RESUMEN
To spread from a localized tumor, metastatic cancer cells must squeeze through constrictions that cause major nuclear deformations. Since chromosome structure affects nucleus stiffness, gene regulation, and DNA repair, here, we investigate the relationship between 3D genome structure and constricted migration in cancer cells. Using melanoma (A375) cells, we identify phenotypic differences in cells that have undergone multiple rounds of constricted migration. These cells display a stably higher migration efficiency, elongated morphology, and differences in the distribution of Lamin A/C and heterochromatin. Hi-C experiments reveal differences in chromosome spatial compartmentalization specific to cells that have passed through constrictions and related alterations in expression of genes associated with migration and metastasis. Certain features of the 3D genome structure changes, such as a loss of B compartment interaction strength, are consistently observed after constricted migration in clonal populations of A375 cells and in MDA-MB-231 breast cancer cells. Our observations suggest that consistent types of chromosome structure changes are induced or selected by passage through constrictions and that these may epigenetically encode stable differences in gene expression and cellular migration phenotype.
Asunto(s)
Lamina Tipo A , Neoplasias , Movimiento Celular/genética , Núcleo Celular/metabolismo , Reparación del ADN , Heterocromatina/metabolismo , Lamina Tipo A/análisis , Lamina Tipo A/metabolismo , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
BACKGROUND: Mutations in LMNA, encoding lamin A/C, lead to a variety of diseases known as laminopathies including dilated cardiomyopathy (DCM) and skeletal abnormalities. Though previous studies have investigated the dysregulation of gene expression in cells from patients with DCM, the role of epigenetic (gene regulatory) mechanisms, such as DNA methylation, has not been thoroughly investigated. Furthermore, the impact of family-specific LMNA mutations on DNA methylation is unknown. Here, we performed reduced representation bisulfite sequencing on ten pairs of fibroblasts and their induced pluripotent stem cell (iPSC) derivatives from two families with DCM due to distinct LMNA mutations, one of which also induces brachydactyly. RESULTS: Family-specific differentially methylated regions (DMRs) were identified by comparing the DNA methylation landscape of patient and control samples. Fibroblast DMRs were found to enrich for distal regulatory features and transcriptionally repressed chromatin and to associate with genes related to phenotypes found in tissues affected by laminopathies. These DMRs, in combination with transcriptome-wide expression data and lamina-associated domain (LAD) organization, revealed the presence of inter-family epimutation hotspots near differentially expressed genes, most of which were located outside LADs redistributed in LMNA-related DCM. Comparison of DMRs found in fibroblasts and iPSCs identified regions where epimutations were persistent across both cell types. Finally, a network of aberrantly methylated disease-associated genes revealed a potential molecular link between pathways involved in bone and heart development. CONCLUSIONS: Our results identified both shared and mutation-specific laminopathy epimutation landscapes that were consistent with lamin A/C mutation-mediated epigenetic aberrancies that arose in somatic and early developmental cell stages.
Asunto(s)
Cardiomiopatía Dilatada/complicaciones , Lamina Tipo A/análisis , Laminopatías/etiología , Cardiomiopatía Dilatada/genética , Metilación de ADN/genética , Metilación de ADN/fisiología , Humanos , Lamina Tipo A/genética , Laminopatías/genéticaRESUMEN
Chromatin organization within the nuclear volume is essential to regulate many aspects of its function and to safeguard its integrity. A key player in this spatial scattering of chromosomes is the nuclear envelope (NE). The NE tethers large chromatin domains through interaction with the nuclear lamina and other associated proteins. This organization is perturbed in cells from Hutchinson-Gilford progeria syndrome (HGPS), a genetic disorder characterized by premature aging features. Here, we show that HGPS-related lamina defects trigger an altered 3D telomere organization with increased contact sites between telomeres and the nuclear lamina, and an altered telomeric chromatin state. The genome-wide replication timing signature of these cells is perturbed, with a shift to earlier replication for regions that normally replicate late. As a consequence, we detected a higher density of replication forks traveling simultaneously on DNA fibers, which relies on limiting cellular dNTP pools to support processive DNA synthesis. Remarkably, increasing dNTP levels in HGPS cells rescued fragile telomeres, and improved the replicative capacity of the cells. Our work highlights a functional connection between NE dysfunction and telomere homeostasis in the context of premature aging.
