RESUMO
For metastasis formation, individual cells from a primary tumor must migrate toward other tissues. The aim of this study was to determine if mesenchymal stromal cells (MSCs) from human bone marrow are able to emit signals that induce this migratory activity in cancer cells. We separated the supernatant of MSCs derived from human bone marrow by size-exclusion and ion-exchange chromatography and have subsequently studied the migratory behavior of the prostate cancer cell line PC3 and the breast cancer cell line MDA-MB-231 toward the respective fractions in a transwell migration assay. We identified the extracellular matrix (ECM) proteins type I collagen, type III collagen, fibronectin, and laminin 421 as potential drivers of cancer cell migration. These results could be reproduced using the corresponding isolated or recombinant ECM proteins. Knockdown of the gene encoding beta 1 integrin, an important cell surface receptor for fibronectin, has led to inhibition of cancer cell migration. This supports the hypothesis that beta 1 integrin signaling represents an initial event that leads to metastasis, and that signaling is triggered by binding of integrin heterodimers to ECM molecules. Further characterization of signaling factors and their respective receptors will have implications for anticancer drug development.
Assuntos
Movimento Celular , Colágeno Tipo III/metabolismo , Colágeno Tipo I/metabolismo , Fibronectinas/metabolismo , Laminina/metabolismo , Células-Tronco Mesenquimais/citologia , Neoplasias/patologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Colágeno Tipo I/genética , Colágeno Tipo III/genética , Fibronectinas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Laminina/genética , Células-Tronco Mesenquimais/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Células Tumorais CultivadasRESUMO
There is a great demand for label-free in vitro assays in a high-throughput context, in order to measure cell viability and analyze cellular functions like cell migration or cell differentiation under noninvasive conditions. Here, we describe impedance measurement to quantify dynamic changes on cell morphology in real time. In order to monitor physiological changes, cells are grown in tissue culture vessels where gold electrodes are incorporated at the bottom. An alternating current signal of several kHz is applied to the electrodes and the resulting voltage is measured to calculate the cellular impedance. Since impedance is closely related to the area of the electrodes covered by the growing cells, parameters such as cell number, size of the cells attached to the electrodes, and cell-cell and cell-substrate/extracellular matrix interactions contribute to the overall impedance values.
Assuntos
Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Impedância Elétrica , Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Contagem de Células , Tamanho Celular , Eletrodos , Células-Tronco de Carcinoma Embrionário/química , Matriz Extracelular/química , Ouro/química , Ensaios de Triagem em Larga Escala , HumanosRESUMO
Decitabine (5-aza-2'-deoxycytidine) is a DNA methyltransferase inhibitor and an archetypal epigenetic drug for the therapy of myeloid leukemias. The mode of action of decitabine strictly depends on the incorporation of the drug into DNA. However, DNA incorporation and ensuing genotoxic effects of decitabine have not yet been investigated in human cancer cell lines or in models related to the approved indication of the drug. Here we describe a robust assay for the quantitative determination of decitabine incorporation rates into DNA from human cancer cells. Using a panel of human myeloid leukemia cell lines we show appreciable amounts of decitabine incorporation that closely correlated with cellular drug uptake. Decitabine incorporation was also detectable in primary cells from myeloid leukemia patients, indicating that the assay is suitable for biomarker analyses to predict drug responses in patients. Finally, we also used next-generation sequencing to comprehensively analyze the effects of decitabine incorporation on the DNA sequence level. Interestingly, this approach failed to reveal significant changes in the rates of point mutations and genome rearrangements in myeloid leukemia cell lines. These results indicate that standard rates of decitabine incorporation are not genotoxic in myeloid leukemia cells.
