RESUMEN

The phenotypic heterogeneity of Lamin A/C (LMNA) variants renders it difficult to classify them. As a consequence, many LMNA variants are classified as variant of unknown significance (VUS). A number of studies reported different types of visible nuclear abnormalities in LMNA-variant carriers, such as herniations, honeycomb-like structures and irregular Lamin staining. In this study, we used lamin A/C immunostaining and nuclear DAPI staining to assess the number and type of nuclear abnormalities in primary dermal fibroblast cultures of laminopathy patients and healthy controls. The total number of abnormal nuclei, which includes herniations, honeycomb-structures, and donut-like nuclei, was found to be the most discriminating parameter between laminopathy and control cell cultures. The percentage abnormal nuclei was subsequently scored in fibroblasts of 28 LMNA variant carriers, ranging from (likely) benign to (likely) pathogenic variant. Using this method, 27 out of 28 fibroblast cell cultures could be classified as either normal (n = 14) or laminopathy (n = 13) and no false positive results were obtained. The obtained specificity was 100% (CI 40-100%) and sensitivity 77% (46-95%). We conclude that assessing the percentage of abnormal nuclei is a quick and reliable method, which aids classification or confirms pathogenicity of identified LMNA variants causing formation of aberrant lamin A/C protein.

Asunto(s)

Núcleo Celular/patología , Fibroblastos/patología , Pruebas Genéticas/métodos , Lamina Tipo A/genética , Células Cultivadas , Citogenética/métodos , Fibroblastos/metabolismo , Humanos

RESUMEN

Erythrokeratodermia variabilis et progressiva (EKV-P) is caused by mutations in either the GJB3 (Cx31) or GJB4 genes (Cx30.3). We identified a rare GJB3 missense mutation, c.134G>A (p.G45E), in two unrelated patients and investigated its cellular characteristics. Expression of Cx31G45E-GFP caused previously undescribed changes within HeLa cells and HaCaT cells, a model human keratinocyte cell line. Cx31WT-GFP localised to the plasma membrane, but expression of Cx31G45E-GFP caused vacuolar expansion of the endoplasmic reticulum (ER), the mutant protein accumulated within the ER membrane and disassembly of the microtubular network occurred. No ER stress responses were evoked. Cx31WT-myc-myc-6xHis and Cx31G45E-GFP co-immunoprecipitated, indicative of heteromeric interaction, but co-expression with Cx31WT-mCherry, Cx26 or Cx30.3 did not mitigate the phenotype. Cx31 and Cx31G45E both co-immunoprecipitated with Cx43, indicating the ability to form heteromeric connexons. WT-Cx31 and Cx43 assembled into large gap junction plaques at points of cell-to-cell contact; Cx31G45E restricted the ability of Cx43 to reach the plasma membrane in both HaCaT cells and HeLa cells stably expressing Cx43 where the proteins strongly co-localised with the vacolourised ER. Cell viability assays identified an increase in cell death in cells expressing Cx31G45E-GFP, which FACS analysis determined was necrotic. Blocking connexin channel function with 18α-glycyrrhetinic acid did not completely rescue necrosis or prevent propidium iodide uptake, suggesting that expression of Cx31G45E-GFP damages the cellular membrane independent of its channel function. Our data suggest that entrapment of Cx43 and necrotic cell death in the epidermis could underlie the EKV skin phenotype.

Asunto(s)

Conexinas/genética , Eritroqueratodermia Variable/genética , Mutación Missense , Muerte Celular , Membrana Celular/efectos de los fármacos , Células Cultivadas , Conexina 43/biosíntesis , Conexina 43/genética , Retículo Endoplásmico/ultraestructura , Epidermis/patología , Eritroqueratodermia Variable/patología , Genes Dominantes , Estudios de Asociación Genética , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/farmacología , Células HeLa , Humanos , Queratinocitos , Necrosis , Transporte de Proteínas

RESUMEN

In adherent cells, the relevance of a physical mechanotransduction pathway provided by the perinuclear actin cap stress fibers has recently emerged. Here, we investigate the impact of a functional actin cap on the cellular adaptive response to topographical cues and uniaxial cyclic strain. Lmna-deficient fibroblasts are used as a model system because they do not develop an intact actin cap, but predominantly form a basal layer of actin stress fibers underneath the nucleus. We observe that topographical cues induce alignment in both normal and Lmna-deficient fibroblasts, suggesting that the topographical signal transmission occurs independently of the integrity of the actin cap. By contrast, in response to cyclic uniaxial strain, Lmna-deficient cells show a compromised strain avoidance response, which is completely abolished when topographical cues and uniaxial strain are applied along the same direction. These findings point to the importance of an intact and functional actin cap in mediating cellular strain avoidance.

