RESUMEN
Despite the identification of numerous key players of the cell death machinery, little is known about their physiological role. Using RNA interference (RNAi) in vivo, we have studied the requirement of all Drosophila caspases and caspase-adaptors in different paradigms of apoptosis. Of the seven caspases, Dronc, drICE, Strica and Decay are rate limiting for apoptosis. Surprisingly, Hid-mediated apoptosis requires a broader range of caspases than apoptosis initiated by loss of the caspase inhibitor DIAP1, suggesting that Hid causes apoptosis not only by antagonizing DIAP1 but also by activating DIAP1-independent caspase cascades. While Hid killing requires Strica, Decay, Dronc/Dark and drICE, apoptosis triggered by DIAP1 depletion merely relied upon Dronc/Dark and drICE. Furthermore, we found that overexpression of DIAP2 can rescue diap1-RNAi-mediated apoptosis, suggesting that DIAP2 regulates caspases directly. Consistently, we show that DIAP2 binds active drICE. Since DIAP2 associates with Hid, we propose a model whereby Hid co-ordinately targets both DIAP1 and DIAP2 to unleash drICE.
Asunto(s)
Apoptosis/fisiología , Caspasas/fisiología , Drosophila/citología , Drosophila/enzimología , Animales , Animales Modificados Genéticamente , Apoptosis/genética , Inhibidores de Caspasas , Caspasas/genética , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Ojo/citología , Ojo/enzimología , Ojo/crecimiento & desarrollo , Genes de Insecto , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/fisiología , Modelos Biológicos , Fenotipo , Interferencia de ARN , Transducción de SeñalAsunto(s)
Ojo/embriología , Animales , Tipificación del Cuerpo , Diferenciación Celular/genética , Drosophila , Inducción Embrionaria , Evolución Molecular , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Proteínas del Ojo , Regulación del Desarrollo de la Expresión Génica , Genes Reguladores , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Morfogénesis , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Células Fotorreceptoras/metabolismo , Proteínas Represoras , VertebradosRESUMEN
The Drosophila eye is widely used as a model system to study neuronal differentiation, survival and axon projection. Photoreceptor differentiation starts with the specification of a founder cell R8, which sequentially recruits other photoreceptor neurons to the ommatidium. The eight photoreceptors that compose each ommatidium exist in two chiral forms organized along two axes of symmetry and this pattern represents a paradigm to study tissue polarity. We have developed a method of fluoroscopy to visualize the different types of photoreceptors and the organization of the ommatidia in living animals. This allowed us to perform an F(1) genetic screen to isolate mutants affecting photoreceptor differentiation, survival or planar polarity. We illustrate the power of this detection system using known genetic backgrounds and new mutations that affect ommatidial differentiation, morphology or chirality.
Asunto(s)
Drosophila/embriología , Drosophila/genética , Ojo/embriología , Neuronas/citología , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo , Diferenciación Celular , Córnea/citología , Córnea/embriología , Cruzamientos Genéticos , Embrión no Mamífero/fisiología , Metanosulfonato de Etilo , Ojo/citología , Femenino , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Masculino , Modelos Neurológicos , Mutagénesis , Neuronas/fisiología , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/embriologíaRESUMEN
In Drosophila the eye-antennal disc gives rise to most adult structures of the fly's head. Yet the molecular basis for its regionalization during development is poorly understood. Here we show that homothorax is required early during development for normal eye development and is necessary for the formation of the ventral head capsule. In the ventral region of the disc only, homothorax and wingless are involved in a positive feedback loop necessary to restrict eye formation. homothorax is able to prevent the initiation and progression of the morphogenetic furrow without inducing wingless, which points to homothorax as a key negative regulator of eye development. In addition, we show that the iroquois-complex genes are required for dorsal head development antagonizing the function of homothorax in this region of the disc.
