RESUMEN
The B-cell translocation gene 2, BTG2, a member of the BTG/TOB (B-cell translocation gene/transducers of ErbB2) gene family, has been implicated in cell cycle regulation, normal development, and possibly tumor suppression. Previously, it was shown that BTG2 expression is lost or down-regulated in human breast cancers. We now report that BTG2 protects human mammary epithelial cells from oxidative stress due to hydrogen peroxide and other oxidants. BTG2 protection against oxidative stress is BRCA1-independent but requires the antioxidant transcription factor NFE2L2 and is associated with up-regulation of the expression of antioxidant enzymes, including catalase and superoxide dismutases 1 and 2. BTG2 stimulation of antioxidant gene expression is also NFE2L2-dependent. We further demonstrate that BTG2 is a binding partner for NFE2L2 and increases its transcriptional activity. In addition, BTG2 is detectable at the antioxidant response element (ARE) of several NFE2L2-responsive genes. Finally, we show that the ability of BTG2 to associate with NFE2L2, to protect cells against oxidative stress, and to stimulate antioxidant gene expression requires box B, a short highly conserved amino acid motif characteristic of BTG2/TOB family proteins, but does not require boxes A or C. These findings suggest a novel role for BTG2 as a co-activator for NFE2L2 in up-regulating cellular antioxidant defenses.
Asunto(s)
Antioxidantes/metabolismo , Células Epiteliales/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Glándulas Mamarias Humanas/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/fisiología , Proteínas Supresoras de Tumor/metabolismo , Secuencias de Aminoácidos , Línea Celular Tumoral , Células Epiteliales/citología , Femenino , Humanos , Peróxido de Hidrógeno/farmacología , Proteínas Inmediatas-Precoces/genética , Glándulas Mamarias Humanas/citología , Factor 2 Relacionado con NF-E2/genética , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Elementos de Respuesta/fisiología , Proteínas Supresoras de Tumor/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
The pathogenesis of Type 2 diabetes mellitus (T2DM) is complex owing to molecular heterogeneity in the afflicted population. Current diagnostic methods rely on blood glucose measurements, which are noninformative with respect to progression of the disease to other associated pathologies. Thus, predicting the risk and development of T2DM-related complications, such as cardiovascular disease, remains a major challenge. We have used a combination of quantitative methods for characterization of circulating serum biomarkers of T2DM using a cohort of nondiabetic control subjects (n = 76) and patients diagnosed with T2DM (n = 106). In this case-control study, the samples were randomly divided as training and validation data sets. In the first step, iTRAQ (isobaric tagging for relative and absolute quantification) based protein expression profiling was performed for identification of proteins displaying a significant differential expression in the two study groups. Five of these protein markers were selected for validation using multiple reaction-monitoring mass spectrometry (MRM-MS) and further confirmed with Western blot and QPCR analysis. Functional pathway analysis identified perturbations in lipid and small molecule metabolism as well as pathways that lead to disruption of glucose homeostasis and blood coagulation. These putative biomarkers may be clinically useful for subset stratification of T2DM patients as well as for the development of novel therapeutics targeting the specific pathology.
Asunto(s)
Biomarcadores/sangre , Proteínas Sanguíneas/metabolismo , Diabetes Mellitus Tipo 2/sangre , Cromatografía Liquida , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masas en TándemRESUMEN
The link between loss or defect in functional BRCA1 and predisposition for development of ovarian and breast cancer is well established. Germ-line mutations in BRCA1 are responsible for both hereditary breast and ovarian cancer, which is around 5-10% for all breast and 10-15% of all ovarian cancer cases. However, majority of cases of ovarian cancer are sporadic in nature. The inactivation of cellular BRCA1 due to mutations or loss of heterozygosity is one of the most commonly observed events in such cases. Complement-resistant retroviral BRCA1 vector, MFG-BRCA1, is the only approved gene therapy for ovarian cancer patients by the Federal and Drug Administration. Given the limited available information, there is a need to evaluate the effects of BRCA1 on the global gene expression pattern for better understanding the etiology of the disease. Here, we use Ingenuity Pathway Knowledge Base to examine the differential pattern of global gene expression due to stable expression of BRCA1 in the ovarian cancer cell line, SKOV3. The functional analysis detected at least five major pathways that were significantly (p < 0.05) altered. These include: cell to cell signaling and interaction, cellular function and maintenance, cellular growth and proliferation, cell cycle and DNA replication, and recombination repair. In addition, we were able to detect several biologically relevant genes that are central for various signaling networks involved in cellular homeostasis; TGF-ß1, TP53, c-MYC, NF-κB and TNF-α. This report provides a comprehensive rationale for tumor suppressor function(s) of BRCA1 in ovarian carcinogenesis.
