ABSTRACT
Bone conduction hearing implants can rehabilitate some types of hearing loss. A hydroxyapatite (HA)-coated skin-penetrating abutment was developed to allow for soft tissue preservation and increased skin-abutment adherence. Inflammation is thought to relate to bacterial infection of pockets around the abutment. Upon integration, the host's ability to cover the abutment surface ("race for the surface"), and thus control and prevent competitive bacteria from colonizing it, is improved. However, the attachment mechanisms behind it are not clear. In this study, we applied two-photon microscopy to visualize tissue attachment on abutments retrieved from patients. Skin integration markers were validated and applied to four HA-coated abutments. Evidence of skin integration was found, including the presence of hemidesmosomes, a basement membrane, dermal collagen and vascularization. Cases with clinical signs of severe inflammation and evident biofilm formation showed limited skin integration based on these indicators, confirming the applicability of the "race for the surface" model.
Subject(s)
Durapatite/chemistry , Microscopy/methods , Skin/metabolismABSTRACT
BACKGROUND: High-risk (hr) human papillomavirus (HPV) infections play a causal role in the development of cervical cancer. The detection of hrHPV is, therefore, advocated in cervical cancer screening programs. OBJECTIVES: The aim of this study was to determine the performance of a novel HPV typing assay, PapillomaFinder® SMART 20. This is a one-tube-per-sample method, to be performed on standard real-time PCR platforms, using melting curve analysis to distinguish targets. The assay detects all 14 hrHPV types, of which 16, 18, 31, 33, 35, 39, 45, 52, 56 and 58 individually. HrHPV types 51, 59, 66 and 68 are detected in an hrHPV pool, and low-risk (lr) HPV types 6, 11, 40, 42, 43 and 44 in an lrHPV pool. STUDY DESIGN: The method was tested on HPV plasmid models, WHO and QCMD proficiency panels and a series of clinical cytological samples (n=45), the latter in comparison with a clinically validated real-time quantitative PCR. RESULTS: Type-specificity of the test was 100% using plasmids, the WHO and QCMD panels. Sensitivity for hrHPV in single infections was 100% using the WHO and QCMD panels and cytological samples, with an analytical sensitivity of 10-25 copies per reaction for all HPV types tested. Of the 34 HPV types present in the 8 multiple infections in the WHO panel, 30 were detected. In all cytological samples at least one hrHPV type was found, in concordance with the clinically validated method. Only when the viral load of the dominant HPV types in multiple infections greatly exceeded that of the other types in the infection, those other types were not always detected. CONCLUSIONS: PapillomaFinder® SMART 20 is a rapid, easy to perform, single tube HPV typing assay. The assay detects the 14 hrHPV types, and the 6 most important lrHPV types with a high sensitivity and type-specificity.
Subject(s)
Genotyping Techniques/methods , Multiplex Polymerase Chain Reaction/methods , Papillomaviridae/classification , Papillomaviridae/genetics , Papillomavirus Infections/diagnosis , Real-Time Polymerase Chain Reaction/methods , Female , Humans , Papillomavirus Infections/virology , Sensitivity and Specificity , Time FactorsABSTRACT
There is growing evidence that laminopathies, diseases associated with mutations in the LMNA gene, are caused by a combination of mechanical and gene regulatory distortions. Strikingly, there is a large variability in disease symptoms between individual patients carrying an identical LMNA mutation. This is why classical genetic screens for mutations appear to have limited predictive value for disease development. Recently, the widespread occurrence of repetitive nuclear ruptures has been described in fibroblast cultures from various laminopathy patients. Since this phenomenon was strongly correlated with disease severity, the identification of biomarkers that report on these rupture events could have diagnostic relevance. One such candidate marker is the PML nuclear body, a structure that is normally confined to the nuclear interior, but leaks out of the nucleus upon nuclear rupture. Here, we show that a variety of laminopathies shows the presence of these cytoplasmic PML particles (PML CPs), and that the amount of these protein aggregates increases with severity of the disease. In addition, between clinically healthy individuals, carrying LMNA mutations, significant differences can be found. Therefore, we postulate that detection of PML CPs in patient fibroblasts could become a valuable marker for diagnosis of disease development.
