DNA METHYLATION REGULATES P27(KIP1) EXPRESSION IN RODENT PITUITARY CELL-LINES

We previously reported loss of expression of p27(Kip1) (p27) protein i n rat GH(3) and mouse GHRH-CL1 pituitary tumor cells compared with nor mal pituitary (NP), The molecular basis for the loss of expression of p27 protein in GH, and GHRH-CLI cells is unknown, To determine the rol e of p7 gene methylation in the regulation of the expression of this c ell cycle protein, the methylation patterns of p27 in normal and neopl astic pituitary cells was analyzed, Inhibition of DNA methyltransferas e (DNA-MTase) with 5-aza-2'-deoxycytidine (AZAdC) induced expression o f both p7 protein and mRNA when GH, and GHRH-CL1 cells were treated fo r 7 days in vitro. DNA methylation correlated inversely with the expre ssion of p27 gene products in NP and pituitary tumor cell lines. Bisul fite genomic sequencing analysis showed that the normally unmethylated cytosines in exon 1 in NP and AtT20 cells were extensively methylated in GH, and GHRH-CL1 cells. After treatment of GH, and GHRH-CL1 cells with 10 mu mol/L AZAdC, there were decreased numbers of methylated cyt osines (by 60% to 90%) with variable methylation patterns observed by bisulfite genomic sequencing, Analysis of genomic DNA with methylation -sensitive enzymes showed that all SmaI, HbaI, and AvaI enzyme sites o f the p27 gene in exon 1 were methylated in GM, cells but not in NP, c onfirming the bisulfite genomic sequencing results. AtT20 cells and a human pituitary null cell adenoma cell line (HP75), which expressed ab undant p7, had a methylation pattern similar to the NP. DNA-MTase acti vity was elevated fourfold in GH, cells and twofold in GHRH-CLI cells compared with DNA-MTase activity in NP and AtT20 cells. These results suggest that increased DNA methylation is another mechanism of silenci ng of the p27 gene in some pituitary tumors and possibly in other type s of neoplasms.