RESUMEN
The classic tumor clonal evolution theory postulates that cancers change over time to produce unique molecular subclones within a parent neoplasm, presumably including regional differences in gene expression. More recently, however, this notion has been challenged by studies showing that tumors maintain a relatively stable transcript profile. To examine these competing hypotheses, we microdissected discrete subregions containing approximately 3000 to 8000 cells (500 to 1500 µm in diameter) from ex vivo esophageal squamous cell carcinoma (ESCC) specimens and analyzed transcriptomes throughout three-dimensional tumor space. Overall mRNA profiles were highly similar in all 59 intratumor comparisons, in distinct contrast to the markedly different global expression patterns observed in other dissected cell populations. For example, normal esophageal basal cells contained 1918 and 624 differentially expressed genes at a greater than twofold level (95% confidence level of <5% false positives), compared with normal differentiated esophageal cells and ESCC, respectively. In contrast, intratumor regions had only zero to four gene changes at a greater than twofold level, with most tumor comparisons showing none. The present data indicate that, when analyzed using a standard array-based method at this level of histological resolution, ESCC contains little regional mRNA heterogeneity.
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Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Carcinoma de Células Escamosas de Esófago , Genes Relacionados con las Neoplasias/genética , Humanos , Microdisección , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/genéticaRESUMEN
Measurement of gene-expression profiles using microarray technology is becoming increasingly popular among the biomedical research community. Although there has been great progress in this field, investigators are still confronted with a difficult question after completing their experiments: how to validate the large data sets that are generated? This review summarizes current approaches to verifying global expression results, discusses the caveats that must be considered, and describes some methods that are being developed to address outstanding problems.
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Perfilación de la Expresión Génica/normas , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Animales , ADN Complementario/genética , Predicción , Perfilación de la Expresión Génica/métodos , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas/genética , Control de Calidad , ARN Mensajero/genética , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Immunohistochemical (IHC) staining is an established technique for visualizing proteins in tissue sections for research studies and clinical applications. IHC is increasingly used as a targeting strategy for procurement of labeled cells via tissue microdissection, including immunodissection, computer-aided laser dissection (CALD), expression microdissection (xMD), and other techniques. The initial antigen retrieval (AR) process increases epitope availability and improves staining characteristics; however, the procedure can damage DNA. To better understand the effects of AR on DNA quality and quantity in immunodissected samples, both clinical specimens (KRAS gene mutation positive cases) and model system samples (lung cancer patient-derived xenograft tissue) were subjected to commonly employed AR methods (heat induced epitope retrieval [HIER], protease digestion) and the effects on DNA were assessed by Qubit, fragment analysis, quantitative PCR, digital droplet PCR (ddPCR), library preparation, and targeted sequencing. The data showed that HIER resulted in optimal IHC staining characteristics, but induced significant damage to DNA, producing extensive fragmentation and decreased overall yields. However, neither of the AR methods combined with IHC prevented ddPCR amplification of small amplicons and gene mutations were successfully identified from immunodissected clinical samples. The results indicate for the first time that DNA recovered from immunostained slides after standard AR and IHC processing can be successfully employed for genomic mutation analysis via ddPCR and next-generation sequencing (NGS) short-read methods.
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Neoplasias Pulmonares , Proteínas Proto-Oncogénicas p21(ras) , Antígenos , ADN/análisis , Epítopos , Genómica , Humanos , Neoplasias Pulmonares/genética , Mutación , Péptido Hidrolasas , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Improving the utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today in the rapidly evolving precision oncology field where tumor genotyping is often essential for the indication of many advanced and targeted therapies. National Comprehensive Cancer Network (NCCN) guidelines now mandate molecular testing for clinically actionable targets in certain malignancies. Utilizing advanced stage lung cancer as an example, an improved genotyping approach for solid tumors is possible. The strategy involves optimization of the microdissection process and analysis of a large number of identical target cells from formalin-fixed paraffin-embedded (FFPE) specimens sharing similar characteristics, in other words, single-cell subtype analysis. The shared characteristics can include immunostaining status, cell phenotype, and/or spatial location within a histological section. Synergy between microdissection and droplet digital PCR (ddPCR) enhances the molecular analysis. We demonstrate here a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time- and cost-efficient manner, using immunostain targeting as an example.
