Effects of von Hippel-Lindau gene mutation and methylation status on expression of transmembrane carbonic anhydrases in renal cell carcinoma

Purpose: Our primary goal was to determine whether increased expression of the transmembrane carbonic anhydrase (CA) isozymes in renal cell carcinoma (RCC) is regulated by the von Hippel-Lindau (VHL) gene. Methods: We studied mRNA expression of all three transmembrane CAs - CA IX. XII. and XIV - in 17 RCC cell lines, transformants of the wild-type VHL gene, and normal kidney tissue cultures, and then compared them with the mutation status in the VHL gene. Results: Northern blot analysis showed no detectable signal for CA XIV mRNA expression in normal and cancer cells. CA XII mRNA was ubiquitously expressed except in two cell lines. Although CA XII expression levels tended to be lower in RCC cell lines without the VHL mutation and in transformants of the wild-type VHL gene, the results were not conclusive. Significant expression of CA IX mRNA was seen in eight of 17 RCC cell lines, Among five cell lines which had no VHL mutation, four lines showed no detectable signal and one cell line showed a low amount of CA IX mRNA expression. In patients with RCC, VHL mutations and significant CA IX expression were seen in established tumor cell lines but not in primary tissue cultures from normal counterparts. Further Study of methylation status showed that the 5' region in the CA9 gene was hypomethylated in all CA IX-positive cell lines and hypermethylated in all CA IX-negative cell lines. Especially, methylation status at -74 and -6 CpG sites perfectly correlated with A IX expression. Conclusions: These findings indicate that VHL suppresses CA IX expression but has no conclusive effect on CA XII and XIV expressions in RCC. CA IX expression is also driven by the methylation status of the CA9 gene.