RESUMEN
Sphingosine 1-phosphate receptor 1 (S1PR1) is a G protein-coupled receptor essential for vascular development and postnatal vascular homeostasis. When exposed to sphingosine 1-phosphate (S1P) in the blood of â¼1 µM, S1PR1 in endothelial cells retains cell-surface localization, while lymphocyte S1PR1 shows almost complete internalization, suggesting the cell-surface retention of S1PR1 is endothelial cell specific. To identify regulating factors that function to retain S1PR1 on the endothelial cell surface, here we utilized an enzyme-catalyzed proximity labeling technique followed by proteomic analyses. We identified Filamin B (FLNB), an actin-binding protein involved in F-actin cross-linking, as a candidate regulating protein. We show FLNB knockdown by RNA interference induced massive internalization of S1PR1 into early endosomes, which was partially ligand dependent and required receptor phosphorylation. Further investigation showed FLNB was also important for the recycling of internalized S1PR1 back to the cell surface. FLNB knockdown did not affect the localization of S1PR3, another S1P receptor subtype expressed in endothelial cells, nor did it affect localization of ectopically expressed ß2-adrenergic receptor. Functionally, we show FLNB knockdown in endothelial cells impaired S1P-induced intracellular phosphorylation events and directed cell migration and enhancement of the vascular barrier. Taken together, our results demonstrate that FLNB is a novel regulator critical for S1PR1 cell-surface localization and thereby proper endothelial cell function.
Asunto(s)
Filaminas , Receptores de Esfingosina-1-Fosfato , Células Endoteliales/metabolismo , Filaminas/genética , Filaminas/metabolismo , Lisofosfolípidos/metabolismo , Proteómica , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Humanos , Técnicas de Silenciamiento del Gen , Células Cultivadas , Transporte de ProteínasRESUMEN
Sphingosine 1-phosphate (S1P) is one of the lipid mediators involved in diverse physiological functions. S1P circulates in blood and lymph bound to carrier proteins. Three S1P carrier proteins have been reported, albumin, apolipoprotein M (ApoM) and apolipoprotein A4 (ApoA4). The carrier-bound S1P exerts its functions via specific S1P receptors (S1PR1-5) on target cells. Previous studies showed several differences in physiological functions between albumin-bound S1P and ApoM-bound S1P. However, molecular mechanisms underlying the carrier-dependent differences have not been clarified. In addition, ApoA4 is a recently identified S1P carrier protein, and its functional differences from albumin and ApoM have not been addressed. Here, we compared the three carrier proteins in the processes of S1P degradation, release from S1P-producing cells and receptor activation. ApoM retained S1P more stable than albumin and ApoA4 in the cell culture medium when compared in the equimolar amounts. ApoM facilitated theS1P release from endothelial cells most efficiently. Furthermore, ApoM-bound S1P showed a tendency to induce prolonged activation of Akt via S1PR1 and S1PR3. These results suggest that the carrier-dependent functional differences of S1P are partly ascribed to the differences in the S1P stability, S1P-releasing efficiency and signaling duration.
Asunto(s)
Lisofosfolípidos , Proteínas Proto-Oncogénicas c-akt , Humanos , Apolipoproteínas M/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Lisofosfolípidos/farmacología , Esfingosina/farmacología , Proteínas Portadoras/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Albúminas/metabolismoRESUMEN
Metformin, which is suggested to have anti-cancer effects, activates KDM2A to reduce rRNA transcription and proliferation of cancer cells. Thus, the specific activation of KDM2A may be applicable to the treatment of cancers. In this study, we screened a food-additive compound library to identify compounds that control cell proliferation. We found that gallic acid activated KDM2A to reduce rRNA transcription and cell proliferation in breast cancer MCF-7 cells. Gallic acid accelerated ROS production and activated AMPK. When ROS production or AMPK activity was inhibited, gallic acid did not activate KDM2A. These results suggest that both ROS production and AMPK activation are required for activation of KDM2A by gallic acid. Gallic acid did not reduce the succinate level, which was required for KDM2A activation by metformin. Metformin did not elevate ROS production. These results suggest that the activation of KDM2A by gallic acid includes mechanisms distinct from those by metformin. Therefore, signals from multiple intracellular conditions converge in KDM2A to control rRNA transcription. Gallic acid did not induce KDM2A-dependent anti-proliferation activity in non-tumorigenic MCF10A cells. These results suggest that the mechanism of KDM2A activation by gallic acid may be applicable to the treatment of breast cancers.
