Methylation of the CA9 promoter can modulate expression of the tumor-associated carbonic anhydrase IX in dense carcinoma cell lines.

CA IX is a tumor-associated transmembrane isoform of the carbonic anhydrase with a high enzyme activity and a functional involvement in the pH regulation and cell adhesion. Expression of CA9 gene in tumor cells is principally regulated by the high cell density and the hypoxia-related VHL-HIF pathway. In renal cell carcinomas with VHL inactivation, CA9 transcription is further controlled by site-specific promoter methylation. Here we explored a possible role of methylation in the non-RCC cell lines represented mainly by HeLa cervical carcinoma cells. Using metabisulfite sequencing and treatment with the methylation inhibitor 5-aza-2'-deoxycytidine we showed that the methylation of a single CpG site at -74 position with respect to the transcription start can down-modulate the expression of CA9 in cells cultivated at high density, but not in cells grown in sparse culture nor in cells exposed to hypoxia. Methylation appears to act in tumor cells expressing intermediate levels of CA IX protein, but not in cell lines expressing high CA IX levels. Our results indicate that promoter methylation is not crucial for the control of CA9 gene expression in the non-RCC cell lines but could represent an accessory mechanism restricting its expression in highly dense carcinoma cell cultures.