Asunto(s)
Proteínas de Drosophila , Drosophila/embriología , Genes Homeobox/fisiología , Proteínas de Homeodominio/fisiología , Células Fotorreceptoras de Invertebrados/embriología , Factores de Transcripción , Alelos , Animales , Clonación Molecular , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox/genética , Proteínas Fluorescentes Verdes , Cabeza/embriología , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Inmunohistoquímica , Proteínas de Insectos/biosíntesis , Proteínas de Insectos/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Modelos Biológicos , Fenotipo , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Factores de Tiempo , Regulación hacia Arriba , Proteína Wnt1RESUMEN
The Drosophila ommatidia contain two classes of photoreceptor cells (PR's), the outer and the inner PR's. We performed an enhancer trap screen in order to target genes specifically expressed in PR's. Using the UAS/GAL4 method with enhanced green fluorescent protein (eGFP) as a vital marker, we screened 180000 flies. Out of 2730 lines exhibiting new eGFP patterns, we focused on 16 lines expressing eGFP in particular subsets of PR's. In particular, we describe three lines inserted near the spalt major, m-spondin and furrowed genes, whose respective expression patterns resemble those genes. These genes had not been reported to be expressed in the adult eye. These examples clearly show the ability of our screen to target genes expressed in the adult Drosophila eye.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insecto , Proteínas Luminiscentes , Células Fotorreceptoras de Invertebrados , Animales , Proteínas de la Matriz Extracelular/genética , Ojo , Femenino , Expresión Génica , Proteínas Fluorescentes Verdes , Proteínas de Homeodominio/genética , Proteínas de Insectos/genética , Operón Lac , Proteínas Luminiscentes/genética , Masculino , Selectinas/genética , Factores de Transcripción/genéticaRESUMEN
Color vision is achieved by comparing the inputs from retinal photoreceptor neurons that differ in their wavelength sensitivity. Recent studies have elucidated the distribution and phylogeny of opsins, the family of light-sensitive molecules involved in this process. Interesting new findings suggest that animals have evolved a strategy to achieve specific sensitivity through the mutually exclusive expression of different opsin genes in photoreceptors.
Asunto(s)
Evolución Biológica , Percepción de Color/fisiología , Animales , Humanos , Insectos/fisiología , Filogenia , Retina/fisiología , Opsinas de Bastones/fisiología , Vertebrados/fisiología , Visión Ocular/fisiologíaRESUMEN
Among the four isoforms of the calcitonin receptor (CTR) described in humans, two differ by the presence of h-CTR1 or absence of h-CTR2 of 16 amino acids in the first intracellular loop. Both receptors are biologically active. The TT cell line derived from a human medullary carcinoma of the thyroid is characterized by the secretion of large amounts of calcitonin. We have recently shown that this cell line expresses h-CTR2. In the present work we have studied the expression of CTR during TT cell proliferation and used dexamethasone to modify calcitonin expression in order to establish if an autocrine regulation involving calcitonin and its receptor was functional in the TT cells. The expression of this receptor and of calcitonin during TT cell proliferation was studied by reverse transcriptase-polymerase chain reaction (RT-PCR). Dexamethasone, a potent inhibitor of TT cell proliferation, levels (day 6 of culture) specifically increased receptor levels from day 8 onwards. CT peptide and CT mRNA levels decreased or were similar during experimental time. CTR regulation by glucocorticoids is suggested in TT cells. Autocrine regulation of CTR is also suggested by relation between CT mRNA levels and CTR mRNA.
Asunto(s)
Comunicación Autocrina/genética , Dexametasona/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Receptores de Calcitonina/genética , Calcitonina/biosíntesis , Calcitonina/genética , Carcinoma Medular/genética , Carcinoma Medular/patología , División Celular , Humanos , ARN Mensajero/análisis , ARN Neoplásico/análisis , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Células Tumorales CultivadasRESUMEN
We recently reported the presence of a truncated form (h-CTR2) of the human calcitonin receptor (CTR) in TT cells, a cell line derived from medullary thyroid carcinoma (MTC). This form (h-CTR2), characterized by the absence of 16 amino acids in the first intracellular domain, was also detected in two cases of MTC. In the present study we determined the expression of CTR mRNA in a larger sample, representative of the different clinical forms of MTC, and in normal thyroid. h-CTR2 was expressed in all MTC specimens (both sporadic and familial) and in the normal thyroid samples. The expression of the receptor mRNA was higher in MTC compared with normal thyroid. Moreover, CT and CTR mRNA levels were modified significantly during proliferation. This result suggests that CT may be involved in proliferation of MTC via autocrine/paracrine regulation. Calcitonin secretion by MTC may play a role in the development and spread of these tumors.
