RESUMO
Deficiency of the extracellular matrix protein latent transforming growth factor-ß (TGF-ß)-binding protein-4 (LTBP4) results in lack of intact elastic fibers, which leads to disturbed pulmonary development and lack of normal alveolarization in humans and mice. Formation of alveoli and alveolar septation in pulmonary development requires the concerted interaction of extracellular matrix proteins, growth factors such as TGF-ß, fibroblasts, and myofibroblasts to promote elastogenesis as well as vascular formation in the alveolar septae. To investigate the role of LTBP4 in this context, lungs of LTBP4-deficient (Ltbp4-/-) mice were analyzed in close detail. We elucidate the role of LTBP4 in pulmonary alveolarization and show that three different, interacting mechanisms might contribute to alveolar septation defects in Ltbp4-/- lungs: 1) absence of an intact elastic fiber network, 2) reduced angiogenesis, and 3) upregulation of TGF-ß activity resulting in profibrotic processes in the lung.
Assuntos
Tecido Elástico/patologia , Fibroblastos/patologia , Fibrose/patologia , Proteínas de Ligação a TGF-beta Latente/fisiologia , Pulmão/patologia , Neovascularização Patológica/patologia , Alvéolos Pulmonares/patologia , Animais , Células Cultivadas , Tecido Elástico/metabolismo , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Fibrose/metabolismo , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Knockout , Neovascularização Patológica/metabolismo , Organogênese/fisiologia , Alvéolos Pulmonares/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Lipid rafts play a key role in the regulation of fundamentally important cellular processes, including cell proliferation, differentiation, and survival. The composition of such detergent-resistant microdomains (DRMs) is altered under pathologic conditions, including cancer. Although DRMs have been analyzed in colorectal carcinoma little information exists about their composition upon treatment with targeted drugs. Hence, a quantitative proteomic profiling approach was performed to define alterations within the DRM fraction of colorectal carcinoma cells upon treatment with the drug U0126, an inhibitor of the mitogen-activated protein kinase pathway. Comparative expression profilings resulted in the identification of 300 proteins, which could partially be linked to key oncogenic signaling pathways and tumor-related cellular features, such as cell proliferation, adhesion, motility, invasion, and apoptosis resistance. Most of these proteins were downregulated upon inhibitor treatment. In addition, quantitative proteomic profilings of cholesterol-depleted versus intact lipid rafts were performed to define, which U0126-regulated target structures represent bona fide raft proteins. Selected differentially abundant raft proteins were validated at the mRNA and/or protein level using U0126- or Trametinib-treated cells. The presented data provide insights into the molecular mechanisms associated with the response to the treatment with MEK inhibitors and might also lead to novel candidates for therapeutic interventions.
Assuntos
Neoplasias do Colo/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteoma/metabolismo , Butadienos/farmacologia , Linhagem Celular Tumoral , Colo/efeitos dos fármacos , Colo/metabolismo , Detergentes/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Nitrilas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteoma/análise , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The clinical outcome of adoptive T cell transfer-based immunotherapies is often limited due to different escape mechanisms established by tumors in order to evade the hosts' immune system. The establishment of an immunosuppressive micromilieu by tumor cells along with distinct subsets of tumor-infiltrating lymphocytes is often associated with oxidative stress that can affect antigen-specific memory/effector cytotoxic T cells thereby substantially reducing their frequency and functional activation. Therefore, protection of tumor-reactive cytotoxic T lymphocytes from oxidative stress may enhance the anti-tumor-directed immune response. In order to better define the key pathways/proteins involved in the response to oxidative stress a comparative 2-DE-based proteome analysis of naïve CD45RA(+) and their memory/effector CD45RO(+) T cell counterparts in the presence and absence of low dose hydrogen peroxide (H(2)O(2)) was performed in this pilot study. Based on the profiling data of these T cell subpopulations under the various conditions, a series of differentially expressed spots were defined, members thereof identified by mass spectrometry and subsequently classified according to their cellular function and localization. Representative targets responding to oxidative stress including proteins involved in signaling pathways, in regulating the cellular redox status as well as in shaping/maintaining the structural cell integrity were independently verified at the transcript and protein level under the same conditions in both T cell subsets. In conclusion the resulting profiling data describe complex, oxidative stress-induced, but not strictly concordant changes within the respective expression profiles of CD45RA(+) and CD45RO(+) T cells. Some of the differentially expressed genes/proteins might be further exploited as potential targets toward modulating the redox capacity of the distinct lymphocyte subsets thereby providing the basis for further studies aiming at rendering them more resistant to tumor micromilieu-induced oxidative stress.