Asunto(s)

Actinas/metabolismo , Lamina Tipo A/deficiencia , Modelos Biológicos , Estrés Mecánico , Estrés Fisiológico , Actinina , Animales , Anisotropía , Forma de la Célula , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Adhesiones Focales/metabolismo , Lamina Tipo A/metabolismo , Ratones , Miosinas/metabolismo , Fosforilación , Fibras de Estrés/metabolismo , Factores de Tiempo

RESUMEN

Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Homólogo de la Proteína 1 Relacionada con la Autofagia , Síndrome de Birt-Hogg-Dubé/metabolismo , Síndrome de Birt-Hogg-Dubé/patología , Proteínas Portadoras/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/deficiencia , Proteína Sequestosoma-1 , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/deficiencia

RESUMEN

The Warburg effect is a tumorigenic metabolic adaptation process characterized by augmented aerobic glycolysis, which enhances cellular bioenergetics. In normal cells, energy homeostasis is controlled by AMPK; however, its role in cancer is not understood, as both AMPK-dependent tumor-promoting and -inhibiting functions were reported. Upon stress, energy levels are maintained by increased mitochondrial biogenesis and glycolysis, controlled by transcriptional coactivator PGC-1α and HIF, respectively. In normoxia, AMPK induces PGC-1α, but how HIF is activated is unclear. Germline mutations in the gene encoding the tumor suppressor folliculin (FLCN) lead to Birt-Hogg-Dubé (BHD) syndrome, which is associated with an increased cancer risk. FLCN was identified as an AMPK binding partner, and we evaluated its role with respect to AMPK-dependent energy functions. We revealed that loss of FLCN constitutively activates AMPK, resulting in PGC-1α-mediated mitochondrial biogenesis and increased ROS production. ROS induced HIF transcriptional activity and drove Warburg metabolic reprogramming, coupling AMPK-dependent mitochondrial biogenesis to HIF-dependent metabolic changes. This reprogramming stimulated cellular bioenergetics and conferred a HIF-dependent tumorigenic advantage in FLCN-negative cancer cells. Moreover, this pathway is conserved in a BHD-derived tumor. These results indicate that FLCN inhibits tumorigenesis by preventing AMPK-dependent HIF activation and the subsequent Warburg metabolic transformation.

Asunto(s)

Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Síndrome de Birt-Hogg-Dubé/etiología , Síndrome de Birt-Hogg-Dubé/genética , Síndrome de Birt-Hogg-Dubé/metabolismo , Línea Celular , Transformación Celular Neoplásica , Metabolismo Energético , Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética

RESUMEN

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder where patients are predisposed to kidney cancer, lung and kidney cysts and benign skin tumors. BHD is caused by heterozygous mutations affecting folliculin (FLCN), a conserved protein that is considered a tumor suppressor. Previous research has uncovered multiple roles for FLCN in cellular physiology, yet it remains unclear how these translate to BHD lesions. Since BHD manifests hallmark characteristics of ciliopathies, we speculated that FLCN might also have a ciliary role. Our data indicate that FLCN localizes to motile and non-motile cilia, centrosomes and the mitotic spindle. Alteration of FLCN levels can cause changes to the onset of ciliogenesis, without abrogating it. In three-dimensional culture, abnormal expression of FLCN disrupts polarized growth of kidney cells and deregulates canonical Wnt signalling. Our findings further suggest that BHD-causing FLCN mutants may retain partial functionality. Thus, several BHD symptoms may be due to abnormal levels of FLCN rather than its complete loss and accordingly, we show expression of mutant FLCN in a BHD-associated renal carcinoma. We propose that BHD is a novel ciliopathy, its symptoms at least partly due to abnormal ciliogenesis and canonical Wnt signalling.

