RESUMEN
Non-negative matrix factorization is a useful tool for reducing the dimension of large datasets. This work considers simultaneous non-negative matrix factorization of multiple sources of data. In particular, we perform the first study that involves more than two datasets. We discuss the algorithmic issues required to convert the approach into a practical computational tool and apply the technique to new gene expression data quantifying the molecular changes in four tissue types due to different dosages of an experimental panPPAR agonist in mouse. This study is of interest in toxicology because, whilst PPARs form potential therapeutic targets for diabetes, it is known that they can induce serious side-effects. Our results show that the practical simultaneous non-negative matrix factorization developed here can add value to the data analysis. In particular, we find that factorizing the data as a single object allows us to distinguish between the four tissue types, but does not correctly reproduce the known dosage level groups. Applying our new approach, which treats the four tissue types as providing distinct, but related, datasets, we find that the dosage level groups are respected. The new algorithm then provides separate gene list orderings that can be studied for each tissue type, and compared with the ordering arising from the single factorization. We find that many of our conclusions can be corroborated with known biological behaviour, and others offer new insights into the toxicological effects. Overall, the algorithm shows promise for early detection of toxicity in the drug discovery process.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Toxicología/métodos , Algoritmos , Animales , Análisis por Conglomerados , Bases de Datos Factuales , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Estadísticos , Familia de Multigenes , Músculos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Activados del Proliferador del Peroxisoma/agonistasRESUMEN
Spectral methods, which use information relating to eigenvectors, singular vectors and generalized singular vectors, help us to visualize and summarize sets of pairwise interactions. In this work, we motivate and discuss the use of spectral methods by taking a matrix computation view and applying concepts from applied linear algebra. We show that this unified approach is sufficiently flexible to allow multiple sources of network information to be combined. We illustrate the methods on microarray data arising from a large population-based study in human adipose tissue, combined with related information concerning metabolic pathways.
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Algoritmos , Biología Computacional/métodos , Animales , Humanos , Redes y Vías MetabólicasRESUMEN
Biomarker identification, using network methods, depends on finding regular co-expression patterns; the overall connectivity is of greater importance than any single relationship. A second requirement is a simple algorithm for ranking patients on how relevant a gene-set is. For both of these requirements discretized data helps to first identify gene cliques, and then to stratify patients.We explore a biologically intuitive discretization technique which codes genes as up- or down-regulated, with values close to the mean set as unchanged; this allows a richer description of relationships between genes than can be achieved by positive and negative correlation. We find a close agreement between our results and the template gene-interactions used to build synthetic microarray-like data by SynTReN, which synthesizes "microarray" data using known relationships which are successfully identified by our method.We are able to split positive co-regulation into up-together and down-together and negative co-regulation is considered as directed up-down relationships. In some cases these exist in only one direction, with real data, but not with the synthetic data. We illustrate our approach using two studies on white blood cells and derived immortalized cell lines and compare the approach with standard correlation-based computations. No attempt is made to distinguish possible causal links as the search for biomarkers would be crippled by losing highly significant co-expression relationships. This contrasts with approaches like ARACNE and IRIS.The method is illustrated with an analysis of gene-expression for energy metabolism pathways. For each discovered relationship we are able to identify the samples on which this is based in the discretized sample-gene matrix, along with a simplified view of the patterns of gene expression; this helps to dissect the gene-sample relevant to a research topic--identifying sets of co-regulated and anti-regulated genes and the samples or patients in which this relationship occurs.