Asunto(s)
Cromatina/ultraestructura , Desoxirribonucleótidos/metabolismo , Lamina Tipo A/fisiología , Lámina Nuclear/patología , Progeria/genética , Homeostasis del Telómero/genética , Telómero/patología , Adulto , Animales , Células Cultivadas , Senescencia Celular/genética , Daño del ADN , Replicación del ADN , Fibroblastos , Genes Reporteros , Proteínas Fluorescentes Verdes , Código de Histonas , Humanos , Recién Nacido , Lamina Tipo A/análisis , Lamina Tipo A/deficiencia , Lamina Tipo A/genética , Lamina Tipo B/análisis , Ratones , Ratones Noqueados , Progeria/patología , Proteínas Recombinantes de Fusión/metabolismo , Piel/patologíaRESUMEN
Lamin A, which is encoded by the LMNA gene, regulates gene expression and genome stability through interactions with a variety of proteins. Mutations in LMNA lead to a diverse set of inherited human diseases, collectively referred to as laminopathies. To gain insight into the protein interactions of lamin A, a yeast twohybrid screen was conducted using the carboxyterminus of lamin A. The screen identified copper metabolism MURR1 domaincontaining 1 (COMMD1) as a novel lamin A binding partner. Colocalization experiments using fluorescent confocal microscopy revealed that COMMD1 colocalized with lamin A in 293 cells. Furthermore, the COMMD1lamin A protein interaction was also demonstrated in coimmunoprecipitation experiments. Collectively, the present study demonstrated a physical interaction between COMMD1 and lamin A, which may aid to elucidate the mechanisms of lamin A in the aging process.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Lamina Tipo A/metabolismo , Proteínas Adaptadoras Transductoras de Señales/análisis , Línea Celular , Humanos , Lamina Tipo A/análisis , Unión Proteica , Mapas de Interacción de Proteínas , Técnicas del Sistema de Dos HíbridosRESUMEN
Nuclear membrane proteins reportedly play important roles in maintaining nuclear structures and coordinating cell activities. Studying profiles of nuclear membrane proteins may help us evaluate the biological and/or clinical nature of malignant tumors. Using immunohistochemistry with antibodies for emerin, lamin A/C, lamin B, and LAP2, we examined 105 lung cancer tissues from 33 small cell lung carcinomas (SCLCs) and 72 non-SCLCs (34 adenocarcinomas, 30 squamous cell carcinomas, and 8 large cell carcinomas). Emerin had negative or local/weak positivity in 79% of SCLCs and 1% of non-SCLCs, and lamin A/C had similar positivity in 91% of SCLCs and 3% of non-SCLCs. LAP2's expression was similar between SCLCs and non-SCLCs. RT-PCR analyses for these four nuclear membrane proteins over 7 cell lines showed that mRNA of emerin and lamin A/C were distinctly downregulated in the SCLC cell lines, supporting the immunohistochemical results. In conclusion, we suggest that downregulation of the nuclear membrane proteins emerin and lamin A/C is characteristic of SCLC cells, and this constitutional abnormality of the nuclear membrane may be related to the biological and/or clinical nature of SCLC. In addition, knowing the nuclear protein profile in SCLC cells may contribute to our understanding of nuclear fragility known as the crush artifact in pulmonary biopsy specimens.
Asunto(s)
Lamina Tipo A/biosíntesis , Neoplasias Pulmonares/patología , Proteínas de la Membrana/biosíntesis , Proteínas Nucleares/biosíntesis , Carcinoma Pulmonar de Células Pequeñas/patología , Biomarcadores de Tumor/análisis , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/biosíntesis , Humanos , Lamina Tipo A/análisis , Lamina Tipo B/análisis , Lamina Tipo B/biosíntesis , Neoplasias Pulmonares/metabolismo , Proteínas de la Membrana/análisis , Proteínas Nucleares/análisis , Carcinoma Pulmonar de Células Pequeñas/metabolismoRESUMEN
The nuclear lamina consists of a dense fibrous meshwork of nuclear lamins, Type V intermediate filaments, and is ~14 nm thick according to recent cryo-electron tomography studies. Recent advances in light microscopy have extended the resolution to a scale allowing for the fine structure of the lamina to be imaged in the context of the whole nucleus. We review quantitative approaches to analyze the imaging data of the nuclear lamina as acquired by structured illumination microscopy (SIM) and single molecule localization microscopy (SMLM), as well as the requisite cell preparation techniques. In particular, we discuss the application of steerable filters and graph-based methods to segment the structure of the four mammalian lamin isoforms (A, C, B1, and B2) and extract quantitative information.