Asunto(s)

Síndrome de Birt-Hogg-Dubé/fisiopatología , Cilios/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Bases , Síndrome de Birt-Hogg-Dubé/genética , Línea Celular , Polaridad Celular , Proliferación Celular , Centrosoma/fisiología , Cilios/patología , Humanos , Riñón/fisiología , Microtúbulos/fisiología , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Vía de Señalización Wnt

RESUMEN

Laminopathies, mainly caused by mutations in the LMNA gene, are a group of inherited diseases with a highly variable penetrance; i.e., the disease spectrum in persons with identical LMNA mutations range from symptom-free conditions to severe cardiomyopathy and progeria, leading to early death. LMNA mutations cause nuclear abnormalities and cellular fragility in response to cellular mechanical stress, but the genotype/phenotype correlations in these diseases remain unclear. Consequently, tools such as mutation analysis are not adequate for predicting the course of the disease.   Here, we employ growth substrate stiffness to probe nuclear fragility in cultured dermal fibroblasts from a laminopathy patient with compound progeroid syndrome. We show that culturing of these cells on substrates with stiffness higher than 10 kPa results in malformations and even rupture of the nuclei, while culture on a soft substrate (3 kPa) protects the nuclei from morphological alterations and ruptures. No malformations were seen in healthy control cells at any substrate stiffness. In addition, analysis of the actin cytoskeleton organization in this laminopathy cells demonstrates that the onset of nuclear abnormalities correlates to an increase in cytoskeletal tension. Together, these data indicate that culturing of these LMNA mutated cells on substrates with a range of different stiffnesses can be used to probe the degree of nuclear fragility. This assay may be useful in predicting patient-specific phenotypic development and in investigations on the underlying mechanisms of nuclear and cellular fragility in laminopathies.

Asunto(s)

Núcleo Celular/metabolismo , Lamina Tipo A/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Línea Celular , Forma del Núcleo Celular/fisiología , Fibroblastos/citología , Fibroblastos/metabolismo , Heterocigoto , Humanos , Lamina Tipo A/genética , Mutación , Progeria/genética , Progeria/metabolismo , Progeria/patología

RESUMEN

Porokeratotic eccrine ostial and dermal duct nevus, or porokeratotic eccrine nevus (PEN), is a hyperkeratotic epidermal nevus. Several cases of widespread involvement have been reported, including one in association with the keratitis-ichthyosis-deafness (KID) syndrome (OMIM #148210), a rare disorder caused by mutations in the GJB2 gene coding for the gap junction protein connexin26 (Cx26). The molecular cause is, as yet, unknown. We have noted that PEN histopathology is shared by KID. The clinical appearance of PEN can resemble that of KID syndrome. Furthermore, a recent report of cutaneous mosaicism for a GJB2 mutation associated with KID describes linear hyperkeratotic skin lesions that might be consistent with PEN. From this, we hypothesized that PEN might be caused by Cx26 mutations associated with KID or similar gap junction disorders. Thus, we analyzed the GJB2 gene in skin samples from two patients referred with generalized PEN. In both, we found GJB2 mutations in the PEN lesions but not in unaffected skin or peripheral blood. One mutation was already known to cause the KID syndrome, and the other had not been previously associated with skin symptoms. We provide extensive functional data to support its pathogenicity. We conclude that PEN may be caused by mosaic GJB2 mutations.

Asunto(s)

Conexinas/genética , Nevo/genética , Poroqueratosis/genética , Neoplasias Cutáneas/genética , Conexina 26 , Análisis Mutacional de ADN , Sordera/genética , Humanos , Ictiosis/genética , Queratitis/genética , Mutación , Nevo/patología , Poroqueratosis/patología , Neoplasias Cutáneas/patología