Asunto(s)
Proteínas F-Box/metabolismo , Ácido Gálico/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Transcripción Genética/efectos de los fármacos , Adenilato Quinasa/metabolismo , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular/genética , Metilación de ADN/genética , Proteínas F-Box/fisiología , Femenino , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/fisiología , Células MCF-7 , Metformina/farmacología , Regiones Promotoras Genéticas/genética , Especies Reactivas de Oxígeno/metabolismo , Transcripción Genética/genéticaRESUMEN
Metformin is used to treat type 2 diabetes. Metformin activates AMP-activated kinase (AMPK), which may contribute to the action of metformin. Metformin also shows anti-proliferation activity. However, the mechanism is remained unknown. We found that treatment of MCF-7 cells with metformin induced the demethylase activity of KDM2A in the rDNA promoter, which resulted in reductions of rRNA transcription and cell proliferation. AMPK activity was required for activation of KDM2A by metformin. Because demethylase activities of JmjC-type enzymes require a side reaction converting α-ketoglutarate to succinate, these organic acids may affect their demethylase activities. We found that metformin did not induce KDM2A demethylase activity in conditions of a reduced level of α-ketoglutarate. A four-hour treatment of metformin specifically reduced succinate, and the replenishment of succinate inhibited the activation of KDM2A by metformin, but did not inhibit the activation of AMPK. Metformin reduced succinate even in the conditions suppressing AMPK activity. These results indicate that metformin activates AMPK and reduces the intracellular succinate level, both of which are required for the activation of KDM2A to reduce rRNA transcription. The results presented here uncover a novel factor of metformin actions, reduction of the intracellular succinate, which contributes to the anti-proliferation activity of metformin.
Asunto(s)
Proteínas F-Box/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Metformina/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN Ribosómico/genética , Diabetes Mellitus Tipo 2/genética , Proteínas F-Box/efectos de los fármacos , Proteínas F-Box/genética , Glucosa/metabolismo , Histonas/metabolismo , Humanos , Hipoglucemiantes/farmacología , Histona Demetilasas con Dominio de Jumonji/efectos de los fármacos , Células MCF-7 , Metformina/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Ribosómico/metabolismo , Ácido Succínico/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Autophagy is an evolutionarily conserved process that degrades subcellular constituents. Mammalian cells undergo two types of autophagy; Atg5-dependent conventional autophagy and Atg5-independent alternative autophagy, and the molecules required for the latter type of autophagy are largely unknown. In this study, we analyzed the molecular mechanisms of genotoxic stress-induced alternative autophagy, and identified the essential role of p53 and damage-regulated autophagy modulator (Dram1). Dram1 was sufficient to induce alternative autophagy. In the mechanism of alternative autophagy, Dram1 functions in the closure of isolation membranes downstream of p53. These findings indicate that Dram1 plays a pivotal role in genotoxic stress-induced alternative autophagy.
RESUMEN
Centrosome number is associated with the chromosome segregation and genomic stability. The ubiquitin-proteasome system is considered to be the main regulator of centrosome number. However, here we show that autophagy also regulates the number of centrosomes. Autophagy-deficient cells carry extra centrosomes. The autophagic regulation of centrosome number is dependent on a centrosomal protein of 63 (Cep63) given that cells lacking autophagy contain multiple Cep63 dots that are engulfed and digested by autophagy in wild-type cells, and that the upregulation of Cep63 increases centrosome number. Cep63 is recruited to autophagosomes via interaction with p62, a molecule crucial for selective autophagy. In vivo, hematopoietic cells from autophagy-deficient and p62-/- mice also contained multiple centrosomes. These results indicate that autophagy controls centrosome number by degrading Cep63.
