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1.
Actas urol. esp ; 38(9): 600-607, nov. 2014. ilus, tab
Artículo en Español | IBECS | ID: ibc-129344

RESUMEN

Introducción: El síndrome de Lynch o cáncer colorrectal hereditario no polipósico se debe a mutaciones en los genes de reparación de ADN, conocidos como mismatch repair (MMR), y se asocian a inestabilidad de microsatélites. El carcinoma urotelial de la pelvis renal también se asocia a este síndrome, e incluso estas anomalías genéticas se han descrito en formas esporádicas de carcinoma urotelial del tracto superior (CU-TUS). Material y método: Estudio descriptivo y análisis de supervivencia en una serie de 80 pacientes con CU-TUS esporádico sin metástasis al diagnóstico (N0/Nx M0) tratados mediante nefroureterectomía exclusivamente. Se evalúa la expresión de genes MMR (hMLH1, hPMS2, hMSH2 y hMSH6) en secciones realizadas con microarray de tejido (TMA) y su asociación con parámetros clínico-patológicos. Se analiza el valor pronóstico de la pérdida de expresión de estos genes en CU-TUS. Resultados: No se detectó pérdida de MSH2 ni de MSH6, pero se evidenció pérdida de MLH1 en 11 casos (13,8%) y de PMS2 en 21 (26,3%). La expresión de hMLH1 y hPMS2 se encuentra fuertemente asociada (p < 0,0001) y este fenotipo de expresión conlleva importantes implicaciones clínicas. Pérdida de MLH1 se asocia con bajo grado (p = 0,02). Pérdida de PMS2 se asocia con menor estadio (p = 0,05), patrón empujante sin borde invasivo (p = 0,008) y menos angiogénesis (p = 0,008). La inactivación dehPMS2 o hMLH1es un factor protector independiente (HR 0,309) y junto con el grado histológico (HR 5,561) define el pronóstico de estos pacientes. Conclusión: En nuestra experiencia la inactivación de hPMS2 o hMLH1 es un marcador independiente de buen pronóstico y sucede en la cuarta parte de los CU-TUS esporádicos. El estudio IHQ de estos pacientes puede emplearse para valorar el cribado de formas ocultas de síndrome de Lynch


Introduction: Lynch syndrome or hereditary nonpolyposis colorectal cancer is caused by mutations in DNA repair genes, known as mismatch repair (MMR) genes, and is associated with microsatellite instability. Urothelial carcinoma of the renal pelvis is also associated with this syndrome. These genetic abnormalities have been described in sporadic forms of upper tract urothelial carcinoma (UTUC). Material and method: This was a descriptive study and survival analysis of a series of 80 patients with sporadic UTUC with no metastases at diagnosis (N0/Nx M0) treated exclusively with nephroureterectomy. We evaluated the expression of MMR genes (hMLH1, hPMS2, hMSH2 and hMSH6) in sections performed with tissue microarray (TMA) and their association with clinical-pathological parameters. We analyzed the prognostic value of the loss of expression of these genes in UTUC. Results: We detected no loss of MSH2 or of MSH6, but there was a loss of MLH1 in 11 cases (13.8%) and of PMS2 in 21 cases (26.3%). The expression of hMLH1 and hPMS2 were strongly associated (P < .0001), and this phenotype expression entails significant clinical implications. The loss of MLH1 was associated with a low grade (P = .02). Loss of PMS2 was associated with a lower stage (P = .05), a pushing pattern with no invasive edges (P = .008) and less angiogenesis (P = .008). The inactivation ofhPMS2 or hMLH1 is an independent protective factor (HR, 0.309) and, along with the histologic grade (HR, 5.561), defines the patients’ prognosis. Conclusion: In our experience, the inactivation of hPMS2 or hMLH1 is an independent marker of good prognosis and occurs in a quarter of sporadic UTUC cases. The immunohistochemical study of these patients can be used to assess the screening of hidden forms of Lynch syndrome


Asunto(s)
Humanos , Reparación del ADN/genética , Reparación del Gen Blanco , Carcinoma de Células Transicionales/genética , Neoplasias Urológicas/genética , Sistema Urinario/patología , Inestabilidad de Microsatélites , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Estudios Prospectivos
2.
Actas Urol Esp ; 38(9): 600-7, 2014 Nov.
Artículo en Inglés, Español | MEDLINE | ID: mdl-24958312

