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Tumor Biology |
Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and basic fibroblast growth factor (3, 4, 5, 6, 7)
. Importantly, breast tumors are not an exception (8
, 9)
. VPF/VEGF is a secreted protein that plays a critical role in tumor-associated microvascular hyperpermeability and angiogenesis (3
, 6)
. Some recent studies have demonstrated the importance of microvessel density for malignant progression in breast cancer, underscoring the importance of angiogenesis in this type of tumor (7
, 10
, 11)
. It has been shown that, in response to hypoxic conditions, human mammary fibroblasts dramatically up-regulate VPF/VEGF mRNA and increase VPF/VEGF protein levels in accordance with the degree of oxygen deprivation (12)
. Oncogenes and antioncogenes also play an important role in regulating VPF/VEGF expression and angiogenesis (13, 14, 15, 16)
. The genetic alterations responsible for oncogenesis and tumor progression may underline the ability of breast tumors to switch to an angiogenic phenotype (17) . The p53 tumor suppressor gene is one of the most frequently mutated genes in human cancers (18) . The loss of p53 function, via somatic mutations or the expression of viral oncoproteins, contributes to the activation of the angiogenic switch during tumorigenesis (13 , 15 , 17 , 19) . We have shown that wt p53 can significantly inhibit the VPF/VEGF transcriptional activation, although the exact mechanism of this inhibition was not explored (13) . Human p53 encodes a multifunctional transcription factor that mediates cellular responses to diverse stimuli, including DNA damage and hypoxia (20) . In addition to being an integral component of the surveillance mechanisms that arrests cell cycle progression under adverse conditions, p53 is also involved in mediating hypoxia-induced apoptosis (21) and inducing inhibitors of angiogenesis such as thrombospondin-1 (22 , 23) . It has been reported that microvessel density, p53 expression, tumor size, and peritumoral lymphatic vessel invasion are relevant prognostic markers in node-negative breast carcinomas (24) .
Amplification and overexpression of the neu (c-erbB2) proto-oncogene has been implicated in the pathogenesis of 20–30% of human breast cancers (25, 26, 27) . Although the activation of Neu receptor tyrosine kinase appears to be a pivotal step during mammary tumorigenesis, the mechanism by which Neu signals cell proliferation is unclear. Molecules bearing a domain shared by the c-Src proto-oncogene (Src homology 2) are thought to be involved in signal transduction from activated receptor tyrosine kinases such as Neu (28) . The polyomavirus middle T oncogene in the mammary epithelium develop multifocal mammary tumors that metastasize with high frequency (29) . It has been reported that the potent transformational activity of polyomavirus middle T antigen is also attributed to its ability to associate with and to activate a number of c-Src family tyrosine kinases (c-Src, c-Yes, and Fyn; Refs. 29 and 30 ). Recent reports have indicated that human breast carcinomas contain functionally activated pp60c-Src (31 , 32) . In previous studies, we have shown that c-Src activation by hypoxia up-regulates VPF/VEGF expression, and constitutive v-Src increased VPF/VEGF mRNA (33) . Thus, Src family kinases seem to play a significant role in the advancement of human breast tumors (31 , 34) . We have also reported that wt p53 and v-Src exert an opposing influence on VPF/VEGF gene expression (13) .
In the present study, we examine in more detail the mechanistic role of p53 and c-Src in regulating the transcriptional expression of VPF/VEGF in breast cancer cell lines MCF-7 and MDA-MB 435 under both normoxic and hypoxic conditions. We demonstrate that p53 makes a complex with Sp1 and thereby inhibits Sp1-mediated VPF/VEGF transcriptional activation. p53 also plays a significant role in down-regulating hypoxia-induced Src kinase activity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmids.
The VPF/VEGF reporter constructs used in transient transfection assays contain sequences derived from human VPF/VEGF promoter driving expression of firefly luciferase. The 0.35- and 0.07-kb deletion mutant constructs were made by PCR from the 2.6-kb promoter fragment and subcloned into pGL-2 basic vector (Promega), as described earlier (14)
. Human cytomegalovirus immediate-early promoter-driven wt and mutant p53 expression plasmids (generously provided by Prof. Arnold J. Levine, Princeton University, Princeton, NJ) were used in this study. wt p53 contains wt p53 cDNA, whereas p53-SCX3 is a single-nucleotide mutation that results in a valine to alanine alteration at codon 143 in p53-wt, and p53–4.2N3 is a mutant human p53 cDNA derived from an epidermoid carcinoma cell line A431 (13)
. This p53 gene encodes an arginine to histidine change at codon 273. Src sense and antisense expression vectors were generous gifts from G. E. Gallick (The University of Texas, M.D. Anderson Cancer Center, Houston, TX; Ref. 35
).
