RESUMEN
Allelic losses on 19q are found in the majority of oligodendroglial tumors and approximately one-third of diffuse astrocytomas. However, the tumor suppressor genes (TSG) on 19q are still elusive. Using cDNA microarray expression profiling, EMP3 at 19q13.3 was among those genes showing the most pronounced expression differences. In line with this, other authors reported EMP3 as being epigenetically silenced in neuroblastomas and astrocytomas. To further investigate EMP3 as a TSG candidate on 19q13.3, we performed molecular analysis of this gene in 162 human gliomas. Mutation analysis did not reveal EMP3 alteration in 132 gliomas. In oligodendroglial tumors, we found that aberrant methylation in the 5'-region of EMP3 was significantly associated with reduced mRNA expression and LOH 19q. In astrocytomas, EMP3 hypermethylation was also paralleled by reduced expression but was independent of the 19q status. EMP3 hypermethylation was detected in more than 80% of diffuse, anaplastic astrocytomas and secondary glioblastomas. Primary glioblastomas, however, mostly lacked EMP3 hypermethylation and frequently overexpressed EMP3. Our data corroborate that oligodendroglial and astrocytic gliomas often show EMP3 hypermethylation and aberrant expression. Furthermore, our findings suggest that primary and secondary glioblastomas are not only characterized by distinct genetic profiles but also differ in their epigenetic aberrations.
Asunto(s)
Neoplasias Encefálicas/genética , Cromosomas Humanos Par 19/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica/genética , Glioma/genética , Glicoproteínas de Membrana/genética , Adulto , Astrocitoma/genética , Astrocitoma/metabolismo , Astrocitoma/fisiopatología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/fisiopatología , Perfilación de la Expresión Génica , Silenciador del Gen/fisiología , Predisposición Genética a la Enfermedad/genética , Glioma/metabolismo , Glioma/fisiopatología , Humanos , Glicoproteínas de Membrana/biosíntesis , Oligodendroglioma/genética , Oligodendroglioma/metabolismo , Oligodendroglioma/fisiopatología , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
To identify novel genes involved in glioma progression we performed suppression subtractive hybridization combined with cDNA array analysis on 4 patients with primary low-grade gliomas of World Health Organization (WHO) grade II that recurred as secondary glioblastomas (WHO grade IV). Eight genes showing differential expression between primary and recurrent tumors in 3 of the 4 patients were selected for further analysis using real-time reverse transcription-PCR on a series of 10 pairs of primary low-grade and recurrent high-grade gliomas as well as 42 astrocytic gliomas of different WHO grades. These analyses revealed that 5 genes, i.e., AMOG (ATP1B2, 17p13.1), APOD (3q26.2-qter), DMXL1 (5q23.1) DRR1 (TU3A, 3p14.2) and PSD3 (KIAA09428/HCA67/EFA6R, 8p22), were expressed at significantly lower levels in secondary glioblastomas as compared to diffuse astrocytomas of WHO grade II. In addition, AMOG, DRR1 and PSD3 transcript levels were significantly lower in primary glioblastomas than in diffuse astrocytomas. Treatment of glioma cell lines with 5-aza-2'-deoxycytidine and trichostatin A resulted in increased expression of AMOG and APOD transcripts. Sequencing of sodium bisulfite-modified DNA demonstrated AMOG promoter hypermethylation in the glioma cell lines and 1 primary anaplastic astrocytoma with low AMOG expression. Taken together, we identified interesting novel candidate genes that likely contribute to glioma progression and provide first evidence for a role of epigenetic silencing of AMOG in malignant glioma cells.
Asunto(s)
Adenosina Trifosfatasas/genética , Astrocitoma/genética , Neoplasias Encefálicas/genética , Proteínas de Transporte de Catión/genética , Moléculas de Adhesión Celular Neuronal/genética , Silenciador del Gen , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Antimetabolitos Antineoplásicos/farmacología , Apolipoproteínas/genética , Apolipoproteínas D , Azacitidina/análogos & derivados , Azacitidina/farmacología , Biomarcadores de Tumor/genética , Metilación de ADN , Decitabina , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen/efectos de los fármacos , Genes Supresores de Tumor , Glicoproteínas/genética , Factores de Intercambio de Guanina Nucleótido , Histona Desacetilasas/genética , Humanos , Ácidos Hidroxámicos/farmacología , Proteínas de Transporte de Membrana/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Hibridación de Ácido Nucleico/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Diffuse astrocytoma of World Health Organization (WHO) grade II has an inherent tendency to spontaneously progress to anaplastic astrocytoma (WHO grade III) and/or glioblastoma (WHO grade IV). The molecular basis of astrocytoma progression is still poorly understood, in particular with respect to the progression-associated changes at the mRNA level. Therefore, we compared the transcriptional profile of approximately 6800 genes in primary WHO grade II gliomas and corresponding recurrent high-grade (WHO grade III or IV) gliomas from eight patients using oligonucleotide-based microarray analysis. We identified 66 genes whose mRNA levels differed significantly (P < 0.01, > or =2-fold change) between the primary and recurrent tumors. The microarray data were corroborated by real-time reverse transcription-polymerase chain reaction analysis of 12 selected genes, including 7 genes with increased expression and 5 genes with reduced expression on progression. In addition, the expression of these 12 genes was determined in an independent series of 43 astrocytic gliomas (9 diffuse astrocytomas, 10 anaplastic astrocytomas, 17 primary, and 7 secondary glioblastomas). These analyses confirmed that the transcript levels of nine of the selected genes (COL4A2, FOXM1, MGP, TOP2A, CENPF, IGFBP4, VEGFA, ADD3, and CAMK2G) differed significantly in WHO grade II astrocytomas as compared to anaplastic astrocytomas and/or glioblastomas. Thus, we identified and validated a set of interesting candidate genes whose differential expression likely plays a role in astrocytoma progression.