Frequent hypermethylation of CpG islands and loss of expression of the 14-3-3 sigma gene in human hepatocellular carcinoma

The 14-3-3 sigma gene has been implicated in G2/M cell cycle arrest by p53, Frequent inactivation of the 14-3-3 sigma gene by hypermethylation of CpG islands has recently been reported in human breast carcinoma. The aim of th is study was to examine the methylation status of CpG islands of the 14-3-3 sigma gene in hepatocellular carcinoma (HCC), The methylation status of th e 14-3-3 sigma gene was evaluated in four normal liver tissues and 19 paire d specimens of carcinoma and adjacent non-tumorous liver tissues using bisu lfite-single strand conformation polymorphism (bisulfite-SSCP), a combinati on of sodium bisulfite modification and fluorescence-based polymerase chain reaction (PCR)-SSCP, The 14-3-3 sigma protein expression was examined by i mmunohistochemical staining. Hypermethylation of CpG islands of the 14-3-3 sigma gene was detected in 89% (17/19) of the HCC tissues but not in any of the four normal liver tissues. All of the 14 methylation-positive HCC samp les analysed by immunohistochemistry showed loss of 14-3-3 sigma expression , while both of the methylation-negative HCC samples retained the expressio n, and a significant correlation was found between methylation and loss of expression. Lower levels of methylation were detected in adjacent non-tumor ous liver tissues (6/16 in cirrhotic tissues and 1/3 in chronic hepatitis t issues), but the 14-3-3 sigma expression was retained in all of these tissu es. In a methylation-positive HCC cell line, HLE, 5-aza-2'-deoxycytidine (5 -aza-dC)-induced demethylation of CpG islands led to reactivation of gene e xpression, indicating that hypermethylation plays a causal role in inactiva tion of the 14-3-3 sigma gene in HCC, Hypermethylation and the resulting lo ss of expression of the 14-3-3 sigma gene corresponds to one of the most co mmon abnormalities reported to date in HCC, suggesting their crucial role i n the development and/or progression of HCC.