Asunto(s)
Lámina Nuclear/química , Lámina Nuclear/ultraestructura , Animales , Núcleo Celular/metabolismo , Tomografía con Microscopio Electrónico/métodos , Humanos , Proteínas de Filamentos Intermediarios/análisis , Filamentos Intermedios/química , Filamentos Intermedios/fisiología , Lamina Tipo A/análisis , Lamina Tipo B/análisis , Laminas/química , Laminas/fisiología , Lámina Nuclear/fisiología , Isoformas de Proteínas/análisisRESUMEN
OBJECTIVE: Nuclear lamina plays important roles in nuclear shape and mechanical stability. Many studies demonstrated that defects of lamin-A were associated with several diseases, but little research was found on its potential roles in ovarian cancer. METHODS: GEPIA and GEO database were used to analyze lamin-A in ovarian tissues, followed by assessing lamin-A and prognosis of ovarian cancer patients with Kaplan-Meier plotter. Then, transient transfected HO-8910 cells with shRNA to knockdown lamin-A. Knockdown efficiency was determined by western blot, qRT-PCR and immunofluorescence. Meanwhile, lamin-A was overexpressed in HO-8910â¯PM cells. Then, 2D migration, 3D migration through 3⯵m and 8⯵m pores were carried out, followed by immunofluorescence and TEM observation. RESULTS: Lamin-A tended to be lower in ovarian cancer, and higher expression of lamin-A was associated with better survival. After lamin-A knockdown, 2D and 3D migration (3⯵m, 8⯵m) abilities of HO-8910 cells were significantly increased (pâ¯<â¯0.001), while overexpression of lamin-A in HO-8910PM impeded migration. Meanwhile, when HO-8910 cells migrated through 3⯵m pores, nuclei became strikingly elongated, and down-regulation of lamin-A promoted nuclear plasticity, making the circularity of nucleus increased. Besides, further knockdown group had the highest proportion of γ-H2AX, with micronuclei forming. Furthermore, western blot showed that the expression of BRCA1, Ku80 and Rad50 decreased significantly after further knockdown, suggesting impairment of DNA damage repair. CONCLUSIONS: Lamin-A was down-regulated in ovarian cancer, and higher lamin-A was associated with better prognosis. Nuclei with high lamin-A were severely deformed through constricted pores. Moderate lamin-A enhanced nuclear plasticity, so as to strengthen migration ability. When lamin-A was further knockdown, ovarian cancer cells that migrated through restricted pores decreased, with DNA damage, genomic instability and impairment of DNA damage repair.
Asunto(s)
Núcleo Celular/patología , Lamina Tipo A/fisiología , Neoplasias Ováricas/patología , Línea Celular Tumoral , Movimiento Celular , Núcleo Celular/ultraestructura , Daño del ADN , Femenino , Histonas/análisis , Humanos , Lamina Tipo A/análisis , Neoplasias Ováricas/genéticaRESUMEN
The illegal use of glucocorticoids (GCs) as growth-promoters (GPs) is prohibited in farm animals in the European Union because the strong pharmacological activity of most synthetic GCs produces residues that are dangerous for human consumption. Among the alternative methods proposed to increase the efficacy of official controls, histology was the technique of choice in Italy on account of its high performance level. The aim of this study was to evaluate the reliability of immunohistochemistry (IHC) using anti-cleaved-Lamin A antibody to enhance the performance of the histological test applied to GC-related microscopic changes in the thymus. Veal calves (VC) and beef cattle (BC) were raised and both underwent different growth-promoting protocols or were left untreated. The morphology of the thymus parenchyma was evaluated for cortical atrophy with concurrent adipose tissue infiltration, and a score of 1 to 3 was attributed. A semi-quantitative IHC analysis was also performed by counting the number of positive thymocytes in 5 randomly selected high-power fields (HPFs). The distribution of the thymus atrophy scores was significantly different among the subgroups in both BC and VC. The IHC values were higher in untreated than in treated animals, for both BC and VC. The association between thymus atrophy score and IHC positivity showed higher median values in control than in treated animals (independently of the treatment protocol), for both BC and VC. Our data shows that IHC against anti-cleaved-Lamin A antibody is a reliable marker to detect illegal GC treatments, administered either alone or in association with other growth promoters, in both BC and VC. Combining IHC with the thymus atrophy score improves the accuracy of the histological method in correctly identifying treated animals and could represent a valuable, reproducible method to be applied to large-scale screening programmes.