Assuntos
Antimetabólitos Antineoplásicos/análise , Azacitidina/análogos & derivados , DNA de Neoplasias/química , Taxa de Mutação , Azacitidina/análise , Linhagem Celular Tumoral , Decitabina , Humanos , Contagem de CintilaçãoRESUMO
The mTOR signaling plays an integral role in cellular homeostasis controlling the transition between the catabolic and anabolic states. Originally approved as immunosuppressive agents preventing allograft rejection, inhibitors of mTOR signaling have recently entered the arena of cancer therapy. Using rapamycin derivative (RAD001) as a prototype inhibitor, we aimed to systematically analyze the molecular mechanisms underlying the pleiotropic effects of mTOR signaling. Using proliferation- and clonogenic survival assays, a preferential sensitivity of microvascular endothelial cells (HDMVEC) followed by fibroblasts and U87 gliblastoma to RAD001 treatment was found. In contrast, lung- and prostate tumor cells demonstrated relative resistance against RAD001 treatment. In co-culture with fibroblasts, RAD001 exerted potent antiangiogenic effects by inhibiting endothelial cell tube formation. Further, RAD001 treatment efficiently prevented tumor growth in U87 tumor xenografts. Integrative transcriptome analysis was performed to decipher the molecular mechanism underlying RAD001 -induced anti-tumor and antiangiogenic effects. The predominant expression pattern was downregulation of genes after RAD001 treatment in all three sensitive cell types. Among the RAD001 downregulated genes, a transcriptional network was discovered enriched for genes related to angiogenesis processes and extracellular matrix remodeling, e.g., VEGF, HIF1A, CXCLs, IL6, FN, PAI-1 or NRP1. Of note, key components of PI3K upstream (PDK1) as well as mTORC2 downstream signaling (SGK1, NDRG) were downregulated by RAD001. Decreased expression of IMPDH and 139 common gene targets between mycophenolic acid and RAD001 suggested in part shared mechanisms underlying their antiangiogenic and immunosuppressive effects. In summary, key genetic participants governing anti-tumor and anti-angiogenic effects of mTOR inhibition were identified.
Assuntos
Biologia de Sistemas , Serina-Treonina Quinases TOR/antagonistas & inibidores , Transcriptoma , Animais , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Perfilação da Expressão Gênica , Humanos , Imunossupressores/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase em Tempo Real , Serina-Treonina Quinases TOR/genéticaRESUMO
Induction of differentiation in cancer stem cells by drug treatment represents an important approach for cancer therapy. The understanding of the mechanisms that regulate such a forced exit from malignant pluripotency is fundamental to enhance our knowledge of tumour stability. Certain nucleoside analogues, such as 2'-deoxy-5-azacytidine and 1ß-arabinofuranosylcytosine, can induce the differentiation of the embryonic cancer stem cell line NTERA 2 D1 (NT2). Such induced differentiation is associated with drug-dependent DNA-damage, cellular stress and the proteolytic depletion of stem cell factors. In order to further elucidate the mode of action of these nucleoside drugs, we monitored differentiation-specific changes of the dielectric properties of growing NT2 cultures using electric cell-substrate impedance sensing (ECIS). We measured resistance values of untreated and retinoic acid treated NT2 cells in real-time and compared their impedance profiles to those of cell populations triggered to differentiate with several established substances, including nucleoside drugs. Here we show that treatment with retinoic acid and differentiation-inducing drugs can trigger specific, concentration-dependent changes in dielectric resistance of NT2 cultures, which can be observed as early as 24 hours after treatment. Further, low concentrations of nucleoside drugs induce differentiation-dependent impedance values comparable to those obtained after retinoic acid treatment, whereas higher concentrations induce proliferation defects. Finally, we show that impedance profiles of substance-induced NT2 cells and those triggered to differentiate by depletion of the stem cell factor OCT4 are very similar, suggesting that reduction of OCT4 levels has a dominant function for differentiation induced by nucleoside drugs and retinoic acid. The data presented show that NT2 cells have specific dielectric properties, which allow the early identification of differentiating cultures and real-time label-free monitoring of differentiation processes. This work might provide a basis for further analyses of drug candidates for differentiation therapy of cancers.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco de Carcinoma Embrionário/efeitos dos fármacos , Células-Tronco de Carcinoma Embrionário/metabolismo , Linhagem Celular Tumoral , Impedância Elétrica , Humanos , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tretinoína/farmacologiaRESUMO