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Biomarcadores , Redes Reguladoras de Genes , Expresión Génica , Humanos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Transcriptional activity of hypoxia-induced factor 1 (HIF1) - a heterodimer of HIF1α and ARNT (HIF1ß) - is essential for cellular adaptation to environmental stress and plays an important role in skin development, wound healing, tumorigenesis and barrier function. Using primary mouse and human epidermal keratinocytes at ambient or hypoxic (1% O(2)) conditions we studied effects of hypoxia upon HIF protein expression. Significant nuclear levels of ARNT and HIF1α along with high HIF1 activity in normoxic keratinocytes suggest an as yet uncharacterised oxygen-independent role for HIF pathway in the epidermis. Acute hypoxia results in an instant but transient increase of HIF1α protein accompanied by a gradual decrease in its mRNA, while ARNT expression remains unchanged. In prolonged (chronic) hypoxia both HIF1α and Arnt are downregulated along with decline of HIF1 activity. However, expression of classical HIF1 targets such as Selenbp1 and Vegfa remains high. Thus, keratinocytes respond to acute hypoxia with immediate block of HIF1α protein degradation and concomitant increase of HIF activity, while under chronic hypoxia pro-angiogenic signalling is maintained through HIF1-independent pathway(s). Decline of HIF1α during chronic exposure is controlled at both mRNA and protein levels, while Arnt is downregulated post-translationally. Distinct transcription levels of Hif1α and Hif3α splice variants under normoxia and their differential response to hypoxia suggest functional diversity of Hif-α isoforms and highlight the complexity of HIF machinery control in epidermal keratinocytes.
Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Epidermis/metabolismo , Hipoxia , Queratinocitos/metabolismo , Empalme Alternativo , Animales , Animales Recién Nacidos , Proteínas Reguladoras de la Apoptosis , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores/metabolismo , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Proteínas Represoras , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND AND AIMS: Familial benign hypocalciuric hypercalcaemia (FBHH) is a benign autosomal dominantly inherited condition which results in elevated serum calcium and low urinary calcium. This condition is of clinical interest because it can be mistakenly diagnosed as primary hyperparathyroidism (PHP). In most cases FBHH can be shown to be due to a mutation in the calcium sensing receptor (CASR) gene and we aimed to find the causative mutation in three Scottish kindreds with FBHH. METHODS: We used a combination of denaturing gradient gel electrophoresis and direct DNA sequencing to detect mutations in the CASR gene. RESULTS: We detected a mutation in the CASR gene in each of the three kindreds. Two different mutations were detected (the same one was present in two kindreds). Neither mutation has been reported previously. All hypercalcaemic individuals from each kindred had the appropriate mutation while all normocalcaemic individuals did not. CONCLUSION: In the vast majority of kindreds with FBHH which have been reported previously, the CASR mutation responsible is private to that kindred. In three Scottish kindreds we have identified two new mutations.
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Calcio/orina , Hipercalcemia/genética , Mutación , Receptores Sensibles al Calcio/genética , Femenino , Humanos , Linaje , EscociaRESUMEN
BACKGROUND AND PURPOSE: The different clinical subtypes of multiple sclerosis (MS) may reflect underlying differences in affected neuroanatomic regions. Our aim was to analyze the effectiveness of jointly using the inferior subolivary medulla oblongata volume (MOV) and the cross-sectional area of the corpus callosum in distinguishing patients with relapsing-remitting multiple sclerosis (RRMS), secondary-progressive multiple sclerosis (SPMS), and primary-progressive multiple sclerosis (PPMS). MATERIALS AND METHODS: We analyzed a cross-sectional dataset of 64 patients (30 RRMS, 14 SPMS, 20 PPMS) and a separate longitudinal dataset of 25 patients (114 MR imaging examinations). Twelve patients in the longitudinal dataset had converted from RRMS to SPMS. For all images, the MOV and corpus callosum were delineated manually and the corpus callosum was parcellated into 5 segments. Patients from the cross-sectional dataset were classified as RRMS, SPMS, or PPMS by using a decision tree algorithm with the following input features: brain parenchymal fraction, age, disease duration, MOV, total corpus callosum area and areas of 5 segments of the corpus callosum. To test the robustness of the classification technique, we applied the results derived from the cross-sectional analysis to the longitudinal dataset. RESULTS: MOV and central corpus callosum segment area were the 2 features retained by the decision tree. Patients with MOV >0.94 cm(3) were classified as having RRMS. Patients with progressive MS were further subclassified as having SPMS if the central corpus callosum segment area was <55.12 mm(2), and as having PPMS otherwise. In the cross-sectional dataset, 51/64 (80%) patients were correctly classified. For the longitudinal dataset, 88/114 (77%) patient time points were correctly classified as RRMS or SPMS. CONCLUSIONS: Classification techniques revealed differences in affected neuroanatomic regions in subtypes of multiple sclerosis. The combination of central corpus callosum segment area and MOV provides good discrimination among patients with RRMS, SPMS, and PPMS.
