Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylation

BMC Cancer. 2005 Jan 20;5:9. doi: 10.1186/1471-2407-5-9.

Abstract

Background: Insulin-like growth factor binding protein (IGFBP)-3 functions as a carrier of insulin-like growth factors (IGFs) in circulation and a mediator of the growth suppression signal in cells. There are two reported p53 regulatory regions in the IGFBP3 gene; one upstream of the promoter and one intronic. We previously reported a hot spot of promoter hypermethylation of IGFBP-3 in human hepatocellular carcinomas and derivative cell lines. As the hot spot locates at the putative upstream p53 consensus sequences, these p53 consensus sequences are really functional is a question to be answered.

Methods: In this study, we examined the p53 consensus sequences upstream of the IGFBP-3 promoter for the p53 induced expression of IGFBP-3. Deletion, mutagenesis, and methylation constructs of IGFBP-3 promoter were assessed in the human hepatoblastoma cell line HepG2 for promoter activity.

Results: Deletions and mutations of these sequences completely abolished the expression of IGFBP-3 in the presence of p53 overexpression. In vitro methylation of these p53 consensus sequences also suppressed IGFBP-3 expression. In contrast, the expression of IGFBP-3 was not affected in the absence of p53 overexpression. Further, we observed by electrophoresis mobility shift assay that p53 binding to the promoter region was diminished when methylated.

Conclusion: From these observations, we conclude that four out of eleven p53 consensus sequences upstream of the IGFBP-3 promoter are essential for the p53 induced expression of IGFBP-3, and hypermethylation of these sequences selectively suppresses p53 induced IGFBP-3 expression in HepG2 cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites
  • Carcinoma, Hepatocellular / genetics
  • Cell Line, Tumor
  • Consensus Sequence
  • DNA Methylation
  • Electrophoretic Mobility Shift Assay
  • Gene Expression Regulation, Neoplastic*
  • Gene Silencing*
  • Humans
  • Insulin-Like Growth Factor Binding Protein 3 / genetics*
  • Liver Neoplasms / genetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Promoter Regions, Genetic*
  • Sequence Deletion
  • Tumor Suppressor Protein p53 / metabolism*

Substances

  • Insulin-Like Growth Factor Binding Protein 3
  • Tumor Suppressor Protein p53