RESUMO
BACKGROUND: Transforming growth factor ß-induced protein (TGFBI) is a secreted protein that mediates cell anchoring to the extracellular matrix. This protein is downregulated in lung cancer, and when overexpressed, contributes to apoptotic cell death. Using a small series of stage IV non-small cell lung cancer (NSCLC) patients, we previously suggested the usefulness of TGFBI as a prognostic and predictive factor in chemotherapy-treated late-stage NSCLC. In order to validate and extend these results, we broaden the analysis and studied TGFBI expression in a large series of samples obtained from stage I-IV NSCLC patients. METHODS: TGFBI expression was assessed by immunohistochemistry in 364 completely resected primary NSCLC samples: 242 adenocarcinomas (ADCs) and 122 squamous cell carcinomas (SCCs). Kaplan-Meier curves, log-rank tests and the Cox proportional hazards model were used to analyse the association between TGFBI expression and survival. RESULTS: High TGFBI levels were associated with longer overall survival (OS, P<0.001) and progression-free survival (PFS, P<0.001) in SCC patients who received adjuvant platinium-based chemotherapy. Moreover, multivariate analysis demonstrated that high TGFBI expression is an independent predictor of better survival in patients (OS: P=0.030 and PFS: P=0.026). CONCLUSIONS: TGFBI may be useful for the identification of a subset of NSCLC who may benefit from adjuvant therapy.
Assuntos
Neoplasias Pulmonares/metabolismo , Fator de Crescimento Transformador beta1/biossíntese , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Idoso , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Quimioterapia Adjuvante , Regulação para Baixo , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Prognóstico , Texas/epidemiologia , Fator de Crescimento Transformador beta1/genética , Resultado do TratamentoRESUMO
TGFbeta-induced protein (TGFBI) is an extracellular protein that mediates cell adhesion to collagen, laminin and fibronectin through its interaction with different beta integrins. We had previously reported that hypoxia-induced TGFBI mRNA expression in lymphatic endothelial cells (LEC). Here, we demonstrate that TGFBI can contribute to hypoxia-induced increases in LEC adhesion to the ECM. We show that while there are no changes in alpha1, alpha4, alphav, beta1, beta2, beta3, alpha5beta1, alphavbeta3, alphavbeta5 integrin expression on the LEC surface after hypoxia exposure, there exists an accumulation of TGFBI adaptor protein in LEC supernatants. We also demonstrate that hypoxia driven TGBFI expression is dependent on TGFbeta production by LEC. Furthermore, we show that TGFBI mediated LEC adhesion and migration through the ECM by its binding to the beta3 integrin. The identification of the specific mechanisms regulating LEC-ECM interactions may help us design new therapeutic applications for diseases in which lymphatic vessel function is compromised.
Assuntos
Adesão Celular/fisiologia , Células Endoteliais/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Sequência de Bases , Hipóxia Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Primers do DNA/genética , Matriz Extracelular/fisiologia , Proteínas da Matriz Extracelular/genética , Humanos , Integrinas/fisiologia , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Regulação para CimaRESUMO
Recent advances in our understanding of the molecular biology of lymphatic endothelial cells have revealed that these vessels, besides their known function in tissue homeostasis and immunity, constitute conduits for the tumor cells to metastasize. One of the factors that contribute to tumor spread is the acquisition of an angiogenic phenotype as a response to the onset of tumor hypoxia. To our knowledge, little is known about the effects of low oxygen levels on the lymphatic vasculature. Therefore, we used cDNA microarrays to study the transcriptional changes occurring in hypoxia exposed lymphatic endothelial cells. Our analysis was then complemented by functional assays showing that these cells responded with increased attachment to the extracellular matrix, delayed proliferation and production of reactive oxygen species. Differential expression of genes involved in these processes such as NADPH oxidase 4, the tissue inhibitor of metalloproteinase 3, and TGFbeta induced protein I, was found. Hypoxia was also found to increase mRNA levels of the cytokine CXCL-12 and its receptor CXCR4. Moreover, adhesion experiments revealed that hypoxia increased the binding of non-small cell lung carcinoma cells to this endothelium in a CXCR4 dependent way. We thus illustrate the response of lymphatic endothelial cells to hypoxia and suggest targets to study tumor metastasis through these vessels.