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Microdisección , Neoplasias , Formaldehído , Humanos , Microdisección/métodos , Adhesión en Parafina/métodos , Reacción en Cadena de la Polimerasa/métodos , Medicina de Precisión , Fijación del Tejido/métodosRESUMEN
Current technologies for measuring protein expression across a tissue section are based on MS or in situ detection such as immunohistochemistry. However, due to the inherent molecular complexity of tissue samples and the large dynamic range of protein expression in cells, current approaches are often unable to measure moderate- and low-abundant proteins. In addition, they do not provide information on the physico-chemical properties of the proteins studied. To address these problems, we are developing a new pre-analytic methodology termed layered electrophoretic transfer (LET) that selectively separates and processes proteins from an intact tissue section without compromising important two-dimensional histological information. LET offers two potential advantages over standard techniques: (i) A reduced complexity of the tissue proteome for subsequent analysis; (ii) An opportunity to assess the biochemical status of proteins as they exist in situ. As an initial proof-of-concept, we demonstrate here that the protein content from a mixture of molecular weight standards, human tissue lysates, and tissue sections can be successfully transferred and separated using LET, and further demonstrate that the method can be coupled with immunoblotting or MS for downstream measurements. LET technology represents a new pre-analytic tool for interrogating the proteome in tissue sections while preserving valuable spatial information.
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Proteínas/análisis , Proteoma/análisis , Proteómica/métodos , Electroforesis en Gel Bidimensional/métodos , Expresión Génica , Técnicas Histológicas , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Immunoblotting/métodos , Inmunohistoquímica/métodos , Espectrometría de Masas , Proteínas/genética , Proteínas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proyectos de InvestigaciónRESUMEN
Measurement of mRNA levels across tissue samples facilitates an understanding of how genes function and what their roles are in disease. Quantifying low-abundance mRNA requires a workflow that preserves spatial information, isolates RNA, and performs reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). This is complex because these steps are typically performed in three separate platforms. In the present study, we describe two-dimensional RT-qPCR (2D-RT-qPCR), a method that quantifies RNA across tissues sections in a single integrated platform. The method uses the grid format of a multi-well plate to macrodissect tissue sections and preserve the spatial location of the RNA; this also eliminates the need for physical homogenization of the tissue. A new lysis and nucleic acid purification protocol is performed in the same multi-well plate, followed by RT-qPCR. The feasibility 2D-RT-qPCR was demonstrated on a variety of tissue types. Potential applications of the technology as a high-throughput tissue analysis platform are discussed.
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Ensayos Analíticos de Alto Rendimiento/métodos , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Perfilación de la Expresión Génica/métodos , Ensayos Analíticos de Alto Rendimiento/instrumentación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/instrumentación , Distribución TisularRESUMEN
BACKGROUND: Esophageal squamous cell carcinomas (ESCC) are usually asymptomatic and go undetected until they are incurable. Cytological screening is one strategy to detect ESCC at an early stage and has shown promise in previous studies, although improvement in sensitivity and specificity are needed. Proteases modulate cancer progression by facilitating tumor invasion and metastasis. In the current study, matrix metalloproteinases (MMPs) were studied in a search for new early detection markers for ESCC. METHODS: Protein expression levels of MMPs were measured using zymography in 24 cases of paired normal esophagus and ESCC, and in the tumor-associated stroma and tumor epithelium in one sample after laser capture microdissection (LCM). MMP-3 and MMP-10 transcripts in both the epithelium and stroma in five cases were further analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). RESULTS: Gelatin zymography showed bands corresponding in size to MMP-2, MMP-3, MMP-9, and MMP-10 enzymes in each of the 24 cancer cases. MMP levels tended to be higher in tumors than paired normal tissue; however, only the 45 kDa band that corresponds to the activated form of MMP-3 and MMP-10 was strongly expressed in all 24 tumors with little or no expression in the paired normal foci. LCM-based analysis showed the 45 kDA band to be present in both the stromal and epithelial components of the tumor microenvironment, and that MMP-3 and MMP-10 mRNA levels were higher in tumors than paired normal tissues for each compartment. CONCLUSIONS: Increased levels of MMPs occur in ESCC suggesting their up-regulation is important in esophageal tumorigenesis. The up-regulated gene products have the potential to serve as early detection markers in the clinic.