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Anatomía Transversal/métodos , Encéfalo/patología , Imagen por Resonancia Magnética/métodos , Esclerosis Múltiple/patología , Fibras Nerviosas Mielínicas/patología , Atrofia/patología , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Hypermethylation of promoter CpG islands is strongly correlated to transcriptional gene silencing and epigenetic maintenance of the silenced state. As well as its role in tumor development, CpG island methylation contributes to the acquisition of resistance to chemotherapy. Differential Methylation Hybridisation (DMH) is one technique used for genome-wide DNA methylation analysis. The study of such microarray data sets should ideally account for the specific biological features of DNA methylation and the non-symmetrical distribution of the ratios of unmethylated and methylated sequences hybridised on the array. We have therefore developed a novel algorithm tailored to this type of data, Methylation Linear Discriminant Analysis (MLDA). RESULTS: MLDA was programmed in R (version 2.7.0) and the package is available at CRAN 1. This approach utilizes linear regression models of non-normalised hybridisation data to define methylation status. Log-transformed signal intensities of unmethylated controls on the microarray are used as a reference. The signal intensities of DNA samples digested with methylation sensitive restriction enzymes and mock digested are then transformed to the likelihood of a locus being methylated using this reference. We tested the ability of MLDA to identify loci differentially methylated as analysed by DMH between cisplatin sensitive and resistant ovarian cancer cell lines. MLDA identified 115 differentially methylated loci and 23 out of 26 of these loci have been independently validated by Methylation Specific PCR and/or bisulphite pyrosequencing. CONCLUSION: MLDA has advantages for analyzing methylation data from CpG island microarrays, since there is a clear rational for the definition of methylation status, it uses DMH data without between-group normalisation and is less influenced by cross-hybridisation of loci. The MLDA algorithm successfully identified differentially methylated loci between two classes of samples analysed by DMH using CpG island microarrays.
Asunto(s)
Algoritmos , Islas de CpG/genética , Metilación de ADN , Análisis Discriminante , Línea Celular Tumoral , Cisplatino/farmacología , Metilasas de Modificación del ADN/metabolismo , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Modelos Lineales , Hibridación de Ácido Nucleico , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genéticaRESUMEN
Peripheral blood as a surrogate tissue for transcriptome profiling holds great promise for the discovery of diagnostic and prognostic disease biomarkers, particularly when target tissues of disease are not readily available. To maximize the reliability of gene expression data generated from clinical blood samples, both the sample collection and the microarray probe generation methods should be optimized to provide stabilized, reproducible and representative gene expression profiles faithfully representing the transcriptional profiles of the constituent blood cell types present in the circulation. Given the increasing innovation in this field in recent years, we investigated a combination of methodological advances in both RNA stabilisation and microarray probe generation with the goal of achieving robust, reliable and representative transcriptional profiles from whole blood. To assess the whole blood profiles, the transcriptomes of purified blood cell types were measured and compared with the global transcriptomes measured in whole blood. The results demonstrate that a combination of PAXgene() RNA stabilising technology and single-stranded cDNA probe generation afforded by the NuGEN Ovation RNA amplification system V2() enables an approach that yields faithful representation of specific hematopoietic cell lineage transcriptomes in whole blood without the necessity for prior sample fractionation, cell enrichment or globin reduction. Storage stability assessments of the PAXgene() blood samples also advocate a short, fixed room temperature storage time for all PAXgene() blood samples collected for the purposes of global transcriptional profiling in clinical studies.