Subject(s)
Cell Nucleus/metabolism , Cytoplasm/metabolism , Fibroblasts/metabolism , Lamin Type A/genetics , Mutation , Nuclear Proteins/metabolism , Skin/metabolism , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Adolescent , Adult , Biomarkers/metabolism , Cell Nucleus/pathology , Cells, Cultured , Child , Child, Preschool , Cytoplasm/pathology , Female , Fibroblasts/pathology , Genotype , Humans , Male , Microscopy, Fluorescence , Middle Aged , Phenotype , Promyelocytic Leukemia Protein , Skin/pathology , Time Factors , Time-Lapse Imaging , TransfectionABSTRACT
During early apoptosis the 33 amino acid C-terminal cytokeratin 18 (CK18) fragment is released by caspase-9 cleavage at the 393DALD/S site. This basic peptide relocates from the cytoskeleton to the nucleoplasm as shown by confocal laser scanning. It is shown that the C-terminal peptide modulates topoisomerase activity as measured by relaxation of plasmid DNA. In an in vitro assay recombinant caspase-induced chromatin condensation is inhibited by the peptide and at the electron microscopical level a clear inhibition of nucleolar breakdown was observed in its presence. We hypothesize that the C-terminal CK18 fragment exerts an effect in the nucleolus by stimulating rRNA transcription and processing via modulation of enzymatic activity of topoisomerase I. This leads to preservation of general transcriptional activity required to exert active steps during early stages of programmed cell death.
Subject(s)
Apoptosis/physiology , Caspase 9/metabolism , DNA Topoisomerases, Type I/metabolism , Keratin-18/metabolism , Peptide Fragments/metabolism , Cell Line, Tumor , Cell Nucleolus/metabolism , Cell Nucleolus/pathology , Chromatin Assembly and Disassembly/physiology , DNA Fragmentation , Electrophoretic Mobility Shift Assay , Humans , Microscopy, Electron, Transmission , Transcription, Genetic/physiologyABSTRACT
The nuclear lamina and the cytoskeleton form an integrated structure that warrants proper mechanical functioning of cells. We have studied the correlation between structural alterations and migrational behaviour in fibroblasts with and without A-type lamins. We show that loss of A-type lamins causes loss of emerin and nesprin-3 from the nuclear envelope, concurring with a disturbance in the connection between the nucleus and the cytoskeleton in A-type lamin-deficient (lmna -/-) cells. In these cells functional migration assays during in vitro wound healing revealed a delayed reorientation of the nucleus and the microtubule-organizing center during migration, as well as a loss of nuclear oscillatory rotation. These observations in fibroblasts isolated from lmna knockout mice were confirmed in a 3T3 cell line with stable reduction of lmna expression due to RNAi approach. Our results indicate that A-type lamins play a key role in maintaining directional movement governed by the cytoskeleton, and that the loss of these karyoskeletal proteins has important consequences for functioning of the cell as a mechanical entity.