Assuntos
Células Endoteliais/metabolismo , Regulação da Expressão Gênica/fisiologia , Vasos Linfáticos/metabolismo , Adesão Celular , Hipóxia Celular , Células Cultivadas , Células Endoteliais/patologia , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Humanos , Metástase Linfática , Vasos Linfáticos/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Espécies Reativas de Oxigênio/metabolismoRESUMO
PCPH was initially defined as a proto-oncogene on the basis of its frequent detection as an activated oncogene in tumorigenic Syrian hamster embryo fibroblast cell lines converted to the neoplastic state by a single treatment with the carcinogen 3-methylcholanthrene (MC). Further studies identified the translation product of the PCPH gene as a ribonucleotide-binding protein with special affinity for ribonucleoside diphosphates. Later, we showed that the PCPH protein was homologous to the product of the yeast GDA1 gene and demonstrated that it had intrinsic guanosine diphosphatase activity, although it did not complement the disrupted phenotype when expressed in gda1 null Saccharomyces cerevisiae strains. These results indicated that the primary function of PCPH was unlikely to be related to the ribonucleotide recycling function that its yeast counterpart performs in the Golgi during the process of protein glycosylation. However, taken together, our data strongly suggested that the normal cellular function of PCPH was related to ribonucleotide metabolism. We now report that PCPH is structurally and functionally identical to the mammalian ectonucleoside triphosphate diphosphohydrolase CD39L4 (ENTPD5), recently described as a member of the lymphoid activation antigen (
Assuntos
Adenosina Trifosfatases/genética , Proteínas Oncogênicas/genética , Adenosina Trifosfatases/química , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Humanos , Dados de Sequência Molecular , Mutação , Proteínas Oncogênicas/química , Monoéster Fosfórico Hidrolases/metabolismo , Biossíntese de Proteínas , Proto-Oncogene Mas , Pirofosfatases , Splicing de RNA , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
No disponible
Assuntos
Feminino , Humanos , Neoplasias da Mama/genética , Proteínas Oncogênicas/genética , Expressão Gênica/genética , Carcinoma/genética , Transformação Celular Neoplásica/genética , CongressoRESUMO
Expression of the Protein Product of the PCPH Proto-oncogene in Human Tumor Cell Lines. Exposure of Syrian hamster embryo fibroblasts to chemical carcinogens resulted in the oncogenic activation of the PCPH proto-oncogene by induction of a single base-pair deletion that generated a truncated PCPH oncoprotein (mutated PCPH). Recently, we isolated and characterized the cDNA for the human PCPH proto-oncogene and determined that in humans PCPH is a single-copy gene located in chromosome 14 (14q24.3). Pilot mRNA expression studies indicated that PCPH was expressed in the majority of normal organs tested, particularly in liver and kidney, but it appeared to be expressed either at low levels or not at all in tumor cells or cell lines derived from the high-expressing tissues. We have generated an antiserum against bacterial recombinant Syrian hamster PCPH. This antiserum recognizes both the normal and truncated, oncogenic Syrian hamster PCPH proteins and cross-reacts with the yeast, mouse, rat and human homologue proteins. Using this antibody, we have performed a study of PCPH expression in a larger sample of human neoplastic cell lines, including some derived from breast, nervous system, colon, lung and pancreas tumors. Results confirmed the frequent lack of PCPH expression in malignant cells and identified several immunoreactive forms of PCPH being differentially expressed in cells of diverse tissue origins.
Assuntos
Transformação Celular Neoplásica/metabolismo , Neoplasias/metabolismo , Proteínas Oncogênicas/biossíntese , Animais , Western Blotting , Transformação Celular Neoplásica/genética , Células Epiteliais/metabolismo , Expressão Gênica , Humanos , Soros Imunes , Neoplasias/genética , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/imunologia , Proto-Oncogene Mas , Pirofosfatases , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Tumorais CultivadasRESUMO
We identified a human cDNA encoding a 47-kDa protein that shares 78% and 87% identity with the products of the Syrian hamster and mouse PCPH proto-oncogenes respectively. The human homolog was localized by radiation-hybrid mapping to chromosome band 14q24.3, a region syntenic to the Pcph location on mouse chromosome 12. Northern analyses revealed that PCPH mRNA was widely distributed in normal human adult tissues, but its expression varied significantly among human tumor cells and cell lines of several tissue types, regardless of the level of expression in the corresponding normal tissues. The highest levels of PCPH mRNA and protein were detected in kidney and liver. However, PCPH was not expressed in the majority of human neoplasms tested, including kidney tumors. These data provide suggestive evidence for a possible association of the lack of PCPH expression to the neoplastic phenotype of human tumor cells. Our results should prove instrumental in designing studies to define the cellular function of the human PCPH proto-oncogene.
Assuntos
Mapeamento Cromossômico , Proteínas Oncogênicas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cricetinae , DNA Complementar , Humanos , Mesocricetus , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , Proto-Oncogene Mas , Homologia de Sequência de AminoácidosRESUMO
Differential-display or RNA fingerprint was applied to identify genes differentially expressed in monocyte maturation induced by an immunomodulating peptide on human peripheral blood mononuclear cells. Two unknown sequences (06c22 and 06c71) and p21 protein (cyclin dependent kinase inhibitor) were repressed, and three genes activated: Cathepsin D, DRP2 (dihydropirimidinase related protein 2), and gp91phox (91-kDa subunit of citochrome b(558)). Phenotype of evolving monocytes was analyzed by flow cytometry and mRNA level of identified genes determined by reverse transcription-PCR. The expression pattern of identified genes seemed to correlate with different monocyte subsets, monocyte-derived cells, and expected functional changes. After peptide addition, immature monocytes were initially activated, increasing the expression of CD25, CD69, and HLA-DR markers. This was accompanied by repression of p21 and the two unknown sequences, along with the simultaneous activation of Cathepsin D and DRP2. Later, the differentiation marker CD16 rose, and gp91phox gene expression activated. Further maturation led certain monocytes to express marker CD23 and gp91phox expression to reach a maximum, while Cathepsin D and DRP2 dropped to preactivation levels. Results reflect part of the evolution of immature monocytes toward macrophages and monocyte-derived dendritic cell precursors.
