may act as chemotherapeutic agents, in this paper we also demonstrate that TSPO ligand-induced cell death is dependent on TSPO density. These findings suggest that the use of TSPO ligands as chemotherapeutic agents could be effective on aggressive tumor cells with a high TSPO expression level.

Asunto(s)

Proteínas Portadoras/genética , Movimiento Celular/genética , Proliferación Celular , Glioma/genética , Receptores de GABA-A/genética , Animales , Antineoplásicos/farmacología , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Glioma/patología , Isoquinolinas/farmacología , Ligandos , Invasividad Neoplásica , Ratas , Receptores de GABA-A/metabolismo , Receptores de GABA-A/fisiología , Transfección , Células Tumorales Cultivadas , Regulación hacia Arriba

RESUMEN

BACKGROUND: Mammalian stem cells are difficult to access experimentally; model systems that can regenerate offer an alternative way to characterize stem cell related genes. Planarian regeneration depends on adult pluripotent stem cells--the neoblasts. These cells can be selectively destroyed using X-rays, enabling comparison of organisms lacking stem cells with wild-type worms. RESULTS: Using a genomic approach we produced an oligonucleotide microarray chip (the Dj600 chip), which was designed using selected planarian gene sequences. Using this chip, we compared planarians treated with high doses of X-rays (which eliminates all neoblasts) with wild-type worms, which led to identification of a set of putatively neoblast-restricted genes. Most of these genes are involved in chromatin modeling and RNA metabolism, suggesting that epigenetic modifications and post-transcriptional regulation are pivotal in neoblast regulation. Comparing planarians treated with low doses of X-rays (after which some radiotolerant neoblasts re-populate the planarian body) with specimens irradiated with high doses and unirradiated control worms, we identified a group of genes that were upregulated as a consequence of low-dose X-ray treatment. Most of these genes encode proteins that are known to regulate the balance between death and survival of the cell; our results thus suggest that genetic programs that control neoblast cytoprotection, proliferation, and migration are activated by low-dose X-rays. CONCLUSION: The broad differentiation potential of planarian neoblasts is unparalleled by any adult stem cells in the animal kingdom. In addition to our validation of the Dj600 chip as a valuable platform, our work contributes to elucidating the molecular mechanisms that regulate the self-renewal and differentiation of neoblasts.

Asunto(s)

Planarias/genética , Células Madre/metabolismo , Animales , Ensamble y Desensamble de Cromatina/genética , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de la radiación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Planarias/citología , Planarias/efectos de la radiación , ARN/metabolismo , Regeneración/genética , Células Madre/citología , Células Madre/fisiología , Rayos X

RESUMEN

Fluorescent ligands for the peripheral-type benzodiazepine receptor (PBR) featuring the 7-nitrobenz-2-oxa-1,3-diazol-4-yl moiety were synthesized, based on N,N-dialkyl-2-phenylindol-3-ylglyoxylamides, a potent, selective class of PBR ligands previously described by us. All the new ligands are moderately to highly potent at the PBR, with a complete selectivity over the central benzodiazepine receptor. Results from fluorescence microscopy showed that these probes specifically labeled the PBR at the mitochondrial level in C6 glioma cells.

Asunto(s)

Colorantes Fluorescentes/síntesis química , Indoles/síntesis química , Receptores de GABA-A/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular Tumoral , Diseño de Fármacos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Técnicas In Vitro , Indoles/química , Indoles/farmacología , Ligandos , Ensayo de Unión Radioligante , Ratas , Espectrofotometría Ultravioleta