RESUMEN

INTRODUCTION: Lynch syndrome or hereditary nonpolyposis colorectal cancer is caused by mutations in DNA repair genes, known as mismatch repair (MMR) genes, and is associated with microsatellite instability. Urothelial carcinoma of the renal pelvis is also associated with this syndrome. These genetic abnormalities have been described in sporadic forms of upper tract urothelial carcinoma (UTUC). MATERIAL AND METHOD: This was a descriptive study and survival analysis of a series of 80 patients with sporadic UTUC with no metastases at diagnosis (N0/Nx M0) treated exclusively with nephroureterectomy. We evaluated the expression of MMR genes (hMLH1, hPMS2, hMSH2 and hMSH6) in sections performed with tissue microarray (TMA) and their association with clinical-pathological parameters. We analyzed the prognostic value of the loss of expression of these genes in UTUC. RESULTS: We detected no loss of MSH2 or of MSH6, but there was a loss of MLH1 in 11 cases (13.8%) and of PMS2 in 21 cases (26.3%). The expression of hMLH1 and hPMS2 were strongly associated (P<.0001), and this phenotype expression entails significant clinical implications. The loss of MLH1 was associated with a low grade (P=.02). Loss of PMS2 was associated with a lower stage (P=.05), a pushing pattern with no invasive edges (P=.008) and less angiogenesis (P=.008). The inactivation of hPMS2 or hMLH1 is an independent protective factor (HR, 0.309) and, along with the histologic grade (HR, 5.561), defines the patients' prognosis. CONCLUSION: In our experience, the inactivation of hPMS2 or hMLH1 is an independent marker of good prognosis and occurs in a quarter of sporadic UTUC cases. The immunohistochemical study of these patients can be used to assess the screening of hidden forms of Lynch syndrome.


Asunto(s)
Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/mortalidad , Reparación del ADN/genética , Neoplasias Renales/genética , Neoplasias Renales/mortalidad , Pelvis Renal , Femenino , Humanos , Masculino , Pronóstico , Estudios Prospectivos , Análisis de Supervivencia
3.
Actas urol. esp ; 37(6): 368-375, jun. 2013. tab, ilus
Artículo en Español | IBECS | ID: ibc-113276

RESUMEN

Objetivos: Realizar una síntesis de los principales avances en el campo del estudio de la epigenética y concretamente la metilación de ADN respecto al diagnóstico de las neoplasias urológicas. Adquisición de evidencia: Revisión de la literatura (PubMed, Medline y Cochrane) sobre el estudio de la metilación del ADN en neoplasias urológicas (cáncer de próstata, cáncer de vejiga, cáncer renal y cáncer testicular) teniendo en cuenta todos los estudios publicados hasta enero de 2013. Síntesis de evidencia: Resulta posible determinar el estado de metilación de un gran número de genes en muestras de tumores, que al compararlo con muestras de tejido sano permite la definición de patrones de metilación aberrantes específicos para cada tipo de tumor. El estudio y la definición de patrones de metilación anómala específicos de cada tipo de tumor es una herramienta de potencial utilidad para el diagnóstico, evaluación, predicción de pronóstico y tratamiento de las diferentes formas de cáncer genitourinario. El análisis de la metilación de genes en la orina, tras la micción o masaje prostático, el semen, el plasma o el líquido de lavado de biopsias prostáticas puede permitir la detección precoz del cáncer vesical, prostático, renal y testicular. En cada una de las neoplasias se ha identificado una firma epigenética que puede detectarse en ADN obtenido de muestras muy escasamente o nada invasivas, con potencial en el diagnóstico y en la evaluación de pronóstico. La validación de estos estudios confirmará la precisión, efectividad y reproducibilidad de los resultados de los que se dispone hasta el momento. No están aún desarrollados criterios que determinen que un panel de genes sea lo suficientemente informativo en la práctica asistencial como para guiar un diagnóstico inequívoco o una conducta terapéutica. Se requiere un mayor número de estudios para contrastar en cada caso la sensibilidad, especificidad, el valor predictivo positivo y negativo de la prueba. Tampoco existen estudios multicéntricos que analicen la reproducibilidad real de estos resultados en un entorno clínico. Conclusiones: El estudio de la metilación aberrante del ADN en muestras biológicas de pacientes tiene enorme potencial para el diagnóstico precoz y cribado de las neoplasias genitourinarias. Se necesita un mayor número de estudios que permitan definir baterías de genes que supongan firmas inequívocas de malignidad. Esta metodología tiene también potencial a la hora de definir grupos pronóstico y potencial de respuesta a diferentes terapias (AU)


