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

Tadashi Hanafusa, Toshiyuki Shinji, Hidenori Shiraha, Kazuhiro Nouso, Yoshiaki Iwasaki, Eichiro Yumoto, Toshiro Ono, Norio Koide

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Article number9
JournalBMC Cancer
Volume5
DOIs
Publication statusPublished - Jan 20 2005

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Insulin-Like Growth Factor Binding Protein 3
Methylation
Consensus Sequence
Neoplasms
Hepatoblastoma
Cell Line
Sequence Deletion
Nucleic Acid Regulatory Sequences
Hep G2 Cells
Electrophoretic Mobility Shift Assay
Somatomedins
Genetic Promoter Regions
Mutagenesis
Electrophoresis
Hepatocellular Carcinoma

ASJC Scopus subject areas

  • Oncology
  • Cancer Research
  • Genetics

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Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylation. / Hanafusa, Tadashi; Shinji, Toshiyuki; Shiraha, Hidenori; Nouso, Kazuhiro; Iwasaki, Yoshiaki; Yumoto, Eichiro; Ono, Toshiro; Koide, Norio.

In: BMC Cancer, Vol. 5, 9, 20.01.2005.

Research output: Contribution to journalArticle

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AU - Shinji, Toshiyuki

AU - Shiraha, Hidenori

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AU - Iwasaki, Yoshiaki

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AU - Koide, Norio

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