RESUMEN
RASSF1A is a tumor suppressor gene that is epigenetically silenced in a wide variety of sporadic human malignancies. Expression of alternative RASSF1 isoforms cannot substitute for RASSF1A-promoted cell-cycle arrest and apoptosis. Apoptosis can be driven by either activating Bax or by activation of MST kinases. The Raf1 proto-oncogene binds to MST2, preventing its activation and proapoptotic signaling. Here we show that key steps in RASSF1A-induced apoptosis are the disruption of the inhibitory Raf1-MST2 complex by RASSF1A and the concomitant enhancement of MST2 interaction with its substrate, LATS1. Subsequently, RASSF1A-activated LATS1 phosphorylates and releases the transcriptional regulator YAP1, allowing YAP1 to translocate to the nucleus and associate with p73, resulting in transcription of the proapoptotic target gene puma. Our results describe an MST2-dependent effector pathway for RASSF1A proapoptotic signaling and indicate that silencing of RASSF1A in tumors removes a proapoptotic signal emanating from p73.
Asunto(s)
Apoptosis , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Modelos Biológicos , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica , Transporte de Proteínas , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Serina-Treonina Quinasa 3 , Transducción de Señal , Factores de Transcripción , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Quiescent and dividing hemopoietic stem cells (HSC) display marked differences in their ability to move between the peripheral circulation and the bone marrow. Specifically, long-term engraftment potential predominantly resides in the quiescent HSC subfraction, and G-CSF mobilization results in the preferential accumulation of quiescent HSC in the periphery. In contrast, stem cells from chronic myeloid leukemia (CML) patients display a constitutive presence in the circulation. To understand the molecular basis for this, we have used microarray technology to analyze the transcriptional differences between dividing and quiescent, normal, and CML-derived CD34+ cells. Our data show a remarkable transcriptional similarity between normal and CML dividing cells, suggesting that the effects of BCR-ABL on the CD34+ cell transcriptome are more limited than previously thought. In addition, we show that quiescent CML cells are more similar to their dividing counterparts than quiescent normal cells are to theirs. We also show these transcriptional differences to be reflected in the altered proliferative activity of normal and CML CD34+ cells. Of the most interest is that the major class of genes that is more abundant in the quiescent cells compared with the dividing cells encodes members of the chemokine family. We propose a role for chemokines expressed by quiescent HSC in the orchestration of CD34+ cell mobilization. Disclosure of potential conflicts of interest is found at the end of this article.
Asunto(s)
Antígenos CD34/genética , Proliferación Celular , Perfilación de la Expresión Génica/métodos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Fase de Descanso del Ciclo Celular/genética , Antígenos CD34/biosíntesis , Ciclo Celular/genética , División Celular/genética , Células Cultivadas , Femenino , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Células Tumorales CultivadasRESUMEN
The APC gene encodes the adenomatous polyposis coli tumour suppressor protein, germline mutation of which characterizes familial adenomatous polyposis (FAP), an autosomal intestinal cancer syndrome. Inactivation of APC is also recognized as the key early event in the development of sporadic colorectal cancers, and its loss results in constitutive activity of the beta-catenin-Tcf4 transcription complex. The proto-oncogene c-MYC has been identified as a target of the Wnt pathway in colorectal cancer cells in vitro, in normal crypts in vivo and in intestinal epithelial cells acutely transformed on in vivo deletion of the APC gene; however, the significance of this is unclear. Therefore, to elucidate the role Myc has in the intestine after Apc loss, we have simultaneously deleted both Apc and Myc in the adult murine small intestine. Here we show that loss of Myc rescued the phenotypes of perturbed differentiation, migration, proliferation and apoptosis, which occur on deletion of Apc. Remarkably, this rescue occurred in the presence of high levels of nuclear beta-catenin. Array analysis revealed that Myc is required for the majority of Wnt target gene activation following Apc loss. These data establish Myc as the critical mediator of the early stages of neoplasia following Apc loss.
