DNA Methylation Regulates p27Kip1 Expression in Rodent Pituitary Cell Lines

Abstract

We previously reported loss of expression of p27Kip1 (p27) protein in rat GH3 and mouse GHRH-CL1 pituitary tumor cells compared with normal pituitary (NP). The molecular basis for the loss of expression of p27 protein in GH3 and GHRH-CL1 cells is unknown. To determine the role of p27 gene methylation in the regulation of the expression of this cell cycle protein, the methylation patterns of p27 in normal and neoplastic pituitary cells was analyzed. Inhibition of DNA methyltransferase (DNA-MTase) with 5-aza-2'-deoxycytidine (AZAdC) induced expression of both p27 protein and mRNA when GH3 and GHRH-CL1 cells were treated for 7 days in vitro. DNA methylation correlated inversely with the expression of p27 gene products in NP and pituitary tumor cell lines. Bisulfite genomic sequencing analysis showed that the normally unmethylated cytosines in exon 1 in NP and AtT20 cells were extensively methylated in GH3 and GHRH-CL1 cells. After treatment of GH3 and GHRH-CL1 cells with 10 micromol/L AZAdC, there were decreased numbers of methylated cytosines (by 60% to 90%/o) with variable methylation patterns observed by bisulfite genomic sequencing. Analysis of genomic DNA with methylation-sensitive enzymes showed that all SmaI, HhaI, and AvaI enzyme sites of the p27 gene in exon 1 were methylated in GH3 cells but not in NP, confirming the bisulfite genomic sequencing results. AtT20 cells and a human pituitary null cell adenoma cell line (HP75), which expressed abundant p27, had a methylation pattern similar to the NP. DNA-MTase activity was elevated fourfold in GH3 cells and twofold in GHRH-CL1 cells compared with DNA-MTase activity in NP and AtT20 cells. These results suggest that increased DNA methylation is another mechanism of silencing of the p27 gene in some pituitary tumors and possibly in other types of neoplasms.

The p21WAF1/CIP1 Promoter Is Methylated in Rat-1 Cells: Stable Restoration of p53-Dependent p21WAF1/CIP1 Expression after Transfection of a Genomic Clone Containing the p21WAF1/CIP1 Gene

“…These results support those obtained with 5-AzaC (above) that p21 WAF1/CIP1 transcriptional expression is functionally inactivated in Rat-1 cells by extensive methylation of the putative CpG island in the 5ЈUTR. Similarly, promoter hypermethylation has also been reported to cause the transcriptional silencing of many genes whose functional inactivation may contribute to the neoplastic process (12,31,33,35,36). To our knowledge, however, this is the first report of functional inactivation of the p21 WAF1/CIP1 gene by this mechanism.…”

“…An increasing amount of evidence suggests that tumor suppressor gene inactivation by promoter methylation may be a frequent occurrence in tumor cells (12,31,33,35,36). Therefore, we wanted to determine whether p21 WAF1/CIP1 expression may be abrogated by this mechanism in Rat-1 cells.…”

Polypyrimidine Tract-Binding Protein Enhances the Internal Ribosomal Entry Site-Dependent Translation of p27Kip1 mRNA and Modulates Transition from G1 to S Phase

“…The p27 protein, a key regulator of cell cycle progression, is regulated both transcriptionally and posttranscriptionally (17,44). Two posttranscriptional steps, translation and protein degradation, are mainly responsible for the oscillation of p27 levels during cell cycle and the induction of p27 in growth-arrested cells (1,15,32,36,38).…”

Microenvironmental Regulation of Proliferation in Multicellular Spheroids Is Mediated through Differential Expression of Cyclin-Dependent Kinase Inhibitors

“…Kip1 gene account for decreased expression in less than 1% of the tumors analyzed (60, 89 -91); the primary mechanisms found to date are enhanced protein degradation via the ubiquitin pathway (92), and gene methylation (93). Although the majority of reports indicate a strong correlation between p27…”