Asunto(s)
Carcinoma Medular/metabolismo , ARN Mensajero/metabolismo , Receptores de Calcitonina/genética , Neoplasias de la Tiroides/metabolismo , Carcinoma Medular/patología , División Celular/fisiología , Espacio Extracelular/metabolismo , Femenino , Humanos , Masculino , Receptores de Calcitonina/metabolismo , Valores de Referencia , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patologíaRESUMEN
The effect of prostaglandin E2 (PGE2) on osteoclast (OC) differentiation is unclear, either stimulator or inhibitor, depending on the in vitro system used. This probably reflects indirect mechanisms through intermediate cells. We have investigated the direct effect of PGE2 on human OC differentiation from cord blood monocytes (CBMs) in the absence of stromal cells. Macrophages and multinucleated cells (MNCs) resembling OCs form in cultures of CBMs stimulated by 1,25-dihydroxyvitamin D3. In the present study, CBMs were cultured for 3 weeks, as previously described, in the presence or absence of PGE2. The number of MNCs was significantly reduced in the presence of PGE2 as was the proliferation of cultured CBMs, assessed on day 7. Immunohistochemistry was performed to evaluate macrophage markers (CD11b and CD14) and OC marker (beta3-chain). PGE2 significantly increased the numbers of CD11b-positive and CD14-positive cells, whereas the number of beta3-chain-positive cells was significantly decreased. beta3-Chain, c-fos, and human calcitonin receptor (h-CTR) messenger RNA (mRNA) expressions were evaluated by reverse transcription-PCR with RNA extracted from cultured CBMs. In the presence of PGE2, expression of beta3-chain and c-fos mRNA was reduced from the first week of culture. h-CTR mRNA expression was also reduced, and only the h-CTR1 isoform was detected in the presence of PGE2. In addition, when PGE2 was added only during the last week of culture, when no CBM proliferation occurred, the number of CD11b- and beta3-positive cells was unchanged compared to that in the control culture, as were the proportion of MNCs, the fusion index, and the expression of c-fos mRNA. In conclusion, our results suggest that PGE2 has an inhibitory effect on human OC differentiation from CBMs, possibly by reducing precursor proliferation in these cultures. We also hypothesize that PGE2 may reduce OC differentiation by increasing the proportion of precursor cells that differentiate into macrophages. In addition, this may be the result of inhibition of the c-fos expression in CBMs.
Asunto(s)
Dinoprostona/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Osteoclastos/citología , Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , Genes fos , Células Madre Hematopoyéticas/citología , Humanos , Receptores de Lipopolisacáridos/análisis , Antígeno de Macrófago-1/análisis , Monocitos/citología , Monocitos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Receptores de Calcitonina/análisis , Receptores de Calcitonina/genéticaRESUMEN
1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces the differentiation of monocytes into macrophage-like cells in vitro. To identify the genes expressed during this process, we performed differential display polymerase chain reaction on RNA extracted from cord blood monocytes (CBMs) treated with 1,25-(OH)2D3. Treated CBMs expressed type-I 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH), the key enzyme of prostaglandin E2 (PGE2) catabolism and a 15-PGDH-related mRNA (15-PGDHr). This newly described 15-PGDH-related mRNA was constitutively expressed in adult monocytes. 15-PGDH gene(s) transcription was accompanied by the appearance of the 15-PGDH activity in treated CBMs. In addition, the cyclooxygenase 2 mRNA level was decreased and PGE2 levels in the culture mediums were lowered (50%). Our results stress that 1,25-(OH)2D3, at least in neonatal monocytes, can exert, directly or indirectly, a dual control on key enzymes of PGE2 metabolism. In conclusion, we suggest that modifications in prostaglandin metabolism, induced by the expression of type-I 15-PGDH and the downregulation of cyclooxygenase 2, could be involved in monocytic differentiation.
Asunto(s)
Calcitriol/farmacología , Sangre Fetal/enzimología , Hidroxiprostaglandina Deshidrogenasas/biosíntesis , Monocitos/enzimología , Adulto , Medios de Cultivo , Ciclooxigenasa 2 , Dinoprostona/análisis , Activación Enzimática , Inducción Enzimática , Sangre Fetal/química , Humanos , Hidroxiprostaglandina Deshidrogenasas/genética , Hidroxiprostaglandina Deshidrogenasas/aislamiento & purificación , Isoenzimas/genética , Proteínas de la Membrana , Monocitos/química , NAD/fisiología , Reacción en Cadena de la Polimerasa , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/análisisRESUMEN
NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH) inactivates prostaglandins. We recently reported an mRNA sequence coding for a predicted isomer (PGDH(rI)) of this enzyme. The TT cell line, derived from medullary thyroid carcinoma (MTC), expresses mRNAs for both isomers. We report here the expression by TT cells and MTC of a third 15-PGDH related mRNA (PGDH(rII)), 241 nt shorter than type-I 15-PGDH. RNase protection assays confirmed that TT cells expressed this mRNA (PGDH(rII)). Thus different splicing patterns could be involved in the post-transcriptional regulation of type-I 15-PGDH gene in MTC.
Asunto(s)
Hidroxiprostaglandina Deshidrogenasas/genética , ARN Mensajero/genética , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Clonación Molecular , ADN Complementario , Humanos , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Distribución Tisular , Células Tumorales CultivadasRESUMEN
The gene encoding the human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase, designated type-I 15-PGDH, was mapped to chromosome 4 by analyzing its segregation in a panel of human-hamster somatic cell hybrids. This gene was further localized to bands 4q34-q35 by in situ hybridization on human chromosomes.
