RESUMEN
BACKGROUND: ß-secretase (BACE1) is a type 1 transmembrane protein implicated in Alzheimer's Disease (AD) pathogenesis. Cleavage of Amyloid Precursor Protein (APP), initiated by BACE1 and followed by γ-secretase, leads to the formation of toxic Aß peptides. Increased levels of BACE1 have been detected in the CSF of AD patients compared to age-matched healthy controls indicating that neurodegenerative conditions induce shedding of BACE1. OBJECTIVE: To mimic such conditions, we examined whether serum deprivation stimulates proteolysis-dependent secretion of BACE1. METHOD: Detection of BACE1 secretion in BACE1 overexpressing cells or ADAM10/ADAM17 knockout fibroblasts cultured under serum deprivation conditions, using western blot analysis. RESULTS: We found that serum deprivation of U251 neuroblastoma or HEK293T cells overexpressing BACE1 stimulated secretion of BACE1. Using ADAM10/ADAM17 knockout fibroblasts and inhibitors of both ADAM10 and ADAM17, we obtained data indicating that these proteases are involved in serum-starvation induced shedding of BACE1. This is unexpected since BACE1 is localized mainly in lipid rafts while ADAM10 is localized mainly in nonlipid raft domains. We hypothesized that serum deprivation results in alterations in the lipid composition of the membrane which can alter the localization of ADAM10 and BACE1. In support, we obtained results indicating that extraction of membrane cholesterol following incubation with methyl ß cyclodextrin potentiated the effect of serum deprivation. Secreted BACE1 was also found to be enzymatically active towards immunoprecipiated full length APP. CONCLUSION: Serum starvation induces ADAM10-mediated BACE1 secretion.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Suero/metabolismo , Estrés Fisiológico/fisiología , Proteína ADAM10/antagonistas & inhibidores , Proteína ADAM10/deficiencia , Proteína ADAM10/genética , Proteína ADAM17/antagonistas & inhibidores , Proteína ADAM17/deficiencia , Proteína ADAM17/genética , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/genética , Línea Celular Tumoral , Colesterol/metabolismo , Medio de Cultivo Libre de Suero , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HEK293 , Humanos , Microdominios de Membrana/química , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Estrés Fisiológico/efectos de los fármacos , Tensoactivos/farmacología , beta-Ciclodextrinas/farmacologíaRESUMEN
We have screened 473 breast/ovarian cancer patients with family history, aiming to define the prevalence and enrich the spectrum of BRCA1/2 pathogenic mutations occurring in the Greek population. An overall mutation prevalence of 32% was observed. Six BRCA1 recurrent/founder mutations dominate the observed spectrum (58.5% of all mutations found). These include three mutations in exon 20 and three large genomic deletions. Of the 44 different deleterious mutations found in both genes, 16 are novel and reported here for the first time. Correlation with available histopathology data showed that 80% of BRCA1 carriers presented a triple-negative breast cancer phenotype while 82% of BRCA2 carriers had oestrogen receptor positive tumours. This study provides a comprehensive view of the frequency, type and distribution of BRCA1/2 mutations in the Greek population as well as an insight of the screening strategy of choice for patients of Greek origin. We conclude that the Greek population has a diverse mutation spectrum influenced by strong founder effects.
Asunto(s)
Efecto Fundador , Genes BRCA1 , Síndrome de Cáncer de Mama y Ovario Hereditario/epidemiología , Síndrome de Cáncer de Mama y Ovario Hereditario/genética , Mutación , Femenino , Genes BRCA2 , Mutación de Línea Germinal , Grecia/epidemiología , Heterocigoto , Humanos , Masculino , Tasa de Mutación , Polimorfismo Genético , PrevalenciaRESUMEN
In this study, we investigated the regulation of FOXM1 expression by estrogen receptor alpha (ERalpha) and its role in hormonal therapy and endocrine resistance. FOXM1 protein and mRNA expression was regulated by ER-ligands, including estrogen, tamoxifen (OHT) and fulvestrant (ICI182780; ICI) in breast carcinoma cell lines. Depletion of ERalpha by RNA interference (RNAi) in MCF-7 cells downregulated FOXM1 expression. Reporter gene assays showed that ERalpha activates FOXM1 transcription through an estrogen-response element (ERE) located within the proximal promoter region. The direct binding of ERalpha to the FOXM1 promoter was confirmed in vitro by mobility shift and DNA pull-down assays and in vivo by chromatin immunoprecipitation (ChIP) analysis. Our data also revealed that upon OHT treatment ERalpha recruits histone deacetylases to the ERE site of the FOXM1 promoter, which is associated with a decrease in histone acetylation and transcription activity. Importantly, silencing of FOXM1 by RNAi abolished estrogen-induced MCF-7 cell proliferation and overcame acquired tamoxifen resistance. Conversely, ectopic expression of FOXM1 abrogated the cell cycle arrest mediated by the anti-estrogen OHT. OHT repressed FOXM1 expression in endocrine sensitive but not resistant breast carcinoma cell lines. Furthermore, qRT-PCR analysis of breast cancer patient samples revealed that there was a strong and significant positive correlation between ERalpha and FOXM1 mRNA expression. Collectively, these results show FOXM1 to be a key mediator of the mitogenic functions of ERalpha and estrogen in breast cancer cells, and also suggest that the deregulation of FOXM1 may contribute to anti-estrogen insensitivity.