Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylation
© Hanafusa et al; licensee BioMed Central Ltd. 2005
Received: 26 August 2004
Accepted: 20 January 2005
Published: 20 January 2005
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.
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.
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.
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.
Insulin-like growth factor binding protein (IGFBP)-3 is a multifunctional protein ferrying insulin-like growth factors (IGFs) in circulation and mediating growth suppression signals in cells. Serum IGFBP-3 protein (< 5000 ng/ml) complexes with IGFs and an acid labile subunit (ALS), to extend the half lives and modulate the bio-availability of IGFs . While a precise mechanism of action is not clear, the growth suppressive activity of IGFBP-3 depends on its nuclear translocation . Other growth suppressors such as p53, retinoic acids, transforming growth factor (TGF)-β, and tumor necrosis factor (TNF)-α induce IGFBP-3 as a mediator of growth suppression [3–6]. The growth suppression by IGFBP-3 is independent from the modulation of IGFs action [7–9]. The functional importance of the IGFBP-3 in the growth suppression is noteworthy.
IGFBP-3 is produced in most tissues, but the main site of production is liver. It is produced by non-parenchymal cells (endothelial and Kupffer cells) while parenchymal cells (hepatocytes) do not produce it under normal condition . We postulate that IGFBP-3 is a gene induced by growth suppression signals such as p53 in hepatocytes. While the growth suppression imported by IGFBP-3 suggests the potential for tumor suppression, polymorphisms, but no significant mutations were observed in a survey of several tumors . As gene silencing may occur without mutations, we recently investigated IGFBP-3 promoter hypermethylation in human hepatocellular carcinoma . These promoter hypermethylations were subsequently reported in other tumors systems [13, 14].
Promoter analysis of IGFBP-3 indicated that the NaB-RE sequence is essential for the sodium butyrate (NaB) induced IGFBP-3 expression , but the importance of the eleven upstream p53 binding sites reported by Bourdon et al. were not confirmed until now . The methylation hot spot we identified exactly matched the putative p53 binding sites that Bourdon et al. indicated. Thus, we postulated that these sites are important for the expression of IGFBP-3 induced by p53. Moreover, we hypothesized that the suppression of apoptosis mediated by IGFBP-3 due to the promoter hypermethylation will be a possible pathway of hepatocarcinogenesis. To explore this possibility, the functions of the promoter upstream binding sites of p53 were examined precisely in this study.
HepG2 cells were obtained from Japanese Cancer Research Resources Bank (Tokyo, Japan), and maintained in D-MEM supplemented with 10 % FCS (Life Technologies, Tokyo, Japan), antibiotic-antimycotics at 37°C in a humidified atmosphere of 95 % air and 5 % CO2.
Oligonucleotide DNAs used for construction of IGFBP-3 promoter mutants.
sense primer** (5' to 3')
antisense primer** (5' to 3')
HepG2 cells were transiently transfected using the FuGENE 6 transfection reagent according to the manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis IN). Cells were seeded at a density of 5 × 104 cells/well in 24-well plates. After 24 hours, cells were transfected with 0.25 μg/well of reporter plasmid DNA in serum-containing medium. Forty-eight hours post transfection, cells were washed twice with PBS and collected for luciferase assays. Transfections were performed in quadruplicate and experiments were performed at least two times.
Luciferase activities of cell lysates were measured according to the manufacturer's instructions (Promega Corp. Madison, WI) using a liquid scintillation counter (Aloka, LSC-700, Tokyo, Japan). Luciferase activities were normalized for total protein determined using the Bradford Assay (Bio-Rad Laboratories, In., Hercules, CA).
EMSA (electrophoresis mobility shift assay)
278 bp of the Mlu I-Bgl II fragment of pGL-210 were labelled using [α-32P]dCTP by end filling with Klenow fragment, and used for EMSA. Oligonucleotide DNAs (BP3WPSF: 5'-GGCTGCAGCG GGCGTGCGCA CGAGGAGCAG GTGCCCGGGC GAGTCTCGAG CTGCACGCCC CCGAGCTCGG-3', BP3WPSR: 5'-CCGAGCTCGG GGGCGTGCAG CTCGAGACTC GCCCGGGCAC CTGCTCCTCG TGCGCACGCC CGCTGCAGCC-3'), comprising the promoter sequence of -210/-149, were custom-made (Sigma-genosys Japan, Ishikari, Japan), annealed one hour at room temperature, and used as cold competitor in the assay. EMSA was performed in a 20 μl reaction containing 10 mM Tris (pH 7.5), 2.5% Glycerol, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, and in the presence of 50 ng/μl of double-stranded poly [d(I-C)], 5000 cpm (2 ng) of 32P-labeled probe DNA, and 2.5 μg of H2O2 treated MCF7 nuclear extract (Active motif LLC, Palomar, CA). The reaction mixture was incubated at 14°C for 20 min. 20 μl of each reaction mixture was then loaded onto a native 4 % polyacrylamide gel containing 0.5 × Tris-Glycine buffer (25 mM Tris, 190 mM Glycine, 1 mM EDTA pH 8.3), and electrophoresed at 14°C, 100 V for 1 hr. For the supershift assay, a p53 antibody (Ab-2, Oncogene Research Products, San Diego, CA) was used.
