RESUMO
Alternative splicing enhances proteome diversity and modulates cancer-associated proteins. To identify tissue- and tumor-specific alternative splicing, we used the GeneChip Human Exon 1.0 ST Array to measure whole-genome exon expression in 102 normal and cancer tissue samples of different stages from colon, urinary bladder, and prostate. We identified 2069 candidate alternative splicing events between normal tissue samples from colon, bladder, and prostate and selected 15 splicing events for RT-PCR validation, 10 of which were successfully validated by RT-PCR and sequencing. Furthermore 23, 19, and 18 candidate tumor-specific splicing alterations in colon, bladder, and prostate, respectively, were selected for RT-PCR validation on an independent set of 81 normal and tumor tissue samples. In total, seven genes with tumor-specific splice variants were identified (ACTN1, CALD1, COL6A3, LRRFIP2, PIK4CB, TPM1, and VCL). The validated tumor-specific splicing alterations were highly consistent, enabling clear separation of normal and cancer samples and in some cases even of different tumor stages. A subset of the tumor-specific splicing alterations (ACTN1, CALD1, and VCL) was found in all three organs and may represent general cancer-related splicing events. In silico protein predictions suggest that the identified cancer-specific splice variants encode proteins with potentially altered functions, indicating that they may be involved in pathogenesis and hence represent novel therapeutic targets. In conclusion, we identified and validated alternative splicing between normal tissue samples from colon, bladder, and prostate in addition to cancer-specific splicing events in colon, bladder, and prostate cancer that may have diagnostic and prognostic implications.
Assuntos
Adenoma/genética , Processamento Alternativo/genética , Neoplasias do Colo/genética , Éxons , Neoplasias da Próstata/genética , Neoplasias da Bexiga Urinária/genética , Actinina/genética , Actinina/metabolismo , Adenoma/metabolismo , Adenoma/patologia , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Modelos Moleculares , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Vinculina/química , Vinculina/genética , Vinculina/metabolismoRESUMO
A cell's ability to remodel adsorbed protein layers on surfaces is influenced by the nature of the protein layer itself. Remodelling is often required to accomplish cellular adhesion and extracellular matrix formation which forms the basis for cell spreading, increased adhesion and expression of different phenotypes. The adhesion of NIH3T3 (EGFP) fibroblasts to serum protein (albumin or fibronectin) precoated tantalum (Ta) and oxidised polystyrene (PS(ox)) surfaces was examined using the quartz crystal microbalance with dissipation (QCM-D) monitoring and fluorescence microscopy. The cells were either untreated or treated with cycloheximide to examine the contribution of endogenous protein production during cell adhesion to the QCM-D response over a period of 2h. Following adsorption of albumin onto Ta and PS(ox) there was no difference detected between the response to seeding untreated and cycloheximide treated cells. The QCM-D was able to detect differences in the untreated cellular responses to fibronectin versus serum precoated Ta and PS(ox) substrates, while cycloheximide treatment of the cells produced the same QCM-D response for fibronectin and serum precoatings on each of the materials. This confirmed that the process of matrix remodelling by the cells is dependent on the underlying substrate and the preadsorbed proteins and that the QCM-D response is dominated by changes in the underlying protein layer. Changes in dissipation correspond to the development of the actin cytoskeleton as visualised by actin staining.
Assuntos
Materiais Revestidos Biocompatíveis/química , Cicloeximida/farmacologia , Matriz Extracelular/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Quartzo , Adsorção , Animais , Bovinos , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibronectinas/química , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Oxirredução , Poliestirenos/química , Soro/química , Soroalbumina Bovina/química , Ovinos , Especificidade por Substrato , Tantálio/química , Fatores de TempoRESUMO
The quartz crystal microbalance with dissipation (QCM-D) (Q-Sense AB, Sweden) has been established as a useful tool for evaluating interactions between various biological and non-biological systems, and there has been increasing interest in using the QCM-D technique for cell monitoring applications. This study investigated the potential of the QCM-D to characterise the initial adhesion and spreading of cells in contact with protein precoated biocompatible surfaces. The QCM-D technique is attractive for monitoring cell adhesion and spreading as it allows in situ real-time measurements. The adhesion of NIH3T3 (EGFP) fibroblasts to tantalum (Ta) and oxidised polystyrene (PS(ox)) surfaces precoated with serum proteins was examined using the QCM-D for a period of either 2 or 4 h. Time-lapse photography was performed at 30 min intervals to visually examine cell adhesion and spreading in order to relate cell morphology to the QCM-D response. Following adsorption of albumin, fibronectin or newborn calf serum onto the surfaces, QCM-D measurements showed that cells adhered and spread on the fibronectin and serum coated surfaces, while few cells adhered to the albumin coated surfaces. Cells adhered to albumin coated surfaces had a rounded morphology. The responses to fibronectin and serum precoated surfaces were quite different for each of the underlying substrates indicating that the process of cell adhesion and spreading elicits different responses depending on both the protein coating composition and the influence of the underlying substrate. The different response may be due to extracellular matrix remodelling as well as cytoskeletal changes. Frequency (f) and dissipation (D) changes associated with cell adhesion were less than would be expected from the Sauerbrey relation due to the viscoelastic properties of the cells.