Subject(s)
Cell Movement , Cell Nucleus/metabolism , Cell Polarity , Fibroblasts/cytology , Lamin Type A/deficiency , 3T3 Cells , Animals , Biological Assay , Fibroblasts/metabolism , Lamin Type A/metabolism , Membrane Proteins/metabolism , Mice , Microtubule-Organizing Center/metabolism , Nuclear Proteins/metabolism , Protein Transport , RNA Interference , Wound HealingABSTRACT
Oncogenic human papillomavirus (HPV) is a causative agent in a subgroup of head and neck carcinomas, particularly tonsillar squamous cell carcinomas (TSCC). This study was undertaken because controversial data exist on the physical status of HPV-DNA and the use of p16(INK4A) overexpression as surrogate HPV marker, and to examine the impact of HPV and tobacco consumption on the clinical course of TSCC. Tissue sections of 81 TSCC were analyzed by HPV 16-specific fluorescence in situ hybridization (FISH) and p16(INK4A)-specific immunohistochemistry. Results were correlated with clinical and demographic data. HPV 16 integration was detected by FISH as punctate signals in 33 out of 81 (41%) TSCC, 32 of which showed p16(INK4A) accumulation. Only 5 out of 48 HPV-negative tumors showed p16(INK4A) immunostaining (p < 0.0001). The presence of HPV furthermore correlates significantly with low tobacco (p = 0.002) and alcohol intake (p = 0.011), poor differentiation grade (p = 0.019), small tumor size (p = 0.024), presence of a local metastasis (p = 0.001) and a decreased (loco)regional recurrence rate (p = 0.039). Statistical analysis revealed that smoking significantly increases the risk of cancer death from TSCC and that non-smoking patients with HPV-containing TSCC show a remarkably better disease-specific survival rate. HPV 16 is integrated in 41% of TSCC and strongly correlates with p16(INK4A) overexpression, implicating the latter to be a reliable HPV biomarker. Patients with HPV-positive tumors show a favorable prognosis as compared to those with HPV-negative tumors, but tobacco use is the strongest prognostic indicator. These findings indicate that oncogenic processes in the tonsils of non-smokers differ from those occurring in smokers, the former being related to HPV 16 infection.
Subject(s)
Carcinoma, Squamous Cell/virology , Papillomaviridae/isolation & purification , Smoking/adverse effects , Survival Rate , Tonsillar Neoplasms/virology , Carcinoma, Squamous Cell/pathology , Cyclin-Dependent Kinase Inhibitor p16/genetics , DNA, Viral/genetics , Female , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Male , Middle Aged , Neoplasm Metastasis , Papillomaviridae/genetics , Polymerase Chain Reaction , Prognosis , Tonsillar Neoplasms/pathologySubject(s)
Biomarkers, Tumor/metabolism , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Keratin-17/metabolism , Uterine Cervical Dysplasia/metabolism , Uterine Cervical Neoplasms/metabolism , Diagnosis, Differential , Female , Humans , Immunohistochemistry/methods , Phenotype , Uterine Cervical Dysplasia/diagnosis , Uterine Cervical Neoplasms/diagnosisABSTRACT
The clinical behavior of endocrine pancreatic tumors (EPTs) is difficult to predict in the absence of metastases or invasion to adjacent organs. Several markers have been indicated as potential predictors of metastatic disease, such as tumor size > or =2 cm, Ki67 proliferative index > or =2%, cytokeratin (CK) 19 status, and recently in insulinomas, chromosomal instability (CIN). The goal of this study was to evaluate the value of these markers, and in particular of the CIN, to predict tumor recurrence or progression and tumor-specific death, using a series of 47 insulinomas and 24 non-insulinoma EPTs. From these EPT cases, a genomic profile has been generated and follow-up data have been obtained. The proliferative index has been determined in 68 tumors and a CK19 expression pattern in 50 tumors. Results are statistically analyzed using Kaplan-Meier plots and the log-rank statistic. General CIN, as well as specific chromosomal alterations such as 3p and 6q loss and 12q gain, turned out to be the most powerful indicators for poor tumor-free survival (P< or =0.0004) and tumor-specific death (P< or =0.0113) in insulinomas. The CIN, chromosome 7q gain, and a proliferative index > or =2% were reliable in predicting a poor tumor-free survival in non-insulinoma EPTs (P< or =0.0181, whereas CK19 expression was the most optimal predictor of tumor-specific death in these tumors. In conclusion, DNA copy number status is the most sensitive and efficient marker of adverse clinical outcome in insulinomas and of potential interest in non-insulinoma EPTs. As a consequence, this marker should be considered as a prognosticator to improve clinical diagnosis, most practically as a simple multi-target test.