To confirm the importance of p53 binding sites between -210 to -150, we abrogated one of p53 binding sites by site-directed mutagenesis (C to T at -179 and G to C at -176, Fig. 3A). In the absence of p53 overexpression, there exist little differences in expression between the wild type and mutant construct (74 % relative to the wild type) (Fig. 3B). However, in the presence of p53 overexpression, IGFBP-3 expression was strongly decreased in the mutant construct (3.4 %) relative to wild type (Fig. 3B). For reasons already mentioned, we did not compare the result between in presence and absence of p53.
Next, we constructed in vitro methylated promoter constructs to evaluate the effect of methylation (Fig. 4A). For methylation, the Mlu I-Xho I fragment of pGL2-210 was methylated with Sss I methylase and reconstituted with unmethylated reporter vector fragment. As we used linear constructs for the transfection, the expression of luciferase was strongly suppressed compared to circular plasmids (0.7 %). But similar patterns compared to site directed mutation were observed. Although the expression of IGFBP-3 was slightly enhanced in the absence of p53 overexpression in the methylated construct, it was decreased in the presence of p53 overexpression in the methylated construct (Fig. 4B). In this experiment, at a glance, we observed induction of IGFBP-3 expression by p53, but the transfection efficiency of linear plasmids is low, while the relative levels of p53 and the availability of putative negative regulators are extremely different from other experiments. Thus, we cannot conclude in this case, whether or not there is induction by p53.
Our observations of promoter hypermethylation in human hepatocellular carcinomas and derivative cell lines (12), and the observations in this report strongly support the notion that IGFBP3 is a true tumor suppressor gene. IGFBP3 is a gene that is silenced by biallelic hypermethylation or hypermethylation and loss of heterogeneity (LOH) in human hepatocellular carcinoma. As reported recently , we have also observed the reduced expression of IGFBP-3 in several tumors such as, breast (9/41), uterus (11/42), ovary (6/16), kidney (6/20), and prostate (1/4) using the cancer profiling array (BD bioscience, data not shown). We therefore postulate that the tumor suppressor role of IGFBP-3 will not be limited to HCCs. In addition, there are also many reports of IGFBP-3 overexpression in tumors from breast , prostate , kidneys , and lung squamous cells , so on. We thus anticipate the existence of additional defects, such as papilloma virus infections that inactivate IGFBP-3 , TGF-β / Rb signalling abnormalities that often coincide with IGFBP-3 overexpression [23–25], or as yet unknown defects in IGFBP-3 receptor function leading to IGFBP-3, for these overexpression in tumors.
IGFBP-3 is a ubiquitous, multifunctional protein, whose importance as a carrier of IGFs is evident. The absence of gross loss-of-function mutations of IGFBP-3 observed to date likely underscores its functional importance. We hypothesize that IGFBP-3 is a gene whose basal level of expression is essential for cell survival, but upon induction by p53, high levels of expression of IGFBP-3 induces apoptosis. Alternatively, IGFBP-3 may be a gene that is essential for cell survival when induced by growth hormones or IGFs, but functions as an apoptotic mediator when induced by p53.
We observed slight base changes within p53 binding sites strongly influenced the induction of IGFBP-3 by p53. As SNPs that change the expression level of IGFBP-3 were within p53 binding sites , and it was reported that the IGFBP-3 is differentially activated by p53 mutants [27, 28], we postulate that the expression and functions of IGFBP-3 is controlled in some way by p53 binding sites in the promoter of IGFBP-3. This may include the intronic p53 binding sites as well as the upstream sites explored here. IGFBP-3 may, therefore, have an important function in tumor development through p53 control.
We identified the MyoD (-195/-186) and WT1 (-164/-156) binding sites as well as p53 binding sites at the hypermethylation hot spot in HCC by promoter analysis using TRANSFAC (v 4.0). As MyoD is a transcription factor that can induce apoptosis, and WT1 is a tumor suppressor gene, the existence of a binding site for these putative regulator genes in the hot spot of the promoter of IGFBP-3 suggest the possibility that these genes also use IGFBP-3 as a mediator of their actions.
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. As IGFBP-3 functions downstream of many growth suppressors and its growth suppression effects are drastic, and as it is a small-sized secreted protein, the use of IGFBP-3 in tumor therapy will be a promising option.
acid labile subunit
Dulbecco's modification of Eagle's medium DTT
- ethylenediaminetetra-acetic acid:
- electrophoresis mobility shift assay:
- fetal calf serum:
- hepatocellular carcinoma:
- Insulin-like growth factor:
- Insulin-like growth factor binding protein-3:
- loss of heterogeneity:
- sodium butyrate:
NaB-RE (sodium butyrate-responsive region), PBS
- phosphate buffered saline:
- polymerase chain reaction:
- transforming growth factor-β:
- tumor necrosis factor-α:
- standard error of mean:
SNP; single nucleotide polymorphism.
We Thank Dr. Y. Oh (Oregon Health Sciences University, Portland, OR) for providing pGL2-IGFBP-3 plasmid and Dr. R. Chaparro (Albert Einstein College of Medicine, New York, NY) for critical reading of this manuscript. This work was supported in part by grants-in aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (#11770273).
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