RESUMEN

The nuclear lamina provides structural support to the nucleus and has a central role in nuclear organization and gene regulation. Defects in its constituents, the lamins, lead to a class of genetic diseases collectively referred to as laminopathies. Using live cell imaging, we observed the occurrence of intermittent, non-lethal ruptures of the nuclear envelope in dermal fibroblast cultures of patients with different mutations of lamin A/C. These ruptures, which were absent in normal fibroblasts, could be mimicked by selective knockdown as well as knockout of LMNA and were accompanied by the loss of cellular compartmentalization. This was demonstrated by the influx of cytoplasmic transcription factor RelA and regulatory protein Cyclin B1 into the nucleus, and efflux of nuclear transcription factor OCT1 and nuclear structures containing the promyelocytic leukemia (PML) tumour suppressor protein to the cytoplasm. While recovery of enhanced yellow fluorescent protein-tagged nuclear localization signal in the nucleus demonstrated restoration of nuclear membrane integrity, part of the mobile PML structures became permanently translocated to the cytoplasm. These satellite PML structures were devoid of the typical PML body components, such as DAXX, SP100 or SUMO1. Our data suggest that nuclear rupture and loss of compartmentalization may add to cellular dysfunction and disease development in various laminopathies.

Asunto(s)

Compartimento Celular , Laminas/metabolismo , Membrana Nuclear/patología , Animales , Proteínas Bacterianas/metabolismo , División Celular , Dextranos/metabolismo , Regulación de la Expresión Génica , Humanos , Lamina Tipo A/metabolismo , Proteínas Luminiscentes/metabolismo , Sustancias Macromoleculares/metabolismo , Ratones , Peso Molecular , Membrana Nuclear/ultraestructura , Señales de Localización Nuclear , Transportador 1 de Catión Orgánico/metabolismo , Transporte de Proteínas

RESUMEN

Mutations in connexin26, a cutaneous gap junction protein, cause a wide variety of skin disorders including keratitis-ichthyosis-deafness syndrome (KID). We previously delineated a phenotype distinct from KID, hypotrichosis-deafness syndrome, caused by the mutation p.Asn14Lys in connexin26. However, a different mutation at the same location, p.Asn14Tyr, was reported to cause a disorder similar to KID. Distinct substitutions cause different conformational changes to the protein, each with unique consequences for its behaviour. This may explain the phenotypic differences. We found the previously described mutation p.Asn14Tyr in connexin26 in two patients from Brazil and Poland, and observe quite distinct phenotypes distinguishable from classical KID syndrome. We assessed functional consequences of p.Asn14Tyr and p.Asn14Lys, using fluorescently labelled proteins and parachute assay, comparing them with the classical KID mutation p.Asp50Asn. Our analyses show that p.Asn14Tyr, p.Asn14Lys and p.Asp50Asn have different consequences for protein localization and gap junction permeability. However, the differences between the phenotypes we observed cannot be readily explained from effects on protein trafficking or gap junction permeability.

Asunto(s)

Asparagina/genética , Conexinas/genética , Mutación Missense/fisiología , Enfermedades de la Piel/genética , Adulto , Ácido Aspártico/genética , Membrana Celular/metabolismo , Niño , Conexina 26 , Conexinas/metabolismo , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Femenino , Fluoresceínas/metabolismo , Uniones Comunicantes/metabolismo , Células HeLa , Pérdida Auditiva/genética , Humanos , Hipertricosis/genética , Hipertricosis/patología , Queratodermia Palmoplantar/tratamiento farmacológico , Queratodermia Palmoplantar/genética , Queratodermia Palmoplantar/patología , Queratosis/tratamiento farmacológico , Queratosis/genética , Queratosis/patología , Lisina/genética , Masculino , Uñas Malformadas/genética , Uñas Malformadas/patología , Transporte de Proteínas/genética , Piel/patología , Enfermedades de la Piel/tratamiento farmacológico , Enfermedades de la Piel/patología , Síndrome , Transfección , Tirosina/genética