Asunto(s)
Análisis de los Alimentos , Glucocorticoides/química , Lamina Tipo A/análisis , Animales , Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo , Bovinos , Glucocorticoides/farmacología , Inmunohistoquímica , Lamina Tipo A/inmunologíaRESUMEN
BACKGROUND: Patients with Marfan (MFS) syndrome and patients with a bicuspid aortic valve (BAV) are more prone to develop aortic dilation and dissection compared to persons with a tricuspid aortic valve (TAV). To elucidate potential common as well as distinct pathways of clinical relevance, we compared the histopathological substrates of aortic pathology. PATIENT AND METHODS: Ascending aortic wall specimen were divided in five groups: BAV (n=36) and TAV (n=23) without and with dilation and non-dilated MFS (n=8). We performed routine histology to study aortic wall features based on the aortic consensus statement. Immunohistological markers for vascular smooth muscle cell (VSMC) maturation, and expression of fibrillin-1 were additionally investigated for the underlying pathogenesis. RESULTS: On basis of the routine histology the aorta in MFS was similar to the aorta in dilated TAVs (overall medial degeneration, elastic fiber fragmentation, loss and disorganization, , and VSMC nuclei loss). The other markers aided in clustering the MFS and BAV patients with a significantly lower fibrillin-1 expression as compared to the TAVs (p<0.05), a lower level of differentiated VSMC markers (p<0.05) and elastic fiber thinning. CONCLUSIONS: Pathogenesis of aortopathy in MFS overlaps with mechanisms seen in BAV and TAV, leading to a so called double hit hypothesis for aortic complications in MFS. The ascending aortic wall in MFS is immature with undifferentiated VSMCs and low levels of fibrillin-1. The immature media becomes even more vulnerable for aortopathy due to other degenerative features which develop probably as a direct consequence of the fibrillin-1 mutation.
Asunto(s)
Aorta/patología , Aneurisma de la Aorta/etiología , Válvula Aórtica/anomalías , Enfermedades de las Válvulas Cardíacas/complicaciones , Síndrome de Marfan/complicaciones , Remodelación Vascular , Actinas/análisis , Adulto , Anciano , Aorta/química , Aneurisma de la Aorta/metabolismo , Aneurisma de la Aorta/patología , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Enfermedad de la Válvula Aórtica Bicúspide , Biomarcadores/análisis , Biopsia , Proteínas del Citoesqueleto/análisis , Dilatación Patológica , Tejido Elástico/patología , Femenino , Fibrilina-1/análisis , Enfermedades de las Válvulas Cardíacas/metabolismo , Enfermedades de las Válvulas Cardíacas/patología , Humanos , Inmunohistoquímica , Lamina Tipo A/análisis , Masculino , Síndrome de Marfan/metabolismo , Síndrome de Marfan/patología , Persona de Mediana Edad , Proteínas Musculares/análisis , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Factores de Riesgo , Adulto JovenRESUMEN
Although most cases of low grade (G1) endometrial cancer (EC) do not behave aggressively, in rare instances, can progress in a highly aggressive manner. In this study we analyzed formalin-fixed, paraffin-embedded (FFPE) EC tissues to find novel clinical and biological features to help diagnosis and treatment of G1 ECs s in order to better stratify patient risk of recurrence. A retrospective cohort of FFPE specimens from patients with EC (n=87) and benign tissue specimens (NE) from patients who underwent a hysterectomy to treat other benign disease (n = 13) were collected. Total RNA and proteins were extracted and analyzed, respectively, by quantitative PCR and western blotting. NF-YAs is expressed and lamin A is down-modulated in all high grade (G2 and G3) ECs. In G1 ECs, NF-YAs expression is heterogeneous being expressed only in a subset of these tumours. Interestingly, the G1 ECs that express NF-YAs display low levels of lamin A similar to those present in G2 and G3 ECs. Of note, this pattern of NF-YAs and lamin A expression correlates with tumor aggressiveness assessed by comparative analysis with estrogen receptor (ER) status and epithelial-mesenchymal transition (EMT) markers thus suggesting its potential role as biomarker of tumour aggressiveness in G1 EC. In all grade ECs, lamin A is strongly downmodulated, being its expression inversely correlated with tumor aggressiveness and its loss of expression. We identified NF-YAs and lamin A expression levels as novel potential biomarkers useful to identify G1 ECs patients with risk of recurrence.
