Clinical implications of promoter hypermethylation in RASSF1A and MGMT in retinoblastoma.
We investigated the epigenetic silencing and genetic changes of the RAS-associated domain family 1A (RASSF1A) gene and the O6-methylguanine-DNA methyltransferase (MGMT) gene in retinoblastoma. We extracted DNA from microdissected tumor and normal retina tissues of the same patient in 68 retinoblastoma cases. Promoter methylation in RASSF1A and MGMT was analyzed by methylation-specific PCR, RASSF1A sequence alterations in all coding exons by direct DNA sequencing, and RASSF1A expression by RT-PCR. Cell cycle staging was analyzed by flow cytometry. We detected RASSF1A promoter hypermethylation in 82% of retinoblastoma, in tumor tissues only but not in adjacent normal retinal tissue cells. There was no expression of RASSF1A transcripts in all hypermethylated samples, but RASSF1A transcripts were restored after 5-aza-2'-deoxycytidine treatment with no changes in cell cycle or apoptosis. No mutation in the RASSF1A sequence was found. MGMT hypermethylation was present in 15% of the retinoblastoma samples, and the absence of MGMT hypermethylation was associated (P = .002) with retinoblastoma at advanced Reese-Ellsworth tumor stage. Our results revealed a high RASSF1A hypermethylation frequency in retinoblastoma. The correlation of MGMT inactivation by promoter hypermethylation with lower-stage diseases indicated that MGMT hypermethylation provides useful prognostic information. Epigenetic mechanism plays an important role in the progression of retinoblastoma.
Choy
Kwong Wai
KW
Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong Eye Hospital, Kowloon, Hong Kong, China. richardchoy@cuhk.edu.hk
Lee
Tom C
TC
Cheung
Kin Fai
KF
Fan
Dorothy S P
DS
Lo
Kwok Wai
KW
Beaverson
Katherine L
KL
Abramson
David H
DH
Lam
Dennis S C
DS
Yu
Christopher B O
CB
Pang
Chi Pui
CP
D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018899:CpG Islands; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D018450:Disease Progression; D005091:Exons; D005434:Flow Cytometry; D006801:Humans; D007834:Lasers; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012160:Retina / Q000378:metabolism; D012175:Retinoblastoma / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D025521:Tumor Suppressor Proteins / Q000235:genetics*
C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins; D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase