RESUMO
The objective was to identify proteins differentially expressed in vaginal cancer to elucidate relevant cancer-related proteins. A total of 16 fresh-frozen tissue biopsies, consisting of 5 biopsies from normal vaginal epithelium, 6 from primary vaginal carcinomas and 5 from primary cervical carcinomas, were analysed using two-dimensional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry. Of the 43 proteins identified with significant alterations in protein expression between non-tumourous and tumourous tissue, 26 were upregulated and 17 were downregulated. Some were similarly altered in vaginal and cervical carcinoma, including cytoskeletal proteins, tumour suppressor proteins, oncoproteins implicated in apoptosis and proteins in the ubiquitin-proteasome pathway. Three proteins were uniquely altered in vaginal carcinoma (DDX48, erbB3-binding protein and biliverdin reductase) and five in cervical carcinoma (peroxiredoxin 2, annexin A2, sarcomeric tropomyosin kappa, human ribonuclease inhibitor and prolyl-4-hydrolase beta). The identified proteins imply involvement of multiple different cellular pathways in the carcinogenesis of vaginal carcinoma. Similar protein alterations were found between vaginal and cervical carcinoma suggesting common tumourigenesis. However, the expression level of some of these proteins markedly differs among the three tissue specimens indicating that they might be useful molecular markers.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma/patologia , Proteínas de Neoplasias/análise , Neoplasias do Colo do Útero/patologia , Neoplasias Vaginais/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Carcinoma/genética , Eletroforese em Gel Bidimensional , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Estadiamento de Neoplasias , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Neoplasias do Colo do Útero/genética , Neoplasias Vaginais/genéticaRESUMO
Two-dimensional gel electrophoresis was used to identify polypeptides differentially expressed between normal and c-jun transformed rat fibroblasts. The level of a 49 kDa polypeptide was 3-fold elevated in c-jun transformed cells. Sequence analysis by ion trap mass spectrometry identified the polypeptide as rat alpha-enolase. Enolase functions as a cell surface receptor for plasminogen, suggesting that upregulation may increase plasminogen activation and cell surface proteolysis important for tumor growth. However, no difference was observed between normal and transformed cells in formation of plasmin, suggesting that upregulation of alpha-enolase may contribute to an increased metabolic capacity, but not to increased plasminogen activation.
Assuntos
Transformação Celular Neoplásica , Genes jun , Fosfopiruvato Hidratase/biossíntese , Plasminogênio/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Linhagem Celular Transformada , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Proteínas Proto-Oncogênicas c-jun/genética , RatosRESUMO
A pair of 35 kDa polypeptides (TAO1/TAO2) are expressed in more than 90% of all primary lung adenocarcinomas but not in other major malignancies. Mass spectrometry of tryptic peptides showed that TAO1/TAO2 is identical to napsin A, a recently described member of the aspartic proteinase family. The site of processing of pronapsin A to the mature form was located. Napsin expression was detected in human lung adenocarcinoma tumors, compatible with the marker nature of TAO1/TAO2 in the diagnosis of primary lung adenocarcinoma. This is important since identification of markers which can distinguish primary lung adenocarcinomas from distant metastases is desirable. Northern blot analysis showed expression of napsin also in normal lung and kidney tissue, and in situ hybridization showed expression in type II alveolar cells of the lung. This protease is concluded to have a specific functional role in the normal alveolar epithelium and is a candidate protease for the proteolytic processing of surfactant precursors.
