MN/CA9 expression and VHL regulation in renal cell carcinoma

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The von Hippel-Lindaw (VHL) gene is a well-known tumor suppressor gene interacting with the hypoxia inducible factor (HIF), which associates with a variety of gene expressions in normal and cancer cells. Frequent gene alterations of the VHL gene, such as deletions or mutations, have been reported in sporadic renal cell carcinoma (RCC), suggesting one of major cause of RCC development. MN/CA9, which is one of the isoenzymes of the carbonic anhydrase family, is frequently over-expressed and considered as a tumor-associated antigen in RCCs. We have previously demonstrated that the RCC cell lines with wild-type VHL gene expression have undetectable or extremely low expressions of MN/CA9 so that the VHL gene alterations may have a role for the MN/CA9 constitutive expression in RCCs. Since the fact that the MN/CA9 expression was induced by hypoxic condition, the VHL-HIF pathway may associate with the MN/CA9 gene regulation in RCCs. To investigate the role of MN/CA9 in VHL-HIF pathway, 6 RCC cell lines were analyzed for MN/CA9 mRNA expression. Despite all these cell lines have the VHL gene alterations, 3 of 6 lines have no expression of MN/CA9. We used other 3 cell lines expressing MN/CA9 for further analysis of VHL-related hypoxia. In nonnoxic condition, the transfectants with the VHL expression vector in these 3 cell lines showed down-regulation of MN/CA9 expression compared to the transfectants with control vector or parental cells. In hypoxic condition, however theMN/CA9 expression was induced in these transfectants with the VHL gene, but not in the control transfectants or parental cells. Furthermore, we used MN/CA9-negative cell line SK-RC 14, which has the VHL gene deletion, to investigate whether MN/CA9-negative cell line can induce MN/CA9 expression in either normoxic or hypoxic condition through VHL dependent pathway. SK-RC14 did not show any induction of MN/CA9. This study demonstrates that regulation of MN/CA9 is not exactly associated with pVHL.