Assuntos
Adesão Celular , Materiais Revestidos Biocompatíveis/metabolismo , Teste de Materiais/métodos , Poliestirenos/metabolismo , Quartzo , Tantálio/metabolismo , Absorção , Albuminas/química , Animais , Bovinos , Materiais Revestidos Biocompatíveis/química , Fibronectinas/química , Camundongos , Células NIH 3T3 , Oxirredução , Poliestirenos/química , Soro/química , Propriedades de SuperfícieRESUMO
The quartz crystal microbalance with dissipation (QCM-D) technique was employed to characterize initial cell adhesion in terms of attachment and spreading of pre-osteoblastic MC3T3-E1 cells on Ta and Cr surfaces. Evaluation of initial cell adhesion established a correlation between input cell number and the shifts in frequency (f) and dissipation (D). The f-shift was found to be much larger in serum-free medium as compared to a medium including serum; hence, initial cell adhesion was subsequently evaluated in serum-free medium. During the first hour of adhesion, we found a positive correlation between the QCM-D f-shift and the average area of the spread cells, as measured by cryo-scanning electron microscopy (cryo-SEM). Finally, the QCM-D technique was used to study cell adhesion on different metal oxide surfaces. Initial cell adhesion on Ta was found to induce a larger f-shift as compared to Cr, indicating larger spreading of cells on Ta. Cryo-SEM data confirmed that spreading of cells on Cr was on average only two-thirds the spreading on Ta. Our results demonstrate that the QCM-D technique is a versatile technique to quickly distinguish initial cell-surface interactions on different biomaterials.
Assuntos
Movimento Celular/fisiologia , Cromo , Osteoblastos/citologia , Osteoblastos/fisiologia , Quartzo , Células-Tronco/citologia , Células-Tronco/fisiologia , Tantálio , Animais , Materiais Biocompatíveis , Técnicas Biossensoriais , Adesão Celular/fisiologia , Linhagem Celular , Microscopia Crioeletrônica , Cristalização , Meios de Cultivo Condicionados , Meios de Cultura Livres de Soro , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica de VarreduraRESUMO
The rare inherited human genetic disorder Cockayne syndrome (CS) is characterized by developmental abnormalities, UV sensitivity and premature aging. The cellular and molecular phenotypes of CS include increased sensitivity to UV-induced and oxidative DNA lesions. Two genes are involved: CSA and CSB. The CS group B (CSB) protein has roles in transcription, transcription-coupled repair, and base excision repair. It is a DNA stimulated ATPase and remodels chromatin in vitro. Here, we have analyzed wild-type (wt) and motif II, V and VI mutant CSB proteins. We find that the mutant proteins display different degrees of ATPase activity deficiency, and in contrast to the in vivo complementation studies, the motif II mutant is more defective than motif V and VI CSB mutants. Furthermore, CSB wt ATPase activity was studied with different biologically important DNA cofactors: DNA with different secondary structures and damaged DNA. The results indicate that the state of DNA secondary structure affects the level of CSB ATPase activity. We find that the CSB protein is phosphorylated in untreated cells and that UV irradiation leads to its dephosphorylation. Importantly, dephosphorylation of the protein in vitro results in increased ATPase activity of the protein, suggesting that the activity of the CSB protein is subject to phosphorylation control in vivo. These observations may have significant implications for the function of CSB in vivo.
Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Mutação , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Sequência de Bases , Células Cultivadas , Sequência Conservada , DNA/química , Dano ao DNA , DNA Helicases/fisiologia , Enzimas Reparadoras do DNA , Humanos , Conformação de Ácido Nucleico , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , Processamento de Proteína Pós-TraducionalRESUMO
INTRODUCTION: Mesenchymal stem cells (MSCs) provide an excellent source of pluripotent progenitor cells for tissue-engineering applications due to their proliferation capacity and differentiation potential. Genetic modification of MSCs with genes encoding tissue-specific growth factors and cytokines can induce and maintain lineage-specific differentiation. Due to anatomical and physiological similarities to humans, porcine research models have been proven valuable for the preclinical testing of tissue engineering protocols in large animals. The aim of this study was to evaluate optimized viral and non-viral ex vivo gene delivery systems with respect to gene transfer efficiency, maintenance of transgene expression, and safety issues using primary porcine MSCs as target cells. MATERIALS AND METHODS: MSCs were purified from bone marrow aspirates from the proximal tibiae of four 3-month-old Danish landrace pigs by Ficoll step gradient separation and polystyrene adherence technique. Vectors expressing enhanced green fluorescent protein (eGFP) and human bone morphogenetic protein-2 (BMP-2) were transferred to the cells by different non-viral methods and by use of recombinant adeno-associated virus (rAAV)-mediated and retroviral gene delivery. Each method for gene delivery was optimized. Gene transfer efficiency was compared on the basis of eGFP expression as assessed by fluorescence microscopy and fluorescence-activated flow cytometry. BMP-2 gene expression and osteogenic differentiation were evaluated by realtime quantitative RT-PCR and histochemical detection of alkaline phosphatase activity, respectively. RESULTS: Non-viral gene delivery methods resulted in transient eGFP expression by less than 2% of the cells. Using high titer rAAV-based vector up to 90% of the cells were transiently transduced. The efficiency of rAAV-mediated gene delivery was proportional to the rAAV vector titer applied. Retroviral gene delivery resulted in long-term transgene expression of porcine MSCs. A 26-fold increase in percentage of eGFP expressing cells (1.7%+/-0.2% versus 44.1% +/-5.0%, mean +/-SD) and a 68-fold increase in mean fluorescence intensity (327.4+/-56.6 versus 4.8+/-1.3) was observed by centrifugation of retroviral particles onto the target cell layer. Porcine MSCs that were BMP-2 transduced by optimized retroviral gene delivery demonstrated a significant increase in BMP-2 gene expression and showed increased osteogenic differentiation. Retrovirally transduced porcine MSCs were furthermore tested free of replication-competent viruses. DISCUSSION: The non-viral gene transfer methods applied were significantly less efficient compared to the viral methods tested. However, due to advantages with respect to safety issues and ease of handling, improvement of non-viral gene delivery to primary MSCs deserves further attention. The high efficiency of rAAV-mediated gene delivery observed at high titers can be explained by the ability of rAAV vector to transduce nondividing cells and by its tropism towards porcine MSCs. rAAV-mediated gene delivery resulted in transient transgene expression due to lack of stable AAV genome integration. MLV-mediated retroviral gene delivery can be considered a safe method for long-term transgene expression by porcine MSCs, and is therefore particularly attractive for advanced tissue engineering strategies requiring extended transgene expression.