Subject(s)
DNA, Neoplasm/analysis , Gene Dosage , Insulinoma/diagnosis , Molecular Diagnostic Techniques/methods , Pancreatic Neoplasms/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Chromosomal Instability , Chromosomes, Human , Female , Follow-Up Studies , Gastrinoma/diagnosis , Gastrinoma/genetics , Gastrinoma/mortality , Gastrinoma/pathology , Humans , Insulinoma/genetics , Insulinoma/mortality , Insulinoma/pathology , Ki-67 Antigen/analysis , Male , Middle Aged , Neoplasm Metastasis , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/mortality , Pancreatic Neoplasms/pathology , Prognosis , Sensitivity and Specificity , Survival AnalysisABSTRACT
Insulinomas are the most common functioning endocrine pancreatic tumors (EPTs). They present with clinical symptoms as a consequence of hypoglycemia induced by inappropriate insulin secretion. The etiology of these tumors is poorly understood. Some tumors may harbor MEN1 gene mutations, the susceptibility gene of the multiple endocrine neoplasia type I syndrome, but most cases show wildtype MEN1. Currently, no reliable clinical tests are available to differentiate benign from malignant tumors. Approximately 30% of the tumors are unresectable, and they often show different growth rates, which hampers treatment. Therefore, a better understanding of the molecular processes underlying the development and progression of insulinomas is required to improve diagnosis, prognosis and therapy. Here we summarize the progress that has been made in insulinoma research in the past decade. We describe the clinical detection, classification and treatment of these tumors, and review the multiplicity of molecular and genetic studies that investigated tumor development and progression using either primary tumors, transgenic mouse models or tumor-derived cell lines. The identification of many interactors of the MEN1 gene product menin, as well as recurrent chromosomal abnormalities that pinpoint candidate genes of interest will likely result in a better understanding of the molecular pathways involved in insulinoma tumorigenesis. In addition, these studies will pave the way for the identification of novel targets for therapeutical intervention and more reliable markers for clinical diagnosis and prognosis.
Subject(s)
Insulinoma/genetics , Pancreatic Neoplasms/genetics , Genetic Markers , Humans , Insulinoma/pathology , Insulinoma/surgery , Neoplasm Invasiveness , Neoplasm Metastasis/genetics , Neoplasm Metastasis/pathology , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/surgery , Signal Transduction/physiologyABSTRACT
The envelope that encapsulates the cell nucleus has recently gained considerable interest, as several clinical syndromes are linked to mutations in its molecular components. Most disorders recognized so far are caused by defects in the nuclear lamins, building blocks of a filamentous network lining the nucleoplasmic side of the inner nuclear membrane. Nuclear lamins are the evolutionary precursors of cytoskeletal intermediate filaments and associate in a head-to-tail manner into a stable lamina at the nuclear periphery and into a more dispersed structure in the nucleoplasm. Lamins have a scaffolding function for several nuclear processes such as transcription, chromatin organization and DNA replication, and maintain nuclear and cellular integrity. Mutations in the LMNA gene, encoding A-type lamins, can cause cardiac and skeletal muscle disease, lipodystrophy and premature ageing phenotypes. Hence, the integrity of the nuclear envelope seems essential for longevity. Furthermore, the laminopathies provide evidence that metabolism and ageing are as tightly linked in humans as they are in model organisms such as C. elegans. In this review, we elaborate on the structure and functions of nuclear lamins, the spectrum of syndromes related to mutations in nuclear envelope components and pathogenic concepts unifying these disorders.
