Renal cell carcinoma-associated G250 methylation and expression: in vivo and in vitro studies.
OBJECTIVE
In renal cell carcinoma (RCC) cell lines, expression of the RCC-associated antigen G250 correlates with hypomethylation of the investigated CpG dinucleotides in the G250 promoter region, despite the absence of a CpG island. To gain insight into the molecular mechanism leading to G250 expression in vivo, we ascertained whether this correlation between G250 gene expression and the methylation status of the G250 gene also existed in primary RCC and normal kidney tissue.


METHODS
G250 mRNA and protein expression was determined by reverse transcriptase-polymerase chain reaction, fluorescence activated cell sorting analysis, and immunohistochemistry in 15 RCC cell lines and 13 paired primary RCC/normal kidney tissue specimens. The methylation status of the G250 gene was determined by bisulfite genomic sequencing.


RESULTS
RCC cell lines revealed a clear correlation between G250 expression and hypomethylation. In contrast, no hypomethylation was observed in primary RCC compared with normal kidney tissue. The CpG dinucleotides investigated were generally completely methylated in RCC, as well as in normal kidney tissue. Furthermore, a primary culture of RCC tissue revealed increasing hypomethylation of the G250 gene in successive passages, suggesting that the G250 hypomethylation observed in vitro is tissue culture induced.


CONCLUSIONS
The methylation status of the G250 gene correlated with G250 expression in vitro but not in vivo.
Experimental Urology, Department of Urology, University Medical Center Nijmegen, Nijmegen, The Netherlands.
Karin
K
Grabmaier
Mirjam
M
de Weijert
Hirotsugu
H
Uemura
Jack
J
Schalken
Egbert
E
Oosterwijk
D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002292:Carcinoma, Renal Cell / Q000235:genetics / Q000378:metabolism*; D018899:CpG Islands; D019175:DNA Methylation; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D007680:Kidney Neoplasms / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D012333:RNA, Messenger; C400566:antigen G250