Asunto(s)
Autofagia , Proteínas de Ciclo Celular/metabolismo , Centrosoma , Proteínas de Neoplasias/metabolismo , Animales , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Humanos , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismoRESUMEN
Mammalian chromosome ends are protected by a specialized nucleoprotein complex called telomeres. Both shelterin, a telomere-specific multi-protein complex, and higher order telomeric chromatin structures combine to stabilize the chromosome ends. Here, we showed that TRF2, a component of shelterin, binds to core histones to protect chromosome ends from inappropriate DNA damage response and loss of telomeric DNA. The N-terminal Gly/Arg-rich domain (GAR domain) of TRF2 directly binds to the globular domain of core histones. The conserved arginine residues in the GAR domain of TRF2 are required for this interaction. A TRF2 mutant with these arginine residues substituted by alanine lost the ability to protect telomeres and induced rapid telomere shortening caused by the cleavage of a loop structure of the telomeric chromatin. These findings showed a previously unnoticed interaction between the shelterin complex and nucleosomal histones to stabilize the chromosome ends.
Asunto(s)
Cromosomas Humanos/metabolismo , Histonas/metabolismo , Homeostasis del Telómero/fisiología , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Animales , Cromosomas Humanos/genética , Células HeLa , Histonas/genética , Humanos , Ratones , Células 3T3 NIH , Dominios Proteicos , Telómero/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
DDHD2 has been reported to exhibit phospholipase A1, triacylglycerol (TG) lipase and diacylglycerol (DG) lipase activities. However, the detailed enzymatic properties of DDHD2 have not yet been elucidated. In the current study, the substrate specificity of DDHD2 towards DG, TG and phosphatidic acid (PA) has been examined using highly purified recombinant rat DDHD2 (rDDHD2) with a liquid chromatography mass spectrometer. The k cat/Km value for DG (18:0/20:4) was much higher than those for TG (18:1/18:1/18:1), and PA (18:0/20:4) in the presence of sodium deoxycholate. The enzyme activity of rDDHD2 towards DG (18:0/20:4) was highest among all of the substrates tested. In addition, rDDHD2 was highly specific to DG substrates with a polyunsaturated fatty acid at their sn-2 position. The levels of 2-arachidonoylglycerol (2-AG) in CHO cells were quantified by gas chromatography-tandem mass spectrometry, showing that CHO cells expressing recombinant rDDHD2 contained higher levels of 2-AG when cells were treated with a monoacylglycerol lipase inhibitor, URB602. These results therefore support the idea that DDHD2 functions as a DG lipase in vivo and produces 2-AG.
Asunto(s)
Ácidos Araquidónicos/química , Ácidos Araquidónicos/metabolismo , Endocannabinoides/química , Endocannabinoides/metabolismo , Glicéridos/química , Glicéridos/metabolismo , Lipoproteína Lipasa/química , Lipoproteína Lipasa/metabolismo , Animales , Compuestos de Bifenilo/farmacología , Células CHO , Cricetinae , Cricetulus , Lipoproteína Lipasa/antagonistas & inhibidores , Lipoproteína Lipasa/genética , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato/efectos de los fármacos , Especificidad por Sustrato/fisiologíaRESUMEN
Diacylglycerol (DG) lipase, which hydrolyses 1-stearoyl-2-arachidonyl-sn-glycerol to produce an endocannabinoid, 2-arachidonoylglycerol, was purified from the soluble fraction of rat brain lysates. DG lipase was purified about 1,200-fold by a sequential column chromatographic procedure. Among proteins identified by mass spectrometry analysis in the partially purified DG lipase sample, only DDHD domain containing two (DDHD2), which was formerly regarded as a phospholipase A1, exhibited significant DG lipase activity. Rat DDHD2 expressed in Chinese hamster ovary cells showed similar enzymatic properties to partially purified DG lipase from rat brain. The source of DG lipase activity in rat brain was immunoprecipitated using anti-DDHD2 antibody. Thus, we concluded that the DG lipase activity in the soluble fraction of rat brain is derived from DDHD2. DDHD2 is distributed widely in the rat brain. Immunohistochemical analysis revealed that DDHD2 is expressed in hippocampal neurons, but not in glia.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Hipocampo/enzimología , Lipoproteína Lipasa , Proteínas del Tejido Nervioso , Neuronas/enzimología , Animales , Ácidos Araquidónicos/genética , Ácidos Araquidónicos/metabolismo , Células CHO , Clonación Molecular , Cricetinae , Cricetulus , Endocannabinoides/genética , Endocannabinoides/metabolismo , Glicéridos/genética , Glicéridos/metabolismo , Lipoproteína Lipasa/biosíntesis , Lipoproteína Lipasa/química , Lipoproteína Lipasa/genética , Lipoproteína Lipasa/aislamiento & purificación , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/aislamiento & purificación , Neuroglía/enzimología , Dominios Proteicos , RatasRESUMEN