Assuntos
Adenocarcinoma/enzimologia , Ácido Aspártico Endopeptidases/biossíntese , Rim/enzimologia , Neoplasias Pulmonares/enzimologia , Pulmão/enzimologia , Proteínas Associadas a Surfactantes Pulmonares , Sequência de Aminoácidos , Apoproteínas/metabolismo , Ácido Aspártico Endopeptidases/genética , Clonagem Molecular , DNA Complementar/análise , Humanos , Dados de Sequência Molecular , Surfactantes Pulmonares/metabolismo , RNA Mensageiro/biossíntese , Distribuição TecidualAssuntos
Mutação de Sentido Incorreto/genética , Neutropenia/congênito , Neutropenia/genética , Proteínas/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Sequência de Bases , Contagem de Células Sanguíneas , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Dados de Sequência Molecular , Linhagem , Proteínas/químicaRESUMO
BACKGROUND AND AIMS: Despite improved techniques, the determination of tumor origin in poorly differentiated adenocarcinomas still remains a challenge for the pathologist. Here we report the use of protein profiling combined with principal component analysis to improve diagnostic decision-making in tumor samples, in which standard pathologic investigations cannot present reliable results. MATERIALS AND METHODS: A poorly differentiated adenocarcinoma of unknown origin located in the pelvis, infiltrating the sigmoid colon as well as the ovary, served as a model to evaluate our proteomic approach. Firstly, we characterized the protein expression profiles from eight advanced colon and seven ovarian adenocarcinomas using two-dimensional gel electrophoresis (2-DE). Qualitative and quantitative patterns were recorded and compared to the tumor of unknown origin. Based on these protein profiles, match sets from the different tumors were created. Finally, a multivariate principal component analysis was applied to the entire 2-DE data to disclose differences in protein patterns between the different tumors. RESULTS: Over 89% of the unknown tumor sample spots could be matched with the colon standard gel, whereas only 63% of the spots could be matched with the ovarian standard. In addition, principal component analysis impressively displayed the clustering of the unknown case within the colon cancer samples, whereas this case did not cluster at all within the group of ovarian adenocarcinomas. CONCLUSION: These results show that 2-DE protein expression profiling combined with principal component analysis is a sensitive method for diagnosing undifferentiated adenocarcinomas of unknown origin. The described approach can contribute greatly to diagnostic decision-making and, with further technical improvements and a higher throughput, become a powerful tool in the armentarium of the pathologist.
Assuntos
Adenocarcinoma/secundário , Diferenciação Celular , Neoplasias do Colo/secundário , Proteínas de Neoplasias/análise , Neoplasias Primárias Desconhecidas/diagnóstico , Neoplasias Ovarianas/secundário , Neoplasias Pélvicas/diagnóstico , Proteômica , Adenocarcinoma/química , Análise por Conglomerados , Neoplasias do Colo/química , Diagnóstico Diferencial , Eletroforese em Gel Bidimensional , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Análise Multivariada , Invasividade Neoplásica , Neoplasias Primárias Desconhecidas/química , Neoplasias Primárias Desconhecidas/patologia , Neoplasias Ovarianas/química , Neoplasias Pélvicas/química , Neoplasias Pélvicas/patologia , Valor Preditivo dos Testes , Análise de Componente Principal , Proteômica/métodos , Reprodutibilidade dos TestesRESUMO
We have used proteomics with cluster analysis for the classification of breast tumour tissues. In our approach, we can distinguish between normal breast, benign breast and breast cancer tissues on the basis of the protein expression profiles. We propose an objective method for the classification of breast tumour specimens.
Assuntos
Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Proteômica , Adolescente , Adulto , Idoso , Mama , Neoplasias da Mama/patologia , Análise por Conglomerados , Diagnóstico Diferencial , Eletroforese em Gel Bidimensional , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
Studies of global protein expression in human tumors have led to the identification of various polypeptide markers, potentially useful as diagnostic tools. Many changes in gene expression recorded between benign and malignant human tumors are due to post-translational modifications, not detected by analyses of RNA. Proteome analyses have also yielded information about tumor heterogeneity and the degree of relatedness between primary tumors and their metastases. Results from our own studies have shown a similar pattern of changes in protein expression in different epithelial tumors, such as decreases in tropomyosin and cytokeratin expression and increases in proliferating cell nuclear antigen (PCNA) and heat shock protein expression. Such information has been used to create artificial learning models for tumor classification. The artificial learning approach has potential to improve tumor diagnosis and cancer treatment prediction.