Objectives: We have synthesized the principal advances in the field of the study of epigenetics and specifically DNA methylation regarding the diagnosis of urological neoplasms. Acquisition of evidence: Review of the literature (PubMed, MEDLINE y COCHRANE) on the study of DNA methylation in urological neoplasms (prostate cancer, bladder cancer, renal cancer and testicular cancer), considering all the studies published up to January 2013. Synthesis of evidence: It was possible to determine the state of methylation of many genes in our tumor samples. When these were compared with healthy tissue samples, it was possible to define the specific aberrant methylation patterns for each type of tumor. The study and definition of specific abnormal methylation patterns of each type of tumor is a tool having potential utility for diagnosis, evaluation, prediction of prognosis and treatment of the different forms of genitourinary cancer. The analysis of gene methylation in urine after micturition or post-prostatic massage urine, semen, in the wash plasma or fluid from prostatic biopsies may allow early detection of bladder, prostate, renal and testicular cancer. In each one of the neoplasms, an epigenetic signature that may be detected in the DNA has been identified, obtained from very scarce or not at all invasive specimens, with potential in the diagnosis and evaluation of prognosis. Validation of these studies will confirm the accuracy, effectiveness and reproducibility of the results available up to now. Criteria have still not been developed that determine if a gene panel provides sufficient information in the health care practice to guide an unequivocal diagnosis or therapeutic conduct. More studies are needed to compare sensitivity, specificity, positive and negative predictive value of the test in each case. Multicenter studies analyzing the real reproducibility of these results in a clinical setting also do not exist. Conclusions: The study of aberrant DNA methylation in biological specimens of patients has an enormous potential for the early diagnosis and screening of genitourinary neoplasms. A larger number of studies is needed to be able to define the series of genes that would mean unequivocal signatures of malignancy. This methodology also has potential when defining prognostic groups and potential of response to different therapies (AU)


Asunto(s)
Humanos , Masculino , Metilación de ADN , Neoplasias Urológicas/genética , Epigénesis Genética/genética , Reacción en Cadena de la Polimerasa/métodos , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Próstata/genética , Neoplasias Renales/genética , Neoplasias Testiculares/genética
4.
Actas Urol Esp ; 37(6): 368-75, 2013 Jun.
Artículo en Inglés, Español | MEDLINE | ID: mdl-23643196

RESUMEN

OBJECTIVES: We have synthesized the principal advances in the field of the study of epigenetics and specifically DNA methylation regarding the diagnosis of urological neoplasms. ACQUISITION OF EVIDENCE: Review of the literature (PubMed, MEDLINE and Cochrane) on the study of DNA methylation in urological neoplasms (prostate cancer, bladder cancer, renal cancer and testicular cancer), considering all the studies published up to January 2013. SYNTHESIS OF EVIDENCE: It was possible to determine the state of methylation of many genes in our tumor samples. When these were compared with healthy tissue samples, it was possible to define the specific aberrant methylation patterns for each type of tumor. The study and definition of specific abnormal methylation patterns of each type of tumor is a tool having potential utility for diagnosis, evaluation, prediction of prognosis and treatment of the different forms of genitourinary cancer. The analysis of gene methylation in urine after micturition or post-prostatic massage urine, semen, in the wash plasma or fluid from prostatic biopsies may allow early detection of bladder, prostate, renal and testicular cancer. In each one of the neoplasms, an epigenetic signature that may be detected in the DNA has been identified, obtained from very scarce or not at all invasive specimens, with potential in the diagnosis and evaluation of prognosis. Validation of these studies will confirm the accuracy, effectiveness and reproducibility of the results available up to now. Criteria have still not been developed that determine if a gene panel provides sufficient information in the health care practice to guide an unequivocal diagnosis or therapeutic conduct. More studies are needed to compare sensitivity, specificity, positive and negative predictive value of the test in each case. Multicenter studies analyzing the real reproducibility of these results in a clinical setting also do not exist. CONCLUSIONS: The study of aberrant DNA methylation in biological specimens of patients has an enormous potential for the early diagnosis and screening of genitourinary neoplasms. A larger number of studies is needed to be able to define the series of genes that would mean unequivocal signatures of malignancy. This methodology also has potential when defining prognostic groups and potential of response to different therapies.