Transfection Assays.
Cells were plated at 2–3 x 105 cells/60-mm dish 1 day before transfection with VPF/VEGF promoter-luciferase construct and expression plasmids using calcium-phosphate precipitation (36)
. The expression was normalized with a control empty-expression vector. Cells were harvested for luciferase assay 40 h after transfection. In all cotransfection experiments, transfection efficiency was normalized by assaying ß-galactosidase activity using the ß-galactosidase gene under control of the cytomegalovirus immediate-early promoter as internal control.
Immunoprecipitations and Western Blot Analyses.
Cells were washed twice with cold PBS, lysed with ice cold lysis buffer [50 mM Tris (pH 7.5), 1% NP40, 150 mM NaCl, 1 mM NaVO4, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 0.5% aprotinin, and 2 mM pepstatin A], incubated for 10 min on ice, and centrifuged for 10 min at 4°C. Immunoprecipitations were carried out at antibody excess using 0.5 mg of total protein with rabbit polyclonal antibodies (1 µg) directed against p53 (Santa Cruz Biotechnology) and Sp1 (Santa Cruz Biotechnology). Immunocomplexes were captured with protein A agarose beads (Pharmacia). After three washes with cell lysis buffer, bead-bound proteins were separated by SDS-PAGE. Western blot analysis was carried out as described earlier (14)
.
Kinase Assay.
Src kinase activity was assayed by measuring the incorporation of 32P into a Src-specific peptide (33)
. The cellular extracts were immunoprecipitated with Src-specific antibody (Santa Cruz Biotechnology) and the immunoprecipitates were incubated in a 25 µl-reaction mixture consisting of 30 mM Tris-HCl (pH 7.5), 0.01% Triton X-100, 10 mM 2-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 10 mM MgCl2, 0.4 mM Src-specific peptide (Upstate Biotechnology, Inc.), and 50 µM [
-32P]ATP for 30 min at 30°C. The reaction was stopped by the addition of ice-cold 25% trichloroacetic acid. Precipitates were collected on phosphocellulose filter paper. The filters were washed with 0.75% phosphoric acid and counted for 32P using liquid scintillation spectroscopy.
Nuclear Extract Preparation and EMSAs.
Nuclear extracts were prepared using MCF-7 cells following a standard protocol, with modifications (14
, 16)
. Cells were washed in cold PBS, suspended in buffer A [10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 10 µg/ml aprotinin, 3 mM DTT, and 0.1 mM phenylmethylsulfonyl fluoride] and incubated for 15 min on ice. Cells were then lysed with 0.5% NP40 and the pellets were resuspended in buffer C [50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% glycerol, 3 mM DTT and 0.1 mM phenylmethylsulfonyl fluoride]. After incubation on a rotating rack for 25 min, samples were centrifuged at 14,000 RPM for 10 min. Clear supernatants containing the nuclear proteins were collected and stored at -70°C.
EMSAs were performed as described previously (14
, 16)
. Briefly, EMSA binding reaction mixtures (25 µl) contained 20 mM HEPES (pH 8.4), 100 mM KCl, 20% glycerol, 0.1 mM EDTA, 0.2 mM ZnSO4, 0.05% NP40, and 1 µg BSA. Extract protein and 200 ng of poly(dA-dT).poly(dA-dT) were added at room temperature 10 min before the addition of 
0.1 ng of radiolabeled oligonucleotide probe. After 20 min incubation at 4°C, samples were run on 7% acrylamide gel in 1x TAE (40 mM Tris-acetate and 1 mM EDTA) buffer.
The radiolabeled oligonucleotide used in EMSA studies was a 188-bp PCR-generated fragment (bp -195 to -7, relative to the transcription start site) of the VPF/VEGF promoter containing the four putative Sp1 binding sites (14 , 16) .
RNA Analysis by Northern Blot Hybridization.
