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


CONCLUSIONS
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.
Hanafusa
Tadashi
T
Okayama University Advanced Science Research Center, Department of Radiation Research, Shikata Laboratory, Shikata-cho 2-5-1, Okayama Japan. hanafusa@cc.okayama-u.ac.jp
Shinji
Toshiyuki
T
Shiraha
Hidenori
H
Nouso
Kazuhiro
K
Iwasaki
Yoshiaki
Y
Yumoto
Eichiro
E
Ono
Toshiro
T
Koide
Norio
N
D001483:Base Sequence; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular / Q000235:genetics; D045744:Cell Line, Tumor; D016384:Consensus Sequence; D019175:DNA Methylation; D024202:Electrophoretic Mobility Shift Assay; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics*; D008113:Liver Neoplasms / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D018972:Insulin-Like Growth Factor Binding Protein 3; D016159:Tumor Suppressor Protein p53