Asunto(s)
Metilación de ADN , ADN de Neoplasias/genética , Neoplasias de los Genitales Masculinos/genética , Neoplasias Urológicas/genética , Islas de CpG , ADN de Neoplasias/análisis , Femenino , Predicción , Genes Relacionados con las Neoplasias , Neoplasias de los Genitales Masculinos/diagnóstico , Neoplasias de los Genitales Masculinos/metabolismo , Humanos , Masculino , Reacción en Cadena de la Polimerasa/métodos , Pronóstico , Análisis de Semen , Sensibilidad y Especificidad , Urinálisis , Neoplasias Urológicas/diagnóstico , Neoplasias Urológicas/orina
5.
Oncogene ; 29(3): 345-55, 2010 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-19838216

RESUMEN

SHP-1, a haematopoietic cell-specific tyrosine phosphatase, is also expressed in human prostate. In this study, we report that SHP-1 depletion in PC-3 cells induced by small interfering RNAs causes G1 phase cell-cycle arrest accompanied by changes in some components of the cell-cycle machinery. SHP-1 knockdown increases p27(Kip1) (p27) protein stability, its nuclear localization and p27 gene transcription. These effects could be mediated by PI3K-AKT pathway as SHP-1 interacts with PI3K regulating its activity and p110 catalytic subunit phosphorylation. The increase in p27 protein stability could also because of reduced cyclin-dependent kinase (CDK2) activity. SHP-1 knockdown decreases the CDK6 levels, inducing retinoblastoma protein hypophosphorylation, downregulation of cyclin E and thereby a decrease in the CDK2 activity. However, the codepletion of SHP-1 and p27 does not produce re-entry into the cycle, implying that p27 is not required to maintain cell-cycle arrest induced by SHP-1 depletion. The maintenance of the PC-3 cell anti-proliferative response after p27 loss could be because of mislocalization of CDK2 induced by SHP-1 knockdown. This study shows that SHP-1 depletion promotes cell-cycle arrest by modulating the activity of cell-cycle regulators and suggests that SHP-1 may be required for the proper functioning of events governing cell-cycle progression.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Interferencia de ARN , Western Blotting , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Ciclina E/genética , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Fase G1 , Regulación Neoplásica de la Expresión Génica , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Fosforilación , Regiones Promotoras Genéticas/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fase S
6.
Oncogene ; 28(11): 1477-84, 2009 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-19169274

RESUMEN

Histone deacetylases (HDACs) play a key role in the regulation of gene expression and chromatin structure, and drugs targeting these enzymes might have an important impact in the treatment of human cancer. Herein, we report the characterization of (1H)-pyrroles as a new subfamily of HDAC inhibitors obtained by computational modeling of class-I human HDACs. From a functional standpoint, (1H)-pyrroles are powerful inductors of acetylation of histones H3 and H4, and restore the expression of growth-inhibitory genes. From a cellular view, these compounds cause a marked decrease in the viability of cancer cells in vitro and in vivo, associated with a cell-cycle arrest at G2/M and an inhibition of angiogenesis. Thus, (1H)-pyrroles emerge as a novel group of HDAC inhibitors with promising antitumoral features.


Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Pirroles/farmacología , Animales , Línea Celular Tumoral , Simulación por Computador , Relación Dosis-Respuesta a Droga , Humanos , Ácidos Hidroxámicos/farmacología , Ratones , Modelos Moleculares , Relación Estructura-Actividad , Vorinostat , Ensayos Antitumor por Modelo de Xenoinjerto
7.
Oncogene ; 28(6): 781-91, 2009 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-19060927

RESUMEN

Sirtuin 1 (Sirt1) and Sirtuin 2 (Sirt2) belong to the family of NAD+ (nicotinamide adenine dinucleotide-positive)-dependent class III histone deacetylases and are involved in regulating lifespan. As cancer is a disease of ageing, targeting Sirtuins is emerging as a promising antitumour strategy. Here we present Salermide (N-{3-[(2-hydroxy-naphthalen-1-ylmethylene)-amino]-phenyl}-2-phenyl-propionamide), a reverse amide with a strong in vitro inhibitory effect on Sirt1 and Sirt2. Salermide was well tolerated by mice at concentrations up to 100 muM and prompted tumour-specific cell death in a wide range of human cancer cell lines. The antitumour activity of Salermide was primarily because of a massive induction of apoptosis. This was independent of global tubulin and K16H4 acetylation, which ruled out a putative Sirt2-mediated apoptotic pathway and suggested an in vivo mechanism of action through Sirt1. Consistently with this, RNA interference-mediated knockdown of Sirt1, but not Sirt2, induced apoptosis in cancer cells. Although p53 has been reported to be a target of Sirt1, genetic p53 knockdowns showed that the Sirt1-dependent proapoptotic effect of Salermide is p53-independent. We were finally able to ascribe the apoptotic effect of Salermide to the reactivation of proapoptotic genes epigenetically repressed exclusively in cancer cells by Sirt1. Taken together, our results underline Salermide's promise as an anticancer drug and provide evidence for the molecular mechanism through which Sirt1 is involved in human tumorigenesis.


Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Naftoles/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Fenilpropionatos/farmacología , Sirtuinas/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Femenino , Genes p53 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Naftoles/química , Fenilpropionatos/química , Sirtuina 1 , Sirtuina 2 , Sirtuinas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo
8.
Oncogene ; 27(28): 4008-12, 2008 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-18264134

RESUMEN

Although disruption of histone modification patterns is a common hallmark of human cancer, our knowledge of the mechanistic role of histone-modifying enzymes in its generation is very limited. We have recently identified an inactivating mutation in the histone deacetylase-2 (HDAC2) in sporadic carcinomas with microsatellite instability and in tumors arising in individuals with hereditary nonpolyposis colorectal cancer syndrome. Since HDAC2 seems to be a central player in epigenetic gene repression, we wondered whether HDAC2-truncating mutations conferred a particular expression signature on these cancer cells. Using unsupervised clustering analysis in microsatellite-unstable colorectal cancer cell lines, we have found that HDAC2 mutant cells (RKO and Co115) show a characteristically different expression microarray signature from HDAC2 wild-type cells (HCT-116, SW48, HCT-15 and LoVo). HDAC2 mutant cells exhibit upregulation of tumor-promoting genes, such as those of tyrosine kinases, mediators of cell cycle progression and angiogenic factors. The overexpression of these genes is associated with a loss of HDAC2 recruitment and a gain of histone H4 hyperacetylation in their particular 5'-end promoters, as observed by chromatin immunoprecipitation. Transfection of wild-type HDAC2 in mutant cells reverted this epigenetic pattern by repressing the transforming genes in association with HDAC2 promoter occupancy. These results suggest a role for HDAC2 mutations in human tumorigenesis through the derepression of key genes from multiple cellular transformation pathways.


Asunto(s)
Epigénesis Genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasas/genética , Histona Desacetilasas/fisiología , Mutación , Neoplasias/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Línea Celular Tumoral , Cromatina/metabolismo , Análisis por Conglomerados , Histona Desacetilasa 2 , Histonas/metabolismo , Humanos , Repeticiones de Microsatélite , Neovascularización Patológica , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo
9.
Oncogene ; 27(25): 3556-66, 2008 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-18223687

RESUMEN

Methyl-cytosine-phosphate-guanine (CpG)-binding domain (MBD) proteins are bound to hypermethylated promoter CpG islands of tumor suppressor genes in human cancer cells, although a direct causal relationship at the genome-wide level between MBD presence and gene silencing remains to be demonstrated. To this end, we have inhibited the expression of MBD proteins in HeLa cells by short hairpin RNAs; and studied the functional consequences of MBD depletion using microarray-based expression analysis in conjunction with extensive bisulfite genomic sequencing and chromatin immunoprecipitation. The removal of MBDs results in a release of gene silencing associated with a loss of MBD occupancy in 5'-CpG islands without any change in the DNA methylation pattern. Our results unveil new targets for epigenetic inactivation mediated by MBDs in transformed cells, such as the cell adhesion protein gamma-parvin and the fibroblast growth factor 19, where we also demonstrate their bona fide tumor suppressor features. Our data support a fundamental role for MBD proteins in the direct maintenance of transcriptional repression of tumor suppressors and identify new candidate genes for epigenetic disruption in cancer cells.


Asunto(s)
Islas de CpG , Epigénesis Genética , Silenciador del Gen , Genes Supresores de Tumor , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Metilación de ADN , Factores de Crecimiento de Fibroblastos/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/metabolismo
10.
Clin. transl. oncol. (Print) ; 8(11): 812-820, nov. 2006. ilus
Artículo en Inglés | IBECS | ID: ibc-126238