Total RNA, isolated by the single-step acid-phenol extraction method (37)
, was separated on a formaldehyde-agarose gel, transferred to a GeneScreen (DuPont) membrane by using 10x SSC, and probed with random-primer-labeled cDNAs in a solution containing 0.5 M sodium phosphate (pH 7.2), 7% SDS, 1% BSA, 1 mM EDTA, and sonicated herring sperm DNA (50 µg/ml) at 68°C. Blots were washed three times with a solution containing 40 mM sodium phosphate (pH 7.2), 0.5% SDS, 0.5% BSA, and 1 mM EDTA at 68°C and autoradiographed.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. To define the region of the VPF/VEGF promoter that is responsive to wt p53, we used two different 5' deletions of the 2.6-kb promoter-reporter vector and cotransfected these deletions with the plasmid expressing wt p53. It has been found that wt p53 decreased the reporter activity by 70% in the 0.35-kb segment of the VPF/VEGF promoter that contains multiple Sp1 binding sites, although there was no change of reporter activity in the 0.07-kb VPF/VEGF promoter having the deleted Sp1 binding site (Fig.1A)
. We also observed a similar result in MDA-MB 435 cells (data not shown). These observations reveal that in breast cancer cells, wt p53 may inhibit VPF/VEGF transcription by interfering with Sp1-mediated promoter activation. Interestingly, when we used two different mutants of wt p53, they could not down-regulate the Sp1-mediated VPF/VEGF promoter activity in MCF-7 cells (Fig.1B)
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To examine the consequences of the association between p53 and Sp1 on VPF/VEGF transcription, we performed EMSA by using a 188-bp VPF/VEGF promoter fragment containing all four Sp1 binding sites, with the nuclear extracts of MCF-7 cells transfected with or without wt p53. As shown in Fig.1D
, wt p53 inhibited the binding of Sp1 to the VPF/VEGF promoter. This specific protein-DNA complex formation was competed away with 10-fold molar excess of Sp1 consensus oligonucleotide. From these results, it seems that wt p53 forms a complex with Sp1 and, thereby, may prevent the VPF/VEGF transcriptional activation by blocking Sp1 and promoter interaction.
p53 Can Also Down-Regulate Hypoxia-induced Activation of VPF/VEGF Transcription.
Hypoxia appears to be an important stimulus in promoting the overexpression of VPF/VEGF. Previous studies have indicated that p53 can down-regulate hypoxia-mediated VPF/VEGF transcription through degradation of HIF-1 (17)
. Here, we attempted to explore whether wt p53 can also inhibit the hypoxic induction of VPF/VEGF transcriptional activation involving Sp1. To this end, MCF-7 cells were transfected with 2.6-kb promoter-reporter construct (containing both HIF-1 and Sp1 binding sites) and wt p53 and then subjected to hypoxic condition. As shown in Fig. 2
, wt p53 clearly down-regulated the hypoxia-mediated activation of VPF/VEGF transcription as compared with the normoxic control. We found a similar result in MDA-MB 435 cells (data not shown). Additionally, we also observed through EMSA that, under hypoxic condition, there was increased binding of Sp1 to the VPF/VEGF promoter and, in the presence of wt p53, this binding was significantly inhibited (Fig. 1D)
. We have also confirmed the binding of Sp1 through antibody supershift study. Thus, from all these observations it is evident that, as under normoxic conditions, wt p53 plays a significant role in regulating Sp1-mediated VPF/VEGF transcription under hypoxic conditions. Interestingly, we did not get any significant hypoxic induction of the 0.35-kb VPF/VEGF promoter sequence, which, in spite of having Sp1 binding sites, does not contain any binding sequence for HIF-1 (data not shown). This observation indicates that a cooperative function of both Sp1 and HIF-1 may be needed for VPF/VEGF transcriptional activation under hypoxic conditions.
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, the substrate was strongly phosphorylated when the cells were exposed to hypoxic condition for 30 min, although the Src protein levels were the same in all of the conditions (Fig. 3B)
. This result suggests that Src, activated by hypoxia, is a key signaling molecule in human breast cancer cells.
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. We also observed a similar type of inhibition of hypoxia-induced VPF/VEGF expression in MDA-MB 435 cells using Src antisense cDNA (data not shown). This result clearly suggests the importance of c-Src in increasing the VPF/VEGF mRNA level in human breast cancer cells under hypoxic conditions.
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shows that in MCF-7 cells cotransfected with 2.6-kb promoter-luciferase construct and Src antisense cDNA, the hypoxia-induced VPF/VEGF transcriptional activation was significantly reduced in comparison to the cells transfected with Src sense cDNA. This result indicates that in breast cancer cells, c-Src activation is critical for hypoxia-mediated up-regulation of VPF/VEGF transcription.