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Carcinoma de Células Escamosas/enzimología , Neoplasias Esofágicas/enzimología , Metaloproteinasas de la Matriz/metabolismo , Adulto , Anciano , Carcinoma de Células Escamosas/patología , Activación Enzimática , Neoplasias Esofágicas/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la PolimerasaRESUMEN
PURPOSE: Over the past 60 years, cytotoxic chemotherapy has targeted the cancer cell. Despite this, there have been few cancer cures. A new approach to cancer therapy is to target the multicellular biological entity of the tumor microenvironment. EXPERIMENTAL DESIGN: Lenalidomide, an immunomodulatory drug, sunitinib, a tyrosine kinase inhibitor, and low-dose metronomic cyclophosphamide, were tested alone and in combination for their abilities to inhibit endothelial cell tube formation, rat aortic ring outgrowth, tumor growth, and metastatic development in mice. In addition, ectopic tumor lysates were evaluated for the presence of proangiogenic proteins. RESULTS: The three agents alone were shown to significantly inhibit endothelial cells' ability to form tubes and significantly inhibit the multicellular microenvironment in the rat aortic ring assay (P < 0.01 and P < 0.001). This effect was also significantly augmented when the agents were combined. Furthermore, the three-drug combination was able halt the progression of tumor growth almost completely in xenograft models of ocular melanoma, colon cancer, pancreatic cancer, and cutaneous melanoma. These agents significantly decrease the number of proliferating cells in tumors, significantly increase the number of cells undergoing active cell death in tumors, and significantly decrease the number of blood vessels in treated tumors (P < 0.05). Combination therapy shows a decrease in the compensatory up-regulation of proangiogenic proteins after treatment when compared with single-agent therapy. CONCLUSIONS: This combination of agents causes an inhospitable microenvironment for tumor cells and shows great promise for use in the clinic.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proliferación Celular/efectos de los fármacos , Neoplasias Experimentales/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Animales , Línea Celular Tumoral , Ciclofosfamida/administración & dosificación , Células Endoteliales/efectos de los fármacos , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Indoles/administración & dosificación , Lenalidomida , Ratones , Neoplasias Experimentales/irrigación sanguínea , Pirroles/administración & dosificación , Ratas , Sunitinib , Talidomida/administración & dosificación , Talidomida/análogos & derivados , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
INTRODUCTION: The development and application of new molecular diagnostic assays based on next-generation sequencing and proteomics require improved methodologies for procurement of target cells from histological sections. Laser microdissection can successfully isolate distinct cells from tissue specimens based on visual selection for many research and clinical applications. However, this can be a daunting task when a large number of cells are required for molecular analysis or when a sizeable number of specimens need to be evaluated. MATERIALS AND METHODS: To improve the efficiency of the cellular identification process, we describe a microdissection workflow that leverages recently developed and open source image analysis algorithms referred to as computer-aided laser dissection (CALD). CALD permits a computer algorithm to identify the cells of interest and drive the dissection process. RESULTS: We describe several "use cases" that demonstrate the integration of image analytic tools probabilistic pairwise Markov model, ImageJ, spatially invariant vector quantization (SIVQ), and eSeg onto the ThermoFisher Scientific ArcturusXT and Leica LMD7000 microdissection platforms. CONCLUSIONS: The CALD methodology demonstrates the integration of image analysis tools with the microdissection workflow and shows the potential impact to clinical and life science applications.
RESUMEN
Layered peptide array is a new methodology for multiplex molecular measurements from two-dimensional life science platforms. The technology can be used in several different configurations depending on the needs of the investigator. Described here is an indirect layered peptide array (iLPA) that is capable of measuring proteins on a solid surface, such as target antigens on a tissue section. A prototype iLPA system was developed and subsequently examined for reproducibility and specificity and then compared with standard immunohistochemistry. Semiquantitative, multiplex proteomic analysis of histological sections was achieved with up to 20 membranes. The experimental variability was 18%. Overall, the data suggest that iLPA technology will be a relatively simple and inexpensive method for molecular measurements from tissue sections.
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Inmunohistoquímica/métodos , Fragmentos de Péptidos/análisis , Análisis por Matrices de Proteínas/métodos , Neoplasias Encefálicas/metabolismo , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Neoplasias del Colon/metabolismo , Estudios de Factibilidad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/metabolismo , Linfoma/metabolismo , Masculino , Melanoma/metabolismo , Modelos Biológicos , Neurilemoma/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias de la Próstata/metabolismo , Análisis de Matrices Tisulares/métodosRESUMEN
The layered peptide array (LPA) is a recently developed technique designed to measure antibody levels in a multiplex, high-throughput manner. LPAs can assess antibody presence either in fluid samples or from tissues while maintaining the two-dimensional orientation of the life science platform. In this manuscript, we evaluated and assessed the performance of the LPA platform, focusing on throughput capability, sensitivity, and specificity of the assay in several different systems.