BACKGROUND AIMS: For their wide mesodermal differentiation potential, mesenchymal stromal/stem cells (MSC) are attractive candidates for tissue engineering. However, standardized quality control assays monitoring differentiation that are non-invasive and continuous over time are lacking. METHODS: We employed a non-invasive assay, using two different systems, to discriminate osteogenic and adipogenic differentiation of MSC by monitoring impedance. Fibroblasts and keratinocytes served as non-specific controls. Impedance profiles were recorded comparing MSC from bone marrow and adipose tissue, either non-induced or induced for osteogenesis or adipogenesis, for 5-14 days, and correlated with differentiation markers assessed by reverse transcription-quantitative polymerase chain reaction and Western blot. Additionally, differentiation modulating effects of extracellular matrix components were analyzed. RESULTS: Adhesion and growth-related impedance profiles of non-induced MSC roughly resembled those of fibroblasts, whereas keratinocytes differed significantly. Distinct from that, osteogenic induction of MSC revealed initially rapid and continuously rising impedance, corresponding to mineralized calcium matrix formation. Conversely, adipogenic induction caused shallower initial slopes and eventually declining profiles, corresponding to more compact, adipocyte-like cells with numerous lipid vacuoles. Pre-coating with either collagen type I or IV apparently favored osteogenesis and fibronectin adipogenesis. Impedance recordings correlated well with the extent of differentiation evaluated by histochemical staining and protein and gene expression. CONCLUSIONS: Overall, our data demonstrate that impedance profiling offers a basis for standardized real-time, non-invasive high-throughput screening of MSC properties. It enables further testing of the influence of diffusible factors or extracellular matrix composites on MSC differentiation or maintenance of stemness, thus substantiating therapeutic application.
Assuntos
Adipogenia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Engenharia Tecidual/normas , Adipogenia/fisiologia , Tecido Adiposo/metabolismo , Antígenos de Diferenciação/metabolismo , Medula Óssea/metabolismo , Adesão Celular , Processos de Crescimento Celular , Células Cultivadas , Impedância Elétrica , Eletrofisiologia/métodos , Estudos de Viabilidade , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Osteogênese/fisiologia , Controle de Qualidade , Engenharia Tecidual/métodos , Ativação TranscricionalRESUMO
Left ventricular hypertrophy (LVH) correlates with chronic renal failure and is one of the most important causes of cardiac mortality. The understanding of the molecular complexity of the disease will help to find biomarkers that open new perspectives about early diagnosis and therapy. This work describes the identification of mediators during pathogenesis relevant for structural remodeling processes of cardiac tissue in uremic LVH. An established rat model of chronic renal failure allowed whole-genome transcriptome analyses as well as the investigation of differential expressed proteins in uremic LVH. The localization of potential biomarkers encoded by candidate genes was done by immunohistochemical analyses of cardiac tissue of the animal model as well as cardiac sections of LVH diseased patients. In addition, the induction of human cardiac fibroblasts (HCF) and human umbilical vein endothelial cells (HUVEC) with the LVH mediator angiotensin II enabled us to investigate uremic LVH progression in vitro. These results point to alterations of myocardial intercellular and cell-matrix contacts in hypertrophic cardiac tissue. Obviously, structural changes of the extracellular matrix are significantly modulated by beta-catenin associated signaling pathways. Interestingly, intracellular translocation of beta-catenin, alpha-actinin and chondroitin sulfate proteoglycan 6 (CSPG6/SMC3) was observed in the animal model and in LVH patients. Our results show that the parallel investigation of rat and human cardiac tissue as well as human cellular models in vitro represents a promising strategy to identify reliable biomarkers of LVH.
Assuntos
Actinina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Miocárdio/metabolismo , beta Catenina/metabolismo , Actinina/genética , Angiotensina II/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas Cromossômicas não Histona/genética , Modelos Animais de Doenças , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Humanos , Hipertrofia Ventricular Esquerda/patologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 7 da Matriz/metabolismo , Miocárdio/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Osteonectina/metabolismo , Ratos , Ratos Sprague-Dawley , Remodelação Ventricular/fisiologia , Versicanas/metabolismo , beta Catenina/genéticaRESUMO
Engrailed transcription factors regulate survival, cell fate decisions and axon pathfinding in central neurons. En-2 can also attenuate Purkinje cell (PC) maturation. Here, we use array analysis to scrutinize gene expression in developing PCs overexpressing Engrailed-2 (L7En-2). The majority (70%) of regulated genes was found down-regulated in L7En-2 cerebella, consistent with the known repressive function of Engrailed-2. Differential gene expression, verified by in situ hybridization or Western blotting, was particularly evident during the first postnatal week, when L7En-2 PCs display conspicuous deficits in dendritogenesis. Functional classification revealed clusters of genes linked to vesicle formation and transport. Consistently, Golgi stacks located at the axonal pole of wild type PC somata were rarely detected in L7En-2 PCs. In addition, long continuous stretches of endoplasmic reticulum typically found around the axonal pole of wild type PCs were less frequently observed in transgenic cells. Engrailed-2 might therefore orchestrate PC survival and process formation as a regulator of subcellular organization.