RESUMEN

Planarian regeneration, based upon totipotent stem cells, the neoblasts, provides a unique opportunity to study in vivo the molecular program that defines a stem cell. In this study, we report the identification of DjPiwi-1, a planarian homologue of Drosophila Piwi. Expression analysis showed that DjPiwi-1 transcripts are preferentially accumulated in small cells distributed along the midline of the dorsal parenchyma. DjPiwi-1 transcripts were not detectable after X-ray irradiation by whole mount in situ hybridization. Real time reverse transcriptase polymerase chain reaction analysis confirmed the significant reduction of DjPiwi-1 expression after X-ray treatment. However, the presence of residual DjPiwi-1 transcription suggests that, although the majority of DjPiwi-1-positive cells can be neoblasts, this gene is also expressed in differentiating/differentiated cells. During regeneration DjPiwi-1-positive cells reorganize along the midline of the stump and no accumulation of hybridization signal was observed either in the blastema area or in the parenchymal region beneath the blastema. DjPiwi-1-positive cells, as well as the DjMCM2-expressing neoblasts located along the midline and those spread all over the parenchyma, showed a lower tolerance to X-ray with respect to the DjMCM2-expressing neoblasts distributed along the lateral lines of the parenchyma. Taken together, these findings suggest the presence of different neoblast subpopulations in planarians.

Asunto(s)

Genes de Helminto , Proteínas del Helminto , Planarias/genética , Planarias/fisiología , Células Madre/fisiología , Secuencia de Aminoácidos , Animales , Supervivencia Celular/efectos de la radiación , Secuencia Conservada , Etiquetas de Secuencia Expresada , Hibridación in Situ , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Planarias/anatomía & histología , Planarias/ultraestructura , Estructura Terciaria de Proteína , Interferencia de ARN , Regeneración/genética , Homología de Secuencia de Aminoácido , Células Madre/metabolismo , Células Madre/ultraestructura , Transcripción Genética , Rayos X

RESUMEN

Peripheral benzodiazepine receptor (PBR) has been considered a promising drug target for cancer therapy, and several ligands have been developed for this purpose. Human T-lymphoma Jurkat cells have been considered as lacking PBR and are often used as negative control to prove the specificity of PBR ligands effects. It is surprising that we evidenced PBR protein expression in this cell line by means of Western blotting and immunocytochemistry assays using specific anti-PBR antibodies. PBR intracellular localization was evidenced in mitochondria and nuclei, as demonstrated by confocal and electron microscopy. The binding of the [(3)H]4'-chloro derivative of diazepam [(3)H]7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864) and the isoquinoline carboxamide derivative [(3)H]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3 isoquinolinecarboxamide (PK11195) evidenced a single class of binding sites with an unusual affinity constant (K(d)) of 1.77 +/- 0.30 and 2.20 +/- 0.20 microM, respectively. The pharmacological profile of the classic ligands showed that PK11195 was the most potent inhibitor in the radioligand binding assays followed by Ro5-4864 and diazepam, whereas clonazepam, a specific ligand for the central-type receptor, showed a K(i) >1.0 x 10(-4) M. By a combined strategy of reverse transcriptase-polymerase chain reaction and Southern blot experiments, we succeeded in isolating and cloning the full-length Jurkat PBR cDNA, called JuPBR. The JuPBR gene showed two single-nucleotide polymorphisms resulting in the two substitutions, Ala147 --> threonine and His162 --> arginine, of PBR amino acidic sequence. In conclusion, for the first time, we demonstrated PBR expression in Jurkat cells: the protein bound classic PBR ligands with micromolar affinity constants and presented a modified amino acidic sequence consequent to the detection of two gene polymorphisms.

Asunto(s)

Linfoma de Células T/metabolismo , Receptores de GABA-A/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Benzodiazepinonas/farmacología , Western Blotting , Clonación Molecular , Cartilla de ADN , ADN Complementario , Humanos , Inmunohistoquímica , Isoquinolinas/farmacología , Células Jurkat , Linfoma de Células T/patología , Microscopía Electrónica , Mutación Puntual , Ensayo de Unión Radioligante , Ratas , Receptores de GABA-A/química , Receptores de GABA-A/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido

RESUMEN

OBJECTIVE: G protein-coupled receptors may up-regulate the inflammatory response elicited by ventilator-induced lung injury but also regulate cell survival via protein kinase B (Akt) and extracellular signal regulated kinases 1/2 (ERK1/2). The G protein-sensitive phosphoinositide-3-kinase gamma (PI3Kgamma) regulates several cellular functions including inflammation and cell survival. We explored the role of PI3Kgamma on ventilator-induced lung injury. DESIGN: Prospective, randomized, experimental study. SETTING: University animal research laboratory. SUBJECTS: Wild-type (PI3Kgamma), knock-out (PI3Kgamma ), and kinase-dead (PI3Kgamma) mice. INTERVENTIONS: Three ventilatory strategies (no stretch, low stretch, high stretch) were studied in an isolated, nonperfused model of acute lung injury (lung lavage) in PI3Kgamma, PI3Kgamma, and PI3Kgamma mice. MEASUREMENTS AND MAIN RESULTS: Reduction in lung compliance, hyaline membrane formation, and epithelial detachment with high stretch were more pronounced in PI3Kgamma than in PI3Kgamma and PI3Kgamma (p < .01). Inflammatory cytokines and IkBalpha phosphorylation with high stretch did not differ among PI3Kgamma, PI3Kgamma, and PI3Kgamma. Apoptotic index (terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling) and caspase-3 (immunohistochemistry) with high stretch were larger (p < .01) in PI3Kgamma and PI3Kgamma than in PI3Kgamma. Electron microscopy showed that high stretch caused apoptotic changes in alveolar cells of PI3Kgamma mice whereas PI3Kgamma mice showed necrosis. Phosphorylation of Akt and ERK1/2 with high stretch was more pronounced in PI3Kgamma than in PI3Kgamma and PI3Kgamma (p < .01). CONCLUSIONS: Silencing PI3Kgamma seems to attenuate functional and morphological consequences of ventilator-induced lung injury independently of inhibitory effects on cytokines release but through the enhancement of pulmonary apoptosis.

Asunto(s)

Citocinas/metabolismo , Enfermedades Pulmonares/enzimología , Lesión Pulmonar , Fosfatidilinositol 3-Quinasas/metabolismo , Respiración Artificial/efectos adversos , Insuficiencia Respiratoria/fisiopatología , Animales , Biomarcadores/análisis , Biopsia con Aguja , Citocinas/análisis , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Pulmón/patología , Enfermedades Pulmonares/fisiopatología , Masculino , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/análisis , Distribución Aleatoria , Valores de Referencia , Pruebas de Función Respiratoria , Sensibilidad y Especificidad , Regulación hacia Arriba

RESUMEN

The ultrastructure of the female gonad of the land planarian Geoplana burmeisteri was investigated by means of electron microscopy and cytochemical techniques. It consists of two small germaria located ventral to the intestine and of two irregular, lateral rows of vitelline follicles, both enveloped by a tunica composed of an extracellular lamina and an inner sheath of accessory cells. Accessory cell projections completely surround developing oocytes and vitellocytes. The main feature of oocyte maturation is the appearance of chromatoid bodies and the development of the rough endoplasmic reticulum (RER) and Golgi complexes. These organelles appear to be correlated with the production of egg inclusions of medium electron density, about 1.5-1.8 microm in diameter, which remain scattered in the ooplasm of mature oocytes. On the basis of cytochemical tests demonstrating their glycoprotein composition, these inclusions were interpreted as residual yolk globules. Vitellocytes are typical secretory cells with well-developed RER and Golgi complexes that are mainly involved in the production of yolk globules and eggshell globules, respectively. Eggshell globules appear to arise from repeated coalescence of small Golgi-derived vesicles and, at an intermediate stage of maturation, show a multigranular pattern. Later, after vesicle fusion, they reach a diameter of 1.3-1.6 microm when completely mature and show a meandering/concentric pattern, as is typical of the situation seen in most Proseriata and Tricladida. The content of yolk globules is completely digested by pronase, while the content of eggshell globules is unaffected. Mature vitellocytes contain, in addition, a large quantity of glycogen and lipid droplets as further reserve material. On the basis of the ultrastructural characteristics of the female gonad described above and in relation to the current literature, we conclude that G. burmeisteri appears to be more closely related to the freshwater triclads, in particular to members of the Dugesiidae, than to the marine triclads.