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Proteína de la Poliposis Adenomatosa del Colon/deficiencia , Proteína de la Poliposis Adenomatosa del Colon/genética , Eliminación de Gen , Genes APC , Genes myc/genética , Intestino Delgado/metabolismo , Proteínas Proto-Oncogénicas c-myc/deficiencia , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/patología , Animales , Enterocitos/metabolismo , Intestino Delgado/citología , Masculino , Ratones , Fenotipo , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
We give a simple and informative derivation of a spectral algorithm for clustering and reordering complementary DNA microarray expression data. Here, expression levels of a set of genes are recorded simultaneously across a number of samples, with a positive weight reflecting up-regulation and a negative weight reflecting down-regulation. We give theoretical support for the algorithm based on a biologically justified hypothesis about the structure of the data, and illustrate its use on public domain data in the context of unsupervised tumour classification. The algorithm is derived by considering a discrete optimization problem and then relaxing to the continuous realm. We prove that in the case where the data have an inherent 'checkerboard' sign pattern, the algorithm will automatically reveal that pattern. Further, our derivation shows that the algorithm may be regarded as imposing a random graph model on the expression levels and then clustering from a maximum likelihood perspective. This indicates that the output will be tolerant to perturbations and will reveal 'near-checkerboard' patterns when these are present in the data. It is interesting to note that the checkerboard structure is revealed by the first (dominant) singular vectors--previous work on spectral methods has focussed on the case of nonnegative edge weights, where only the second and higher singular vectors are relevant. We illustrate the algorithm on real and synthetic data, and then use it in a tumour classification context on three different cancer data sets. Our results show that respecting the two-signed nature of the data (thereby distinguishing between up-regulation and down-regulation) reveals structures that cannot be gleaned from the absolute value data (where up- and down-regulation are both regarded as 'changes').
Asunto(s)
Algoritmos , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis por Conglomerados , Interpretación Estadística de Datos , Regulación hacia Abajo/genética , Humanos , Funciones de Verosimilitud , Neoplasias Pulmonares/clasificación , Neoplasias Pulmonares/genética , Linfoma/clasificación , Linfoma/genética , Masculino , Neoplasias/genética , Neoplasias de la Próstata/clasificación , Neoplasias de la Próstata/genética , Regulación hacia Arriba/genéticaRESUMEN
Most head and neck squamous cell carcinoma (HNSCC) patients present with late-stage cancers, which are difficult to treat. Therefore, early diagnosis of high-risk premalignant lesions and incipient cancers is important. HNSCC is currently perceived as a single progression mechanism, resulting in immortal invasive cancers. However, we have found that approximately 40% of primary oral SCCs are mortal in culture, and these have a better prognosis. About 60% of oral premalignancies (dysplasias) are also mortal. The mortal and immortal tumors are generated in vivo as judged by p53 mutations and loss of p16(INK4A) expression being found only in the original tumors from which the immortal cultures were derived. To investigate the relationships of dysplasias to SCCs, we did microarray analysis of primary cultures of 4 normal oral mucosa biopsies, 19 dysplasias, and 16 SCCs. Spectral clustering using the singular value decomposition and other bioinformatic techniques showed that development of mortal and immortal SCCs involves distinct transcriptional changes. Both SCC classes share most of the transcriptional changes found in their respective dysplasias but have additional changes. Moreover, high-risk dysplasias that subsequently progress to SCCs more closely resemble SCCs than nonprogressing dysplasias. This indicates for the first time that there are divergent mortal and immortal pathways for oral SCC development via intermediate dysplasias. We believe that this new information may lead to new ways of classifying HNSCC in relation to prognosis.