Assuntos
Cerebelo/fisiologia , Proteínas de Homeodomínio/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Células de Purkinje/fisiologia , Vesículas Sinápticas/genética , Vesículas Sinápticas/metabolismo , Animais , Animais Recém-Nascidos , Sobrevivência Celular/genética , Cerebelo/citologia , Regulação para Baixo/genética , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Transporte Proteico/genética , Células de Purkinje/citologiaRESUMO
The relative risk for the development of malignancies following solid organ transplantation seems to be decreased in patients treated with the immunosuppressive agent mycophenolic acid (MPA). However, the molecular mechanisms of the antineoplastic effects of MPA are not completely understood. Here, we report that human endothelial cells and fibroblasts are highly sensitive to MPA treatment. We found that U87 glioblastoma cells were resistant to MPA treatment in vitro. However, U87 tumor growth was markedly inhibited in vivo in BALB/c nude mice, suggesting that MPA exerted its antitumor effects via modulation of the tumor microenvironment. Accordingly, microvascular density and pericyte coverage were markedly reduced in MPA-treated tumors in vivo. Using functional in vitro assays, we showed that MPA potently inhibited endothelial cell and fibroblast proliferation, invasion/migration, and endothelial cell tube formation. To identify the genetic participants governing the antiangiogenic and antifibrotic effects of MPA, we performed genome-wide transcriptional analysis in U87, endothelial and fibroblast cells at 6 and 12 h after MPA treatment. Network analysis revealed a critical role for MYC signaling in endothelial cells treated with MPA. Moreover, we found that the antiangiogenic effects of MPA were organized by coordinated communications between MYC and NDRG1, YYI, HIF1A, HDAC2, CDC2, GSK3B, and PRKACB signaling. The regulation of these "hub nodes" was confirmed by real-time quantitative reverse transcription-PCR and protein analysis. The critical involvement of MYC in the antiangiogenic signaling of MPA was further shown by gene knockdown experiments. Together, these data provide a molecular basis for the antiangiogenic and antifibrotic effects of MPA, which warrants further clinical investigations.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Ácido Micofenólico/farmacologia , Animais , Linhagem Celular , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Neoplasias/irrigação sanguínea , Neoplasias/genética , Neovascularização Patológica/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The homeodomain transcription factors PHOX2A and PHOX2B are vital for development of the autonomic nervous system. Their spatial and temporal expression at the neural crest is instrumental in determining neuronal precursor fate, and by regulating DbetaH expression, the enzyme catalysing noradrenaline synthesis from dopamine, they also play a role in determination of noradrenergic phenotype. Disturbing this finely regulated process leads to disruption of autonomic development and autonomic dysfunction syndromes such as DbetaH deficiency. As it had previously been shown that the catecholamine system is responsive to ELF-EMF, and as this has also been linked to various pathologies and to certain types of cancer, we wondered whether exposure to this type of radiation could affect the expression of PHOX2A, PHOX2B and DbetaH, also during differentiation triggered by retinoic acid. To investigate this possibility we exposed the human SH-SY5Y neuroblastoma cell line to 50 Hz power-line magnetic field at various flux densities and for various exposure times. We measured gene expression in exposed cells compared to control cells and also investigated any changes at protein level. Using our exposure protocol, we found no changes at either transcript or protein level of these important components of the autonomic nervous system and catecholaminergic system.
