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. 2006 Jan 31;103(5):1406-11.
doi: 10.1073/pnas.0508103103. Epub 2006 Jan 23.

A SNP in the flt-1 promoter integrates the VEGF system into the p53 transcriptional network

Daniel Menendez  1 , Oliver Krysiak, Alberto Inga, Bianca Krysiak, Michael A Resnick, Gilbert Schönfelder
Affiliations

A SNP in the flt-1 promoter integrates the VEGF system into the p53 transcriptional network

Daniel Menendez et al. Proc Natl Acad Sci U S A. .

Abstract

The VEGF system is essential for angiogenesis. VEGF overexpression frequently correlates with increased microvascularity and metastasis and decreased spontaneous apoptosis. Although a precise mechanism has not been established, studies suggest that VEGF expression is negatively regulated by p53, a master regulator and tumor suppressor. There are no reports of additional components of the VEGF signal transduction pathway being part of the p53 transcriptional network. A target of VEGF, the VEGF receptor 1/flt-1, can regulate growth and migration of endothelial cells and modulate angiogenesis. VEGF appears to be up-regulated in various cancers in which flt-1 may have a role in tumor progression and metastasis. We identified a C-to-T SNP upstream of the transcriptional start site in approximately 6% of the people examined. The SNP is located within a putative p53 response element. Only the promoter with the T SNP (FLT1-T) was responsive to p53 when examined with reporter assays or by endogenous gene expression analysis in cell lines with different SNP status. In response to doxorubicin-induced DNA damage, there was clear allele discrimination based on p53 binding at the FLT1-T but not FLT1-C promoters as well as p53-dependent induction of flt-1 mRNA, which required the presence of FLT1-T. Our results establish that p53 can differentially stimulate transcription at a polymorphic variant of the flt-1 promoter and directly places the VEGF system in the p53 stress-response network via flt-1 in a significant fraction of the human population. We suggest that the p53-VEGF-flt-1 interaction is relevant to risks in angiogenesis-associated diseases, including cancer.

