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Updated: Sep 26, 2026

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
Published on: August 5, 2022
Down-regulation of connexin 32 gene expression through DNA methylation in a human renal cell carcinoma cell
Ayako Hirai1, Tomohiro Yano, Kazuhiro Nishikawa
1Department of Veterinary Pharmacology, College of Bioresource Sciences, Nihon University, Kanagawa, Japan.
Background:
We have recently reported that connexin (Cx) 32 is down-regulated in a human renal cell carcinoma (RCC) cell (Caki-2 cell).
Hypothesis:
We postulated that the down-regulation of Cx32 gene in the RCC cell is due to hypermethylation of its promoter region.
Methods:
We estimated methylation status in the promoter region of Cx32 gene in the RCC cell by DNA digestion with methylation-sensitive restriction enzyme and PCR, and methylation-specific PCR (MSP). We also checked the recovery of Cx32 gene expression in the RCC cell treated with a DNA methyltranferase inhibitor, 5-Aza-2'-deoxycytidine (5-Aza-CdR).
Results:
Treatment with 5-Aza-CdR resulted in induction of Cx32 expression in the RCC cell. Hypermethylation of the Cx32 promoter region in the RCC cell was confirmed by DNA digestion with methylation-sensitive restriction enzyme and PCR, and MSP.
Conclusion:
We suggest that hypermethylation in the promoter region is a mechanism for the Cx32 gene repression in the RCC cell.
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