Assuntos
Campos Eletromagnéticos/efeitos adversos , Expressão Gênica/efeitos da radiação , Proteínas de Homeodomínio/efeitos da radiação , Fatores de Transcrição/efeitos da radiação , Linhagem Celular Tumoral , Dopamina beta-Hidroxilase/efeitos da radiação , Humanos , Neuroblastoma/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos da radiação , Tretinoína/farmacologiaRESUMO
Possible biological effects of mobile phone microwaves were investigated in vitro. In this study, which was part of the 5FP EU project REFLEX (Risk Evaluation of Potential Environmental Hazards From Low-Energy Electromagnetic Field Exposure Using Sensitive in vitro Methods), six human cell types, immortalized cell lines and primary cells, were exposed to 900 and 1800 MHz. RNA was isolated from exposed and sham-exposed cells and labeled for transcriptome analysis on whole-genome cDNA arrays. The results were evaluated statistically using bioinformatics techniques and examined for biological relevance with the help of different databases. NB69 neuroblastoma cells, T lymphocytes, and CHME5 microglial cells did not show significant changes in gene expression. In EA.hy926 endothelial cells, U937 lymphoblastoma cells, and HL-60 leukemia cells we found between 12 and 34 up- or down-regulated genes. Analysis of the affected gene families does not point towards a stress response. However, following microwave exposure, some but not all human cells might react with an increase in expression of genes encoding ribosomal proteins and therefore up-regulating the cellular metabolism.
Assuntos
Telefone Celular , Regulação para Baixo/efeitos da radiação , Micro-Ondas , Regulação para Cima/efeitos da radiação , Linhagem Celular , HumanosRESUMO
This study focused on the cell activating capacity of extremely low frequency magnetic fields (ELF-MF) on human umbilical cord blood-derived monocytes. Our results confirm the previous findings of cell activating capacity of ELF-MF (1.0 mT) in human monocytes, which was detected as an increased ROS release. Furthermore, gene expression profiling (whole-genome cDNA array Human Unigene RZPD-2) was performed to achieve a comprehensive view of involved genes during the cell activation process after 45 min ELF-MF exposure. Our results indicate the alteration of 986 genes involved in metabolism, cellular physiological processes, signal transduction and immune response. Significant regulations could be analyzed for 5 genes (expression >2- or <0.5-fold): IL15RA (Interleukin 15 receptor, alpha chain), EPS15R (Epidermal growth factor receptor pathway substrate 15 - like 1), DNMT3A (Hypothetical protein MGC16121), DNMT3A (DNA (cytosine-5) methyltransferase 3 alpha), and one gene with no match to known genes, DKFZP586J1624. Real-time RT-PCR analysis of the kinetic of the expression of IL15RA, and IL10RA during 45 min ELF-MF exposure indicates the regulation of cell activation via the alternative pathway, whereas the delayed gene expression of FOS, IL2RA and the melatonin synthesizing enzyme HIOMT suggests the suppression of inflammatory processes. Accordingly, we suggest that ELF-MF activates human monocytes via the alternative pathway.
Assuntos
Campos Eletromagnéticos , Perfilação da Expressão Gênica , Monócitos/metabolismo , Células Cultivadas , DNA Metiltransferase 3A , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Humanos , CinéticaRESUMO
BACKGROUND: Sarcoidosis is a systemic autoimmune disease where an inflammatory reaction involving alveolar macrophages, T-helper lymphocytes, and epitheloid cells is mounted against unknown antigens. A genetic predisposition for sarcoidosis is supposed by studies in twins, by geographical and racial distribution. In the current investigation we compared the expression patterns between slow onset and acute sarcodosis using a whole-genome cDNA array. METHODS: Bronchoalveolar lavage was performed in six patients with slow onset sarcoidosis and four patients with acute sarcoidosis (Löfgren's disease) and obtained cells were used for gene expression profiling. The results were confirmed by RT- and Taqman-PCR. In addition, protein expression was examined on paraffin sections of sarcoid granulomas by immunohistochemistry. RESULTS: In T-helper lymphocytes and alveolar macrophages we found an upregulation of genes belonging to the phosphoinositol-3-kinase/v-akt murine thymoma viral oncogene homolog/signal transducer and activator of transcription 3 pathway, as well as a downregulation of genes of the extrinsic and intrinsic apoptotic signaling cascades. In addition an upregulation of the genes encoding fatty acid binding protein 4 and 5, as well as peroxisome proliferative activated receptor delta in Löfgren's disease was detected. Differences in gene expression between slow onset sarcoidosis and Löfgren's syndrome were found mainly within genes of the major histocompatibility complex. CONCLUSIONS: In sarcoidosis enhanced cell proliferation and decreased apoptosis result in accumulation and prolonged survival of antigen-primed T-helper lymphocytes and activated macrophages. This is enhanced in Löfgren's disease, probably by hyper-stimulation via the peroxisome proliferation signaling, providing a larger pool of antigen-primed immune cells.