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Anticuerpos/análisis , Péptidos/análisis , Análisis por Matrices de Proteínas , Anticuerpos/sangre , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Péptidos/sangreRESUMEN
Characterization of gene expression profiles in tumor cells and the tumor microenvironment is an important step in understanding neoplastic progression. To date, there are limited data available on expression changes that occur in the tumor-associated stroma as either a cause or consequence of cancer. In the present study, we employed a 54,000 target oligonucleotide microarray to compare expression profiles in the 4 major components of the microenvironment: tumor epithelium, tumor-associated stroma, normal epithelium, and normal stroma. Cells from 5 human, whole-mount prostatectomy specimens were microdissected and the extracted and amplified mRNA was hybridized to an Affymetrix Human Genome U133 Plus 2.0 GeneChip. Using the intersection of 2 analysis methods, we identified sets of differentially expressed genes among the 4 components. Forty-four genes were found to be consistently differentially expressed in the tumor-associated stroma; 35 were found in the tumor epithelium. Interestingly, the tumor-associated stroma showed a predominant up-regulation of transcripts compared with normal stroma, in sharp contrast to the overall down-regulation seen in the tumor epithelium relative to normal epithelium. These data provide insight into the molecular changes occurring in tumor-associated stromal cells and suggest new potential targets for future diagnostic, imaging, or therapeutic intervention.
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Perfilación de la Expresión Génica , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Epitelio/metabolismo , Humanos , Masculino , Células del Estroma/metabolismoRESUMEN
With the advent of the genomic era, there is an increasing use of high-throughput techniques to generate transcriptome- and proteome-based profiles of biological specimens. Each of these methodologies offers a unique window into the inner workings of cell and tissue samples. Often, these studies generate large data sets and provide investigators with a substantial number of candidate dysregulated genes and pathways. Follow-up studies are then undertaken to independently validate the original findings and to extend the study to additional samples or more quantitative measurements. Although there are several methods available for these validation efforts, they are often tedious and laborious to perform; thus, additional tools that enable this task are needed. One such approach is layered expression scanning (LES), a new technique developed via a cooperative research and development agreement (CRADA) between the National Cancer Institute and 20/20 GeneSystems, Inc. The technique is based on the movement of biomolecules from a two-dimensional life science platform (histological tissue section, electrophoresis gel, multi-well plate, etc.) through a set of analysis membranes while maintaining the original distribution pattern of the molecules. Each membrane measures one analyte and the data are then mapped back to the original specimen, permitting each component of the life science platform to be studied in detail. LES can be configured in several different ways depending on the goals of the study. In this review, we summarize the use of the LES technique for a variety of biological applications.
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Membranas Artificiales , Ácidos Nucleicos/análisis , Péptidos/análisis , Mapeo de Interacción de Proteínas/métodos , Proteómica/métodos , Mapeo de Interacción de Proteínas/instrumentación , Proteómica/instrumentaciónRESUMEN
BACKGROUND: A functional blood supply is essential for tumor growth and proliferation. However, the mechanism of blood vessel recruitment to the tumor is still poorly understood. Ideally, a thorough molecular assessment of blood vessel cells would be critical in our comprehension of this process. Yet, to date, there is little known about the molecular makeup of the endothelial cells of tumor-associated blood vessels, due in part to the difficulty of isolating a pure population of endothelial cells from the heterogeneous tissue environment. METHODS: Here we describe the use of a recently developed technique, Expression Microdissection, to isolate endothelial cells from the tumor microenvironment. The methylation status of the dissected samples was evaluated for GSTP1 and RARbeta2 promoters via the QMS-PCR method. RESULTS: Comparing GSTP1 and RARbeta2 promoter methylation data, we show that 100% and 88% methylation is detected, respectively, in the tumor areas, both in epithelium and endothelium. Little to no methylation is observed in non-tumor tissue areas. CONCLUSION: We applied an accurate microdissection technique to isolate endothelial cells from tissues, enabling DNA analysis such as promoter methylation status. The observations suggest that epigenetic alterations may play a role in determining the phenotype of tumor-associated vasculature.