Asunto(s)
Carcinoma de Células Escamosas/genética , Neoplasias de la Boca/genética , Lesiones Precancerosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Perfilación de la Expresión Génica , Humanos , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Transcripción Genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Bacterial comparative genomics has been revolutionized by microarrays, but the power of any microarray is dependent on the number and diversity of gene reporters it contains. Staphylococcus aureus is an important human pathogen causing a wide range of invasive and toxin-mediated diseases, and more than 20% of the genome of any isolate consists of variable genes. Seven whole-genome sequences of S. aureus are available, and we exploited this rare opportunity to design, build, and validate a comprehensive, nonredundant PCR product microarray carrying reporters that represent every predicted open reading frame (3,623 probes). Such a comprehensive microarray necessitated a novel design strategy. Validation with the seven sequenced strains showed correct identification of 93.9% of genes present or absent/divergent but was dependent on the method of analysis chosen. Microarray data were highly reproducible, reducing the need for many replicate slides. Interpretation of microarray data was enhanced by focusing on the major areas of variation--the presence or absence of mobile genetic elements (MGEs). We compiled "composite genomes" of every individual MGE and visualized their distribution. This allowed the sensitive discrimination of related isolates, including the first clear description of how isolates of the same clone of epidemic methicillin-resistant S. aureus differ substantially in their carriage of MGEs. These MGEs carry virulence and resistance genes, suggesting differences in pathogenic potential. The novel methods of design and interpretation of data generated from this microarray will enable further studies of S. aureus evolution, epidemiology, and pathogenesis.
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Genómica , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa/métodos , Proyectos de Investigación , Staphylococcus aureus/clasificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Genoma Bacteriano , Humanos , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Staphylococcus aureus/genéticaRESUMEN
OBJECTIVE: Methylation of a CpG island within the Methylation controlled DNAJ (MCJ) gene results in loss of expression in normal and neoplastic cells. Normal ovarian surface epithelial cells are methylated at the MCJ CpG island and do not express the MCJ gene. Furthermore, re-expression of the MCJ gene, in ovarian cancer cell lines, has been correlated with increased sensitivity to several important chemotherapeutic drugs. The objective of this study was to determine the extent of MCJ promoter methylation in epithelial ovarian cancer patients and address the possible role of MCJ methylation levels in response to chemotherapy in ovarian cancer patients. METHODS: The methylation status of 35 CpG sites within the MCJ CpG island was determined by sequencing of sodium bisulfite modified tumor DNA in 41 patients with stage III/IV epithelial ovarian tumors. Levels of methylation of the MCJ CpG island were then compared with response to therapy and overall survival in the patients. RESULTS: The analysis identified frequent loss of MCJ methylation in ovarian tumors, with only a subset retaining high methylation levels. While 93% (38/41) of tumors examined showed some level of MCJ methylation, only 17% (7/41) retained very high levels (>90% methylation). The presence of such high levels of CpG island methylation correlated significantly with poor response of patients' tumors to therapy (P = 0.027) and poor overall survival (P = 0.023, hazard ratio = 2.9). CONCLUSIONS: These results suggest that MCJ methylation may be useful as a marker of response to chemotherapy in ovarian cancer and are consistent with previous in vitro data linking loss of MCJ expression with drug resistance.
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Metilación de ADN , Proteínas de Choque Térmico/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Secuencia de Bases , Islas de CpG , Femenino , Proteínas del Choque Térmico HSP40 , Humanos , Datos de Secuencia Molecular , Estadificación de Neoplasias , Neoplasias Ováricas/patología , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Tasa de SupervivenciaRESUMEN
PURPOSE: : The greater accuracy of apical dissection and reconstruction in our first 100 patients undergoing transperitoneal laparoscopic radical prostatectomy (TLRP) was not matched by a proportionate increase in the rate of return to normal continence compared with our prior open prostatectomy experience. We postulated that greater bladder dysfunction due to the almost total bladder dissection mandated by TLRP might be responsible and this might be rectified by the adoption of laparoscopic radical prostatectomy using an extraperitoneal approach (ELRP). MATERIALS AND METHODS: : A total of 100 patients undergoing TLRP were compared with 100 undergoing ELRP. The groups were subdivided into halves to investigate the influence of any learning curve effect. All patients had clinical stage T3aN0M0 or less prostate cancer and they were operated on by a single surgeon. RESULTS: : Mean operative time (238.9 vs 190.6 minutes), blood loss (310.5 vs 201.5 ml), postoperative hospitalization (3.8 vs 2.6 nights) and catheterization duration (11.3 vs 10.1 days) were significantly greater in the TLRP group. After the first 50 cases were excluded in each group statistical significance persisted only for operative time (218.3 vs 184.2 minutes) and hospitalization (3.5 vs 2.5 nights). The pad-free rate was significantly lower 3 months following ELRP (80% vs 56%, p = 0.02). The overall 12-month pad-free rate for TLRP and ELRP was 90% and 96%, respectively. The overall 12-month erection rate for TLRP and ELRP was 61% and 82%, respectively. CONCLUSIONS: : ELRP is superior to TLRP with respect to operative time, hospitalization and early continence.