Asunto(s)

Gónadas/ultraestructura , Platelmintos/ultraestructura , Animales , Diferenciación Celular , Femenino , Gónadas/química , Microscopía Electrónica de Transmisión , Oocitos/fisiología , Oocitos/ultraestructura , Oogénesis

RESUMEN

Mitochondrial benzodiazepine-receptor (mBzR) ligands constitute a heterogeneous class of compounds that show a pleiotropic spectrum of effects within the cells, including the modulation of apoptosis. In this paper, a novel synthetic 2-phenylindol-3-ylglyoxylamide derivative, N,N-di-n-butyl-5-chloro-2-(4-chlorophenyl)indol-3-ylglyoxylamide (PIGA), which shows high affinity and selectivity for the mBzR, is demonstrated to induce apoptosis in rat C6 glioma cells. PIGA was able to dissipate mitochondrial transmembrane potential (DeltaPsim) and to cause a significant cytosolic accumulation of cytochrome c. Moreover, typical features of apoptotic cell death, such as caspase-3 activation and DNA fragmentation, were also detected in PIGA-treated cells. Our data expand the knowledge on mBzR ligand-mediated apoptosis and suggest PIGA as a novel proapoptotic compound with therapeutic potential against glial tumours, in which apoptosis resistance has been reported to be involved in carcinogenesis.

Asunto(s)

Apoptosis/efectos de los fármacos , Glioma/patología , Indoles/síntesis química , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Receptores de GABA-A/metabolismo , Animales , Antineoplásicos/farmacología , Caspasa 3 , Caspasas/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Citosol/metabolismo , Daño del ADN , Indoles/farmacología , Ligandos , Potenciales de la Membrana/fisiología , Mitocondrias/metabolismo , Ratas , Factores de Tiempo

RESUMEN

As stem cells are rare and difficult to study in vivo in adults, the use of classical models of regeneration to address fundamental aspects of the stem cell biology is emerging. Planarian regeneration, which is based upon totipotent stem cells present in the adult--the so-called neoblasts--provides a unique opportunity to study in vivo the molecular program that defines a stem cell. The choice of a stem cell to self-renew or differentiate involves regulatory molecules that also operate as translational repressors, such as members of PUF proteins. In this study, we identified a homologue of the Drosophila PUF gene Pumilio (DjPum) in the planarian Dugesia japonica, with an expression pattern preferentially restricted to neoblasts. Through RNA interference (RNAi), we demonstrate that gene silencing of DjPum dramatically reduces the number of neoblasts, thus supporting the intriguing hypothesis that stem cell maintenance may be an ancestral function of PUF proteins.

Asunto(s)

Proteínas de Drosophila/genética , Silenciador del Gen , Filogenia , Planarias/genética , Planarias/fisiología , Regeneración/genética , Células Madre/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Citometría de Flujo , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Planarias/ultraestructura , Interferencia de ARN , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Células Madre/metabolismo