Asunto(s)
Biomarcadores de Tumor/análisis , Factor de Unión a CCAAT/análisis , Neoplasias Endometriales/química , Lamina Tipo A/análisis , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Factor de Unión a CCAAT/genética , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Neoplasias Endometriales/cirugía , Transición Epitelial-Mesenquimal , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Histerectomía , Lamina Tipo A/genética , Persona de Mediana Edad , Clasificación del Tumor , Isoformas de Proteínas , ARN Mensajero/genética , Receptores de Estrógenos/análisis , Estudios RetrospectivosRESUMEN
This study combined cytotoxicity assays with proteomic analysis to characterize the unique biological responses of the A549 human lung epithelial cell line to two physicochemically distinct respirable particles titanium dioxide (TiO2) and carbon black (CB). Cellular LDH, ATP, BrdU incorporation and resazurin reduction indicated that CB was more potent than TiO2. Proteomic analysis was done using 2D-GE and MALDI-TOF-TOF-MS. Proteomic changes reflected common and particle-specific responses. Particle-specific proteomic responses were associated with cell death (necrosis and apoptosis), viability and proliferation pathways. Our results suggested that these pathways were consistent with the cytotoxicity data. For instance, increased expressions of anti-proliferative proteins LMNA and PA2G4 were in agreement with the decreased BrdU incorporation in A549 cells after exposure to CB. Similarly, increased expression of HSPA5 that is associated with ATPase activity was consistent with decreased cellular ATP levels in these cells. These findings reveal that proteomic changes can explain the cellular cytotoxicity characteristics of the particles. In essence, our results demonstrate that the in vitro toxicoproteomic approach is a promising tool to gain insight into molecular mechanisms underlying particle exposure-specific cytotoxicity. BIOLOGICAL SIGNIFICANCE: In this study we have shown that toxicoproteomics is a sensitive and informative method to resolve the toxicity characteristics of particles with different physicochemical properties. This approach can be useful in the investigation of molecular mechanisms underpinning cellular cytotoxic responses elicited by particle exposures. Thus, the toxicoproteomic approach can be valuable in assessing the risk associated with particle exposures in vitro.
Asunto(s)
Células Epiteliales Alveolares/efectos de los fármacos , Proteínas/análisis , Proteómica/métodos , Hollín/toxicidad , Protectores Solares/toxicidad , Titanio/toxicidad , Células A549 , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Epiteliales Alveolares/metabolismo , Análisis de Varianza , Supervivencia Celular/efectos de los fármacos , Electroforesis en Gel Bidimensional , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/análisis , Proteínas de Choque Térmico/metabolismo , Humanos , Lamina Tipo A/análisis , Lamina Tipo A/metabolismo , Tamaño de la Partícula , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Pruebas de ToxicidadRESUMEN
At present time, relationships between lamins and processes leading to aging are established. Mutations of genes of lamins lead to diseases, one of them is progeria. This disease is caused by violation of splaysing of lamin A gene and accumulation the farnezylated prelamin A (progerin) in the nucleus. LAP-2 is an important factor which regulates and stabilizes the lamin A. However, roles of lamin A and LAP-2 in behavior of population of dermal fibroblasts in relation to age were not examined. The aim of this research was to study A type lamin and LAP-2 in human skin at different ages. Lamin A and LAP-2 were detected in sections of the skin by indirect immunohistochemistry. The number of fibroblasts containing lamin A was gradually decreased from 90,4 to 76,9 % from 20 weeks of gestation to 85 years old. There were 32 % of dermal fibroblasts with positive staining for LAP-2 at the period from 20 weeks of gestation to 20 years old. From 21 to 40 years, 37,8 % of fibroblasts containing lamin A were found in the dermis. In age interval 41-85 years, 49-51 % of dermal fibroblasts had a positive staining for LAP-2. Content of lamin A in the nuclei of fibroblasts was almost constant from 20 weeks of gestation to 85 years old. Expression of LAP-2 in the nuclei of fibroblasts was reduced from birth to 20 years old but increased from 21 years old. Number of fibroblasts and PCNA+ fibroblasts in dermis was diminished with age. The most significant decrease in the number of fibroblasts was observed from 20 weeks of gestation to 20 years old. Results allow to assume the participation of lamin A and LAP-2 in triggering age-dependent decrease in the number of fibroblasts in the dermis in humans.