Assuntos
Apoptose/genética , Líquido da Lavagem Broncoalveolar , Perfilação da Expressão Gênica , Sarcoidose Pulmonar/genética , Doença Aguda , Adulto , Lavagem Broncoalveolar , Proliferação de Células , Doença Crônica , Feminino , Humanos , Ativação de Macrófagos/genética , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Fator de Transcrição STAT3/genética , Sarcoidose Pulmonar/patologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Colorectal cancer arises after a series of mutational events in the colon epithelia and is often used as a model of the multistep progression of tumorigenesis. Mutations in Ki-Ras have been detected in some 50% of cases and are thought to occur at an early stage. Almost never do mutations arise in the loci of other Ras isoforms (Ha- and N-), leading to the assumption that Ki-Ras plays a unique role in tumorigenesis. In order to examine the distinctive function that Ki-Ras plays in cancer development in the colon, we introduced constitutively active mutant Ki- and Ha-Ras genes into an intermediate-stage colon adenoma cell line (Caco-2). We found that mutant active Ha-RasV12 was more efficient at transforming these colon epithelial cells as assessed by anchorage-independent growth, tumor formation in SCID mice and the development of mesenchymal morphology compared to transformation by Ki-RasV12. We conducted microarray analysis in an attempt to reveal the genes whose aberrant expression is a direct result of overexpression of either Ki-RasV12 or Ha-RasV12. We used Clontech's Atlas cancer cDNA (588 genes) and RZPD's Onco Set 1 (1,544 genes) arrays. We identified fewer genes that were commonly regulated than were differentially expressed between Ki- and Ha-RasV12 isoforms. Specifically, we found that Ki-RasV12 regulated genes involved in cytokine signaling, cell adhesion and colon development, whereas Ha-RasV12 mainly regulated genes involved in controlling cell morphology, correlating to an epithelial-mesenchymal transition only observed in these cells. Our results demonstrate how 2 Ras isoforms regulate disparate biologic processes, revealing a number of genes whose deregulated expression may influence colon carcinogenesis (supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html).
Assuntos
Adenocarcinoma/genética , Transformação Celular Neoplásica/genética , Neoplasias Colorretais/genética , Genes ras/genética , Mutação/genética , Adenocarcinoma/metabolismo , Animais , Neoplasias Colorretais/metabolismo , DNA de Neoplasias/análise , Perfilação da Expressão Gênica , Humanos , Mesoderma , Camundongos , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Isoformas de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Whole-genome analyses become more and more necessary for pharmaceutical research. DNA chip hybridizations are an important tool for monitoring gene expression profiles during diseases or medical treatment. However, drug target identification and validation as well as an increasing number of antibodies and other polypeptides tested as potential drugs produce an increasing demand for genome-wide functional assays. Protein arrays are an important step into this direction. Peptide arrays and protein expression libraries are useful for the identification of antibodies and for epitope mapping. Antibody arrays allow protein quantification, protein binding studies, and protein phosphorylation assays. Tissue micro-arrays give a detailed information about the localization of macromolecules. More complex interactions can be addressed in cells spotted in array format. Finally, microfluidics chips enable us to describe the communication between cells in a tissue. In this review, possibilities, limitations and chances of different protein array techniques are discussed.