RESUMEN

OBJECTIVE: Peripheral benzodiazepine receptors (PBRs) are part of the mitochondrial permeability transition pore, and their activation may induce cell death. PBRs are expressed in human pancreatic islets, and cytokine-induced damage is accompanied by changes in their properties. We hypothesized that PBRs can have a role in human islet physiopathology, and evaluated the effects of prolonged exposure to two specific PBR ligands, PK11195 and Ro5-4864 on the function and survival of isolated human islets. DESIGN: Isolated human islets were prepared from the pancreas of 25 multiorgan cadaveric donors and incubated for 12 h in the presence of PK11195 or Ro5-4864. Insulin secretion studies and apoptosis experiments were then performed, together with assessment of intracellular pathways involved in islet cell function and survival. METHODS: Islets were prepared by enzymatic digestion and density gradient purification. Insulin secretion was assessed by the batch incubation method, and glucose oxidation was evaluated by the use of D-[U-(14)C]glucose. Apoptosis was studied using the TUNEL technique, ELISA methods, and electron microscopy evaluation. PCR experiments were performed by the use of specific primers. RESULTS: Glucose-stimulated insulin release was significantly lower after exposure to PK11195 than after exposure to Ro5-4864. This was accompanied by reduced glucose oxidation and no major change of insulin or GLUT-1 mRNA expression. Apoptosis was higher in PK11195-exposed islets, and electron microscopy demonstrated the involvement of beta-cells. The apoptotic effects were prevented by bongkrekic acid and low-dose cyclosporin A, which stabilize the mitochondrial membrane, and were associated with no evident change of inducible nitric oxide synthase (iNOS), B-cell leukemia/lymphoma-2 (Bcl-2) or Bcl-2-associated X protein (Bax) expression. Caspase inhibition markedly reduced the amount of apoptosis, and the role of these proteases was confirmed by the increased activity of caspase-3 and caspase-9. CONCLUSIONS: Prolonged binding to PBRs may cause human beta-cells functional damage and apoptosis, a phenomenon which is prevented by stabilizing the mitochondrial membrane; occurs without changes of iNOS, Bax and Bcl-2 mRNA expression; and involves caspase activation. These results suggest an involvement of PBRs in human pancreatic beta-cell function and survival.

Asunto(s)

Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Receptores de GABA-A/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Benzodiazepinonas/metabolismo , Benzodiazepinonas/farmacología , Caspasa 3 , Caspasa 9 , Inhibidores de Caspasas , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Transportador 2 de Aminoácidos Excitadores/genética , Expresión Génica/efectos de los fármacos , Humanos , Hipolipemiantes/metabolismo , Hipolipemiantes/farmacología , Insulina/genética , Islotes Pancreáticos/efectos de los fármacos , Isoquinolinas/metabolismo , Isoquinolinas/farmacología , Ligandos , Mitocondrias/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína X Asociada a bcl-2

RESUMEN

The peripheral benzodiazepine receptor (PBR) is a component of a multiprotein complex, located at the contact site between the inner and outer mitochondrial membranes, which constitutes the mitochondrial permeability transition (MPT)-pore. The opening of the MPT-pore, leading to the transmembrane mitochondrial potential (DeltaPsi(m)) dissipation, is a critical event in the mechanism of apoptosis. In the present work, we investigated the ability of the specific PBR ligands, PK 11195 or Ro5-4864, to affect mitochondrial potential and to induce apoptotic cell death in rat C6 glioma cells. Both specific ligands inhibited cell survival in a dose- and time-dependent manner, as assessed by MTS conversion assay, whereas the non-site selective ligand Diazepam or the low-affinity benzodiazepine Clonazepam showed no significant effects. After cell exposure to PK 11195 or Ro5-4864 we evidenced typical alterations of apoptotic cell death such as DNA fragmentation and chromatin condensation assessed by flow cytometric and transmission electron microscopy (TEM) analysis, respectively. Activation of the "effector" caspase-3 confirmed the ability of specific PBR ligands to induce apoptosis. Moreover, PK 11195 and Ro5-4864 induced a decrease of DeltaPsi(m), as evidenced by JC-1 flow cytometry analysis. Our data demonstrate the pro-apoptotic effects of specific PBR ligands on rat C6 glioma cells.

