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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.

SUBMITTER: Qian X

PROVIDER: S-EPMC1853401 | biostudies-literature | 1998 Nov

REPOSITORIES: biostudies-literature

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DNA methylation regulates p27kip1 expression in rodent pituitary cell lines.

Qian X X Jin L L Kulig E E Lloyd R V RV

The American journal of pathology 19981101 5

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) ...[more]

PMID: 9811339

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Project description:Abnormal activation of the Wnt signaling pathway is found in 90% of colorectal cancers (CRCs). Secreted frizzled-related protein 4 (sFRP4) serves as an antagonist of the canonical Wnt signaling pathway. Epigenetic alterations, including changes in DNA methylation and histone methylation, may influence the expression of sFRP4. Polycomb group (PcG) proteins are epigenetic transcriptional repressors that selectively repress gene expression by forming polycomb repressive complexes (PRCs). Enhancer of zeste homolog 2 (EZH2), the core component of PRC2, is a histone-lysine N-methyltransferase that interacts with DNA methyltransferases. In the present study, the promoter DNA methylation status of sFRP4 in CRC cell lines was analyzed and the underlying mechanisms of action governing this modification was investigated. Firstly, the DNA methylation status of the sFRP4 promoter in CRC cell lines was assessed using methylation-specific PCR. Subsequently, the mRNA and protein levels of sFRP4 were measured using real-time qPCR and western blot analysis, respectively, to determine whether the DNA methylation status of the sFRP4 promoter is correlated with its transcriptional levels. To screen for important epigenetic modifiers that may regulate the promoter DNA methylation status of sFRP4, the expression levels of PcG proteins were examined by gene array analysis. ChIP-qPCR was performed to test whether the selected PcG proteins directly bind the promoter region of sFRP4. Finally, the downregulated PcG proteins EZH2, chromobox 7 (CBX7) and jumonji and AT-rich interaction domain containing 2 (JARID2) were identified and their association with sFRP4 expression levels and Wnt/?-catenin signaling pathway activity were investigated. The present study revealed that sFRP4 was hypermethylated in the promoter region and downregulated during the progression of the CRC cell lines from Dukes A to Dukes C. Expression levels of PcG proteins EZH2, CBX7 and JARID2 were upregulated and positively associated with the aberrantly activated Wnt signaling pathway in the CRC cell lines. EZH2, CBX7 and JARID2 were all enriched in the sFRP4 promoter region in CRC cells. EZH2 downregulation did not affect the promoter DNA methylation status of sFRP4 but increased its expression levels and decreased CRC cell proliferation. DNA methylation controls the expression of sFRP4. EZH2 regulates sFRP4 expression without affecting the DNA hypermethylation of the sFRP4 promoter and influences CRC cell proliferation and Wnt/?-catenin signaling pathway activities.

Dataset Information

DNA methylation regulates p27kip1 expression in rodent pituitary cell lines.

Publications

DNA methylation regulates p27kip1 expression in rodent pituitary cell lines.

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