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PURPOSE: After an initial response to androgen ablation, most prostate tumors recur, ultimately progressing to highly aggressive androgen-independent cancer. The molecular mechanisms underlying progression are not well known in part due to the rarity of androgen-independent samples from primary and metastatic sites. EXPERIMENTAL DESIGN: We compared the gene expression profiles of 10 androgen-independent primary prostate tumor biopsies with 10 primary, untreated androgen-dependent tumors. Samples were laser capture microdissected, the RNA was amplified, and gene expression was assessed using Affymetrix Human Genome U133A GeneChip. Differential expression was examined with principal component analysis, hierarchical clustering, and Student's t testing. Analysis of gene ontology was done with Expression Analysis Systematic Explorer and gene expression data were integrated with genomic alterations with Differential Gene Locus Mapping. RESULTS: Unsupervised principal component analysis showed that the androgen-dependent and androgen-independent tumors segregated from one another. After filtering the data, 239 differentially expressed genes were identified. Two main gene ontologies were found discordant between androgen-independent and androgen-dependent tumors: macromolecule biosynthesis was down-regulated and cell adhesion was up-regulated in androgen-independent tumors. Other differentially expressed genes were related to interleukin-6 signaling as well as angiogenesis, cell adhesion, apoptosis, oxidative stress, and hormone response. The Differential Gene Locus Mapping analysis identified nine regions of potential chromosomal deletion in the androgen-independent tumors, including 1p36, 3p21, 6p21, 8p21, 11p15, 11q12, 12q23, 16q12, and 16q21. CONCLUSIONS: Taken together, these data identify several unique characteristics of androgen-independent prostate cancer that may hold potential for the development of targeted therapeutic intervention.
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Andrógenos/metabolismo , Antineoplásicos Hormonales/farmacología , Regulación Neoplásica de la Expresión Génica , Regulación de la Expresión Génica , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/terapia , Anciano , Antagonistas de Andrógenos/metabolismo , Biopsia , Adhesión Celular , Deleción Cromosómica , Mapeo Cromosómico , Cromosomas/ultraestructura , Análisis por Conglomerados , Progresión de la Enfermedad , Regulación hacia Abajo , Eliminación de Gen , Genoma , Humanos , Interleucina-6/metabolismo , Rayos Láser , Masculino , Persona de Mediana Edad , Modelos Estadísticos , Metástasis de la Neoplasia , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , Análisis de Componente Principal , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN/metabolismo , Transducción de Señal , Regulación hacia ArribaRESUMEN
Precision medicine promises to enhance patient treatment through the use of emerging molecular technologies, including genomics, transcriptomics, and proteomics. However, current tools in surgical pathology lack the capability to efficiently isolate specific cell populations in complex tissues/tumors, which can confound molecular results. Expression microdissection (xMD) is an immuno-based cell/subcellular isolation tool that procures targets of interest from a cytological or histological specimen. In this study, we demonstrate the accuracy and precision of xMD by rapidly isolating immunostained targets, including cytokeratin AE1/AE3, p53, and estrogen receptor (ER) positive cells and nuclei from tissue sections. Other targets procured included green fluorescent protein (GFP) expressing fibroblasts, in situ hybridization positive Epstein-Barr virus nuclei, and silver stained fungi. In order to assess the effect on molecular data, xMD was utilized to isolate specific targets from a mixed population of cells where the targets constituted only 5% of the sample. Target enrichment from this admixed cell population prior to next-generation sequencing (NGS) produced a minimum 13-fold increase in mutation allele frequency detection. These data suggest a role for xMD in a wide range of molecular pathology studies, as well as in the clinical workflow for samples where tumor cell enrichment is needed, or for those with a relative paucity of target cells.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Microdisección/métodos , Animales , Núcleo Celular/metabolismo , Epitelio/metabolismo , Humanos , Ratones , Células 3T3 NIH , Coloración y EtiquetadoRESUMEN
High-throughput methods to detect and quantify antibodies in sera and other patient specimens have use for many clinical and laboratory studies, including those associated with cancer detection, microbial exposures, and autoimmune diseases. We developed a new technique, termed layered peptide array (LPA), to serve as a screening tool to detect antibodies in a highly multiplexed format. We demonstrate here that a prototype LPA was capable of producing approximately 5000 measurements per experiment and appeared to be scalable to higher throughput levels. Sera and saliva from Sjögren's syndrome patients served as a test set to examine antibody titers in clinical samples. The LPA platform exhibited both a high sensitivity (100%) and high specificity (94%) for correctly identifying SSB antigen-positive samples. The multiplex capability of the platform was also confirmed when serum and saliva samples were analyzed for antibody reactivity to several peptides, including Sjögren's syndrome antigens A and B. The data indicate that LPA analysis will be a useful method for a number of screening applications.