Efficient apoptotic cell engulfment is important for both tissue homeostasis and immune response in mammals. In the present study, we report that Beclin 1 (a regulator of autophagy) is required for apoptotic cell engulfment. The engulfment process was largely abolished in Beclin 1 knock-out cells, and Beclin 1 knockdown significantly decreased apoptotic cell internalization in macrophage and fibroblast cell lines. Beclin 1 was recruited to the early phagocytic cup along with the generation of phosphatidylinositol 3-phosphate and Rac1, which regulates actin dynamics in lamellipodia. No lamellipodia were formed in Beclin 1 knock-out cells, and Beclin 1 knockdown completely inhibited the promotion of engulfment by ectopic expression of Rac1. Beclin 1 was co-immunoprecipitated with Rac1. These data indicate that Beclin 1 coordinates actin dynamics and membrane phospholipid synthesis to promote efficient apoptotic cell engulfment.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Células Madre Embrionarias/citología , Proteínas de la Membrana/metabolismo , Neuropéptidos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Autofagia , Beclina-1 , Biblioteca de Genes , Humanos , Procesamiento de Imagen Asistido por Computador , Células Jurkat , Ratones , Microscopía Electrónica/métodos , Células 3T3 NIH , Fagocitosis , Plásmidos/metabolismoRESUMEN
TRF2 is a component of shelterin, the telomere-specific protein complex that prevents DNA damage signaling and inappropriate repair at the natural ends of mammalian chromosomes. We describe a temperature-sensitive (ts) mutation in the Myb/SANT DNA-binding domain of TRF2 that allows controlled and reversible telomere deprotection. At 32 degrees C, TRF2ts was functional and rescued the lethality of TRF2 deletion from conditional TRF2(F/-) mouse embryonic fibroblasts (MEFs). When shifted to the nonpermissive temperature (37 degrees C), TRF2ts cells showed extensive telomere damage resulting in activation of the ATM kinase and nonhomologous end-joining (NHEJ) of chromosome ends. The inactivation of TRF2ts at 37 degrees C was rapid and reversible, permitting induction of short periods (3-6 h) of telomere dysfunction in the G0, G1, and S/G2 phases of the cell cycle. The results indicate that both the induction of telomere dysfunction and the re-establishment of the protected state can take place throughout interphase. In contrast, the processing of dysfunctional telomeres by NHEJ occurred primarily in G1, being repressed in S/G2 in a cyclin-dependent kinase (CDK)-dependent manner.
Asunto(s)
Ciclo Celular/genética , Mutación , Recombinación Genética/genética , Telómero/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Alelos , Secuencia de Aminoácidos , Animales , Ciclo Celular/fisiología , Células Cultivadas , Inmunoprecipitación de Cromatina , Electroforesis en Gel de Campo Pulsado , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Fase G1/genética , Fase G1/fisiología , Fase G2/genética , Fase G2/fisiología , Immunoblotting , Hibridación Fluorescente in Situ , Ratones , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Fase de Descanso del Ciclo Celular/genética , Fase de Descanso del Ciclo Celular/fisiología , Fase S/genética , Fase S/fisiología , Homología de Secuencia de Aminoácido , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/química , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , TemperaturaRESUMEN
Thymic carcinoids are classified into three categories: (1) nonsecretory tumors, (2) hormonal secretory tumors, and (3) tumors associated with multiple endocrine neoplasia type 1. We report a rare case with adrenocorticotropic hormone secreting thymic carcinoid with multiple endocrine neoplasia type 1. Radiologic examination showed an anterior mediastinal mass and a parathyroid tumor. Blood analysis revealed high levels of parathyroid hormone and adrenocorticotropic hormone. Urine cortisol and 17-hydroxycorticoids levels were also elevated. Extended thymectomy was performed. Subsequently adjuvant radiation therapy and parathyroid tumor resection were performed. A germline mutation of exon 7 in the multiple endocrine neoplasia type 1 gene was detected and a somatic mutation of exon 9 was demonstrated in the thymic tumor.