Subject(s)
Gene Expression Regulation , Lamins/genetics , Lamins/physiology , Nuclear Envelope/physiology , Aging, Premature/genetics , Animals , Bone Diseases, Developmental/genetics , Cardiomyopathy, Dilated/genetics , Cell Differentiation/physiology , Cell Survival/physiology , Charcot-Marie-Tooth Disease/genetics , DNA Repair/physiology , Evolution, Molecular , Hereditary Central Nervous System Demyelinating Diseases/genetics , Heterochromatin/physiology , Humans , Lamins/biosynthesis , Lipodystrophy/genetics , Muscular Dystrophies/genetics , Nuclear Envelope/pathology , Nuclear Envelope/ultrastructure , Nuclear Lamina/physiologyABSTRACT
The response of individual cells to cellular stress is vital for cellular functioning. A large network of physically interconnected cellular components, starting from the structural components of the cells' nucleus, via cytoskeleton filaments to adhesion molecules and the extracellular matrix, constitutes an integrated matrix that functions as a scaffold allowing the cell to cope with mechanical stress. Next to a role in mechanical properties, this network also has a mechanotransductional function in the response to mechanical stress. This signaling route does not only regulate a rapid reorganization of structural components such as actin filaments, but also stimulates for example gene activation via NFkappaB and other transcription factors. The importance of an intact mechano-signaling network is illustrated by the physiological consequences of several genetic defects of cellular network components e.g. actin, dystrophin, desmin and lamins. These give rise to an impaired response of the affected cells to mechanical stress and often result in dystrophy of the affected tissue. Recently, the importance of the cell nucleus in cellular strength has been established. Several new interconnecting proteins, such as the nesprins that link the nuclear lamina to the cytoskeleton, have been identified. Furthermore, the function of nuclear lamins in determining cellular strength and nuclear stability was illustrated in lamin-knock-out cells. Absence of the A-type lamins or mutations in these structural components of the nuclear lamina lead to an impaired cellular response to mechanical stress and disturbances in cytoskeletal organization. In addition, laminopathies show clinical phenotypes comparable to those seen for diseases resulting from genetic defects in cytoskeletal components, further indicating that lamins play a central role in maintaining the mechanical properties of the cell.
Subject(s)
Cell Physiological Phenomena , Cytoskeleton/physiology , Nuclear Lamina/physiology , Animals , Cells/cytology , Embryo, Mammalian , Fibroblasts/cytology , Fibroblasts/physiology , Humans , Mice , Models, Biological , Stress, MechanicalABSTRACT
Insulinomas represent the predominant syndromic subtype of endocrine pancreatic tumors (EPTs). Their metastatic potential cannot be predicted reliably using histopathological criteria. In the past few years, several attempts have been made to identify prognostic markers, among them TP53 mutations and immunostaining of p53 and recently cytokeratin 19 (CK19). In a previous study using conventional comparative genomic hybridization (CGH) we have shown that chromosomal instability (CIN) is associated with metastatic disease in insulinomas. It was our aim to evaluate these potential parameters in a single study. For the determination of CIN, we applied CGH to microarrays because it allows a high-resolution detection of DNA copy number changes in comparison with conventional CGH as well as the analysis of chromosomal regions close to the centromeres and telomeres, and at 1pter-->p32, 16p, 19 and 22. These regions are usually excluded from conventional CGH analysis, because they may show DNA gains in negative control hybridizations. Array CGH analysis of 30 insulinomas (15 tumors of benign, eight tumors of uncertain and seven tumors of malignant behavior) revealed that >or=20 chromosomal alterations and >or=6 telomeric losses were the best predictors of malignant progression. A subset of 22 insulinomas was further investigated for TP53 exon 5-8 gene mutations, and p53 and CK19 expression. Only one malignant tumor was shown to harbor an arginine 273 serine mutation and immunopositivity for p53. CK19 immunopositivity was detected in three malignant tumors and one tumor with uncertain behavior. In conclusion, our results indicate that CIN as well as telomeric loss are very powerful indicators for malignant progression in sporadic insulinomas. Our data do not support a critical role for p53 and CK19 as molecular parameters for this purpose.
