Aberrant DNA methylation of cyclin D2 and p27 genes in rodent pituitary tumor cell lines correlates with specific gene expression

We previously reported that increased DNA methylation was an important mech anism of silencing the p27 gene in some pituitary tumor cell lines [1]. DNA methylation correlated inversely with p27 gene expression. The p27 and cyc lin D2 genes are located in the same region of mouse chromosome 6, rat chro mosome 4, and human chromosome 12p13. Because both genes are located in the same gene cluster, we investigated whether methylation was a principal mec hanism regulating cyclin D2 as well as p27 expression in rodent pituitary c ell lines. Bisulfite genomic sequencing showed that the normally unmethylat ed cytosines of the p27 gene in normal pituitary (NP) were extensively meth ylated in GH3 and GHRH-CL1 cells, but not in AtT 20, alpha T-3-1 and L beta T-2 cells; but cyclin D2 was extensively inactivated in various pituitary tumor cell lines by increased DNA methylation. These abnormalities of methy lation in p27 and cyclin D2 genes occurred with different frequencies in fi ve pituitary tumor cell lines with 100% (5/5) methylation of the cyclin D2 gene and 40% (2/5) methylation of the p27 gene. Treatment with the methyl t ransferase inhibitor 5'-aza-2'-deoxycytidine (AZAdC) increased expression o f cyclin D2 and p27 in GH3 and GHRH-CL1 pituitary tumor cells. There was a correlation between hypermethylation and gene expression. GH, tumors implan ted into Wistar-Furth rats in vivo did not change the methylation status of the p27 and cyclin D2 genes. These data indicate a coordinately reduced ex pression of these two linked genes in most rodent pituitary tumor cell line s and suggest that methylation of cyclin D2 and p27 might occur in a "hot s pot" in this gene-rich cluster.