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. The mutant p53 could not decrease either normoxic or hypoxic induction of Src kinase activity (Fig. 6)
. This result clearly suggests that p53 may play an important role in regulating tumor angiogenesis by inhibiting Src kinase activity under both normoxic and hypoxic conditions.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The tumor suppressor gene p53 has been found to be mutated in most human cancers (17
, 18
, 43)
. It inhibits the proliferation of normal as well as transformed cells by interacting with viral and cellular oncoproteins (38)
. Overexpression of wt p53 can inhibit transcription of many cellular and viral promoters. We have previously shown that wt p53 can inhibit the transcription of VPF/VEGF (13)
. The present study elucidates a novel mechanism by which p53 inhibits the transcription of VPF/VEGF in an Sp1-dependent manner in the two breast cancer cell lines MCF-7 and MDA-MB 435 (Fig. 1, A and B)
. p53 was able to inhibit the VPF/VEGF promoter activity only if its Sp1 binding sites were intact (Fig.1A)
. There was no change in the activity of the promoter with the deleted Sp1 binding sites (Fig. 1A)
. It has been shown that p53 interacts with the transcription factor Sp1 and thereby prevents its binding to the VPF/VEGF promoter for its transcriptional activation (Fig. 1C and D)
. Thus, Sp1 seems to be the key target molecule for p53 in regulating VPF/VEGF transcription.
Breast tumors, being solid in nature, contain regions of vascular deficiency or defective microcirculation and thereby are deprived of oxygen, glucose, and other nutrients (10
, 11)
. Hypoxic regions of these solid breast tumors produce the powerful and directly acting angiogenic protein VPF/VEGF (8
, 9)
. It is known that HIF-1, a heterodimeric transcription factor, is the key molecule that generally regulates cellular energy metabolism and angiogenesis in response to oxygen deprivation (17
, 44)
. Some earlier studies have shown that wt p53 does not repress hypoxia-induced transcription of VPF/VEGF (45)
. Interestingly, some later studies have demonstrated that p53 promotes the proteasomal degradation of the subunit of HIF-1 and thereby inhibits angiogenesis by down-regulating VPF/VEGF transcription (17
, 46)
. In this study, we report that, like HIF-1, Sp1 also plays a significant role in promoting VPF/VEGF transcription in breast cancer cells under hypoxic conditions (Fig. 1D)
. We have shown that, in this condition, there is increased binding of Sp1 to the VPF/EGF promoter (Fig. 1D)
. We have also demonstrated that, as under normoxic conditions, p53 can also down-regulate the hypoxic induction of VPF/VEGF transcriptional activation in breast cancer cells by preventing the binding of Sp1 to its promoter (Figs. 1D
and 2
). The exact role of HIF-1 in this situation needs to be explored.
It has been reported that higher activity of the proto-oncogene c-Src is necessary for the increased expression of VPF/VEGF in different tumor cell lines, including glioma (47)
, colon carcinoma (35)
, ovarian carcinoma (48)
, lung carcimoma (49)
, and also in primary culture like mouse brown adipocytes (50)
or in retinal vascular disorder (51)
. This phenomenon was observed not only in hypoxia but also in growth factor-regulated or X-ray- or C-ion-induced VPF/VEGF expression (49
, 50
, 52)
. In the present paper, we report increased c-Src kinase activity in MDA-MB 435 cells under hypoxia (Fig. 3)
. In breast cancer cell lines, c-Src is an important modulator of VPF/VEGF transcription under hypoxia because, in the presence of the antisense of Src, there was a down-regulation of the total mRNA level and also of the promoter activity (Fig. 4
and 5
). Previously, we have reported that v-Src in presence of wt p53 was unable to activate the transcription of the VPF/VEGF promoter (13)
. The present study suggests that wt p53, apart from its inhibitory role on the transcription factor Sp1, may also regulate c-Src activity and thereby down-regulate VPF/VEGF transcription, either directly or indirectly (Fig. 6)
. To understand the function of p53 and c-Src in breast cancer cells, we transiently overexpressed wt p53 as well as its dominant-negative mutant in MCF-7 and MDA-MB 435 cell lines, and we determined their Src kinase activity in both normoxic and hypoxic conditions (Fig. 6)
. The observation of down-regulation of c-Src kinase activity, only through wt p53, has led us to conclude that p53 has a more direct effect on the inhibition of VPF/VEGF transcription by c-Src, and that this inhibitory function is probably different from its effect on Sp1.