Asunto(s)
Cromosomas Humanos Par 4 , Hidroxiprostaglandina Deshidrogenasas/genética , Bandeo Cromosómico , Células HL-60 , Humanos , Células Híbridas , Hibridación in SituAsunto(s)
Hidroxiprostaglandina Deshidrogenasas/biosíntesis , Isoenzimas/biosíntesis , Macrófagos/citología , Osteoclastos/citología , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Colecalciferol/farmacología , Ciclooxigenasa 2 , Inducción Enzimática , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Proteínas de la Membrana , Modelos Biológicos , Osteoclastos/efectos de los fármacos , Osteoclastos/enzimología , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Transcripción Genética/efectos de los fármacosRESUMEN
Osteoclasts (OCs), which form by fusion of hematopoietic precursor cells, are typically present in large numbers in giant cell tumors of bone (GCTBs). These tumors may, therefore, contain cells which secrete factors that stimulate recruitment and differentiation of OC precursors. Multinucleated cells resembling OCs also form in cultures of human cord blood monocytes (CBMs) stimulated by 1.25 dihydroxyvitamin D3, but these cells lack the ability to form bone resorption pits, the defining functional characteristic of mature OCs. CBMs may thus require additional stimulation to form OCs; we therefore investigated whether GCTBs are a source of such a stimulus. CBMs were stimulated in long term (21 day) culture by medium conditioned by explants of GCTBs; media collected within 15 days of explant (early-CM) and after 15 days (late-CM) were employed. We also cocultured CBMs with primary GCTB-derived stromal cells as well as immortalized bone marrow stroma-derived cells. CBMs stimulated by early-CM formed resorption pits on cortical bone slices; however, stimulation by late-CM resulted in virtually no resorption. Both early-CM and late-CM increased CBM proliferation, but not the proportion of vitronectin receptor positive or multinucleated cells. Coculture of CBMs with stromal cells of GCTBs or bone marrow did not result in bone resorption, although these stromal cells (most expressing alkaline phosphatase but progressively losing parathyroid hormone receptor expression) expressed mRNA for cytokines involved in OC differentiation, including macrophage-CSF, granulocyte-macrophage-CSF, IL-11, IL-6, and stem cell factor. Our results indicate that CBMs are capable of terminal OC differentiation in vitro, a process requiring 1,25 dihydroxyvitamin D3 as well as diffusible factor(s) which can be derived from GCTB. Stromal cells of GCTB may produce such factors in vivo, but do not support OC differentiation in vitro, possibly through phenotypic instability in culture.
Asunto(s)
Neoplasias Óseas/patología , Sangre Fetal/citología , Tumores de Células Gigantes/patología , Monocitos/citología , Osteoclastos/citología , Fosfatasa Ácida/metabolismo , Fosfatasa Alcalina/metabolismo , Secuencia de Bases , Resorción Ósea , Diferenciación Celular , Citocinas/genética , Cartilla de ADN/química , Humanos , Datos de Secuencia Molecular , Osteocalcina/metabolismo , Osteólisis , Receptores de Hormona Paratiroidea/fisiología , Células Tumorales CultivadasRESUMEN
We amplified, using the polymerase chain reaction (PCR) and NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH)-specific primers, RNA extracted from the HL-60 cell line. Two bands, differing in size by approx. 160 bp, were detected with ethidium bromide staining after electrophoresis of amplification products and hybridization with a 15-PGDH-specific probe. Sequencing these DNA bands revealed that the largest corresponded to the 15-PGDH cloned from human placenta [Ensor et al., J. Biol. Chem. 265 (1990) 14888-14891]. The smaller sequence coded for a predicted C-terminal-truncated form of 15-PGDH. This subtype of the type-I 15-PGDH mRNA was also found using RT-PCR in human liver, placenta and a cell line derived from a human medullary thyroid carcinoma (TT cells). Hybridization studies using specific probes indicated that this new mRNA form probably corresponded to the 3.4-kb mRNA, one of the two 15-PGDH mRNAs previously detected in Northern blot analysis.
Asunto(s)
Hidroxiprostaglandina Deshidrogenasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Expresión Génica , Células HL-60 , Humanos , Isoenzimas/genética , Datos de Secuencia Molecular , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
We amplified, using the polymerase chain reaction and calcitonin receptor (CTR) specific primers, RNA extracted from medullary thyroid carcinoma (MTC) and the derived TT cell line. Both secrete large amounts of calcitonin. Electrophoresis of amplification products revealed, in both cases, an ethidium bromide-stained band that hybridized to a CTR probe. Sequencing the band amplified from TT cells revealed an open reading frame identical to the sequence of H-CTR but lacking 16 amino acids in the first intracellular loop. This demonstrates the existence of an mRNA coding for a subtype of H-CTR which is expressed in TT cells and MTC.