RESUMEN

BACKGROUND: Data derived from epidemiological and experimental studies suggest that alphalinolenic acid (ALA; 18:3n-3), the main omega-3 polyunsaturated fatty acid (PUFA) present in the Western diet, may have protective effects in breast cancer risk and metastatic progression. A recent pilot clinical trial assessing the effects of ALA-rich dietary flaxseed on tumor biological markers in postmenopausal patients with primary breast cancer demonstrated significant reductions in tumor growth and in HER2 (erbB-2) oncogene expression. HYPOTHESIS: The molecular mechanism by which ALA inhibits breast cancer cell growth and metastasis formation may involve a direct regulation of HER2, a well-characterized oncogene playing a key role in the etiology, progression and response to some chemo- and endocrine therapies in approximately 20% of breast carcinomas. METHODS: Using HER2-specific ELISA, flow cytometry, immunofluorescence microscopy, Western blotting, RT-PCR and HER2 promoter-reporter analyses, we characterized the effects of exogenous supplementation with ALA on the expression of HER2 oncogene, a master key player in the onset and metastasis formation of breast cancer disease. Metabolic status (MTT) assays were performed to evaluate the nature of the cytotoxic interaction between ALA and the humanized anti-HER2 monoclonal antibody trastuzumab (Herceptin). To study these issues we used BT-474 and SKBr-3 breast cancer cells, which naturally exhibit amplification of the HER2 oncogene. RESULTS: ALA treatment dramatically suppressed the expression of HER2-coded p185Her-2/neu oncoprotein as determined by ELISA, flow cytometry, immunofluorescence microscopy and immunoblotting techniques. Interestingly, ALA-induced down-regulation of p185Her-2/neu correlated with a transcriptional response as no HER2 mRNA signal could be detected by RT-PCR upon treatment with optimal concentrations of ALA (up to 20 microM). Consistent with these findings, ALA exposure was found to dramatically repress the activity of a Luciferase reporter gene driven by the HER2 promoter. Moreover, the nature of the cytotoxic interaction between ALA and trastuzumab (Herceptin) revealed a significant synergism as assessed by MTT-based cell viability assays. CONCLUSIONS: i) These findings reveal that the omega-3 PUFA ALA suppresses overexpression of HER2 oncogene at the transcriptional level, which, in turn, interacts synergistically with anti-HER2 trastuzumab- based immunotherapy. ii) Our results molecularly support a recent randomized double-blind placebo-controlled clinical trial suggesting that ALA may be a potential dietary alternative or adjunct to currently used drugs in the management of HER2-positive breast carcinomas. iii) Considering our previous findings demonstrating the <> of the omega-6 PUFA linolenic acid (LA; 18:2n-6) and the <> of the omega-3 PUFA docosahexaenoic acid (DHA; 22:6n-3) and of the omega-9 monounsaturated fatty acid oleic acid (OA; 18:1n-9), it is reasonable to suggest that a low omega-6/omega-3 PUFA ratio and elevated MUFA levels, the two prominent <> of the <>, should be extremely efficient at blocking HER2 expression in breast cancer cells (AU)


Asunto(s)
Humanos , Femenino , Anticuerpos Monoclonales Humanizados , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/prevención & control , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Carcinoma Ductal de Mama/prevención & control , Genes erbB-2 , /antagonistas & inhibidores , /biosíntesis , Línea Celular Tumoral , Línea Celular Tumoral/metabolismo , Dieta Mediterránea , Grasas de la Dieta , Ácidos Grasos , Inducción Enzimática
11.
Carcinogenesis ; 27(5): 1099-104, 2006 May.
Artículo en Inglés | MEDLINE | ID: mdl-16410262

RESUMEN

The transcription factor aryl hydrocarbon receptor (AhR) has relevant functions in cell proliferation. Interestingly, the AhR can either promote or inhibit proliferation depending on the cell phenotype. Although recent data reveal potential pathways for AhR signaling in cell proliferation, the mechanisms that regulate its activity in tumor cells remain unknown. Here, we have analyzed promoter hypermethylation as a potential mechanism controlling AhR expression in human tumor cells. AhR promoter CpG methylation was sporadic in a panel of 19 tumor cell lines except for the chronic myeloid leukemia (CML) K562 and the acute lymphoblastic leukemia (ALL) REH. When compared with normal lymphocytes, REH had very low constitutive AhR expression that could be attributed to promoter hypermethylation since treatment with the DNA demethylating agent 5-aza-2'-deoxycitidine (AZA) significantly increased AhR mRNA and protein. These results in leukemia-derived cell lines were further confirmed in primary ALL, where 33% of the patients (7/21) had AhR promoter hypermethylation. Chromatin immunoprecipitation (ChIP) showed that methylation impaired binding of the transcription factor Sp1 to the AhR promoter, thus providing a mechanism for AhR downregulation in REH cells. Therefore, promoter hypermethylation represents a novel epigenetic mechanism downregulating AhR activity in hematological malignancies such as ALL.