Subject(s)
Computational Biology/methods , Insulinoma/diagnosis , Insulinoma/pathology , Keratin-19/genetics , Pancreatic Neoplasms/genetics , Tumor Suppressor Protein p53/genetics , Chromosomal Instability , DNA Mutational Analysis , Disease Progression , Exons , Female , Humans , Male , Mutation , Oligonucleotide Array Sequence Analysis , Pancreatic Neoplasms/pathology , PrognosisABSTRACT
Insulinomas represent the predominant syndromic subtype of endocrine pancreatic tumours. Previous molecular studies have shown that gain of chromosome 9q rather than MEN1 gene mutation is an important early event in tumour development and that chromosomal instability is associated with metastatic disease. In order to identify new gene loci and to define further the critical genetic events in insulinoma tumourigenesis, 27 insulinomas were investigated by array-based comparative genomic hybridization (array CGH) on 3.7 k genomic BAC arrays (resolution < or =1 Mb). Fluorescence in situ hybridization was used to validate alterations in a subset of tumours. Array CGH most frequently detected loss of chromosomes 11q and 22q and gains of chromosome 9q. The chromosomal regions of interest (CRI) included 11q24.1 (56%), 22q13.1 (67%), 22q13.31 (56%), and 9q32 (63%). Evaluation of the simultaneous occurrence of these aberrations in the individual tumours revealed that gain of 9q32 and loss of 22q13.1 are early genetic events in insulinomas, occurring independently of the other alterations. In tumours with increased genomic complexity, these alterations were often detected simultaneously, occurring in the same tumour cells. Losses of 11q24.1 and 22q13.31 were also associated with these more advanced tumour cases. The CRIs identified most likely harbour crucial candidate genes important in insulinoma tumourigenesis.
Subject(s)
Chromosome Aberrations , Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 22/genetics , Genes, Tumor Suppressor , Insulinoma/genetics , Pancreatic Neoplasms/genetics , Chromosomal Instability/genetics , Chromosome Deletion , Chromosomes, Human, Pair 4/genetics , Chromosomes, Human, Pair 9/genetics , DNA, Neoplasm/genetics , Female , Humans , In Situ Hybridization, Fluorescence/methods , Male , Middle Aged , PloidiesABSTRACT
Recently proposed events associated with the progression of cervical intraepithelial neoplasia (CIN) 2/3 to cervical carcinoma include integration of human papillomavirus (HPV) into the host genome, genomic instability, and an increase in chromosome 3q copy number. In particular, the gene coding for the RNA component of telomerase (TERC) at 3q26 has been implicated as a possible candidate gene. Since it is not known to date how these events are temporally related during cervical carcinogenesis, the aim of the present study was to assess the correlation between TERC gene copy number and the physical status of HPV during progression in cervical neoplasia. Solitary precursor lesions of the uterine cervix (CIN 2/3, n = 17), lesions associated with a micro-invasive carcinoma (CIN 3&mCA, n = 13), and advanced invasive carcinomas (invCA, n = 7) were analysed by fluorescence in situ hybridization (FISH) to determine the physical status of the virus and TERC gene copy number. The TERC gene was increasingly gained with progression of CIN 2/3 (3 of 17) through CIN 3&mCA (7 of 13) to invCA (5 of 7). In the lesions exhibiting gain of TERC, the virus was predominantly integrated. This was seen in eight of ten diploid lesions, indicating that these events can occur prior to aneuploidization and are strongly associated with the progression of CIN 3 to mCA and invCA (p < 0.001). With progression to carcinoma, a number of these lesions show polyploidization, resulting in aneuploidy and high TERC gene copy numbers. In conclusion, genomic integration of oncogenic HPV and gain of the human telomerase gene TERC appear to be important associated genetic events in the progression of uterine cervical dysplasia to invasive cancer.