Assuntos
Biomarcadores Tumorais/análise , Proteínas de Neoplasias/análise , Proteoma/análise , Bases de Dados Factuais , Eletroforese em Gel Bidimensional/métodos , Previsões , Expressão Gênica , Humanos , Neoplasias/classificação , Neoplasias/diagnósticoRESUMO
Protein patterns in six samples from primary vaginal cancers, in five from normal vaginal tissue and in five primary cervical cancers, were analysed using two-dimensional polyacrylamide gel electrophoresis (2-DE). Protein expression profile was evaluated by computer-assisted image analysis (PDQUEST) and proteins were subsequently identified using matrix-assisted laser desorption/ionisation mass spectrometry. The aim was to analyse the protein expression profiles using the hierarchical clustering method in vaginal carcinoma and to compare them with the protein pattern in cervical carcinoma in order to find a helpful tool for correct classification and for increased biomedical knowledge. Protein expression data of a distinct set of 33 protein spots were differentially expressed. These differences were statistically significant (Mann-Whitney signed-Ranked Test, P<0.05) between normal tissue, vaginal and cervical cancer. Furthermore, protein profiles of pairs of primary vaginal and cervical cancers were found to be very similar. Some of the protein spots that have so far been identified include Tropomyosin 1, cytokeratin 5, 15 and 17, Apolipoprotein A1, Annexin V, Glutathione-S-transferase. Others are the stress-related proteins, calreticulin, HSP 27 and HSP 70. We conclude that cluster analysis of proteomics data allows accurate discrimination between normal vaginal mucosa, primary vaginal and primary cervical cancer. However, vaginal and cervical carcinomas also appear to be relatively homogeneous in their gene expression, indicating similar carcinogenic pathways. There might, further, be a possibility to identify tumour-specific markers among the proteins that are differentially expressed. The results from this study have to be confirmed by more comprehensive studies in the future.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Neoplasias/metabolismo , Proteoma , Vagina/química , Neoplasias Vaginais/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Biomarcadores Tumorais/isolamento & purificação , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Eletroforese em Gel Bidimensional/métodos , Feminino , Humanos , Proteínas de Neoplasias/isolamento & purificação , Mapeamento de Peptídeos , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Neoplasias Vaginais/patologiaRESUMO
Biochemical and genetic strategies have implied that aberrant signaling in the extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase pathway contributes significantly to transformed phenotypes. Using PD98059, an inhibitor of the ERK-kinase MEK1, we have here assessed the effects of ERK inhibition on the pattern of protein expression in the metastatic human breast cancer cell line MDA-MB-231. At a concentration of inhibitor which did not significantly affect cell growth, PD98059 induced large changes in the expression of MDA-MB-231 polypeptides. The majority of these changes were due to decreased expression of low-abundance proteins. Decreases of more abundant proteins such as glutathione-S-transferase pi, hsp80 and hsp100 were also recorded. The levels of a few proteins increased, among them cytokeratin 8. We conclude that PD98059 treatment of MDA-MB-231 cells induces large changes in protein expression.
Assuntos
Neoplasias da Mama/enzimologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eletroforese em Gel Bidimensional , Feminino , Perfilação da Expressão Gênica , Humanos , MAP Quinase Quinase 1 , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Metástase Neoplásica , Proteínas de Neoplasias/fisiologia , Peptídeos/análise , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Técnica de Subtração , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologiaRESUMO
The process of tumor progression leads to the emergence of multiple clones, and to the development of tumor heterogeneity. One approach to the study of the extent of such heterogeneity is to examine the expression of marker proteins in different tumor areas. Two-dimensional gel electrophoresis (2-DE) is a powerful tool for such studies, since the expression of a large number of polypeptide markers can be evaluated. In the present study, tumor cells were prepared from human ovarian tumors and analyzed by 2-DE and PDQUEST. As judged from the analysis of two different areas in each of nine ovarian tumors, the intratumoral variation in protein expression was low. In contrast, large differences were observed when the protein profiles of different tumors were compared. The differences in gene expression between pairs of malignant carcinomas were slightly larger than the differences observed between pairs of benign tumors. We conclude that 2-DE analysis of intratumoral heterogeneity in ovarian cancer tissue indicates a low degree of heterogeneity.
Assuntos
Cistadenoma Mucinoso/química , Cistadenoma Seroso/química , Eletroforese em Gel Bidimensional/métodos , Processamento de Imagem Assistida por Computador , Proteínas de Neoplasias/análise , Neoplasias Ovarianas/química , Cistadenoma Mucinoso/classificação , Cistadenoma Mucinoso/genética , Cistadenoma Mucinoso/patologia , Cistadenoma Seroso/classificação , Cistadenoma Seroso/genética , Cistadenoma Seroso/patologia , Feminino , Heterogeneidade Genética , Humanos , Processamento de Imagem Assistida por Computador/métodos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/classificação , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/classificação , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , SoftwareRESUMO
Cells were collected from prostate hyperplasias (n = 6) and prostate carcinomas (n = 6) and subjected to two-dimensional gel electrophoresis (2-DE). The resulting polypeptide patterns were analysed with the PDQUEST computer software. Malignant tumors showed significant increases in the level of expression of proliferating cell nuclear antigen (PCNA), calreticulin, HSP 90 and pHSP 60, oncoprotein 18(v), elongation factor 2, glutathione-S-transferase pi (GST-pi), superoxide dismutase and triose phosphate isomerase. In addition, decreases in the levels of tropomyosin-1 and 2 and cytokeratin 18 were observed in prostate carcinomas compared to prostate hyperplasias. This pattern of alterations is similar to that observed in other carcinomas in our previous studies. All malignant tumors showed simultaneous alterations in 5 or more of 9 markers studied, whereas only one case of benign hyperplasia showed alterations in 5 markers. The EST-data base for prostate tumors available from NCI (CGAP) was searched for the expression of the mRNAs corresponding to proteins identified in our gels. Large differences in the relative expression of mRNAs and proteins were observed. Our data show alterations in the pattem of polypeptide expression in prostate carcinomas which are similar to those observed in other carcinomas.
Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/biossíntese , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica/métodos , Proteínas de Neoplasias/biossíntese , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/metabolismo , Adenocarcinoma/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Hiperplasia Prostática/genética , Neoplasias da Próstata/genéticaRESUMO
Surfactant protein A (SP-A), a major protein component of natural pulmonary surfactant, is absent in exogenous surfactants currently used in clinical practice. We investigated the physical and physiological properties of one of these modified natural surfactants (Curosurf) after enrichment with 5% SP-A (SP-A-Curosurf). A pulsating bubble system was used for in vitro assessments and ventilated newborn rabbits for evaluation of in vivo effects. In the presence of various potential inhibitors (meconium 5 mg.mL-1, fibrinogen 5 mg.mL-1, albumin 25 mg.mL-1, or whole serum proteins 25 mg.mL-1), Curosurf at a concentration of 5 mg.mL-1 was inactivated while SP-A-Curosurf and natural porcine surfactant at the same concentration had normal maximum and minimum surface tension. This protective effect of SP-A was calcium dependent. In immature newborn rabbits, the improvement of lung-thorax compliance observed after treatment with 100 mg.kg-1 of SP-A-Curosurf was equivalent to that obtained with 200 mg.kg-1 of Curosurf. Similarly, in near-term newborn rabbits with respiratory failure induced by instillation of fibrinogen via the airways, the increase in compliance after administration of 100 mg.kg-1 of SP-A-Curosurf corresponded to that seen after treatment with 200 mg.kg-1 of Curosurf, whereas Curosurf at a dose of 100 mg.kg-1 had no substantial effect. Our data thus indicate that surfactant protein A increases the resistance of Curosurf to inactivation under in vivo conditions.