Assuntos
Genoma , Análise Serial de Proteínas/métodos , Anticorpos/análise , Indústria Farmacêutica , Imuno-Histoquímica , Microeletrodos , Técnicas Analíticas Microfluídicas/métodos , Sondas Moleculares/análise , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Peptídeos/análise , Ligação Proteica , Especificidade da EspécieRESUMO
Chronic myelogenous leukemia (CML) is a malignant disorder of the hematopoietic stem cell characterized by the BCR-ABL oncogene. We examined gene expression profiles of highly enriched CD34(+) hematopoietic stem and progenitor cells from patients with CML in chronic phase using cDNA arrays covering 1.185 genes. Comparing CML CD34(+) cells with normal CD34(+) cells, we found 158 genes which were significantly differentially expressed. Gene expression patterns reflected BCR-ABL-induced functional alterations such as increased cell-cycle and proteasome activity. Detoxification enzymes and DNA repair proteins were downregulated in CML CD34(+) cells, which might contribute to genetic instability. Decreased expression of junction plakoglobulin and CXC chemokine receptor 4 (CXCR-4) might facilitate the release of immature precursors from bone marrow in CML. GATA-2 was upregulated in CML CD34(+) cells, suggesting an increased self-renewal in comparison with normal CD34(+) cells. Moreover, we found upregulation of the proto-oncogene SKI and of receptors for neuromediators such as opioid mu1 receptor, GABA B receptor, adenosine A1 receptor, orexin 1 and 2 receptors and corticotropine-releasing hormone receptor. Treatment of CML progenitor cells with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) resulted in a dose-dependent significant inhibition of clonogenic growth by 40% at a concentration of 10(-5) M, which could be reversed by the equimolar addition of the receptor agonist 2-chloro-N6-cyclopentyladenosine (P<0.05). The incubation of normal progenitor cells with DPCPX resulted in an inhibition of clonogenic growth to a significantly lesser extent in comparison with CML cells (P<0.05), suggesting that the adenosine A1 receptor is of functional relevance in CML hematopoietic progenitor cells.
Assuntos
Antígenos CD34/análise , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células-Tronco Hematopoéticas/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Adulto , Idoso , Regulação para Baixo , Citometria de Fluxo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Proto-Oncogene Mas , Receptores Acoplados a Proteínas G/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
Recently, overlapping molecular phenotypes of hematopoietic and neuropoietic cells were described in mice. Here, we examined primary human CD34(+) hematopoietic stem and progenitor cells applying specialized cDNA arrays, real-time reverse-transcriptase-polymerase chain reaction (RT-PCR), and fluorescent-activated cell sorter (FACS) analysis focusing on genes involved in neurobiologic functions. We found expression of vesicle fusion and motility genes, ligand- and voltage-gated ion channels, receptor kinases and phosphatases, and, most interestingly, mRNA as well as protein expression of G protein-coupled receptors of neuromediators (corticotropin-releasing hormone 1 [CRH 1] and CRH 2 receptors, orexin/hypocretin 1 and 2 receptors, GABAB receptor, adenosine A(2)B receptor, opioid kappa 1 and mu 1 receptors, and 5-HT 1F receptor). As shown by 2-color immunofluorescence, the protein expression of these receptors was higher in the more immature CD38(dim) than in the CD38(bright) subset within the CD34(+) population, and completely absent in fully differentiated blood cells, suggesting that those receptors play a role in developmentally early CD34(+) stem and progenitor cells. The intracellular concentration of cyclic adenosine monophosphate (cAMP) in CD34(+) cells was diminished significantly upon stimulation of either CRH or orexin receptors, indicating that those are functionally active and coupled to inhibitory G proteins in human hematopoietic cells. In conclusion, these findings suggest a molecular interrelation of neuronal and hematopoietic signaling mechanisms in humans.
Assuntos
Antígenos CD34/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Receptores de Neurotransmissores/biossíntese , Adesão Celular , Movimento Celular , Proteínas do Citoesqueleto/biossíntese , Citoesqueleto/genética , Citoesqueleto/metabolismo , Distroglicanas , Espaço Extracelular/metabolismo , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Espaço Intracelular/metabolismo , Canais Iônicos/biossíntese , Canais Iônicos/genética , Glicoproteínas de Membrana/biossíntese , Proteínas de Membrana/biossíntese , Fatores de Crescimento Neural/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas R-SNARE , RNA Mensageiro/biossíntese , Receptores de Neurotransmissores/genéticaRESUMO
It is here demonstrated that the set of gene expressions underlying the angiogenic balance in tissues can be molecularly reset en masse by a single protein. Using genome-wide expression profiling, coupled with RT-PCR and phosphorylation analysis, we show that the endogenous angiogenesis inhibitor endostatin downregulates many signaling pathways in human microvascular endothelium associated with proangiogenic activity. Simultaneously, endostatin is found to upregulate many antiangiogenic genes. The result is a unique alignment between the direction of gene regulation and angiogenic status. Profiling further reveals the regulation of genes not heretofore associated with angiogenesis. Our analysis of coregulated genes shows complex interpathway communications in an intricate signaling network that both recapitulates and extends on current understanding of the angiogenic process. More generally, insights into the nature of genetic networking from the cell biologic and therapeutic perspectives are revealed.