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Adenocarcinoma/cirugía , Laparoscopía/métodos , Prostatectomía/métodos , Neoplasias de la Próstata/cirugía , Anciano , Humanos , Laparoscopía/efectos adversos , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/epidemiología , Complicaciones Posoperatorias/etiología , Prostatectomía/efectos adversosRESUMEN
Invasion is generally perceived to be a late event during the progression of human cancer, but to date there are no consistent reports of alterations specifically associated with malignant conversion. We provide evidence that the v-Fos oncogene induces changes in gene expression that render noninvasive normal human diploid fibroblasts highly invasive, without inducing changes in growth factor requirements or anchorage dependence for proliferation. Furthermore, v-Fos-stimulated invasion is independent of the pRb/p16(INK4a) and p53 tumor suppressor pathways and telomerase. We have performed microarray analysis using Affymetrix GeneChips, and the gene expression profile of v-Fos transformed cells supports its role in the regulation of invasion, independent from proliferation. We also demonstrate that invasion, but not proliferation, is dependent on the activity of the up-regulated epidermal growth factor receptor. Taken together, these results indicate that AP-1-directed invasion could precede deregulated proliferation during tumorigenesis and that sustained activation of AP-1 could be the epigenetic event required for conversion of a benign tumor into a malignant one, thereby explaining why many malignant human tumors present without an obvious premalignant hyperproliferative dysplastic lesion.
Asunto(s)
Transformación Celular Neoplásica/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Invasividad Neoplásica/patología , Proteínas Oncogénicas v-fos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , División Celular , Transformación Celular Neoplásica/genética , Daño del ADN , Receptores ErbB/genética , Receptores ErbB/metabolismo , Perfilación de la Expresión Génica , Humanos , Cariotipificación , Ratones , Invasividad Neoplásica/genética , Proteínas Oncogénicas v-fos/genética , Transducción de Señal , Telomerasa/genética , Telomerasa/metabolismo , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba , Cicatrización de HeridasRESUMEN
Over 50% of human genes are associated with CpG islands and DNA methylation within such CpG islands has been clearly correlated with inhibition of expression. Whereas changes in DNA methylation play a key role in a number of human diseases, in particular cancer, in normal DNA CpG islands are nearly always methylation free, regardless of the expression status of the associated gene. Only limited evidence supports a role for DNA methylation in controlling tissue-specific expression in adult somatic tissue. Loss of expression of the MCJ gene has previously been linked to increased chemotherapeutic drug resistance in ovarian cancer. We report that loss of expression of MCJ in drug-resistant ovarian cancer cell lines depends on methylation of a CpG island within its first exon, but is independent of methylation within the promoter region. Furthermore, cell type-specific expression of the MCJ gene in normal cells also depends on the methylation status of the CpG island within its first exon. The MCJ CpG island is methylated and the gene is not expressed in cells of epithelial origin, but unmethylated and expressed in cells of lymphocyte or fibroblast origin. Chromatin immunoprecipitation assays determined that MCJ CpG island methylation was associated with loss of histone acetylation in ovarian epithelial cells compared with unmethylated fibroblast cells. Reduced acetylation was observed not only within the CpG island, but also within the promoter region, suggesting that CpG island methylation may direct alterations in chromatin structure within the promoter region, leading to gene inactivation.