RESUMEN

A thorough understanding of fat cell biology is necessary to counter the epidemic of obesity. Although molecular pathways governing adipogenesis are well delineated, the structure of the nuclear lamina and nuclear-cytoskeleton junction in this process are not. The identification of the 'linker of nucleus and cytoskeleton' (LINC) complex made us consider a role for the nuclear lamina in adipose conversion. We herein focused on the structure of the nuclear lamina and its coupling to the vimentin network, which forms a cage-like structure surrounding individual lipid droplets in mature adipocytes. Analysis of a mouse and human model system for fat cell differentiation showed fragmentation of the nuclear lamina and subsequent loss of lamins A, C, B1 and emerin at the nuclear rim, which coincides with reorganization of the nesprin-3/plectin/vimentin complex into a network lining lipid droplets. Upon 18 days of fat cell differentiation, the fraction of adipocytes expressing lamins A, C and B1 at the nuclear rim increased, though overall lamin A/C protein levels were low. Lamin B2 remained at the nuclear rim throughout fat cell differentiation. Light and electron microscopy of a subcutaneous adipose tissue specimen showed striking indentations of the nucleus by lipid droplets, suggestive for an increased plasticity of the nucleus due to profound reorganization of the cellular infrastructure. This dynamic reorganization of the nuclear lamina in adipogenesis is an important finding that may open up new venues for research in and treatment of obesity and nuclear lamina-associated lipodystrophy.

Asunto(s)

Adipocitos/citología , Adipogénesis , Citoesqueleto/metabolismo , Lámina Nuclear/metabolismo , Células 3T3-L1 , Adipocitos/metabolismo , Adipocitos/ultraestructura , Animales , Células Cultivadas , Citoesqueleto/ultraestructura , Humanos , Inmunohistoquímica , Laminas/análisis , Laminas/biosíntesis , Ratones , Lámina Nuclear/ultraestructura

RESUMEN

Dunnigan-type familial partial lipodystrophy (FPLD) is a laminopathy characterized by an aberrant fat distribution and a metabolic syndrome for which oxidative stress has recently been suggested as one of the disease-causing mechanisms. In a family affected with FPLD, we identified a heterozygous missense mutation c.1315C>T in the LMNA gene leading to the p.R439C substitution. Cultured patient fibroblasts do not show any prelamin A accumulation and reveal honeycomb-like lamin A/C formations in a significant percentage of nuclei. The mutation affects a region in the C-terminal globular domain of lamins A and C, different from the FPLD-related hot spot. Here, the introduction of an extra cysteine allows for the formation of disulphide-mediated lamin A/C oligomers. This oligomerization affects the interaction properties of the C-terminal domain with DNA as shown by gel retardation assays and causes a DNA-interaction pattern that is distinct from the classical R482W FPLD mutant. Particularly, whereas the R482W mutation decreases the binding efficiency of the C-terminal domain to DNA, the R439C mutation increases it. Electron spin resonance spectroscopy studies show significantly higher levels of reactive oxygen species (ROS) upon induction of oxidative stress in R439C patient fibroblasts compared to healthy controls. This increased sensitivity to oxidative stress seems independent of the oligomerization and enhanced DNA binding typical for R439C, as both the R439C and R482W mutants show a similar and significant increase in ROS upon induction of oxidative stress by H2O2.

Asunto(s)

Lamina Tipo A/fisiología , Lipodistrofia Parcial Familiar/metabolismo , Mutación Missense , Proteínas Nucleares/metabolismo , Estrés Oxidativo , Precursores de Proteínas/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Predisposición Genética a la Enfermedad , Humanos , Peróxido de Hidrógeno/farmacología , Lamina Tipo A/genética , Lipodistrofia Parcial Familiar/genética , Complejos Multiproteicos , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

LMNA-associated progeroid syndromes have been reported with both recessive and dominant inheritance. We report a 2-year-old boy with an apparently typical Hutchinson-Gilford progeria syndrome (HGPS) due to compound heterozygous missense mutations (p.T528M and p.M540T) in LMNA. Both mutations affect a conserved region within the C-terminal globular domain of A-type lamins, defining a progeria hot spot. The nuclei of the patient showed no prelamin A accumulation. In general, the nuclear phenotype did not correspond to that previously described for HGPS. Instead, honeycomb figures predominated and nuclear blebs with reduced/absent expression of B-type lamins could be detected. The healthy heterozygous parents showed similar nuclear changes, although in a smaller percentage of nuclei. Treatment with a farnesylation inhibitor resulted in accumulation of prelamin A at the nuclear periphery, in annular nuclear membrane plaques and in intra/trans-nuclear membrane invaginations. In conclusion, these findings suggest a critical role for the C-terminal globular lamin A/C region in nuclear structure and support a major contribution of abnormal assembly to the progeroid phenotype. In contrast to earlier suggestions, we show that prelamin A accumulation is not the major determinant of the progeroid phenotype.

