DNA Methylation Is Not Involved in Growth Regulation of Gene Expression of Proliferating Cell Nuclear Antigen

Abstract

Expression of proliferating cell nuclear antigen (PCNA) gene is growth regulated. By Southern blot analysis with restriction enzymes such as Hpa II and MspI, no change of DNA methylation state in this gene was detected during rat liver regeneration. There is only one/ HpaII site located in the promoter region of rat PCNA gene and this HpaII site was found to be demethylated in both normal and regenerating livers. Human hepatoma cell lines Hep G2 and Hep 3B were used to study the relation of PCNA gene expression and the DNA methylation. There are 16 HpaII sites in human PCNA gene, and according to the HpaII and MspI restriction patterns, many of the HpaII sites were methylated in vivo. Upon serum stimulation of quiescent cells, no change of DNA methylation state in the Hpa II and HhaI sites was found. Demethylation by the methylation inhibitor, 5-azacytidine, did not affect the expression of PCNA gene in the hepatoma cells. With the human primary fibroblasts, Y2, the demethylation by 5-azacytidine did not seem to change the growth dependence of PCNA gene expression. This is consistent with the observation that the expression of PCNA gene of some cultured cell lines such as CHO.K1, in which the PCNA gene was unmethylated, showed growth dependence. Also, no variation in methylation pattern of PCNA gene was found among the different rat tissues in which the expression of PCNA varies. Therefore, we conclude that DNA methylation is not involved in growth regulation of the PCNA gene expression.