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Anticuerpos/análisis , Anticuerpos/inmunología , Tamizaje Masivo/métodos , Péptidos/inmunología , Análisis por Matrices de Proteínas/métodos , Análisis por Conglomerados , Humanos , Tamizaje Masivo/instrumentación , Péptidos/química , Análisis por Matrices de Proteínas/instrumentación , Saliva/inmunología , Síndrome de Sjögren/sangre , Síndrome de Sjögren/diagnóstico , Síndrome de Sjögren/inmunologíaRESUMEN
The solution structure of the mouse pro-hormone convertase (PC) 1 pro-domain was determined using heteronuclear NMR spectroscopy and is the first structure to be obtained for any of the domains in the convertase family. The ensemble of NMR-derived structures shows a well-ordered core consisting of a four-stranded antiparallel beta-sheet with two alpha-helices packed against one side of this sheet. Sequence homology suggests that the other eukaryotic PC pro-domains will have the same overall fold and most of the residues forming the hydrophobic core of PC1 are highly conserved within the PC family. However, some of the core residues are predicted by homology to be replaced by polar amino acid residues in other PC pro-domains and this may help to explain their marginal stability. Interestingly, the folding topology observed here is also seen for the pro-domain of bacterial subtilisin despite little or no sequence homology. Both the prokaryotic and eukaryotic structures have hydrophobic residues clustered on the solvent-accessible surface of their beta-sheets although the individual residue types differ. In the bacterial case this region is buried at the binding interface with the catalytic domain and, in the eukaryotic PC family, these surface residues are conserved. We therefore propose that the hydrophobic patch in the PC1 pro-domain is involved in the binding interface with its cognate catalytic domain in a similar manner to that seen for the bacterial system. The PC1 pro-domain structure also reveals potential mechanisms for the acid-induced dissociation of the complex between pro- and catalytic domains.
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Ácido Aspártico Endopeptidasas/química , Proproteína Convertasa 1 , Secuencia de Aminoácidos , Animales , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Péptidos/química , Proproteína Convertasas , Pliegue de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Soluciones , Subtilisina/químicaRESUMEN
Proteomic analysis of clinical tissue specimens is a difficult undertaking. Described here is a multiplex study of protein expression levels in histological sections of human prostate that addresses many of the associated challenges. Whole-mount sections from 10 prostatectomy specimens were studied using 15 antibodies, immunohistochemical staining, digital imaging, and mathematical analysis of the data sets. The approach was successful in stratifying cell lineages present in the samples based on proteomic patterns, including differentiating normal epithelium from cancer. This strategy likely will be a useful method for extending the number of proteins that can be analyzed in clinical cancer specimens using currently available laboratory techniques.
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Próstata/metabolismo , Proteínas/metabolismo , Células Epiteliales/metabolismo , Epitelio/metabolismo , Epitelio/patología , Estudios de Factibilidad , Humanos , Inmunohistoquímica , Masculino , Matemática , Análisis de Componente Principal , Próstata/citología , Próstata/patología , Hiperplasia Prostática/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ProteómicaRESUMEN
Laser microdissection (LM) offers a relatively rapid and precise method of isolating and removing specified cells from complex tissues for subsequent analysis of their RNA, DNA, protein or metabolite content, thereby allowing assessment of the role of different cell types in the normal physiological or disease processes being studied. In this unit, protocols for the preparation of mammalian frozen tissues, fixed tissues, and cytologic specimens for LM, including tissue freezing, tissue processing and paraffin embedding, histologic sectioning, cell processing, hematoxylin and eosin staining, immunohistochemistry, and image-guided cell targeting are presented. Also provided are recipes for generating lysis buffers for the recovery of nucleic acids and proteins. The Commentary section addresses the types of specimens that can be utilized for LM and approaches to staining of specimens for cell visualization. Emphasis is placed on the preparation of tissue or cytologic specimens as this is critical to effective LM.