Asunto(s)

Lamina Tipo A/genética , Mutación , Proteínas Nucleares/metabolismo , Progeria/etiología , Progeria/genética , Precursores de Proteínas/metabolismo , Adulto , Huesos/diagnóstico por imagen , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Preescolar , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/ultraestructura , Heterocigoto , Humanos , Imagenología Tridimensional , Inmunohistoquímica , Lamina Tipo A/análisis , Lovastatina/farmacología , Masculino , Metionina/análogos & derivados , Metionina/farmacología , Modelos Moleculares , Progeria/diagnóstico , Progeria/diagnóstico por imagen , Prenilación de Proteína/efectos de los fármacos , Radiografía

RESUMEN

Although there is consensus that HPV integration is common in invasive cervical carcinomas and uncommon or absent in low-grade uterine cervical intraepithelial neoplasia (CIN I), estimates for HPV integration in CIN II/III range from 5 to 100% using different PCR-based and in situ hybridization (ISH) approaches. It has been suggested that HPV integration can be identified using ISH by scoring of punctate signals. The increased sensitivity of fluorescence ISH (FISH) methods, allowing the detection of single copies of HPV, complicates the distinction between integrated and episomal HPV. Recently it has been suggested that, in such assays, the signals originating from integrated virus can be hidden in a background of episomal HPV. We therefore compared 2 different FISH protocols for the detection of integrated HPV in a series of CIN II/III lesions: 1) a mild protocol in which episomal HPV and RNA is retained and 2) a harsh protocol that extensively extracts proteins and RNA, and which promotes the partial loss of episomal HPV but not integrated HPV. A series of 28 HPV 16/18 positive CIN II/III lesions (17 solitary lesions and 11 lesions adjacent to microinvasive carcinoma) were studied. A punctate signal pattern was identified in 7 of these lesions with both protocols. Punctate signal was also present in control samples from lesions that are known to be associated with HPV integration (invasive squamous cell carcinoma (n = 3), adenocarcinoma in situ (n = 3), and invasive adenocarcinoma (n = 1). HPV RNA contributed significantly to the intensity of punctate FISH signal, especially when applying the mild protocol, as shown by omitting DNA denaturation, including RNase pretreatment steps and measuring the fluorescence signal intensity. Also, HPV RNA was frequently detected in addition to episomal/integrated HPV DNA in the majority of the other 21 CIN II/III lesions; this resulted in intense granular/diffuse FISH signals throughout the epithelium. However, in 7 of these lesions, the harsh protocol gave a more consistent punctate pattern in cells throughout the full thickness of the epithelium. This supports the hypothesis that the harsh protocol unmasks integrated HPV more efficiently by extracting RNA and episomal HPV. Overall, with this harsh protocol, a clonally expanded population of cells containing punctate HPV signals was found in 5 of 17 (29%) solitary CIN II/III lesions and in 9 of 11 (88%) CIN II/III lesions associated with microinvasive carcinoma. Combining these data with the results from our previous study, with the harsh protocol in 7 of 40 (18%) solitary CIN II/III lesions and 19/21 (90%) CIN II/III lesions associated with microinvasive carcinoma (p < 0.001), this pattern was found. This indicates that, when robustly defined, a punctate HPV pattern in CIN II/III lesions is associated with the presence of an invasive carcinoma.