Assuntos
Técnicas de Transferência de Genes , Células-Tronco Mesenquimais/citologia , Animais , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Proteínas de Fluorescência Verde/metabolismo , Humanos , Vírus da Leucemia Murina/metabolismo , Camundongos , Células NIH 3T3 , Retroviridae/metabolismo , Suínos , Tíbia/patologia , Engenharia Tecidual , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , TransgenesRESUMO
PURPOSE: Epigenetic alterations are common and can now be addressed in a parallel fashion. We investigated the methylation in bladder cancer with respect to location in genome, consistency, variation in metachronous tumors, impact on transcripts, chromosomal location, and usefulness as urinary markers. EXPERIMENTAL DESIGN: A microarray assay was utilized to analyze methylation in 56 samples. Independent validation was conducted in 63 samples by a PCR-based method and bisulfite sequencing. The methylation levels in 174 urine specimens were quantified. Transcript levels were analyzed using expression microarrays and pathways were analyzed using dedicated software. RESULTS: Global methylation patterns were established within and outside CpG islands. We validated methylation of the eight tumor markers genes ZNF154 (P < 0.0001), HOXA9 (P < 0.0001), POU4F2 (P < 0.0001), EOMES (P = 0.0005), ACOT11 (P = 0.0001), PCDHGA12 (P = 0.0001), CA3 (P = 0.0002), and PTGDR (P = 0.0110), the candidate marker of disease progression TBX4 (P < 0.04), and other genes with stage-specific methylation. The methylation of metachronous tumors was stable and targeted to certain pathways. The correlation to expression was not stringent. Chromosome 21 showed most differential methylation (P < 0.0001) and specifically hypomethylation of keratins, which together with keratin-like proteins were epigenetically regulated. In DNA from voided urine, we detected differential methylation of ZNF154 (P < 0.0001), POU4F2 (P < 0.0001), HOXA9 (P < 0.0001), and EOMES (P < 0.0001), achieving 84% sensitivity and 96% specificity. CONCLUSIONS: We initiated a detailed mapping of the methylome in metachronous bladder cancer. Novel genes with tumor, chromosome, as well as pathway-specific differential methylation in bladder cancer were identified. The methylated genes were promising cancer markers for early detection of bladder cancer.
Assuntos
Biomarcadores Tumorais/urina , Metilação de DNA , Genes Neoplásicos , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Cromossomos Humanos Par 21/genética , Ilhas de CpG , Feminino , Humanos , Queratinas/metabolismo , Masculino , Metilação , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Bexiga Urinária/urinaRESUMO
INTRODUCTION: The establishment of Danish CancerBiobank to hold frozen blood and tissues from patients with primary cancer was made possible by a donation from the Ministry of Health and Prevention. MATERIAL AND METHODS: Danish departments of pathology already have a long experience with biobanks as they have handled paraffin-embedded tissues and have undertaken national registration of data in the nationwide pathological databank, Patobanken, for years. Furthermore, departments of clinical biochemistry are experienced in ensuring the quality of biobanks including that of blood samples. Knowledge from these two areas together with visits to biobanks in other countries was used to design and establish the Danish CancerBiobank. Five centres at the university hospitals in Aalborg, Herlev, Odense, Copenhagen and Arhus received the donation. RESULTS: A group of experts was established and made specifications for data registration and technical advice for the handling of blood and tissue and produced a draft handbook on quality assurance. CONCLUSION: The Danish CancerBiobank is unique nationally and internationally owing to 1) national recommendations for handling the materials included in the biobank, 2) centralized nationwide data registration, 3) correlation between information on blood and tissue for the single patient and 4) linking of the information to the final diagnosis through Patobanken. Furthermore, in the future it will be possible to connect to the nationwide clinical databases and other national registries. The final criteria of success for Danish CancerBiobank will be the publication of international research results of clinical importance for the treatment of patients diagnosed with cancer.
Assuntos
Bancos de Espécimes Biológicos , Neoplasias , Bancos de Espécimes Biológicos/organização & administração , Bancos de Espécimes Biológicos/normas , Pesquisa Biomédica , Biópsia , Coleta de Amostras Sanguíneas , Dinamarca , Humanos , Neoplasias/diagnóstico , Neoplasias/patologia , Neoplasias/terapia , Guias de Prática Clínica como Assunto , Controle de Qualidade , Sistema de Registros/normas , Manejo de Espécimes/normasRESUMO
Nano- and microstructured surfaces are known to impact on the binding and differentiation of cells, but the detailed basic understanding of the underlying regulatory mechanisms is still scarce, which impedes the rational design of smart biomaterials. Towards a comprehensive analysis of the interplay between topographical parameters such as feature design and lateral and vertical dimensions we here report on a combinatorial screening approach, BioSurface Structure Array (BSSA) of test squares each with a distinct topography. Using such BSSA libraries of 504 topographically distinct surface structures, we have identified combinations of size, gap and height of structures which enhance mineralization as well as the expression of osteogenic markers of a preosteoblastic murine cell line. This generic BSSA screening platform is a versatile technology for the systematic identification of surfaces with specific biological properties, and it may for example be useful for optimizing the design of biomaterials for regulating cellular behaviour.