Abstract
BackgroundEpigenetic silencing of the RAS association domain family 1A (RASSF1A) tumor suppressor gene promoter has been demonstrated in renal cell carcinoma (RCC) as a result of promoter hypermethylation. Contradictory results have been reported for RASSF1A methylation in normal kidney, thus it is not clear whether a significant difference between RASSF1A methylation in normal and tumor cells of the kidney exists. Moreover, RASSF1A expression has not been characterized in tumors or normal tissue as yet.ResultsUsing combined bisulfite restriction analysis (COBRA) we compared RASSF1A methylation in 90 paired tissue samples obtained from primary kidney tumors and corresponding normal tissue. Bisulfite sequence analysis was carried out using both pooled amplicons from the tumor and normal tissue groups and subclones obtained from a single tissue pair. Expression of RASSF1A was analyzed by the use of tissue arrays and immunohistochemistry. We found significantly increased methylation in tumor samples (mean methylation, 20%) compared to corresponding normal tissues (mean methylation, 11%; P < 0.001). Densely methylated sequences were found both in pooled and individual sequences of normal tissue. Immunohistochemical analysis revealed a significant reduced expression of RASSF1A in most of the tumor samples. Heterogeneous expression patterns of RASSF1A were detected in a subgroup of histologically normal tubular epithelia.ConclusionOur methylation and expression data support the hypothesis that RASSF1A is involved in early tumorigenesis of renal cell carcinoma.
Highlights
Epigenetic silencing of the RAS association domain family 1A (RASSF1A) tumor suppressor gene promoter has been demonstrated in renal cell carcinoma (RCC) as a result of promoter hypermethylation
Quantitation of RASSF1A methylation in paired tumoral and normal tissue samples Methylation was first quantitatively analyzed in tumor vs. paired normal tissue from each individual patient using combined bisulfite restriction analysis (COBRA) (Fig. 2), Fig. 3a)
Findings regarding RASSF1A methylation in histopathologically normal kidney tissue isolated adjacent to tumor tissue or from autopsy samples [8,18,19] might be indicative for an involvement of RASSF1A in the early tumorigenesis of clear cell RCC (CC-RCC)
Summary
Epigenetic silencing of the RAS association domain family 1A (RASSF1A) tumor suppressor gene promoter has been demonstrated in renal cell carcinoma (RCC) as a result of promoter hypermethylation. At least two tumor suppressors are localized on 3p213p25, the von Hippel-Lindau (VHL) and the RAS association domain family 1A (RASSF1A) genes, both found to be altered in sporadic RCC. Chromosomal alterations, point mutations and epigenetic silencing have been described to affect VHL function [5,6,7], while RASSF1A has frequently been detected to undergo promoter hypermethylation and epigenetic silencing in CC-RCC [8,9,10,11]. Depletion of RASSF1A is associated with accelerated mitotic progression and an increased risk for chromosomal defects [13,14,15], enhanced cellular motility [16] and with increased tumor susceptibility in knock-out mice [17]
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