Frequent promoter hypermethylation of RASSF1A and CASP8 in neuroblastoma

Abstract

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.

Conclusion: 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.

Figure 1

Loss of heterozygosity study at D3S3522 polymorphic marker in neuroblastic tumors. PCR products were run in a 15% polyacrylamide gel and visualized after ethidium bromide staining (0.5 μg/ml) T: DNA from tumor tissue; N: DNA from peripheral blood of the same patient; M1/M2: 10 bp DNA ladder/1 Kb Plus DNA ladder (Invitrogen™ Life and Technologies, Carlsbad, CA, USA). Sample number 64 showed microsatellite instability.

Figure 2

Promoter methylation analysis of RASSF1A (A), NORE1A (B), BLU (C) and CASP8 (D) in neuroblastic tumors (B, C and D) and cell lines (A), determined by MSP and visualized in 2.5% agarose gels stained with 0.1 μg/ml ethidium bromide. U/M: unmethylated/methylated; M: 1 Kb Plus DNA Ladder (Invitrogen™ Life and Technologies, Carlsbad, CA, USA); B: DNA obtained from blood of a normal donor; IMD (in vitro methylated DNA): positive control, CpGenome™ Universal Methylated DNA (Chemicon International, Inc., Temecula, CA, USA);-: water (no DNA negative control). A) 2: SH-SY5Y; 3: SK-N-SH; 4: SK-N-MC; 5: IMR-32; 6: MHH-NB-11; 7: SK-N-Be(2); 8: SK-N-DZ; 9: BE(2)C. B), C), and D) Numbers on top of each well correspond to DNA from different neuroblastic tumors analyzed.

Figure 3

Expression of RASSF1A, NORE1A, BLU and CASP8 in neuroblastoma cell lines determined by RT-PCR and visualized in 2% agarose gels stained with 0.1 μg/ml ethidium bromide. M: 1 Kb Plus DNA Ladder (Invitrogen™ Life and Technologies, Carlsbad, CA, USA); 1: Normal human lung cDNA; 2: Kelly; 3: BE(2)-C; 4: SK-N-MC; 5: SK-N-FI; 6: SK-N-Be(2); 7: IMR-32; 8: MC-IXC; 9: SH-SY5Y; 10: SK-N-SH; 11: MHH-NB-11; 12: SIMA; 13: SK-N-DZ; 14: genomic DNA; 15: water. A fragment of the transferrin receptor gene was amplified as an internal control.