Assuntos
Catepsina D/genética , Regulação da Expressão Gênica , Leucócitos Mononucleares/fisiologia , Glicoproteínas de Membrana/genética , Proteínas/genética , Sequência de Bases , Proteínas Sanguíneas/genética , Catepsina D/sangue , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/sangue , Ciclinas/genética , Primers do DNA , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Leucócitos Mononucleares/citologia , Glicoproteínas de Membrana/sangue , NADPH Oxidase 2 , NADPH Oxidases/sangue , NADPH Oxidases/genética , Proteínas do Tecido Nervoso , Fenótipo , Proteínas/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNARESUMO
Human monocyte subsets, isolated from cultures of mononuclear cells, or freshly obtained from patients with multiple sclerosis, Graves' disease or pemphigus vulgaris, differed in phenotype, apoptotic features, mRNA levels of arginase II (A-II) and the inducible form of nitric oxide synthase (iNOS). Liver-type arginase I mRNA was present in all subsets. Apoptosis was followed by the expression of T cell intracellular antigen (TIA) and the simultaneous detection of DNA stainability by propidium iodine and annexin V binding. Apoptosis was practically absent both in activated CD14(++)CD33(++)DR(++)CD25(++)CD69(++)CD71(++/+) CD16(-) cells, expressing A-II mRNA and having arginase activity, but not iNOS mRNA, and in not fully mature large CD14(++)CD16(+)CD23(+)DR(++) monocytes, expressing simultaneously both mRNAs and having both enzyme activities. However, differentiated small CD14(+/++)CD16(+)CD69(+)CD25(+/-)CD71(++)CD23(+) DR(++) monocytes, expressing high levels of iNOS mRNA, exhibited apoptotic signs. Amounts of NO synthesised by monocytes co-expressing iNOS and arginase changed with the addition of arginine or an iNOS inhibitor; in that case a correlation of NO production and apoptotic features was observed. Data suggest a regulatory role for endogenous NO in apoptosis of stimulated and differentiated monocytes, and also that iNOS and A-II, when simultaneously present, could control the production of NO as a consequence of their competition for arginine.
Assuntos
Apoptose/fisiologia , Arginase/biossíntese , Monócitos/enzimologia , Óxido Nítrico Sintase/biossíntese , Antígenos CD/análise , Antígenos CD/genética , Arginase/análise , Citometria de Fluxo , Expressão Gênica , Doença de Graves/sangue , Humanos , Esclerose Múltipla/sangue , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase Tipo II , Pênfigo/sangue , Fenótipo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Activated and differentiated human monocytes with a CD14+CD16+ phenotype were found to contain a DNase activity associated with secretion granules. Activated cells were obtained from patients with autoimmune diseases. Activation and differentiation of monocytes were also achieved after incubation of PBMC from healthy subjects with protein A (SpA) or immunopotentiating peptides. DNase activity corresponded to a 66-kDa protein, similar to that described in granules from T lymphocytes, active preferentially on double-strand DNA. DNA fragmentation activity increased when NO donors were present; the activity was higher in the presence of Ca2+, and at low pH values. The Ca2+-dependent activity was inhibited by Zn2+. NO-dependent activity was additive with that of Ca2+-dependent and it was not inhibited by Zn2+. Dithiothreitol did not modify the effect of NO on DNase activity. Incubation of PBMC in the presence of NMLA, an inhibitor of NO synthases, decreased this DNase activity. Data reported clearly suggest a regulatory role of NO in granule-associated DNase activity.
Assuntos
Doenças Autoimunes/enzimologia , Grânulos Citoplasmáticos/enzimologia , Desoxirribonucleases/metabolismo , Monócitos/enzimologia , Óxido Nítrico/farmacologia , Adulto , Cálcio/farmacologia , Células Cultivadas , Ditiotreitol/farmacologia , Ativação Enzimática , Inibidores Enzimáticos , Feminino , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase/antagonistas & inibidores , Zinco/farmacologia , ômega-N-Metilarginina/farmacologiaRESUMO
In order to dissect the molecular mechanisms of monocytic differentiation we have developed a subtractive hybridisation method based on a simplified 'representational difference analysis'. We have selected 16 sequences and confirmed their down-regulation along the TPA-induced monocytic differentiation of HL60 cells. Among these sequences we have identified the alpha-tubulin, the TaxREB protein and two ribosomal protein sequences which had not been previously described as differentially expressed. These results add to our knowledge about the molecules implicated along the monocytic differentiation and growth arrest of leukemic cells and provide a first step in the study of their respective roles.