Assuntos
Endostatinas/fisiologia , Células Endoteliais/metabolismo , Neovascularização Fisiológica/genética , Proteínas Repressoras , Transdução de Sinais/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Endostatinas/genética , Efrinas/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Proteína 1 Inibidora de Diferenciação , NF-kappa B/genética , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets , RNA Mensageiro/metabolismo , Fator de Transcrição STAT1 , Transativadores/genética , Fator de Transcrição AP-1/genética , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/genética , Regulação para Cima/genéticaRESUMO
Cardiac remodelling with interstitial fibrosis in renal failure, which so far is only poorly understood on the molecular level, was investigated in the rat model by a global gene expression profiling analysis. Sprague-Dawley rats were subjected to subtotal nephrectomy (SNX) or sham operation (sham) and followed for 2 and 12 weeks, respectively. Heart-specific gene expression profiling, with RZPD Rat Unigene-1 cDNA arrays containing about 27 000 gene and EST sequences revealed substantial changes in gene expression in SNX compared to sham animals. Motor protein genes, growth and differentiation markers, and extracellular matrix genes were upregulated in SNX rats. Obviously, not only genes involved in cardiomyocyte hypertrophy, but also genes involved in the expansion of non-vascular interstitial tissue are activated very early in animals with renal failure. Together with earlier findings in the SNX model, the present data suggest the hypothesis that the local renin-angiotensin system (RAS) may be activated by at least two pathways: (a) via second messengers and Gproteins (short-term signalling); and (b) via motor proteins, actins and integrins (longterm signalling). The study documents that complex hybridization analysis yields reproducible and promising results of patterns of gene activation pointing to signalling pathways involved in cardiac remodelling in renal failure. The complete array data are available via http://www.rzpd.de/cgi-bin/services/exp/viewExpressionData.pl.cgi.
RESUMO
BACKGROUND: Sarcoidosis is a systemic granulomatous multiorgan disease of unknown cause. Some facts point to a genetically determined predisposition, like associations with certain Major Histocompatibility Antigens Class I- and II-types (MHC). Mycobacterial DNA has been found in about 30% of sarcoidosis patients within the granulomas, which has been interpreted as a probable trigger mechanism. METHODS: Cells from bronchoalveolar lavage (BAL) of sarcoidosis patients were stimulated with dead Mycobacterium avium and gene expression was studied after six hours. Gene expression was profiled by hybridization to PCR filters (Human UniGene Set RZPD-1). To avoid the detection of effects related to secondary and tertiary inflammatory messages, we compared gene expression in sarcoidosis with that of tuberculosis and extrinsic allergic alveolitis. After normalization genes up- or downregulated for a factor of at least 1.8 were further analyzed. Genes differentially expressed in sarcoidosis as well as in tuberculosis or EAA, respectively, were subtracted. RESULTS AND CONCLUSIONS: Four different genes were exclusively upregulated in sarcoidosis: fatty acid binding protein 4 (FABP4), B-MYB, and two EST's. FABP4 has already been described in some autoimmune diseases, but its function in sarcoidosis is unknown. B-MYB is a potent growth factor for haematopoietic cells including lymphocytes. It might be one of the central genes upregulated in sarcoidosis and by that inducing clonal expansion of sarcoidosis-antigen primed T-helper-lymphocytes. Clonal selection of T-helper cells might be facilitated by c-Akt together with Tcl-1, both also upregulated in sarcoidosis. For the two ESTs neither function nor location is known.