Subject(s)
Papillomaviridae/genetics , RNA/genetics , Telomerase/genetics , Uterine Cervical Dysplasia/genetics , Uterine Cervical Neoplasms/genetics , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Centromere/genetics , Cervix Uteri/pathology , Chromosome Aberrations , Chromosomes, Human, Pair 3/genetics , Chromosomes, Human, Pair 7/genetics , DNA, Neoplasm/genetics , Female , Gene Amplification/genetics , Humans , In Situ Hybridization, Fluorescence/methods , Neoplasm Invasiveness , Ploidies , Uterine Cervical Neoplasms/pathology , Uterine Cervical Dysplasia/pathologyABSTRACT
It has been demonstrated that nuclear lamins are important proteins in maintaining cellular as well as nuclear integrity, and in maintaining chromatin organization in the nucleus. Moreover, there is growing evidence that lamins play a prominent role in transcriptional control. The family of laminopathies is a fast-growing group of diseases caused by abnormalities in the structure or processing of the lamin A/C (LMNA) gene. Mutations or incorrect processing cause more than a dozen different inherited diseases, ranging from striated muscular diseases, via fat- and peripheral nerve cell diseases, to progeria. This broad spectrum of diseases can only be explained if the responsible A-type lamin proteins perform multiple functions in normal cells. This review gives an overview of current knowledge on lamin structure and function and all known diseases associated with LMNA abnormalities. Based on the knowledge of the different functions of A-type lamins and associated proteins, explanations for the observed phenotypes are postulated. It is concluded that lamins seem to be key players in, among others, controlling the process of cellular ageing, since disturbance in lamin protein structure gives rise to several forms of premature ageing.
Subject(s)
Aging, Premature/pathology , Aging, Premature/physiopathology , Lamins/physiology , Nuclear Lamina/pathology , HumansABSTRACT
Diseases caused by mutations in lamins A and C (laminopathies) suggest a crucial role for A-type lamins in different cellular processes. Laminopathies mostly affect tissues of mesenchymal origin. As transforming growth factor-beta1 (TGF-beta1) signalling impinges on the retinoblastoma protein (pRB) and SMADs, we tested the hypothesis that lamins modulate cellular responses to TGF-beta1 signalling, via the regulation of these transcription factors in mesenchymal cells. Here, we report that A-type lamins are essential for the inhibition of fibroblast proliferation by TGF-beta1. TGF-beta1 dephosphorylated pRB through PP2A, both of which, we show, are associated with lamin A/C. In addition, lamin A/C modulates the effect of TGF-beta1 on collagen production, a marker of mesenchymal differentiation. Our findings implicate lamin A/C in control of gene activity downstream of TGF-beta1, via nuclear phosphatases such as PP2A. This biological function provides a novel explanation for the observed mesenchymal dysfunction in laminopathies.
Subject(s)
Lamin Type A/physiology , Phosphoprotein Phosphatases/metabolism , Retinoblastoma Protein/metabolism , Transcription Factors/metabolism , Transforming Growth Factor beta/pharmacology , Animals , Cell Proliferation , Cells, Cultured , Fibroblasts/metabolism , Lamin Type A/genetics , Lamin Type A/metabolism , Mice , Mice, Mutant Strains , Phosphorylation , Signal Transduction , Transforming Growth Factor beta1ABSTRACT
Recent insights into the cell biology of the epidermis and its appendages are transforming our understanding of the pathogenesis of basal cell carcinoma (BCC). The significant progress that has been made warrants a comprehensive review of the molecular and cellular pathology of BCC. The items addressed include environmental and genetic risk factors, the biology of the putative precursor cell(s), and the contribution of aberrations in processes such as apoptosis, cell proliferation, differentiation and signalling to carcinogenesis. Furthermore, established and novel treatment modalities are discussed with particular attention to future biological approaches.