Asunto(s)
Síndrome de ACTH Ectópico/etiología , Tumor Carcinoide/metabolismo , Neoplasia Endocrina Múltiple Tipo 1/complicaciones , Neoplasias del Timo/metabolismo , Síndrome de ACTH Ectópico/sangre , Antineoplásicos Hormonales/uso terapéutico , Tumor Carcinoide/sangre , Tumor Carcinoide/genética , Tumor Carcinoide/terapia , Terapia Combinada , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Exones/genética , Rubor/etiología , Genes Supresores de Tumor , Humanos , Hipercalcemia/etiología , Masculino , Persona de Mediana Edad , Neoplasia Endocrina Múltiple Tipo 1/sangre , Octreótido/uso terapéutico , Hormona Paratiroidea/sangre , Hormona Paratiroidea/metabolismo , Neoplasias de las Paratiroides/genética , Neoplasias de las Paratiroides/metabolismo , Neoplasias de las Paratiroides/terapia , Paratiroidectomía , Neoplasias Pleurales/secundario , Neoplasias Pleurales/cirugía , Radioterapia Adyuvante , Timectomía , Neoplasias del Timo/sangre , Neoplasias del Timo/genética , Neoplasias del Timo/terapiaRESUMEN
Epidermal growth factor receptor (EGFR) gene mutations have been found in a subset of non-small cell lung cancer (NSCLC) with good clinical response to gefitinib therapy. A quick and sensitive method with large throughput is required to utilize the information to determine whether the molecular targeted therapy should be applied for the particular NSCLC patients. Using probes for the 13 different mutations including 11 that have already been reported, we have genotyped the EGFR mutation status in 94 NSCLC patients using the TaqMan PCR assay. We have also genotyped the EGFR mutations status in additional 182 NSCLC patients, as well as 63 gastric, 95 esophagus and 70 colon carcinoma patients. In 94 NSCLC samples, the result of the TaqMan PCR assay perfectly matched with that of the sequencing excluding one patient. In one sample in which no EGFR mutation was detected by direct sequencing, the TaqMan PCR assay detected a mutation. This patient was a gefitinib responder. In a serial dilution study, the assay could detect a mutant sample diluted in 1/10 with a wild-type sample. Of 182 NSCLC samples, 46 mutations were detected. EGFR mutation was significantly correlated with gender, smoking status, pathological subtypes, and differentiation of lung cancers. There was no mutation detected by the TaqMan PCR assay in gastric, esophagus and colon carcinomas. TaqMan PCR assay is a rapid and sensitive method of detection of EGFR mutations with high throughput, and may be useful to determine whether gefitinib should be offered for the treatment of NSCLC patients. The TaqMan PCR assay can offer us a complementary and confirmative test.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutación , Reacción en Cadena de la Polimerasa/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias del Colon/genética , Clorhidrato de Erlotinib , Neoplasias Esofágicas/genética , Femenino , Gefitinib , Genotipo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Quinazolinas/uso terapéutico , Sensibilidad y Especificidad , Neoplasias Gástricas/genéticaRESUMEN
Mitotic catastrophe occurs as a result of the uncoupling of the onset of mitosis from the completion of DNA replication, but precisely how the ensuing lethality is regulated or what signals are involved is largely unknown. We demonstrate here the essential role of the ATM/ATR-p53 pathway in mitotic catastrophe from premature mitosis. Chk1 deficiency resulted in a premature onset of mitosis because of abnormal activation of cyclin B-Cdc2 and led to the activation of caspases 3 and 9 triggered by cytoplasmic release of cytochrome c. This deficiency was associated with foci formation by the phosphorylated histone, H2AX (gammaH2AX), specifically at S phase. Ectopic expression of Cdc2AF, a mutant that cannot be phosphorylated at inhibitory sites, also induced premature mitosis and foci formation by gammaH2AX at S phase in both embryonic stem cells and HCT116 cells. Depletion of ATM and ATR protected against cell death from premature mitosis. p53-deficient cells were highly resistant to lethality from premature mitosis as well. Our results therefore suggest that ATM/ATR-p53 is required for mitotic catastrophe that eliminates cells escaping Chk1-dependent mitotic regulation. Loss of this function might be important in mammalian tumorigenesis.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclina B/metabolismo , Mitosis/fisiología , Proteínas Quinasas/deficiencia , Animales , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada , Secuencia de Bases , Proteína Quinasa CDC2/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Citocromos c/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Humanos , Ratones , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , Transfección , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
PURPOSE: Recently, somatic mutations of the epidermal growth factor receptor (EGFR) gene were found in approximately 25% of Japanese lung cancer patients. These EGFR mutations are reported to be correlated with clinical response to gefitinib therapy. However, DNA sequencing using the PCR methods described to date is time-consuming and requires significant quantities of DNA; thus, this existing approach is not suitable for a routine pretherapeutic screening program. EXPERIMENTAL DESIGN: We have genotyped EGFR mutation status in Japanese lung cancer patients, including 102 surgically treated lung cancer cases from Nagoya City University Hospital and 16 gefitinib-treated lung cancer cases from Kinki-chuo Chest Medical Center. The presence or absence of three common EGFR mutations were analyzed by real-time quantitative PCR with mutation-specific sensor and anchor probes. RESULTS: In exon 21, EGFR mutations (CTG --> CGG; L858R) were found from 8 of 102 patients from Nagoya and 1 of 16 from Kinki. We also detected the deletion mutations in exon 19 from 7 of 102 patients from Nagoya (all were deletion type 1a) and 4 of 16 patients from Kinki (one was type 1a and three were type 1b). In exon 18, one example of G719S mutation was found from both Nagoya and Kinki. The L858R mutation was significantly correlated with gender (women versus men, P < 0.0001), Brinkman index (600 < or = versus 600, P = 0.001), pathologic subtypes (adenocarcinoma versus nonadenocarcinoma, P = 0.007), and differentiation status of the lung cancers (well versus moderately or poorly, P = 0.0439), whereas the deletion mutants were not. EGFR gene status, including the type of EGFR somatic mutation, was correlated with sensitivity to gefitinib therapy. For example, some of our gefitinib-responsive patients had L858R or deletion type 1a mutations. On the other hand, one of our gefitinib-resistant patients had a G719S mutation. CONCLUSIONS: Using the LightCycler PCR assay, the EGFR L858R mutation status might correlate with gender, pathologic subtypes, and gefitinib sensitivity of lung cancers. However, further genotyping studies are needed to confirm the mechanisms of EGFR mutations for the sensitivity or resistance of gefitinib therapy for the lung cancer.
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Análisis Mutacional de ADN/instrumentación , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutación , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/cirugía , ADN de Neoplasias/química , ADN de Neoplasias/genética , Femenino , Gefitinib , Frecuencia de los Genes , Genotipo , Humanos , Japón , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/cirugía , Masculino , Persona de Mediana Edad , Mutación Missense , Reacción en Cadena de la Polimerasa/métodos , Quinazolinas/uso terapéuticoAsunto(s)
Apoptosis/genética , Apoptosis/efectos de la radiación , Daño del ADN/efectos de la radiación , Animales , Permeabilidad de la Membrana Celular/genética , Permeabilidad de la Membrana Celular/fisiología , Histonas/fisiología , Proteínas de la Membrana/fisiología , Mitocondrias/genética , Mitocondrias/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteína p53 Supresora de Tumor/fisiología , Proteína Destructora del Antagonista Homólogo bcl-2RESUMEN
Using the enzyme immunoassay for epidermal growth factor receptor (EGFR), we investigated whether serum EGFR levels could be used as a predictor of the development and extension of thymoma. Serum samples were collected from 31 patients with thymoma and 16 patients with nonmalignant thoracic disease before clinical treatment. There was no difference between the serum EGFR levels of the patients with thymoma and the nonmalignant controls, being 49.1 +/- 136.3 and 22.6 +/- 7.3 fm/ml, respectively ( P = 0.11). However, patients with stage IV thymoma had significantly higher EGFR levels than those with stage I or stage II thymoma, the respective values being 127.8 +/- 243.9, 10.9 +/- 9.2 ( P = 0.02), and 19.7 +/- 10.6 ( P = 0.0433) fm/ml. The serum EGFR levels were similar in the pathological subtypes. These findings suggest that serum EGFR levels may serve as a marker that could be used as a diagnostic indicator of the invasion of thymoma.