Asunto(s)
Proteína BRCA1/metabolismo , Neoplasias Ováricas/metabolismo , Transducción de Señal , Proteína BRCA1/genética , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Análisis por Conglomerados , Reparación del ADN , Replicación del ADN , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Mapas de Interacción de ProteínasRESUMEN
Follistatin (FST), a folliculogenesis regulating protein, is found in relatively high concentrations in female ovarian tissues. FST acts as an antagonist to Activin, which is often elevated in human ovarian carcinoma, and thus may serve as a potential target for therapeutic intervention against ovarian cancer. The breast cancer susceptibility gene 1 (BRCA1) is a known tumor suppressor gene in human breast cancer; however its role in ovarian cancer is not well understood. We performed microarray analysis on human ovarian carcinoma cell line SKOV3 that stably overexpress wild-type BRCA1 and compared with the corresponding empty vector-transfected clones. We found that stable expression of BRCA1 not only stimulates FST secretion but also simultaneously inhibits Activin expression. To determine the physiological importance of this phenomenon, we further investigated the effect of cellular BRCA1 on the FST secretion in immortalized ovarian surface epithelial (IOSE) cells derived from either normal human ovaries or ovaries of an ovarian cancer patient carrying a mutation in BRCA1 gene. Knock-down of BRCA1 in normal IOSE cells demonstrates down-regulation of FST secretion along with the simultaneous up-regulation of Activin expression. Furthermore, knock-down of FST in IOSE cell lines as well as SKOV3 cell line showed significantly reduced cell proliferation and decreased cell migration when compared with the respective controls. Thus, these findings suggest a novel function for BRCA1 as a regulator of FST expression and function in human ovarian cells.
Asunto(s)
Proteína BRCA1/metabolismo , Folistatina/metabolismo , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/metabolismo , Activinas/genética , Activinas/metabolismo , Proteína BRCA1/genética , Western Blotting , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Movimiento Celular/genética , Células Cultivadas , Análisis por Conglomerados , Femenino , Folistatina/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Glandulares y Epiteliales/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
BACKGROUND: Topo-poisons can produce an enzyme-DNA complex linked by a 3'- or 5'-phosphotyrosyl covalent bond. 3'-phosphotyrosyl bonds can be repaired by tyrosyl DNA phosphodiesterase-1 (TDP1), an enzyme known for years, but a complementary human enzyme 5'-tyrosyl DNA phosphodiesterase (hTDP2) that cleaves 5'-phosphotyrosyl bonds has been reported only recently. Although hTDP2 possesses both 3'- and 5'- tyrosyl DNA phosphodiesterase activity, the role of Mg2+ in its activity was not studied in sufficient details. RESULTS: In this study we showed that purified hTDP2 does not exhibit any 5'-phosphotyrosyl phosphodiesterase activity in the absence of Mg2+/Mn2+, and that neither Zn2+ or nor Ca2+ can activate hTDP2. Mg2+ also controls 3'-phosphotyrosyl activity of TDP2. In MCF-7 cell extracts and de-yolked zebrafish embryo extracts, Mg2+ controlled 5'-phosphotyrosyl activity. This study also showed that there is an optimal Mg2+ concentration above which it is inhibitory for hTDP2 activity. CONCLUSION: These results altogether reveal the optimal Mg2+ requirement in hTDP2 mediated reaction.
Asunto(s)
Embrión no Mamífero/enzimología , Proteínas de Peces/metabolismo , Magnesio/metabolismo , Manganeso/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Pez Cebra/metabolismo , Animales , Calcio/metabolismo , Extractos Celulares/química , ADN/metabolismo , Proteínas de Unión al ADN , Electroforesis en Gel de Poliacrilamida , Embrión no Mamífero/embriología , Activación Enzimática , Escherichia coli/genética , Proteínas de Peces/aislamiento & purificación , Humanos , Células MCF-7 , Proteínas Nucleares/genética , Proteínas Nucleares/aislamiento & purificación , Oligonucleótidos/metabolismo , Hidrolasas Diéster Fosfóricas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Extractos de Tejidos/química , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación , Pez Cebra/embriología , Zinc/metabolismoRESUMEN
Posttranslational modifications (PTMs) modulate protein function in most eukaryotes and have a ubiquitous role in diverse range of cellular functions. Identification, characterization, and mapping of these modifications to specific amino acid residues on proteins are critical towards understanding their functional significance in a biological context. The interpretation of proteome data obtained from the high-throughput methods cannot be deciphered unambiguously without a priori knowledge of protein modifications. An in-depth understanding of protein PTMs is important not only for gaining a perception of a wide array of cellular functions but also towards developing drug therapies for many life-threatening diseases like cancer and neurodegenerative disorders. Many of the protein modifications like ubiquitination play a decisive role in various drug response(s) and eventually in disease prognosis. Thus, many commonly observed PTMs are routinely tracked as disease markers while many others are used as molecular targets for developing target-specific therapies. In this paper, we summarize some of the major, well-studied protein alterations and highlight their importance in various chronic diseases and normal development. In addition, other promising minor modifications such as SUMOylation, observed to impact cellular dynamics as well as disease pathology, are mentioned briefly.