RESUMEN
Deletions of chromosomal arms 1p and 19q are frequent in oligodendroglial tumours and linked to radio- and chemotherapy response as well as longer survival. The molecular mechanisms underlying this clinically important association are as yet unknown. Here, we studied the peroxiredoxin 1 (PRDX1) gene at 1p34.1 for promoter methylation and expression in primary gliomas and investigated its role in radio- and chemosensitivity of glioma cells in vitro. In total, we screened primary glioma tissues from 93 patients for methylation of the 5'-CpG island of PRDX1 by sodium bisulfite sequencing. PRDX1 mRNA and protein expression levels were determined in subsets of the tumours by quantitative PCR and western blot analysis, respectively. PRDX1 hypermethylation and reduced expression were frequently detected in oligodendroglial tumours and secondary glioblastomas, but not in primary glioblastomas. In oligodendroglial tumours, both PRDX1 hypermethylation and reduced mRNA expression were significantly associated with 1p/19q-deletion. Stable knockdown of PRDX1 by lentiviral transduction of short-hairpin (sh)RNA constructs significantly increased apoptosis and reduced cell viability of Hs683 glioma cells exposed to ionizing irradiation or temozolomide in vitro. Taken together, our findings indicate that epigenetic silencing of PRDX1 is frequent in 1p/19q-deleted oligodendroglial tumours and likely contributes to radio- and chemosensitivity of these tumours.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 1/genética , Glioma/patología , Oligodendroglía/metabolismo , Peroxirredoxinas/genética , Regiones Promotoras Genéticas/genética , Tolerancia a Radiación/genética , Adulto , Anciano , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Islas de CpG/genética , Metilación de ADN/efectos de los fármacos , Metilación de ADN/efectos de la radiación , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Regulación hacia Abajo/genética , Femenino , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/radioterapia , Humanos , Isocitrato Deshidrogenasa/genética , Masculino , Persona de Mediana Edad , Mutación , Oligodendroglía/efectos de los fármacos , Oligodendroglía/patología , Oligodendroglía/efectos de la radiación , Peroxirredoxinas/deficiencia , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de la radiación , Temozolomida , Adulto JovenRESUMEN
Large-gel two-dimensional gel electrophoresis (2-DE) is the method of choice for high-resolution proteome analysis of complex protein mixtures. Until now, however, the advantages of large 2-DE in combination with multiplexed fluorescence dye protein labelling has been complicated by the separate handling and analysis of the second-dimension gels. Therefore, we adapted the large 2-DE procedure allowing us to run "one-piece" large 2-DE gels (40 cm x 30 cm) in the second dimension for high resolution proteome analysis. Here, we show that in combination with fluorescence dye protein saturation labelling "one-piece" large 2-DE enables analysis of small amounts of sample (3 microg protein) for high-resolution proteome analysis.