Assuntos
Perfilação da Expressão Gênica , Estresse Oxidativo/genética , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Antígenos Comuns de Leucócito/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Mapeamento de Peptídeos , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Coloração e Rotulagem , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologiaRESUMO
The "two-signal paradigm" in T cell activation predicts that the cooperation of "signal 1," provided by the T cell receptor (TCR) through engagement of major histocompatility complex (MHC)-presented peptide, with "signal 2â³ provided by costimulatory molecules, the prototype of which is CD28, is required to induce T cell effector functions. While the individual signalling pathways are well understood, little is known about global changes in the proteome pattern during TCR/CD28-mediated activation. Therefore, comparative 2-DE-based proteome analyses of CD3(+) CD69(-) resting T cells versus cells incubated with (i) the agonistic anti-CD3 antibody OKT3 mimicking signal 1 in absence or presence of IL-2 and/or with (ii) the agonistic antibody 15E8 triggering CD28-mediated signaling were performed. Differentially regulated spots were defined leading to the identification of proteins involved in the regulation of the metabolism, shaping and maintenance of the cytoskeleton and signal transduction. Representative members of the differentially expressed protein families, such as calmodulin (CALM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), L-lactate dehydrogenase (LDH), Rho GDP-dissociation inhibitor 2 (GDIR2), and platelet basic protein (CXCL7), were independently verified by flow cytometry. Data provide a detailed map of individual protein alterations at the global proteome level in response to TCR/CD28-mediated T cell activation.
Assuntos
Proteoma/metabolismo , Linfócitos T/metabolismo , Anticorpos/imunologia , Anticorpos/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Citoesqueleto , Eletroforese em Gel Bidimensional , Citometria de Fluxo , Humanos , Interleucina-2/metabolismo , Lectinas Tipo C/metabolismo , Ativação Linfocitária , Muromonab-CD3/imunologia , Muromonab-CD3/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
The presentation of tumor antigen-derived peptides by human leukocyte antigen (HLA) class I surface antigens on tumor cells is a key prerequisite to trigger effective T-cell responses in cancer patients. Multiple complementary strategies like cDNA and serological expression cloning, reverse immunology and different 'ome'-based methods have been employed to identify potential T-cell targets. This report focuses on a ligandomic profiling approach leading to the identification of 49 naturally processed HLA class I peptide ligands presented on the cell surface of renal cell carcinoma (RCC) cells. The source proteins of the defined HLA ligands are classified according to their biological function and subcellular localization. Previously established cDNA microarray data of paired tissue specimen of RCC and renal epithelium assessed the transcriptional regulation for 28 source proteins. In addition, HLA-A2-restricted, peptide-specific T cells directed against a HLA ligand derived from sulfiredoxin-1 (SRXN1) were generated, which were able to recognize and lyse ligand-presenting target cells in a HLA class I-restricted manner. Furthermore, tumor-infiltrating T cells isolated from a RCC patient were also able to kill SRXN1 expressing tumor cells. Thus, this experimental strategy might be suited to define potential candidate biomarkers and novel targets for T-cell-based immunotherapies of this disease.