Asunto(s)
Envejecimiento/fisiología , Proteínas de Unión al ADN , Fibroblastos , Lamina Tipo A , Proteínas de la Membrana , Envejecimiento de la Piel , Piel , Adulto , Anciano de 80 o más Años , Niño , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Femenino , Desarrollo Fetal/fisiología , Feto/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Inmunohistoquímica , Lactante , Lamina Tipo A/análisis , Lamina Tipo A/metabolismo , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Piel/metabolismo , Piel/patología , Envejecimiento de la Piel/patología , Envejecimiento de la Piel/fisiologíaRESUMEN
Various types of somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells. Somatic stem cells may generate iPS cells more efficiently than do differentiated cells. We show that granulosa cells exhibit characteristic of somatic stem cells and can be reprogrammed to iPS cells more efficiently or with few factors. Here, we describe generation of mouse and pig iPS cells from granulosa cells with high efficiency.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Técnicas de Reprogramación Celular/métodos , Reprogramación Celular , Células de la Granulosa/citología , Células Madre Pluripotentes Inducidas/citología , Animales , Células Cultivadas , Criopreservación/métodos , Medios de Cultivo/farmacología , Cuerpos Embrioides , Femenino , Fibroblastos/citología , Vectores Genéticos/genética , Gonadotropinas Equinas/farmacología , Células de la Granulosa/química , Células de la Granulosa/efectos de los fármacos , Células HEK293 , Humanos , Lamina Tipo A/análisis , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/genética , Retroviridae/genética , Sus scrofa , Porcinos , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Prostate cancer (PCa) is the most common cancer among men in western countries. While active surveillance is increasingly utilized, the majority of patients are currently treated with radical prostatectomy. In order to avoid over-treatment, there is an indisputable need for reliable biomarkers to identify the potentially aggressive and lethal cases. Nuclear intermediate filament proteins called lamins play a role in chromatin organization, gene expression and cell stiffness. The expression of lamin A is associated with poor outcome in colorectal cancer but to date the prognostic value of the lamins has not been tested in other solid tumors. METHODS: We studied the expression of different lamins with immunohistochemistry in a tissue microarray material of 501 PCa patients undergoing radical prostatectomy and lymph node dissection. Patients were divided into two staining categories (low and high expression). The correlation of lamin expression with clinicopathological variables was tested and the association of lamin status with biochemical recurrence (BCR) and disease specific survival (DSS) was further analyzed. RESULTS: Low expression of lamin A associated with lymph node positivity (p<0.01) but not with other clinicopathological variables and low expression had a borderline independent significant association with DSS (HR = 0.4; 95% CI 0.2-1.0; p = 0.052). Similarly, low lamin C expression associated with poorer survival (HR = 0.2; 95% CI 0.1-0.6; p = 0.004). Lamin B1 expression did not associate with clinicopathological variables but high expression independently predicted BCR in multivariable Cox regression analysis (HR = 1.8; 95% CI 1.1-2.9; p = 0.023). Low expression of lamin B2 correlated with lymph node positivity (p<0.01) and predicted unfavorable DSS (HR = 0.4; 95% CI 0.2-1.0; p = 0.047). CONCLUSIONS: These results suggest differential roles for lamins in PCa progression. Reduced amounts of lamin A/C and B2 increase risk for lymph node metastasis and disease specific death possibly through increased nuclear deformability while high expression of lamin B1 predicts disease recurrence.
Asunto(s)
Carcinoma/patología , Núcleo Celular/metabolismo , Lamina Tipo A/análisis , Lamina Tipo B/análisis , Neoplasias de la Próstata/patología , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma/metabolismo , Núcleo Celular/química , Diagnóstico Diferencial , Progresión de la Enfermedad , Humanos , Inmunohistoquímica , Lamina Tipo A/metabolismo , Lamina Tipo B/metabolismo , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Pronóstico , Neoplasias de la Próstata/metabolismoRESUMEN
Emerin, lamin A/C, lamin B, and lamin-associated polypeptide 2 (LAP2) are nuclear membrane proteins that play an important role in maintaining nuclear structure and coordinating cell activity. We studied the expression and significance of nuclear membrane proteins in neoplastic thyroid cells by immunohistochemistry, RT-PCR, and real-time PCR. In papillary carcinomas (PCs), the nuclear proteins most frequently expressed at high levels were emerin (82 % positive), lamin A/C (64 %), and LAP2 (82 %). Follicular carcinomas (FCs) most frequently expressed lamin B, while none of the undifferentiated carcinomas (UCs) showed strong expression of emerin or lamin A/C. In all medullary carcinomas (MCs), intermediate to high levels of expression of lamin A/C and LAP2 were found. By RT-PCR analysis, messenger RNA (mRNA) expression of all nuclear membrane proteins except emerin was higher in PC than in normal tissue. Real-time PCR analysis showed that mRNA expression of nuclear membrane protein varied between cell lines. Our findings suggest that expression of nuclear membrane proteins may be related to follicular function in normal and hyperplastic follicles, and we hypothesize that they are also involved in the proliferation and differentiation of neoplastic thyroid cells. We suggest that they reflect the biological nature and/or function of normal, hyperplastic, and neoplastic thyroid cells and may have some value in diagnosing thyroid tumors.