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Figures

Fig. 1.
Fig. 1.
Identification of a SNP in the flt-1 promoter region. (A) SSCP analysis of a 200-bp flt-1 promoter fragment (positions 465-665; GenBank accession no. D640169) from healthy volunteers (lanes 1-8). An extra band is seen in lane 2. Sequence analysis revealed a single heterozygous C/T nucleotide polymorphism (C519T) located 677 bases upstream of the TSS (position 519, GenBank accession no. D64016). Bioinformatic analysis predicted that this SNP falls within a potential p53 RE. (B) RFLP analysis of HMEC, MCF-7, and HCT116 cells revealed that only HCT116 cells harbor a heterozygous C/T allele, whereas HMEC and MCF-7 cells are homozygous for the C allele. The T allele SNP results in an NSP1 site that enables the T (+) and C (-) to be distinguished. A DNA sample from an individual with heterozygous C/T alleles was used as a positive control. (C) Schematic representation of the putative p53 consensus RE containing the C/T SNP in the promoter sequence of the FLT-1 gene. The asterisk indicates the location of SNP c519t, and the underlined region corresponds to the spacer sequence. The second half site contains three mismatches from the consensus (lowercase letters).
Fig. 2.
Fig. 2.
Characterization of the promoter activity of FLT1-C and FLT1-T alleles from different cell lines. (A) A description of the T and C alleles (FLT1-T and FLT1-C) of the flt-1 promoter placed into the luciferase reporter vector pGL3-basic. (B) To assess the potential of the T allele within the flt-1 promoter within different human cell lines, the ability of the FLT1-T and FLT1-C promoter alleles to support transcription was determined after transfections into HMEC and MCF-7 cells (homozygous for the C allele). There was an at least 8-fold increase of transcriptional activity of the T allele promoter (FLT1-T) when compared with FLT1-C in these p53+/+ cell lines. (C) MCF-7 cells were also transiently cotransfected with vectors containing wtp53 or a truncation of the human p53 (Q331stop) under the control of a CMV promoter. Cotransfection with pSV-β-galactosidase control vector was carried out to standardize for transfection efficiency. Luciferase activity of each FLT1-C or FLT1-T reporter construct was compared with the empty pGL3-promoter vector (control). Data represent the averages and standard deviations for three experiments.
Fig. 3.
Fig. 3.
Transactivation of FLT1-C/T alleles by transfected wtp53 in SaOS2 cells. The ability of the FLT1-T and FLT1-C alleles to support transactivation by p53 was examined after transfection into SaOS2 p53-/- cells. (A) SaOS2 cells were transiently cotransfected with a vector containing wtp53, and proteins were determined 24 h after transfection by Western blot. (B) Luciferase activity from the FLT1-T and FLT1-C luciferase reporter plasmids relative to the empty pGL3-promoter vector in the presence or absence of p53. Cells were also transformed with pSV-RLV40 vector to standardize for transfection efficiency. The P21-5′-p53 RE-luciferase reporter is a positive control for p53 induction. Presented are the averages and standard deviations for three experiments. (C) p53 occupancy on FLT1-C and FLT1-T promoters. p53-deficient SaOs2 cells were transfected with the empty vector (control), wild-type FLT1-C and FLT1-T reporter constructs, and a p53-producing plasmid. ChIP was performed with antibodies (Ab) against p53. p53 binding was determined by using flt-1-specific primers in the PCR analysis. “Input” corresponds to total lysate used as a control for PCR amplification. “Ab” indicates samples immunoprecipitated with p53 antibody. “No Ab” corresponds to the amplification from cell extracts that were immunoprecipitated with only the control anti-mouse secondary antibody. (D) RFLP analysis of chromatin immunoprecipitated DNA from SaOS2 cells. Digestion of DNA samples with NspI restriction enzyme (+) is presented. Restriction-digested FLT1-C and FLT1-T promoters were used as internal controls.
Fig. 4.
Fig. 4.
The endogenous FLT1-T allele in HCT116 (FLT1-C/T) is specifically induced by doxorubicin via p53. (A) Western blots of p53 from HCT116 p53+/+ and p53-/- cell lines that are heterozygous for the FLT1-C/T alleles after 24 h of doxorubicin treatment (0.3 μg/ml). Expression is normalized to β-actin levels. (B) flt-1 mRNA is regulated by p53 in HCT116 cells harboring a heterozygous C/T allele. Shown are the relative flt-1 mRNA expression levels as analyzed by real-time PCR analyses after 24 h of doxorubicin treatment (0.3 μg/ml). Expression is normalized to GAPDH levels. p53-dependent flt-1 mRNA expression was compared in HCT116 p53+/+ and HCT116 p53-/- cells. The mean of the control gene expression level was set as 1. Each bar represents the mean of six independent experiments ± SD. (C) After exposure to doxorubin, ChIP was performed on extracts with antibodies against p53. Total lysate was used as a control for PCR amplification (input). PCR was performed with gene-specific primers. p53 binding was shown to be much greater to the T allele than to the C allele in the promoters of the endogenous genes. There was no flt-1 or p21 DNA recovered by ChIP from HCT116 p53-/- cells. Presented are the averages and standard deviations for three independent experiments. (D) RFLP analysis of the PCR products from the ChIP experiment involving p53+/+ cells in C. Presented as controls (first three columns) are restriction analyses of PCR products of the FLT1-T and FLT1-C reporter constructs with and without treatment by NspI as well as PCR products of the endogenous flt-1 promoter region (HCT116). +, NspI restriction enzyme.
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References

    1. Luttun, A., Autiero, M., Tjwa, M. & Carmeliet, P. (2004) Biochim. Biophys. Acta 1654, 79-94. - PubMed
    1. Hoeben, A., Landuyt, B., Highley, M. S., Wildiers, H., Van Oosterom, A. T. & De Bruijn, E. A. (2004) Pharmacol. Rev. 56, 549-580. - PubMed
    1. Bellamy, W. T. (2002) Curr. Opin. Oncol. 14, 649-656. - PubMed
    1. Ferrara, N. (2004) Endocr. Rev. 25, 581-611. - PubMed
    1. Shibuya, M. (2001) Cell. Struct. Funct. 26, 25-35. - PubMed

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