Asunto(s)

Apoptosis , Agonistas de Receptores de GABA-A , Isoquinolinas/farmacología , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Benzodiazepinonas/farmacología , Caspasa 3 , Caspasas/metabolismo , Glioma , Ligandos , Potenciales de la Membrana/fisiología , Mitocondrias/fisiología , Ratas , Células Tumorales Cultivadas

RESUMEN

A conserved network of nuclear proteins is crucial to eye formation in both vertebrates and invertebrates. The finding that freshwater planarians can regenerate eyes without the contribution of Pax6 suggests that alternative combinations of regulatory elements may control the morphogenesis of the prototypic planarian eye. To further dissect the molecular events controlling eye regeneration in planarians, we investigated the role of eyes absent (Djeya) and six-1 (Djsix-1) genes in Dugesia japonica. These genes are expressed in both regenerating eyes and in differentiated photoreceptors of intact adults. Through RNAi studies, we show that Djsix-1 and Djeya are both critical for the regeneration of normal eyes in planarians and genetically cooperate in vivo to establish correct eye cell differentiation. We further demonstrate that the genetic interaction is mediated by physical interaction between the evolutionarily conserved domains of these two proteins. These data indicate that planarians use cooperatively Djsix-1 and Djeya for the proper specification of photoreceptors, implicating that the mechanism involving their evolutionarily conserved domains can be very ancient. Finally, both Djsix-1 and Djeya double-stranded RNA are substantially more effective at producing no-eye phenotypes in the second round of regeneration. This is probably due to the significant plasticity of the planarian model system, based on the presence of a stable population of totipotent stem cells, which ensure the rapid cell turnover of all differentiated cell types.

Asunto(s)

Genes Reguladores/fisiología , Proteínas de Homeodominio/fisiología , Planarias/embriología , Planarias/genética , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Secuencia Conservada , Datos de Secuencia Molecular , Morfogénesis , Interferencia de ARN , Regeneración

RESUMEN

A(2A) adenosine receptor-mediated signaling affects a variety of important processes in the central nervous system both in physiological and pathological conditions, and has been indicated as possible novel therapeutic target in several nervous system diseases. In the present work, cell death induction was investigated after neuronal PC 12 cell treatment with proinflammatory cytokines and adenosine receptor ligands. Interleukin-1-beta (IL-1-beta, 500 U/mL), tumor necrosis factor-alpha (TNF-alpha, 1000 U/mL) and the non selective adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), caused a significant reduction of cell viability with a maximal effect within 3-48 hr. Moreover, an addictive effect was detected when the cells were simultaneously treated with Interleukin-1-beta and NECA for 3 hr. To investigate the adenosine receptor subtypes involved in PC 12 cell death, the effects of several adenosine receptor agonists/antagonists were evaluated. The endogenous nucleoside, adenosine, and the selective A(2A) adenosine receptor agonist, 2-(carboxyethylphenylethylamino)adenosine-5'-carboxamide (CGS21680) reduced PC 12 cell viability. This effect was counteracted by the selective A(2A) adenosine receptor antagonist, 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3e]-1,2,4-triazolo[1,5c]pyrimidine (SCH58261), but not by selective A(2B) adenosine receptor antagonist N-(4-acethylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS1706), suggesting the specific involvement of A(2A) adenosine receptor subtype in adenosine-mediated cytotoxicity. Moreover, the selective A(1) adenosine receptor agonist, N(6)-cyclohexyladenosine (CHA), did not induce any significant effect on cell viability. By ELISA immunoassay cell death detection and transmission electron microscopy (TEM) we demonstrated that A(2A) adenosine receptor ligands and cytokines induced cell death through an apoptotic pathway. In conclusion, our results showed that A(2A) adenosine receptors are involved in the control of PC 12 cell survival/death and may contribute to modulate cellular activity in response to tissue damage associated with inflammatory mediator production.

Asunto(s)

Agonistas del Receptor de Adenosina A2 , Adenosina-5'-(N-etilcarboxamida)/farmacología , Adenosina/análogos & derivados , Apoptosis , Citocinas/farmacología , Glicina/análogos & derivados , Adenosina/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Ratas