Asunto(s)
Proteínas Nucleares/análisis , Glándula Tiroides/química , Neoplasias de la Tiroides/química , Línea Celular Tumoral , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Células Epiteliales/química , Humanos , Hiperplasia , Inmunohistoquímica , Lamina Tipo A/análisis , Lamina Tipo A/genética , Lamina Tipo B/análisis , Lamina Tipo B/genética , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Proteínas Nucleares/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Glándula Tiroides/patologíaRESUMEN
INTRODUCTION: Nuclear accumulation of a mutant form of the nuclear protein Lamin-A, called Progerin (PG) or Lamin AΔ50, occurs in Hutchinson-Gilford Progeria Syndrome (HGPS) or Progeria, an accelerated aging disease. One of the main symptoms of this genetic disorder is a loss of sub-cutaneous fat due to a dramatic lipodystrophy. METHODS: We stably induced the expression of human PG and GFP -Green Fluorescent Protein- as control in 3T3L1 cells using a lentiviral system to study the effect of PG expression in the differentiation capacity of this cell line, one of the most used adipogenic models. Quantitative proteomics (iTRAQ) was done to study the effect of the PG accumulation. Several of the modulated proteins were validated by immunoblotting and real-time PCR. Mitochondrial function was analyzed by measurement of a) the mitochondrial basal activity, b) the superoxide anion production and c) the individual efficiency of the different complex of the respiratory chain. RESULTS: We found that over-expression PG by lentiviral gene delivery leads to a decrease in the proliferation rate and to defects in adipogenic capacity when compared to the control. Quantitative proteomics analysis showed 181 proteins significantly (p<0.05) modulated in PG-expressing preadipocytes. Mitochondrial function is impaired in PG-expressing cells. Specifically, we have detected an increase in the activity of the complex I and an overproduction of Superoxide anion. Incubation with Reactive Oxygen Species (ROS) scavenger agents drives to a decrease in autophagic proteolysis as revealed by LC3-II/LC3-I ratio. CONCLUSION: PG expression in 3T3L1 cells promotes changes in several Biological Processes, including structure of cytoskeleton, lipid metabolism, calcium regulation, translation, protein folding and energy generation by the mitochondria. Our data strengthen the contribution of ROS accumulation to the premature aging phenotype and establish a link between mitochondrial dysfunction and loss of proteostasis in HGPS.
Asunto(s)
Lamina Tipo A/análisis , Mitocondrias/metabolismo , Proteómica , Especies Reactivas de Oxígeno/metabolismo , Células 3T3-L1 , Animales , Autofagia , Cromatografía Líquida de Alta Presión , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lentivirus/genética , Ratones , Proteolisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Low voltage transmission electron microscopy (LVTEM) was employed to examine biological tissues with accelerating voltages as low as 5kV. Tissue preparation was modified to take advantage of the low-voltage techniques. Treatments with heavy metals, such as post-fixation with osmium tetroxide, on block and counterstaining were omitted. Sections (40nm) were thinner than usual and generated highly contrasted images. General appearance of the cells remains similar to that of conventional TEM. New features were however revealed. The matrix of the pancreatic granules displays heterogeneity with partitions that may correspond to the inner-segregation of their secretory proteins. Mitochondria revealed the presence of the ATP synthase granules along their cristea. The nuclear dense chromatin displayed a honeycomb organization while distinct beads, nucleosomes, aligned along thin threads were seen in the dispersed chromatin. Nuclear pore protein complexes revealed their globular nature. The intercalated disks in cardiac muscle displayed their fine structural organization. These features correlate well with data described or predicted by cell and molecular biology. These new aspects are not revealed when thicker and conventionally osmicated tissue sections were examined by LVTEM, indicating that major masking effects are associated with standard TEM techniques. Immunogold was adapted to LVTEM further enhancing its potential in cell biology.