Assuntos
Antígenos de Neoplasias/imunologia , Carcinoma de Células Renais/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Neoplasias Renais/imunologia , Proteínas de Neoplasias/imunologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/imunologia , Linfócitos T Citotóxicos/imunologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Cromatografia Líquida , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Epitopos/imunologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoterapia/métodos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Ligantes , Espectrometria de Massas , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Linfócitos T Citotóxicos/metabolismo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Despite recent advances in the understanding of the biology of renal cell carcinoma (RCC) and the implementation of novel targeted therapies, the overall 5 years' survival rate for RCC patients remains disappointing. Late presentation, tumor heterogeneity and in particular the lack of molecular biomarkers for early detection and classification represent major obstacles. Global, untargeted comparative analysis of RCC vs tumor adjacent renal epithelium (NN) samples by high throughput analyses both at the transcriptome and proteome level have identified signatures, which might further clarify the molecular differences of RCC subtypes and might allow the identification of suitable therapeutic targets and diagnostic/prognostic biomarkers, but none thereof has yet been implemented in routine clinical use. The increasing knowledge regarding the functional role of noncoding microRNA (miR) in physiological, developmental, and pathophysiological processes by shaping the protein expression profile might provide an important link to improve the definition of disease-relevant regulatory networks. Taking into account that miR profiling of RCC and NN provides robust signatures discriminating between malignant and normal tissues, the concept of evaluating and scoring miR/protein pairs might represent a strategy for the selection and prioritization of potential biomarkers and their translation into practical use.
Assuntos
Biomarcadores Tumorais , Carcinoma de Células Renais , Neoplasias Renais , MicroRNAs , Proteômica/métodos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Bases de Dados Genéticas , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
The prevention of mammary carcinoma by immunological strategies targeting the HER-2/neu receptor has proved to be effective in preclinical models. Thus, a well-characterized HER-2/neu oncogene-driven mammary carcinogenesis model was analysed by various profiling strategies following "triplex" vaccination to identify new candidate targets for breast cancer immunoprevention. 2-DE-based proteomic profiling of preneoplastic and tumour lesions versus normal and aged mammary tissue demonstrated that tumour progression was associated with an up-regulation of molecular chaperones including glucose-regulated protein (GRP)78 and of proteins favouring cell motility, which was in line with the corresponding transcriptomic profiling data. Furthermore, PROTEOMEX analyses suggested that naturally induced autoantibody responses occur during early phases of mammary cancer progression. Most of the cancer progression-induced antibodies targeted proteins of normal and preneoplastic mammary glands. However, three proteins were only recognized by sera obtained from vaccinated mice, including 2 isoforms of annexin A6. The distinct expression patterns for annexin A6 and GRP78 during tumour progression were further verified by western blot and/or immunoprecipitation. In addition, an inhibitor-mediated blockade of GRP78 expression in a model cell line caused a reduced cell growth. Thus, the proteome-based approaches applied in the murine BALB-NeuT model might indeed provide candidates for immunoprevention strategies in breast cancer.
Assuntos
Modelos Animais de Doenças , Neoplasias Mamárias Experimentais/metabolismo , Proteômica/métodos , Receptor ErbB-2/genética , Animais , Anticorpos Antineoplásicos/sangue , Autoanticorpos/sangue , Vacinas Anticâncer/farmacologia , Linhagem Celular Tumoral , Análise por Conglomerados , Progressão da Doença , Eletroforese em Gel Bidimensional , Chaperona BiP do Retículo Endoplasmático , Feminino , Perfilação da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Immunoblotting , Imunoglobulina G/sangue , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Análise de Componente Principal , Receptor ErbB-2/biossíntese , Receptor ErbB-2/metabolismo , Reprodutibilidade dos TestesRESUMO
Proteome-based technologies represent powerful tools for the analysis of protein expression profiles, including the identification of potential cancer candidate biomarkers. Thus, here we provide a comprehensive protein expression map for clear cell renal cell carcinoma established by systematic comparative two-dimensional gel electrophoresis-based protein expression profiling of 16 paired tissue systems comprising clear cell renal cell carcinoma lesions and corresponding tumor-adjacent renal epithelium using overlapping narrow pH gradients. This approach led to the mapping of 348 distinct spots corresponding to 248 different protein identities. By implementing restriction criteria concerning their detection frequency and overall regulation mode, 28 up- and 56 down-regulated single target spots were considered as potential candidate biomarkers. Based on their gene ontology information, these differentially expressed proteins were classified into distinct functional groups and according to their cellular distribution. Moreover, three representative members of this group, namely calbindin, gelsolin, and heart fatty acid-binding protein, were selected, and their expression pattern was analyzed by immunohistochemistry using tissue microarrays. Thus, this pilot study provides a significant update of the current renal cell carcinoma map and defines a number of differentially expressed proteins, but both their potential as candidate biomarkers and clinical relevance has to be further explored in tissues and for body fluids like serum and urine.