Subject(s)
Carcinoma, Basal Cell/genetics , Skin Neoplasms/genetics , Apoptosis , Carcinoma, Basal Cell/pathology , Carcinoma, Basal Cell/therapy , Cell Differentiation , Cell Proliferation , Epidermis/metabolism , Epidermis/pathology , Genetic Predisposition to Disease , Humans , Immunosuppression Therapy , Immunotherapy , Risk Factors , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Ultraviolet Rays/adverse effects , Virus Diseases/complications , Virus Diseases/pathologyABSTRACT
Endocrine pancreatic tumors (EPTs) comprise a highly heterogeneous group of tumors with different clinical behavior and genetic makeup. Insulinomas represent the predominant syndromic subtype of EPTs. The metastatic potential of insulinomas can frequently not be predicted using histopathological criteria, and also molecular markers indicating malignant progression are unreliable because of the small number of cases per subtype studied so far. For the identification of reliable indicators of metastatic disease, we investigated 62 sporadic insulinomas (44 benign and 18 tumors with metastases) by means of comparative genomic hybridization (CGH). In addition, the role of MEN1 (multiple endocrine neoplasia type 1) gene mutations was determined to assess specific chromosomal alterations associated with dysfunction of this endocrine tumor-related tumor suppressor gene. Only one case with a somatic MEN1 mutation was identified (1527del7bp), indicating that the MEN1 gene plays a minor pathogenic role in sporadic insulinomas. CGH analysis revealed that the total number of aberrations per tumor differs strongly between the benign and the malignant group (4.2 vs 14.1; P<0.0001). Furthermore, chromosome 9q gain was found to be the most frequent aberration in both benign and malignant insulinomas, whereas chromosome 6q losses and 12q, 14q and 17pq gains are strongly associated with metastatic disease. Our study shows that chromosomal instability, as defined by > or =5 gains together with > or =5 losses, or total number of gains and losses > or =8, rather than parameters such as tumor size and proliferation index, is the most powerful indicator for the development of metastatic disease in patients with sporadic insulinoma.
Subject(s)
Chromosomal Instability/genetics , Insulinoma/diagnosis , Pancreatic Neoplasms/diagnosis , Proto-Oncogene Proteins/genetics , Adult , Diagnosis, Differential , Female , Humans , Insulinoma/genetics , Insulinoma/pathology , Male , Middle Aged , Mutation , Neoplasm Metastasis , Nucleic Acid Hybridization , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , PrognosisABSTRACT
We have applied the fluorescence loss of intensity after photobleaching (FLIP) technique to study the molecular dynamics and organization of nuclear lamin proteins in cell lines stably transfected with green fluorescent protein (GFP)-tagged A-type lamin cDNA. Normal lamin A and C proteins show abundant decoration of the inner layer of the nuclear membrane, the nuclear lamina, and a generally diffuse localization in the nuclear interior. Bleaching studies revealed that, while the GFP-tagged lamins in the lamina were virtually immobile, the intranuclear fraction of these molecules was partially mobile. Intranuclear lamin C was significantly more mobile than intranuclear lamina A. In search of a structural cause for the variety of inherited diseases caused by A-type lamin mutations, we have studied the molecular organization of GFP-tagged lamin A and lamin C mutants R453W and R386K, found in Emery-Dreifuss muscular dystrophy (EDMD), and lamin A and lamin C mutant R482W, found in patients with Dunnigan-type familial partial lipodystrophy (FPLD). In all mutants, a prominent increase in lamin mobility was observed, indicating loss of structural stability of lamin polymers, both at the perinuclear lamina and in the intranuclear lamin organization. While the lamin rod domain mutant showed overall increased mobility, the tail domain mutants showed mainly intranuclear destabilization, possibly as a result of loss of interaction with chromatin. Decreased stability of lamin mutant polymers was confirmed by flow cytometric analyses and immunoblotting of nuclear extracts. Our findings suggest a loss of function of A-type lamin mutant proteins in the organization of intranuclear chromatin and predict the loss of gene regulatory function in laminopathies.