RASSF1A promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis.
BACKGROUND
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. 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.


RESULTS
Using 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.


CONCLUSIONS
Our methylation and expression data support the hypothesis that RASSF1A is involved in early tumorigenesis of renal cell carcinoma.
Peters
Inga
I
Department of Urology, Medizinische Hochschule Hannover, Hannover, Germany. inga-peters@gmx.de
Rehmet
Kristina
K
Wilke
Nadine
N
Kuczyk
Markus A
MA
Hennenlotter
Jörg
J
Eilers
Tyark
T
Machtens
Stefan
S
Jonas
Udo
U
Serth
Jürgen
J
D002292:Carcinoma, Renal Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D019175:DNA Methylation; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism / Q000473:pathology; D007680:Kidney Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods; D013447:Sulfites / Q000737:chemistry; D046888:Tissue Array Analysis; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
C411762:RASSF1 protein, human; D013447:Sulfites; D025521:Tumor Suppressor Proteins; C042345:hydrogen sulfite