Asunto(s)
Biomarcadores de Tumor/sangre , Receptores ErbB/sangre , Timoma/sangre , Neoplasias del Timo/sangre , Humanos , Invasividad Neoplásica , Estadificación de Neoplasias , Timoma/patología , Neoplasias del Timo/patologíaRESUMEN
Intravenous endostatin gene transfection results in tumor suppression in a murine pulmonary metastasis model. We transfected the endostatin gene at different times, in order to achieve an optimal protective effect. pST2-Endo encoding murine endostatin was injected in a complex with cationic lipid. Pulmonary metastases were caused by intravenous injection of murine fibrosarcoma cells. Mice were observed for 14 days following fibrosarcoma cell inoculation (FSI). In the study groups, the animals were transfected with pST2-Endo at three different times: 2 days before and 3 and 7 days after FSI. In the group transfected with pST2-Endo 2 days before FSI, the weights of the lungs and tumor-occupied area ratio were significantly less than in the other groups. Significant inhibition of tumor neovascularization was documented by means of CD31 immunohistochemistry. The effect of repeated endostatin transfection on survival after FSI was determined. Animals repeatedly transfected with the endostatin gene survived significantly longer than the groups treated with a single endostatin gene transfection. A stable endostatin-expressing fibrosarcoma transfectant was created and tested for migration and invasion. Compared with controls, endostatin expression reduced migration and invasion by 15%. It is concluded that endostation gene transfection before FSI and repeated transfection thereafter results in significant tumor suppression.
Asunto(s)
Endostatinas/genética , Fibrosarcoma/genética , Terapia Genética , Lípidos , Neoplasias Pulmonares/genética , Transfección , Animales , Supervivencia Celular/genética , Endostatinas/biosíntesis , Endostatinas/inmunología , Fibrosarcoma/patología , Fibrosarcoma/terapia , Vectores Genéticos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Endogámicos C3H , Neovascularización Patológica/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunología , Transfección/métodosRESUMEN
Recently Smac/DIABLO (second mitochondria-derived activator of caspase) has been identified as a proapoptotic protein that inhibits IAPs (inhibitors of apoptosis proteins). Smac is expressed ubiquitously in many organs, including the lung. Here we evaluated the expression of Smac mRNA with real-time reverse-transcription PCR in 88 primary lung cancers and matched normal tissues. Smac mRNA expression in tumor tissues was significantly lower than that in normal tissues (p<0.0001). In squamous cell carcinomas, Smac mRNA expression was significantly lower than that in adenocarcinomas (p=0.0072). Smac mRNA expression in T2-T4 tumors was significantly lower than that in T1 tumors (p=0.0006). The expression of Smac in the tumors of smokers was lower than that in the tumors of non-smokers (p=0.0011). The prognosis of patients with a tumor exhibiting a low expression of Smac mRNA was worse than that in those with a tumor exhibiting high Smac mRNA expression (log-rank test, p=0.047). These results indicate that Smac expression may play a role in the carcinogenesis, progression, and prognosis of primary lung cancer.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Proteínas Portadoras/metabolismo , Neoplasias Pulmonares/diagnóstico , Proteínas Mitocondriales/metabolismo , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/genética , Femenino , Expresión Génica , Humanos , Inmunoquímica , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Pulmonares/patología , Masculino , Proteínas Mitocondriales/genética , Pronóstico , ARN Mensajero/metabolismo , Estudios RetrospectivosRESUMEN
It is poorly understood how apoptotic signals arising from DNA damage are transmitted to mitochondria, which release apoptogenic factors into the cytoplasm that activate downstream destruction programs. Here, we identify histone H1.2 as a cytochrome c-releasing factor that appears in the cytoplasm after exposure to X-ray irradiation. While all nuclear histone H1 forms are released into the cytoplasm in a p53-dependent manner after irradiation, only H1.2, but not other H1 forms, induced cytochrome c release from isolated mitochondria in a Bak-dependent manner. Reducing H1.2 expression enhanced cellular resistance to apoptosis induced by X-ray irradiation or etoposide, but not that induced by other stimuli including TNF-alpha and UV irradiation. H1.2-deficient mice exhibited increased cellular resistance in thymocytes and the small intestine to X-ray-induced apoptosis. These results indicate that histone H1.2 plays an important role in transmitting apoptotic signals from the nucleus to the mitochondria following DNA double-strand breaks.