Asunto(s)
Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Regiones Promotoras Genéticas , Receptores de Hidrocarburo de Aril/genética , Factor de Transcripción Sp1/genética , Azacitidina/análogos & derivados , Azacitidina/farmacología , Secuencia de Bases , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Islas de CpG , Decitabina , Humanos , Datos de Secuencia Molecular , Unión Proteica
13.
Rev. senol. patol. mamar. (Ed. impr.) ; 14(1): 8-9, ene. 2001. tab, ilus, graf
Artículo en Es | IBECS | ID: ibc-683

RESUMEN

La amplificación del oncogén HER2 fue descrita en humanos en 1985; detectándose en el 20-30 por ciento de cánceres de mama. En 1987 se describió el valor pronóstico de la amplificación en cáncer de mama, y más tarde de la sobreexpresión del RNAm y de la proteína. Dos de las aplicaciones clínicas actuales del oncogén HER2 son el valor pronóstico y el valor predictivo de la sobreexpresión del gen en cáncer de mama. Una tercera aplicación de la oncoproteína HER2 es su papel como diana terapéutica para los nuevos tratamientos que están dirigidos contra la proteína HER2.En la actualidad existe el consenso de que la expresión de HER2 en carcinomas de mama tiene un valor pronóstico adverso, aunque en general es de menor importancia que el valor pronóstico que confieren indicadores clásicos como el tamaño tumoral o el número de ganglios axilares afectados.Respecto al valor predictivo de HER2, todos los estudios clínicos realizados, salvo uno, han mostrado que existe una asociación entre la expresión de HER2 y la eficacia de los tratamientos hormonales. La diferencia entre la respuesta clínica al tamoxifeno u otros tratamientos endocrinos es, en general, muy marcada, y en varios de los estudios clínicos la eficacia de la hormonoterapia en las pacientes con sobreexpresión de HER2 es de la mitad o menos de la mitad que en las pacientes sin sobreexpresión de HER2. Las investigaciones realizadas en cáncer de mama primario o metastásico sugieren que HER2 puede tener valor predictivo de resistencia a la quimioterapia. La asociación de HER2 con la resistencia a la quimioterapia es especialmente notable en tres estudios que se han realizado en pacientes con cáncer avanzado de mama, en las que se ha evaluado la expresión del dominio extracelular de HER2 en el suero, y que han mostrado que la eficacia de la quimioterapia puede verse reducida a menos de la mitad en los casos HER2 positivos. Aunque en este momento no se pueden efectuar recomendaciones terapéuticas definitivas basadas en los datos de los que se disponen, sí debe recomendarse la participación de las pacientes con sobreexpresión de HER2 en ensayos clínicos que investiguen el uso de los tratamientos estándar con o sin la adición de tratamientos dirigidos contra HER2, como el anticuerpo monoclonal Herceptin (AU)


Asunto(s)
Femenino , Humanos , Genes erbB-2/genética , Amplificación de Genes/métodos , Neoplasias de la Mama/genética , Genes erbB-2 , Terapia de Reemplazo de Hormonas , Pronóstico , Congreso , Valor Predictivo de las Pruebas , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/tratamiento farmacológico , Tamoxifeno/farmacología
14.
Endocrinology ; 141(3): 1093-9, 2000 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-10698185

RESUMEN

Previous results from our group have indicated that arachidonic acid decrease cAMP production through a modification of heterotrimeric G proteins. In the present study, we have characterized the high affinity GTPase activity present in Leydig cell membranes and its regulation by fatty acids. The high-affinity GTPase activity, measured as [gamma32P] GTP hydrolysis rate, was both time and protein concentration dependent. Arachidonic acid elicited a dose-dependent inhibition of enzyme activity with an IC50 = 26.7+/-1.1 microM. The existence of only two double bonds in linoleic acid is reflected by a decrease in its inhibitory activity (IC50 = 34+/-2.3 microM). Saturated fatty acids showed no effect at this level. The kinetic analysis as interpreted by Lineweaver-Burk plots, indicated that 50 microM arachidonic acid had no effect on the apparent affinity for GTP, but resulted in a 40% decreases in the maximal velocity of the reaction. Arachidonic acid modulation of GTPase activity was not attenuated by blocking eicosanoid metabolism with inhibitors of 5'-lipoxygenase, cyclooxygenase, or epoxygenase P-450. The addition of arachidonic acid to pertussis toxin-treated membranes had no effect on the enzyme activity, indicating that arachidonic acid does not modify the GTPase activity present in Galphas protein. However, ADP-ribosylation with cholera toxin followed by arachidonic acid treatment led to a further 40% inhibition when compared with cholera toxin treatment alone. These results allowed us to postulate that arachidonic acid inhibits the GTPase activity of Gi protein family. To further analyze the mechanism of arachidonic acid inhibition of GTPase activity, the effect of arachidonic acid on the [35S]GTPgammaS binding was studied. No effect of this fatty acid on GTP binding was found. Combining our previous results with those found here, we can conclude that arachidonic acid maintains Gi proteins in their active state, which in turn inhibit adenylate cyclase and results in decrease cAMP levels.