Assuntos
Carcinoma de Células Renais/metabolismo , Eletroforese em Gel Bidimensional/métodos , Neoplasias Renais/metabolismo , Proteínas de Neoplasias/metabolismo , Proteômica/métodos , Calbindinas , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Epitélio/metabolismo , Epitélio/patologia , Proteína 3 Ligante de Ácido Graxo , Proteínas de Ligação a Ácido Graxo/metabolismo , Gelsolina/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Renais/genética , Neoplasias Renais/patologia , Proteínas de Neoplasias/genética , Projetos Piloto , Reprodutibilidade dos Testes , Proteína G de Ligação ao Cálcio S100/metabolismo , Análise Serial de TecidosRESUMO
Results obtained from expression profilings of renal cell carcinoma using different "ome"-based approaches and comprehensive data analysis demonstrated that proteome-based technologies and cDNA microarray analyses complement each other during the discovery phase for disease-related candidate biomarkers. The integration of the respective data revealed the uniqueness and complementarities of the different technologies. While comparative cDNA microarray analyses though restricted to up-regulated targets largely revealed genes involved in controlling gene/protein expression (19%) and signal transduction processes (13%), proteomics/PROTEOMEX-defined candidate biomarkers include enzymes of the cellular metabolism (36%), transport proteins (12%), and cell motility/structural molecules (10%). Candidate biomarkers defined by proteomics and PROTEOMEX are frequently shared, whereas the sharing rate between cDNA microarray and proteome-based profilings is limited. Putative candidate biomarkers provide insights into their cellular (dys)function and their diagnostic/prognostic value but still warrant further validation in larger patient numbers. Based on the fact that merely three candidate biomarkers were shared by all applied technologies, namely annexin A4, tubulin alpha-1A chain, and ubiquitin carboxyl-terminal hydrolase L1, the analysis at a single hierarchical level of biological regulation seems to provide only limited results thus emphasizing the importance and benefit of performing rather combinatorial screenings which can complement the standard clinical predictors.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Perfilação da Expressão Gênica/métodos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Proteoma/análise , Idoso , Eletroforese em Gel Bidimensional , Epitélio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Humanos , Imuno-Histoquímica , Rim/metabolismo , Rim/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/classificação , Reprodutibilidade dos TestesRESUMO
Although the human genome has been decoded, the knowledge about the pathogenesis of diseases including cancer is still limited. By focusing on renal cell carcinoma (RCC) we here summarize the data of various research groups analyzing the protein/peptide expression profiles of tumor lesions/cell lines or serum obtained from patients and respective controls. Different powerful approaches such as 2-DE, PROTEOMEX/SERPA/SPEARS, and T cell epitope discovery upon elution of MHC class I-bound peptides in combination with MS/LC-MS/MS revealed 500 differentially expressed proteins. The overlap in target recognition limits the pool to 299 unique protein identities, but only few thereof (12%) have been validated. The management, analysis, and interpretation of the distinct data sets derived from 27 publications required bioinformatic restructuring of the results. However, the comprehensive analysis of the results expands the knowledge about the pathophysiology of RCC in particular of the most prominent clear cell subtype by providing information on the differentially expressed proteins, their regulation status in RCC compared to normal kidney epithelium next to additional information on MHC-presented T cell epitopes and on serological targets. Despite the low number of validated differentially expressed proteins some of them might serve as candidate biomarkers for the diagnosis and/or as therapeutic targets.