Asunto(s)
Islas de CpG , Metilación de ADN , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Proteínas de Choque Térmico/genética , Intrones/genética , Neoplasias Ováricas/genética , Acetilación , Antineoplásicos/farmacología , Secuencia de Bases , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Cisplatino/farmacología , Células Epiteliales , Femenino , Fibroblastos , Proteínas del Choque Térmico HSP40 , Histona Desacetilasas , Histonas/química , Histonas/metabolismo , Humanos , Linfocitos , Datos de Secuencia Molecular , Neoplasias Ováricas/patología , Ovario/fisiología , Pruebas de Precipitina , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Gastric carcinoma is the fourth most common cause of cancer death worldwide but its molecular biology is poorly understood. We catalogued the genes expressed in two gastric adenocarcinomas and normal stomach, using serial analysis of gene expression (SAGE), and compared the profiles on-line with other glandular epithelia. Candidates were validated by Northern blotting and immunohistochemistry. A total of 29 480 transcripts, derived from 10 866 genes, were identified. In all, 1% of the genes were differentially expressed (>/=fivefold difference plus P-value =0.01) between cancers and normal stomach. The most abundant transcripts included ribosomal and mitochondrial proteins, of which most were upregulated in the tumours, as were other widely expressed genes including transcription factors, signalling molecules (serine/threonine protein kinases), thymosin beta 10 and collagenase I. Transcripts abundant in normal stomach were functionally important, including gastrin, immunoglobulin alpha, lysozyme, MUC5, pS2 and pepsinogens, which were among 55 gastric-specific genes. Many transcripts were minimally characterized or new, some cancer-associated genes reflected their intestinal morphology, and some normal gastric genes had previously been considered as pancreatic carcinoma markers. The gastric carcinoma profiles resembled other tumours', supporting the existence of common cancer-associated targets. These data provide a catalogue from which to develop markers for better diagnosis and therapy of gastric carcinoma.
Asunto(s)
Carcinoma/metabolismo , Mucosa Gástrica/metabolismo , Regulación Neoplásica de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Neoplasias Gástricas/metabolismo , Northern Blotting , Carcinoma/genética , Biblioteca de Genes , Humanos , Inmunohistoquímica , ARN/metabolismo , ARN Mensajero/metabolismo , Neoplasias Gástricas/genética , Células Tumorales CultivadasRESUMEN
AIMS: The incidence, bone marrow morphology and genetic features of bcr+ essential thrombocythaemia were investigated. METHODS AND RESULTS: Sixty-four consecutive patients meeting the criteria of essential thrombocythaemia have been investigated for bcr-abl rearrangement and chimera mRNA expression. Reverse transcriptase-polymerase chain reaction indicated bcr-abl expression in six patients, in two of whom large fraction of the blood and bone marrow cells proved to be positive for Philadelphia chromosome (Ph) by fluorescent in-situ hybridization (FISH) and conventional cytogenetic analysis. In the remaining four patients FISH analysis could not detect Ph+ cells among the blood cells, but in one of these four patients conventional cytogenetic analysis indicated a very small fraction (2%) of Ph+ mitoses in the bone marrow (bcr+ essential thrombocythaemia patients). In three of these four patients, X-chromosome-linked clonality assay showed that the disease is of uncommitted stem cell origin. During an average of 57 month long follow-up no transformation to chronic myeloid leukaemia type of disease or acceleration/blastic crisis could be observed in the four bcr+ essential thrombocythaemia patients. They did not differ significantly from typical essential thrombocythaemia patients in quantitative indices of bone marrow cellularity or the size of megakaryocytes. In these two parameters as well as in the total nucleolus organizer region area per nucleus, however, significant differences could be detected between these four as well as typical chronic myeloid leukaemia patients. Statistical analysis of the morphometric data obtained from all six Ph+ and bcr+ essential thrombocythaemia patients combined indicated a shift of the bone marrow morphology towards the chronic myeloid leukaemia type of myeloproliferation. CONCLUSIONS: These investigations indicate that bcr+ essential thrombocythaemia is infrequent among essential thrombocythaemia patients, and this condition resembles essential thrombocythaemia more than chronic myeloid leukaemia. Various expansions of the Ph+ clone appear to lead to either essential thrombocythaemia or, rather, chronic myeloid leukaemia type of myeloproliferation; however, data in the present study do not indicate that bcr+ essential thrombocythaemia would be a form fruste variant of chronic myeloid leukaemia.