Asunto(s)
Ácido Araquidónico/farmacología , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Células Intersticiales del Testículo/enzimología , Toxina de Adenilato Ciclasa , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Ciguatoxinas/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Trifosfato/metabolismo , Cinética , Células Intersticiales del Testículo/efectos de los fármacos , Masculino , Toxina del Pertussis , Ratas , Ratas Sprague-Dawley , Factores de Virulencia de Bordetella/farmacología
15.
J Cell Biochem ; 76(3): 368-75, 2000 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-10649434

RESUMEN

The Galpha subunits of heterotrimeric G proteins are constituted by a conserved GTPase "Ras-like" domain (RasD) and by a unique alpha-helical domain (HD). Upon GTP binding, four regions, called switch I, II, III, and IV, have been identified as undergoing structural changes. Switch I, II, and III are located in RasD and switch IV in HD. All Galpha known functions, such as GTPase activity and receptor, effector, and Gbetagamma interaction sites have been found to be localized in RasD, but little is known about the role of HD and its switch IV region. Through the construction of chimeras between human and Xenopus Gsalpha we have previously identified a HD region, encompassing helices alphaA, alphaB, and alphaC, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEBS Lett 340:249-254). Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gsalpha proteins, in the present work we constructed two human Gsalpha mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGsalphaalphaS133N, M135P, P138K, P143S) and M17 (hGsalphaalphaS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and characterized by their ability to bind GTPgammaS, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP release, GTPgammaS binding, and GTP hydrolysis was observed for both mutants, M17 having considerably slower kinetics than M15 for all functions tested. Reconstituted adenylyl cyclase activity with both mutants showed normal activation in the presence of AlF(4)(-), but a decreased activation with GTPgammaS, which is consistent with the lower GDP dissociating rate they displayed. These data provide new evidence on the role that HD is playing in modulating the GDP/GTP exchange of the Gsalpha subunit.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Adenilil Ciclasas/metabolismo , Secuencia de Bases , Cartilla de ADN/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Expresión Génica , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Técnicas In Vitro , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tripsina
16.
FEBS Lett ; 401(1): 68-72, 1997 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-9003808

RESUMEN

We show that the levels and activity of the alpha-subunits of Gs and Gi proteins in plasma membrane of GH4C1 cells are regulated by the availability of mevalonate (MVA), and not by changes in cholesterol cell content. Changes in the levels of MVA, induced by modulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, determine the amount of both membrane-bound G alpha-subunits, which correlated with the activity of their effector adenylyl cyclase. Lipoprotein deficient serum (LPDS) decreases cholesterol content and increases both HMG-CoA reductase activity and G alpha-subunits in the membrane. Cholesterol and 25-hydroxycholesterol (25-HC) each repress HMG-CoA reductase and diminish G alpha-subunit levels. However, while cholesterol cell content is also decreased by 25-HC, exogenous cholesterol increases it. In addition, the decrease of both G alpha-subunits is reversed by the presence of MVA. This regulation appears to be mediated by nonsterol products generated from MVA. We assume that the first is the prenylation of the gamma-subunits, since the attachment of G alpha-subunits to the membrane is dependent on this modification. However, as neither of our treatments completely abolished protein prenylation, we conclude that another MVA derivative is required in addition to prenyl residues to the presence and activity of alpha-subunits in the membrane.


Asunto(s)
Proteínas de Unión al GTP/metabolismo , Ácido Mevalónico/metabolismo , Sangre , Línea Celular , Membrana Celular/metabolismo , Colesterol/farmacología , Hidroxicolesteroles/farmacología , Transducción de Señal
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