RESUMEN
Exposure of the developing or adult brain to ionizing radiation (IR) can cause cognitive impairment and/or brain cancer, by targeting neural stem/progenitor cells (NSPCs). IR effects on NSPCs include transient cell cycle arrest, permanent cell cycle exit/differentiation, or cell death, depending on the experimental conditions. In vivo studies suggest that brain age influences NSPC response to IR, but whether this is due to intrinsic NSPC changes or to niche environment modifications remains unclear. Here, we describe the dose-dependent, time-dependent effects of X-ray IR in NSPC cultures derived from the mouse foetal cerebral cortex. We show that, although cortical NSPCs are resistant to low/moderate IR doses, high level IR exposure causes cell death, accumulation of DNA double-strand breaks, activation of p53-related molecular pathways and cell cycle alterations. Irradiated NSPC cultures transiently upregulate differentiation markers, but recover control levels of proliferation, viability and gene expression in the second week post-irradiation. These results are consistent with previously described in vivo effects of IR in the developing mouse cortex, and distinct from those observed in adult NSPC niches or in vitro adult NSPC cultures, suggesting that intrinsic differences in NSPCs of different origins might determine, at least in part, their response to IR.
Asunto(s)
Corteza Cerebral/citología , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de la radiación , Animales , Biomarcadores/metabolismo , Ciclo Celular/efectos de la radiación , Muerte Celular/efectos de la radiación , Diferenciación Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Relación Dosis-Respuesta en la Radiación , Histonas/metabolismo , Cinética , Ratones , Modelos Biológicos , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de la radiación , Rayos XRESUMEN
Background: KDM5 enzymes are H3K4 specific histone demethylases involved in transcriptional regulation and DNA repair. These proteins are overexpressed in different kinds of cancer, including breast, prostate and bladder carcinomas, with positive effects on cancer proliferation and chemoresistance. For these reasons, these enzymes are potential therapeutic targets. Methods: In the present study, we analyzed the effects of three different inhibitors of KDM5 enzymes in MCF-7 breast cancer cells over-expressing one of them, namely KDM5B/JARID1B. In particular we tested H3K4 demethylation (western blot); radio-sensitivity (cytoxicity and clonogenic assays) and damage accumulation (COMET assay and kinetics of H2AX phosphorylation). Results: we show that all three compounds with completely different chemical structures can selectively inhibit KDM5 enzymes and are capable of increasing sensitivity of breast cancer cells to ionizing radiation and radiation-induced damage. Conclusions: These findings confirm the involvement of H3K4 specific demethylases in the response to DNA damage, show a requirement of the catalytic function and suggest new strategies for the therapeutic use of their inhibitors.
Asunto(s)
Neoplasias de la Mama/enzimología , Histona Demetilasas/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/genética , Proteínas Nucleares/genética , Fármacos Sensibilizantes a Radiaciones/farmacología , Proteínas Represoras/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Células MCF-7 , Modelos Moleculares , Estructura Molecular , Proteínas Nucleares/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/química , Proteínas Represoras/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
JARID1B/KDM5B histone demethylase's mRNA is markedly overexpressed in breast cancer tissues and cell lines and the protein has been shown to have a prominent role in cancer cell proliferation and DNA repair. However, the mechanism of its post-transcriptional regulation in cancer cells remains elusive. We performed a computational analysis of transcriptomic data from a set of 103 breast cancer patients, which, along with JARID1B upregulation, showed a strong downregulation of 2 microRNAs (miRNAs), mir-381 and mir-486, potentially targeting its mRNA. We showed that both miRNAs can target JARID1B 3'UTR and reduce luciferase's activity in a complementarity-driven repression assay. Moreover, MCF7 breast cancer cells overexpressing JARID1B showed a strong protein reduction when transfected with mir-486. This protein's decrease is accompanied by accumulation of DNA damage, enhanced radiosensitivity and increase of BRCA1 mRNA, 3 features previously correlated with JARID1B silencing. These results enlighten an important role of a miRNA's circuit in regulating JARID1B's activity and suggest new perspectives for epigenetic therapies.
