RESUMEN
Wilms' tumor 1 (WT1) is a transcription factor with a multitude of downstream targets that have wide-ranging effects in non-glioma cell lines. Though its expression in glioblastomas is now well-documented, the role of WT1 in these tumors remains poorly defined. We hypothesized that WT1 functions as an oncogene to enhance glioblastoma viability and chemoresistance. WT1's role was examined by studying the effect of WT1 silencing and overexpression on DNA damage, apoptosis and cell viability. Results indicated that WT1 silencing adversely affected glioblastoma viability, at times, in synergy with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and cisplatin. To investigate other mechanisms through which WT1 could affect viability, we measured cell cycle distribution, senescence, and autophagy. WT1 silencing had no effect on these processes. Lastly, we examined WT1 regulation of IGF-1R expression. Counterintuitively, upregulation of IGF-1R was evident after WT1 silencing. In conclusion, WT1 functions as a survival factor in glioblastomas, possibly through inhibition of IGF-1R expression.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Encefálicas/patología , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Silenciador del Gen/efectos de los fármacos , Glioblastoma/patología , Receptor IGF Tipo 1/metabolismo , Proteínas WT1/genética , Apoptosis/efectos de los fármacos , Western Blotting , Neoplasias Encefálicas/genética , Carmustina/administración & dosificación , Ciclo Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Humanos , Técnicas In Vitro , Regiones Promotoras Genéticas/genética , ARN Interferente Pequeño/genética , Receptor IGF Tipo 1/genética , Células Tumorales CultivadasRESUMEN
The Wilms' tumor 1 (WT1) gene is overexpressed in human glioblastoma and correlates with wild-type p53 status. In other cell types, WT1 inhibits p53-mediated apoptosis in response to DNA damaging agents. However, neither this interaction nor the relationship between WT1 and radiosensitivity has been studied in glioblastoma. To study this interaction, we generated LN-229 glioma cell lines (p53 mutant) stably expressing WT1 isoforms and induced apoptosis by transfecting with different doses of wild-type p53 plasmid expression vector. Constitutive expression of WT1 did not protect against exogenous p53-mediated apoptosis. Likewise, WT1 expression did not protect against endogenous p53-mediated cell death induced by radiotherapy in U87MG cells, which contain functional wild-type p53. We then tested the efficacy of WT1 siRNA in inhibiting WT1 expression and its effect on radiosensitivity. In T98G and LN-18 glioma cells, which possess p53 mutations, WT1 siRNA decreased WT1 protein to almost undetectable levels by 96-h post-transfection. Furthermore, WT1 siRNA transfection caused a significantly larger decrease in viability following irradiation than was seen in untransfected cells in both cell lines after treatment with ED50 of ionizing radiation. In conclusion, WT1 overexpression did not protect against p53-mediated apoptosis or ionizing radiation induced cell death. WT1 siRNA increased the radiosensitivity of two human glioma cell lines independently of p53. Anti-WT1 strategies may, therefore, prove useful in improving the response of glioblastoma to radiotherapy, thus potentially improving patient survival.