Assuntos
Produtos Biológicos , Fosfolipídeos , Proteolipídeos/farmacologia , Surfactantes Pulmonares/farmacologia , Síndrome do Desconforto Respiratório do Recém-Nascido/fisiopatologia , Albuminas/farmacologia , Animais , Animais Recém-Nascidos , Ácido Edético/farmacologia , Fibrinogênio/farmacologia , Frequência Cardíaca , Humanos , Concentração de Íons de Hidrogênio , Recém-Nascido , Complacência Pulmonar , Mecônio , Peso Molecular , Proteolipídeos/química , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/química , Coelhos , Propriedades de Superfície , SuínosRESUMO
Results of two-dimensional electrophoresis (2-DE) analyses of human breast carcinoma are described. Tumor cells were extracted and purified from breast carcinomas with different proliferative indeces and degrees of genomic stability. Cells purified from fibroadenoma tissue served as controls for benign cells. The following results were observed: (i) Analysis of samples from different areas of the same tumor showed a high degree of similarity in the pattern of polypeptide expression. Similarly, analysis of two tumors and their metastases revealed similar 2-DE profiles. (ii) In contrast, large variations were observed between different lesions with comparable histological characteristics. Larger differences in polypeptide expression were observed between potentially highly malignant carcinomas compared to comparisons of less malignant lesions. These differences were in the same order of magnitude as those observed comparing a breast carcinoma to a lung carcinoma. (iii) The levels of all cytokeratin forms resolved (CK7, CK8, CK15, and CK18) were significantly lower in carcinomas compared to fibroadenomas. (iv) The levels of high molecular weight tropomyosins (1-3) were lower in carcinomas compared to fibroadenomas. The expression of tropomyosin-1 was found to be 1.7-fold higher in primary tumors with metastatic spread to axillar lymph nodes compared to primary tumors with no evidence of metastasis (p < 0.05). (v) The expression of proliferating cell nuclear antigen (PCNA) and some members of the stress protein family (pHSP60, HSP90, and calreticulin) were higher in carcinomas. We conclude that malignant progression of breast carcinomas results in large heterogeneity in polypeptide expression between different tumors, but that some common themes such as decreased expression of cytokeratin and tropomyosin polypeptides can be discerned.
Assuntos
Neoplasias da Mama/química , Proteínas de Neoplasias/análise , Animais , Neoplasias da Mama/patologia , Linhagem Celular , Chaperonina 60/análise , Regulação para Baixo , Feminino , Fibroadenoma/química , Proteínas de Choque Térmico HSP90/análise , Humanos , Queratinas/análise , Metástase Neoplásica , Peptídeos/análise , Ratos , Tropomiosina/análiseRESUMO
Large amounts of data on quantitative gene expression are generated by procedures such as 2-DE analysis of proteins or cDNA microarrays. Quantitative molecular variation may potentially be used for the development of methods for the classification of tumors. We used here the statistical concepts of principal components analysis (PCA) and partial least square analysis (PLS) in an attempt to type ovarian tumors. Using a set of 170 polypeptides, 22 tumors were used to establish a model ("learning set") for classification into 3 groups (benign/borderline/malignant). Eighteen tumors were then used to test the model. Six of 8 carcinomas and 3 of 4 borderline tumors were correctly classified. Two of 6 benign lesions were correctly classified, 3 were classified as borderline and 1 as carcinoma. We conclude that it may be possible to classify tumors according to their constitutive protein expression profile using multivariate analysis, thus making classification by artificial intelligence a future possibility.
Assuntos
Neoplasias Ovarianas/classificação , Peptídeos/análise , Neoplasias da Mama/química , Eletroforese em Gel Bidimensional/métodos , Feminino , Humanos , Análise Multivariada , Neoplasias Ovarianas/química , Neoplasias Ovarianas/metabolismo , Mapeamento de Peptídeos , Células Tumorais CultivadasRESUMO
Sample preparation procedures are critical for obtaining high quality two-dimensional electrophoresis (2-DE) separations from clinical tumor materials. In this communication, we describe procedures which we have found satisfactory for preparing various tumor samples for 2-DE. By these procedures, tumor cells are enriched from serum proteins and contaminating stromal cells. Tumors can be prepared with good reproducibility to obtain high resolution using these techniques.