Asunto(s)
Microscopía Electrónica de Transmisión/métodos , Microtomía/métodos , Miocardio/ultraestructura , Páncreas/ultraestructura , Amilasas/análisis , Amilasas/metabolismo , Animales , Inmunohistoquímica/métodos , Lamina Tipo A/análisis , Lamina Tipo A/metabolismo , Microscopía Electrónica de Transmisión/instrumentación , Mitocondrias/ultraestructura , Miocardio/metabolismo , Proteínas de Complejo Poro Nuclear/ultraestructura , Nucleosomas/ultraestructura , Páncreas/metabolismo , Ratas , Ratas Sprague-Dawley , Vesículas Secretoras/ultraestructura , Adhesión del Tejido/métodos , Fijación del Tejido/métodosRESUMEN
Nuclear lamins contact the genome at the nuclear periphery through large domains and are involved in chromatin organization. Among broad peak calling algorithms available to date, none are suited for mapping lamin-genome interactions genome wide. We disclose a novel algorithm, enriched domain detector (EDD), for analysis of broad enrichment domains from chromatin immunoprecipitation (ChIP)-seq data. EDD enables discovery of genomic domains interacting with broadly distributed proteins, such as A- and B-type lamins affinity isolated by ChIP. The advantages of EDD over existing broad peak callers are sensitivity to domain width rather than enrichment strength at a particular site, and robustness against local variations.
Asunto(s)
Algoritmos , Cromatina/química , Genoma Humano , Células Cultivadas , Inmunoprecipitación de Cromatina , Humanos , Lamina Tipo A/análisis , Lamina Tipo B/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcripción GenéticaRESUMEN
BACKGROUND INFORMATION: The optimal repair of DNA lesions is fundamental for physiological processes. We asked whether the recruitment of HP1ß, 53BP1 and BMI1 proteins to ultraviolet (UVA)-induced DNA lesions requires functional A-type lamins. RESULTS: We found that UVA irradiation of nuclear lamina abolished the fluorescence of mCherry-tagged A-type lamins and destroyed the nuclear lamina as also observed by electron microscopy studies. Similarly, an absence of endogenous A- and B-type lamins was found in irradiated regions by UVA. However, irradiation did not affect the recruitment of HP1ß, 53BP1 and BMI1 to DNA lesions. The UVA-induced shrinkage of the nuclear lamina, which anchors chromatin, explains why UVA-micro-irradiated chromatin is relaxed. Conversely, additional experiments with γ-irradiation showed that the nuclear lamina remained intact and the genome-wide level of HP1ß was stable. Fluorescence intensity of HP1ß and BMI1 in UVA-induced DNA lesions and level of HP1ß after γ-irradiation were unaffected by deficiency in A-type lamins, whereas those parameters of 53BP1 were changed. CONCLUSIONS: We conclude that only the 53BP1 status in DNA lesions, induced by UVA or γ-rays, is affected by A-type lamin deficiency, which was not observed for heterochromatin-related proteins HP1ß and BMI1.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Daño del ADN/efectos de la radiación , Lamina Tipo A/metabolismo , Células 3T3 , Animales , Proteínas Cromosómicas no Histona/análisis , ADN/genética , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Lamina Tipo A/análisis , Ratones , Complejo Represivo Polycomb 1/análisis , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53 , Rayos UltravioletaRESUMEN
Mutations in the human LMNA gene, encoding A-type lamins, give rise to laminopathies, which include several types of muscular dystrophy. Here, heterozygous sequence variants in LMNA, which result in single amino-acid substitutions, were identified in patients exhibiting muscle weakness. To assess whether the substitutions altered lamin function, we performed in vivo analyses using a Drosophila model. Stocks were generated that expressed mutant forms of the Drosophila A-type lamin modeled after each variant. Larvae were used for motility assays and histochemical staining of the body-wall muscle. In parallel, immunohistochemical analyses were performed on human muscle biopsy samples from the patients. In control flies, muscle-specific expression of the wild-type A-type lamin had no apparent affect. In contrast, expression of the mutant A-type lamins caused dominant larval muscle defects and semi-lethality at the pupal stage. Histochemical staining of larval body wall muscle revealed that the mutant A-type lamin, B-type lamins, the Sad1p, UNC-84 domain protein Klaroid and nuclear pore complex proteins were mislocalized to the cytoplasm. In addition, cytoplasmic actin filaments were disorganized, suggesting links between the nuclear lamina and the cytoskeleton were disrupted. Muscle biopsies from the patients showed dystrophic histopathology and architectural abnormalities similar to the Drosophila larvae, including cytoplasmic distribution of nuclear envelope proteins. These data provide evidence that the Drosophila model can be used to assess the function of novel LMNA mutations and support the idea that loss of cellular compartmentalization of nuclear proteins contributes to muscle disease pathogenesis.