Activation of theMN/CA9 gene is associated with hypomethylation in human renal cell carcinoma cell lines

Abstract

The MN/CA9 (G250) gene expressed in the normal alimentary tract in a tissue-specific manner is often activated in renal cell carcinomas. To cast light on the activation mechanism, we examined the methylation status of this gene in seven human renal cell carcinoma cell lines (SKRC-01, -06, -10, -12, -14, -44, and -59) and three normal kidney tissue samples by using the bisulfite genomic sequencing protocol. CpG methylation was measured at seven locations in the MN/CA9 5' region. MN/CA9 transcripts were detected by reverse transcription-polymerase chain reaction in five of the renal cell carcinoma cell lines (SKRC-01, -06, -10, -44, and -59). These MN/CA9 positive cell lines showed hypomethylation, whereas the remaining two cell lines (SKRC-12, and -14), and three normal kidney tissue samples without transcripts demonstrated hypermethylation. Treatment with the demethylating agent 5-aza-2'-deoxycytidine resulted in activation of the MN/CA9 gene in the negative cell lines (SKRC-12 and -14). These data suggest that hypomethylation in the 5' region may have a major role in expression of the MN/CA9 gene in renal cell carcinoma cells.

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

“…To address the former possibility, we further studied the methylation status of the CA9 gene in RCC cell lines. The results clearly showed that CA IX expression was down-regulated by methylation in the 5' region of the CA9 gene as previously reported (Cho et al 2000). Although the biological activity and expression levels of normal VHL in RCC cell lines are still unknown, the results obtained in this study indicate CA IX expression is under the dual-regulation by methylation on its gene and VHL.…”

“…DNA was resuspended in water and stored at )20°C until later use. Sequencing of the bisulfite-treated DNA was carried out to investigate the CpG methylation status at the 5'-end of the coding region of the CA9 gene as previously reported Cho et al 2000). DNA fragments (nt -142 to +267) were amplified using the nested PCR technique.…”

“…The 5' region (nt )142 to +267) of the CA9 gene encompassed seven CpG sites that were numbered from #1 to #7 starting from the 5' terminus (for their location in the sequence, see Cho et al 2000). Typical profiles of bisulfite genomic sequencing are shown in Fig.…”

MAD2 downregulation in hypoxia is independent of promoter hypermethylation

“…Interestingly, aberrant methylation of CAIX has been demonstrated in renal cell carcinoma. [48][49][50] Supporting this, we also found indirect evidence for CAIX epigenetic regulation in this study with the re-expression of CAIX protein in the OVCA432 and UPN251 following chromatin modifying treatment with TSA.…”

A Genetic Approach to Cancer Epigenetics

“…This loss of methylation has important consequences. It can lead to increased expression of dominantly acting oncogenes, such as R-ras in gastrointestinal cancer (Nishigaki et al 2005) and cyclinD2 and maspin in pancreatic cancer (Cho et al 2000;Akiyama et al 2003). This hypomethylation-associated gene activation received comparatively less attention than gene silencing until recently, but it is increasingly clear that these changes are as important as those associated with gene silencing.…”