Assuntos
Eletroforese em Gel Bidimensional , Proteínas de Neoplasias/análise , Manejo de Espécimes/métodos , Humanos , Reprodutibilidade dos TestesRESUMO
Malignant progression of tumour cells is caused by the accumulation of genetic defects, which when combined will generate a large phenotypic diversity. Simultaneous quantitation of a large number of gene products in tumour cells is desirable, but difficult to achieve. We have here quantitated the levels of a number of abundant polypeptides in human breast carcinoma cells using two-dimensional gel electrophoresis (2-DE; PDQUEST). For this purpose, tumour cells were prepared from the tissue of 17 breast carcinomas. Fibroadenoma tissue was used as reference for benign cells. An increase of the spot density of the PCNA polypeptide was observed in rapidly proliferating tumour cells, confirming the validity of the procedures used. In the set of 24 polypeptide spots with known identity, decreases in cytokeratin and tropomyosin levels were observed. The levels of all cytokeratin forms resolved (CK7, CK8, CK15 and CK18) were significantly lower in carcinomas than in fibroadenomas. The levels of tropomyosin 2 and 3 were lower in carcinomas than in fibroadenomas. In contrast, the levels of some members of the stress protein family (pHSP60, HSP90 and calreticulin) were higher in carcinomas. Furthermore, changes in the expression of lactate dehydrogenase and GT-pi, but not in nm23, were observed. We conclude that simultaneous analysis of multiple polypeptides in human carcinomas can be achieved by 2-DE and may be useful in prognostic studies, and that malignant progression of breast carcinomas results in the decreased expression of cytokeratin polypeptides. This phenomenon must be considered in studies where cytokeratins are used as markers to identify the epithelial cell compartment in breast carcinomas.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Regulação para Baixo/fisiologia , Fibroadenoma/metabolismo , Queratinas/metabolismo , Proteínas Monoméricas de Ligação ao GTP , Proteínas de Neoplasias/metabolismo , Núcleosídeo-Difosfato Quinase , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Eletroforese em Gel Bidimensional , Feminino , Glutationa Transferase/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Nucleosídeo NM23 Difosfato Quinases , Fatores de Transcrição/metabolismoRESUMO
Studies of multiple markers in tumors are required for adequate biological characterization. We have characterized the expression of multiple proteins in human ovarian tumors using the technique of 2-dimensional gel electrophoresis (2-DE/PDQUEST). Tumor cells were prepared from the tissue of 22 ovarian tumors. Large variations were observed between tumors in the expression of various polypeptides, indicating heterogeneity in gene expression. An increase in the spot density of 2 cell-cycle-related proteins, PCNA and OP18/stathmin, was observed in carcinomas. Borderline tumors expressed low levels of these proteins. Significant increases in the levels of nm23, GST-pi, elongation factor 2 and triose phosphate isomerase were recorded in ovarian carcinomas. Furthermore, decreases in the levels of tropomyosin-2 and lamin C were observed in malignant as compared with benign tumors. The pattern of expression of 9 protein markers was examined in individual tumors. All malignant tumors showed simultaneous alterations in the expression of 5 or more of these proteins, whereas no benign tumor showed alterations in the expression of more than 3 polypeptides. Borderline tumors showed alterations in 0 to 6 markers. We conclude that the simultaneous analysis of multiple polypeptides, which can be achieved by 2-DE, is useful for characterization of gene expression and diagnostic studies in ovarian tumors.