Asunto(s)

Hibridación Fluorescente in Situ , Invasividad Neoplásica/patología , Papillomaviridae/genética , Infecciones por Papillomavirus/genética , Neoplasias del Cuello Uterino/genética , Integración Viral/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma/virología , Adulto , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Transformación Celular Neoplásica/patología , Aberraciones Cromosómicas , ADN Viral/análisis , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Persona de Mediana Edad , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Plásmidos/genética , ARN Viral/análisis , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/genética , Displasia del Cuello del Útero/patología , Displasia del Cuello del Útero/virología

RESUMEN

An unusual haplotype was detected in a family of a caucasian transplant patient. Human leukocyte antigen (HLA) analysis of the family demonstrated the absence of HLA-A on one of the haplotypes present in two family members. One was serologically typed A24, the other A2. Because they had one haplotype in common, the HLA-A allele of the shared haplotype was supposed to be a null allele. Different molecular typing methods identified only one allele in both individuals. The results suggest a deletion of the complete HLA-A gene or a major part of it. For confirmation, microsatellite analysis of the HLA-A region was performed with six microsatellite markers. Both family members were heterozygous for all markers, and a deletion of HLA-A could not be proven. Fluorescent in situ hybridization (FISH) was performed with cosmid and PAC probes encompassing the HLA-A gene. Both probes demonstrated an identical normal distribution pattern for diploid results. The absence of any serologic and molecular reaction with the results of the microsatellite and FISH analysis make a deletion of a narrow region, encompassing the HLA-A gene, the most plausible explanation.

Asunto(s)

Eliminación de Gen , Antígenos HLA-A/genética , Haplotipos , Cartilla de ADN , Femenino , Humanos , Hibridación Fluorescente in Situ , Masculino , Repeticiones de Microsatélite , Linaje , Reacción en Cadena de la Polimerasa , Población Blanca

RESUMEN

Carcinoma in situ (CIS) of the urinary bladder is a flat, aggressive lesion and may be the most common precursor of invasive bladder cancer. Although chromosome 9 alterations are among the earliest and most prevalent genetic alterations in bladder cancer, discrepancy exists about the frequency of chromosome 9 losses in CIS. We analyzed 22 patients with CIS of the bladder (15 patients with isolated CIS, 7 patients combined with synchronous pTa or pT1 carcinomas) for gains and losses of chromosome (peri)centromere loci 1q12, 7p11-q11, 9p11-q12, and 9p21 harboring the INK4A/ARF locus (p16(INK4A)/p14(ARF)) and INK4B (p15(INK4B)) by multiple-target fluorescence in situ hybridization, and for p53 protein accumulation by immunohistochemistry. In 15 of 20 (75%) CIS lesions analyzed p53 overexpression was detected, whereas aneusomy for chromosomes 1 and 7 was identified in 20 of 22 (91%) CIS. In 13 of 22 (60%) CIS cases analyzed, 12 of which were not associated with a synchronous pTa or pT1 carcinoma, no numerical losses for chromosome 9 (p11-q12 and 9p21) were detected as compared with chromosomes 1 and 7. Furthermore 6 of 12 (50%) patients showed a metachronous invasive carcinoma within 2 years. In the remaining nine biopsies CIS lesions (40%) were recognized that showed losses of chromosome 9p11-q12 and 9p21, six of these were associated with a synchronous pTa or pT1 carcinoma. Three of these carcinomas were pTa and exhibited loss of 9q12 as well as a homozygous deletion of 9p21. The others were invasive carcinomas in which CIS lesions were also recognized that showed no numerical loss of chromosome 9, but did show an accumulation of p53. In conclusion our data demonstrate that predominantly isolated CIS lesions contained cells with no specific loss of chromosome 9, as opposed to CIS lesions with synchronous carcinomas that showed evidence of chromosome 9 loss. Furthermore our data strengthen the proposition that p53 mutations (p53 overexpression) precede loss of chromosomes 9 and 9p21 in CIS as precursor for invasive bladder cancer, as opposed to noninvasive carcinomas where chromosome 9 (9p11-q12) losses are early and frequently combined with homozygous deletions of 9p21.

Asunto(s)

Carcinoma in Situ/genética , Carcinoma/genética , Aberraciones Cromosómicas , Cromosomas Humanos Par 9 , Pérdida de Heterocigocidad , Proteína p53 Supresora de Tumor/genética , Neoplasias de la Vejiga Urinaria/genética , Biopsia , Carcinoma/patología , Carcinoma in Situ/patología , Mapeo Cromosómico , Cromosomas Humanos Par 1 , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Estudios Retrospectivos , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias de la Vejiga Urinaria/patología