Frequent promoter hypermethylation of RASSF1A and CASP8 in neuroblastoma.
BACKGROUND
Epigenetic alterations and loss of heterozygosity are mechanisms of tumor suppressor gene inactivation. A new carcinogenic pathway, targeting the RAS effectors has recently been documented. RASSF1A, on 3p21.3, and NORE1A, on 1q32.1, are among the most important, representative RAS effectors.


METHODS
We screened the 3p21 locus for the loss of heterozygosity and the hypermethylation status of RASSF1A, NORE1A and BLU (the latter located at 3p21.3) in 41 neuroblastic tumors. The statistical relationship of these data was correlated with CASP8 hypermethylation. The expression levels of these genes, in cell lines, were analyzed by RT-PCR.


RESULTS
Loss of heterozygosity and microsatellite instability at 3p21 were detected in 14% of the analyzed tumors. Methylation was different for tumors and cell lines (tumors: 83% in RASSF1A, 3% in NORE1A, 8% in BLU and 60% in CASP8; cell lines: 100% in RASSF1A, 50% in NORE1A, 66% in BLU and 92% in CASP8). In cell lines, a correlation with lack of expression was evident for RASSF1A, but less clear for NORE1A, BLU and CASP8. We could only demonstrate a statistically significant association between hypermethylation of RASSF1A and hypermethylation of CASP8, while no association with MYCN amplification, 1p deletion, and/or aggressive histological pattern of the tumor was demonstrated.


CONCLUSIONS
1) LOH at 3p21 appears in a small percentage of neuroblastomas, indicating that a candidate tumor suppressor gene of neuroblastic tumors is not located in this region. 2) Promoter hypermethylation of RASSF1A and CASP8 occurs at a high frequency in neuroblastomas.
Lázcoz
Paula
P
Departamento de Ciencias de la Salud, Universidad Pública de Navarra, Pamplona, Spain. paula.lazcoz@unavarra.es
Muñoz
Jorge
J
Nistal
Manuel
M
Pestaña
Angel
A
Encío
Ignacio
I
Castresana
Javier S
JS
D048868:Adaptor Proteins, Signal Transducing; D000293:Adolescent; D051017:Apoptosis Regulatory Proteins; D053181:Caspase 8 / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002648:Child; D002675:Child, Preschool; D043171:Chromosomal Instability; D002893:Chromosomes, Human, Pair 3; D003598:Cytoskeletal Proteins; D019175:DNA Methylation; D015972:Gene Expression Regulation, Neoplastic; D016147:Genes, Tumor Suppressor; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D019656:Loss of Heterozygosity; D018895:Microsatellite Repeats; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D003598:Cytoskeletal Proteins; C411762:RASSF1 protein, human; C454042:RASSF5 protein, human; D025521:Tumor Suppressor Proteins; C472720:ZMYND10 protein, human; C505530:CASP8 protein, human; D053181:Caspase 8; D020559:Monomeric GTP-Binding Proteins