In conclusion, here we define a central inhibitory role of p53 on the transcriptional regulation of the angiogenic growth factor VPF/VEGF in two different breast cancer cell lines. p53 can directly regulate the transcriptional activity of Sp1 by inhibiting its association to the promoter region of the VPF/VEGF gene, under both normoxic and hypoxic conditions. p53 also regulates c-Src kinase activity and thereby inhibits c-Src-induced VPF/VEGF expression under the above conditions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by NIH Grant CA78383, Massachusetts Public Health, and the Department of the Army Breast Cancer Research Grants DAMD17-00-1-0153 and DAMD17-00-1-0607 (to D. M.). D. M. is the recipient of the Eugene P. Schonfeld Medical Research Award from the National Kidney Cancer Association. 
2 The first two authors have contributed equally to this work.
3 To whom requests for reprints should addressed, at Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, RN 270H, Boston, MA 02215. Phone: (617) 667-7853; Fax: (617) 667-3591; E-mail; dmukhopa{at}caregroup.harvard.edu
4 The abbreviations used are: VPF/VEGF, vascular permeability factor/vascular endothelial growth factor; wt, wild type; EMSA, electrophoretic mobility shift analysis; HIF-1, hypoxia-inducible factor 1.
Received 2/27/01. Accepted 7/16/01.
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REFERENCES |
|---|
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Genes Dev., 14: 34-44, 2000. in common human cancers and their metastases. Cancer Res., 59: 5830-5835, 1999.This article has been cited by other articles:
|
|
 |
|
  D. M. Birk, J. Barbato, L. Mureebe, and R. A. Chaer Basic Science Review: Current Insights on the Biology and Clinical Aspects of VEGF Regulation Vascular and Endovascular Surgery, January 1, 2009; 42(6): 517 - 530. [Abstract] [PDF]
|
|
|
|
 |
|
 B. Nie, Z. Shen, J.-B. Wen, O. G.-W. Wong, W. D. Hsueh, L.-F. Huo, H.-F. Kung, B. Jiang, and M. C.M. Lin AAV-HGFK1 and Ad-p53 cocktail therapy prolongs survival of mice with colon cancer Mol. Cancer Ther., September 1, 2008; 7(9): 2855 - 2865. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Guo, V. Gokhale, L. H. Hurley, and D. Sun Intramolecularly folded G-quadruplex and i-motif structures in the proximal promoter of the vascular endothelial growth factor gene Nucleic Acids Res., August 1, 2008; 36(14): 4598 - 4608. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Finn Targeting Src in breast cancer Ann. Onc., August 1, 2008; 19(8): 1379 - 1386. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Sun, W.-J. Liu, K. Guo, J. J. Rusche, S. Ebbinghaus, V. Gokhale, and L. H. Hurley The proximal promoter region of the human vascular endothelial growth factor gene has a G-quadruplex structure that can be targeted by G-quadruplex-interactive agents Mol. Cancer Ther., April 1, 2008; 7(4): 880 - 889. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. K. Choi, S. H. Roh, H. G. Kim, E. H. Han, H. G. Jeong, and K. W. Kang Enhanced expression of aromatase in p53-inactivated mammary epithelial cells Endocr. Relat. Cancer, March 1, 2008; 15(1): 139 - 147. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Harper, L. Yan, R. M. Loureiro, I. Wu, J. Fang, P. A. D'Amore, and M. A. Moses Repression of Vascular Endothelial Growth Factor Expression by the Zinc Finger Transcription Factor ZNF24 Cancer Res., September 15, 2007; 67(18): 8736 - 8741. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Goukassian, G. Qin, C. Dolan, T. Murayama, M. Silver, C. Curry, E. Eaton, C. Luedemann, H. Ma, T. Asahara, et al. Tumor Necrosis Factor-{alpha} Receptor p75 Is Required in Ischemia-Induced Neovascularization Circulation, February 13, 2007; 115(6): 752 - 762. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Peng, D. Wei, L. Wang, H. Tang, J. Zhang, X. Le, Z. Jia, Q. Li, and K. Xie RUNX3 Inhibits the Expression of Vascular Endothelial Growth Factor and Reduces the Angiogenesis, Growth, and Metastasis of Human Gastric Cancer. Clin. Cancer Res., November 1, 2006; 12(21): 6386 - 6394. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Datta, J. A. Flaxenburg, S. Laxmanan, C. Geehan, M. Grimm, A. M. Waaga-Gasser, D. M. Briscoe, and S. Pal Ras-induced Modulation of CXCL10 and Its Receptor Splice Variant CXCR3-B in MDA-MB-435 and MCF-7 Cells: Relevance for the Development of Human Breast Cancer Cancer Res., October 1, 2006; 66(19): 9509 - 9518. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S M Hyder Sex-steroid regulation of vascular endothelial growth factor in breast cancer. Endocr. Relat. Cancer, September 1, 2006; 13(3): 667 - 687. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Gonzalez, M. T. Agullo-Ortuno, J. M. Garcia-Martinez, A. Calcabrini, C. Gamallo, J. Palacios, A. Aranda, and J. Martin-Perez Role of c-Src in Human MCF7 Breast Cancer Cell Tumorigenesis J. Biol. Chem., July 28, 2006; 281(30): 20851 - 20864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-i. Yamagishi, K. Nakamura, T. Matsui, Y. Inagaki, K. Takenaka, Y. Jinnouchi, Y. Yoshida, T. Matsuura, I. Narama, Y. Motomiya, et al. Pigment Epithelium-derived Factor Inhibits Advanced Glycation End Product-induced Retinal Vascular Hyperpermeability by Blocking Reactive Oxygen Species-mediated Vascular Endothelial Growth Factor Expression J. Biol. Chem., July 21, 2006; 281(29): 20213 - 20220. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Qin, R. Kishore, C. M. Dolan, M. Silver, A. Wecker, C. N. Luedemann, T. Thorne, A. Hanley, C. Curry, L. Heyd, et al. Cell cycle regulator E2F1 modulates angiogenesis via p53-dependent transcriptional control of VEGF PNAS, July 18, 2006; 103(29): 11015 - 11020. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Sun, K. Guo, J. J. Rusche, and L. H. Hurley Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents Nucleic Acids Res., October 20, 2005; 33(18): 6070 - 6080. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Laxmanan, D. Datta, C. Geehan, D. M. Briscoe, and S. Pal CD40: A Mediator of Pro- and Anti-Inflammatory Signals in Renal Tubular Epithelial Cells J. Am. Soc. Nephrol., September 1, 2005; 16(9): 2714 - 2723. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Vidal, S. Zacharoulis, W. Guo, D. Shaffer, F. Giancotti, A. H. Bramley, C. de la Hoz, K. K. Jensen, D. Kato, D. D. MacDonald, et al. p130Rb2 and p27kip1 cooperate to control mobilization of angiogenic progenitors from the bone marrow PNAS, May 10, 2005; 102(19): 6890 - 6895. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. I. Rini and E. J. Small Biology and Clinical Development of Vascular Endothelial Growth Factor-Targeted Therapy in Renal Cell Carcinoma J. Clin. Oncol., February 10, 2005; 23(5): 1028 - 1043. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Hicklin and L. M. Ellis Role of the Vascular Endothelial Growth Factor Pathway in Tumor Growth and Angiogenesis J. Clin. Oncol., February 10, 2005; 23(5): 1011 - 1027. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. K. Kairaitis, Y. Wang, M. Gassmann, Y.-C. Tay, and D. C. H. Harris HIF-1{alpha} expression follows microvascular loss in advanced murine adriamycin nephrosis Am J Physiol Renal Physiol, January 1, 2005; 288(1): F198 - F206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kaluzova, S. Kaluz, M. I. Lerman, and E. J. Stanbridge DNA Damage Is a Prerequisite for p53-Mediated Proteasomal Degradation of HIF-1{alpha} in Hypoxic Cells and Downregulation of the Hypoxia Marker Carbonic Anhydrase IX Mol. Cell. Biol., July 1, 2004; 24(13): 5757 - 5766. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Flaxenburg, M. Melter, P. H. Lapchak, D. M. Briscoe, and S. Pal The CD40-Induced Signaling Pathway in Endothelial Cells Resulting in the Overexpression of Vascular Endothelial Growth Factor Involves Ras and Phosphatidylinositol 3-Kinase J. Immunol., June 15, 2004; 172(12): 7503 - 7509. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Wei, L. Wang, Y. He, H. Q. Xiong, J. L. Abbruzzese, and K. Xie Celecoxib Inhibits Vascular Endothelial Growth Factor Expression in and Reduces Angiogenesis and Metastasis of Human Pancreatic Cancer via Suppression of Sp1 Transcription Factor Activity Cancer Res., March 15, 2004; 64(6): 2030 - 2038. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zheng, C. Wasylyk, A. Ayadi, J. Abecassis, J. A Schalken, H. Rogatsch, N. Wernert, S.-M. Maira, M.-C. Multon, and B. Wasylyk The transcription factor Net regulates the angiogenic switch Genes & Dev., September 15, 2003; 17(18): 2283 - 2297. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Huang, T. Tyler, N. Saadatmandi, C. Lee, P. Borgstrom, and R. A. Gjerset Enhanced Tumor Suppression by a p14ARF/p53 Bicistronic Adenovirus through Increased p53 Protein Translation and Stability Cancer Res., July 1, 2003; 63(13): 3646 - 3653. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Abe, K. Terada, H. Wakimoto, R. Inoue, E. Tyminski, R. Bookstein, J. P. Basilion, and E. A. Chiocca PTEN Decreases in Vivo Vascularization of Experimental Gliomas in Spite of Proangiogenic Stimuli Cancer Res., May 1, 2003; 63(9): 2300 - 2305. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. M. J. J. Berns, J. G. M. Klijn, M. P. Look, N. Grebenchtchikov, R. Vossen, H. Peters, A. Geurts-Moespot, H. Portengen, I. L. van Staveren, M. E. Meijer-van Gelder, et al. Combined Vascular Endothelial Growth Factor and TP53 Status Predicts Poor Response to Tamoxifen Therapy in Estrogen Receptor-positive Advanced Breast Cancer Clin. Cancer Res., April 1, 2003; 9(4): 1253 - 1258. [Abstract] [Full Text] [PDF]
|
|
|
|
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H. F. Dvorak Vascular Permeability Factor/Vascular Endothelial Growth Factor: A Critical Cytokine in Tumor Angiogenesis and a Potential Target for Diagnosis and Therapy J. Clin. Oncol., November 1, 2002; 20(21): 4368 - 4380. [Abstract] [Full Text] [PDF]
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A. Takahashi, Y. Kureishi, J. Yang, Z. Luo, K. Guo, D. Mukhopadhyay, Y. Ivashchenko, D. Branellec, and K. Walsh Myogenic Akt Signaling Regulates Blood Vessel Recruitment during Myofiber Growth Mol. Cell. Biol., July 1, 2002; 22(13): 4803 - 4814. [Abstract] [Full Text] [PDF]
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, empty expression vector; 
, wt p53 expression. B, MCF-7 cells were cotransfected with 0.35-kb VPF/VEGF promoter-luciferase construct (1.0 µg) and mutant p53 (p53-SCX3 and p53–4.2N3) expression vectors. After harvesting the cells, luciferase activity was measured as described above. C, extracts were prepared from MCF-7 and MDA-MB 435 cells and transfected with wt p53 expression vector. Both the transfected and untransfected cell lysates were immunoprecipitated with a polyclonal antibody directed against wt p53. All immunoprecipitates were then captured by protein A-Sepharose beads. After thorough washings, the Sepharose beads were boiled in SDS buffer and separated by SDS-PAGE. Western blottings (Blot) were performed by using Sp1 polyclonal antibody. Lane 1, transfected with wt p53 expression vector; Lane 2, transfected with empty expression vector; Lanes 3 and 4: control cell lysates, immunoprecipitated with polyclonal antibodies, directed against Sp1 and IGF-1 respectively. D, by using a 188-bp VPF/VEGF promoter fragment (having all of the four Sp1 binding sites) as the probe, EMSA was performed with partially purified nuclear extracts of MCF-7 cells, transfected with wt p53 (0.5 µg), and subjected to both normoxic (I) and hypoxic conditions (II). In hypoxic conditions, the cells were exposed to hypoxia overnight. Nuclear extracts were prepared from the transfected cells 40 h after transfection. Lane 1, probe control, without any nuclear extract. In Lane 2 of the normoxic condition, the unradiolabeled Sp1 consensus oligonucleotide (oligo; 10-fold molar excess) was added to the binding reaction mixture of the control sample (without any transfection) to show that it can compete away the specific protein-DNA complexes. In Lane 5 of the hypoxic condition, Sp1 polyclonal antibody (2 µg) was added to the reaction mixture of the hypoxic sample to show the supershift of the Sp1 band.