Assuntos
Proteínas de Neoplasias/análise , Neoplasias Ovarianas/química , Fragmentos de Peptídeos/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Eletroforese em Gel Bidimensional , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia , Mapeamento de Peptídeos , FenótipoRESUMO
High molecular weight tropomyosins (tms) are commonly down-regulated in fibroblasts transformed by oncogenes. Previous studies have also demonstrated that specific tm isoforms are down-regulated in human breast carcinoma cell lines. We examined tropomyosin isoforms in cells prepared from non-cancerous breast lesions and primary human breast carcinomas. The average level of expression of all three high molecular weight tm isoforms (tm 1-3) in carcinomas was generally found to be less than 25% of that observed in non-cancerous breast lesions. Interestingly, the expression of tm 1 was found to be 1.7-fold higher in primary tumours with metastatic spread to axillary lymph nodes compared with primary tumours with no evidence of metastasis (p<0.05). Similarly, tm 1 expression was higher in two 12V-H-ras transformed fibroblast cell lines capable of experimental metastasis compared with three weakly metastatic cell lines. We conclude from these studies that expression of high molecular weight tm isoforms is low in primary breast carcinomas, and that metastatic tumours express relatively high levels of tm 1.
Assuntos
Neoplasias da Mama/metabolismo , Mama/metabolismo , Proteínas de Neoplasias/biossíntese , Tropomiosina/biossíntese , Mama/anormalidades , Mama/patologia , Transformação Celular Neoplásica/genética , Feminino , Fibroadenoma/metabolismo , Fibroblastos/metabolismo , Genes ras , Hamartoma/metabolismo , Humanos , Hiperplasia/metabolismo , Isomerismo , Metástase Linfática , Tropomiosina/metabolismo , Células Tumorais CultivadasRESUMO
Protein patterns in cells collected from benign prostatic tissues and prostate carcinomas were analyzed using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Polypeptide expression was evaluated by computer-assisted image analysis (PDQUEST). Proteins expressed by prostate tumors were identified via in-gel digestion and subsequent matrix-assisted laser desorption/ionization mass spectrometry. In addition to cytoskeletal and mitochondrial proteins, a 40-kDa protein was identified as prostatic acid phosphatase (PAP). PAP expression decreased approximately twofold between benign and malignant tissue. Increased expression of heat shock protein 70 and decreased expression of tropomyosin 1 were also observed in the malignant tissue. The analysis of prostate material by two-dimensional gel electrophoresis and mass spectrometry shows that particular proteins are of interest as markers of disease.
Assuntos
Proteínas de Drosophila , Eletroforese em Gel Bidimensional/métodos , Proteínas de Neoplasias/análise , Neoplasias da Próstata/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fosfatase Ácida/análise , Adenocarcinoma/química , Idoso , Idoso de 80 Anos ou mais , Proteínas de Choque Térmico HSP70/análise , Humanos , Masculino , Pessoa de Meia-Idade , Hiperplasia Prostática/metabolismo , Tropomiosina/análiseRESUMO
Two-dimensional polyacrylamide gel electrophoresis combined with a non-enzymatic sample preparation technique is useful for analysing clinical tumour material. Using these techniques, we analysed the relationship between the histopathological findings in primary lung malignancies and the expression of a number of unidentified polypeptides that were detected in the molecular weight region 20-35 kDa. In this study 45 cases of primary lung cancer (PLC) (21 cases of adenocarcinoma, ten cases of squamous cell carcinoma, five cases of large-cell carcinoma, one case of adenosquamous cell carcinoma, five cases of small-cell carcinoma and three cases of carcinoid tumour) were examined. For reference, a human diploid fibroblast cell line (W138) and normal peripheral lymphocytes were used. Sixteen polypeptides were judged to be associated with histopathological features. These polypeptides seem to be valuable as differentiation markers. The simultaneous evaluation of these polypeptides and some other proliferation markers (e.g. PCNA, PCNA 'satellite', Numatin/protein B23 and lamin B) seems to clarify the characteristics of each case of PLC. Furthermore, it is possible to classify PLC based on the two-dimensional electrophoresis findings, and this classification of PLC is suggested to reflect the biological features of the tumour more precisely than that based only on morphology.