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[Cancer Research 60, 3655-3661, July 1, 2000]
© 2000 American Association for Cancer Research


Tumor Biology

Wild-Type p53 Suppresses Angiogenesis in Human Leiomyosarcoma and Synovial Sarcoma by Transcriptional Suppression of Vascular Endothelial Growth Factor Expression1

Lianglin Zhang, Dihua Yu, Mei Hu, Shunbin Xiong, Aiqing Lang, Lee M. Ellis and Raphael E. Pollock2

Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030


    ABSTRACT
 Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our recent studies (R. Pollock et al., Clin. Cancer Res., 4: 1985–1994, 1998; M. Milas et al., Cancer Gene Ther., in press, 2000) have shown that the restoration of wild-type (wt) p53 enhances cell cycle control in vitro and inhibits the growth of human soft-tissue sarcoma in severe combined immunodeficient mice. We hypothesized that the antitumor effect of wt p53 overexpression in sarcoma cells is attributable not only to enhanced cell cycle control but also to inhibition of angiogenesis. We evaluated the effect of restoring wt p53 function on angiogenesis in human soft-tissue sarcoma harboring mutant p53. Restoration of wt p53 expression in human leiomyosarcoma SKLMS-1 cells that contain mutant p53 markedly inhibited angiogenesis induced by tumor cells in vivo. Angiogenesis assays using an in vivo Matrigel plug assay demonstrated that less neovascularization in severe combined immunodeficient mice was observed with conditioned medium (CM) from human synovial sarcoma cells expressing wt p53 compared with CM from human synovial sarcoma cells expressing mutant p53. Microvessel density and microvessel counts were lower in tumor xenografts from cells containing wt p53 than in tumor xenografts from cells containing mutant p53. The growth and migration of murine lung endothelial cells were decreased when cells were treated with CM from sarcoma cells expressing wt p53 compared with CM from sarcoma cells expressing mutant p53. The introduction of wt p53 into sarcoma cells containing mutant p53 significantly reduced the expression of vascular endothelial growth factor (VEGF), which is a key mediator of tumor angiogenesis. Stimulation of endothelial cell migration by CM from cells expressing mutant p53 was significantly reduced after anti-VEGF neutralizing antibody was added to the CM. Using luciferase as the reporter of VEGF promoter activity, we found that wt p53 inhibited VEGF promoter activity in SKLMS-1 cells. Deletion analysis defined an 87-bp region (bp -135 to -48) in the VEGF promoter that is necessary for inhibiting VEGF promoter activity by wt p53. The transcription factor Sp1 may be involved in the repression of VEGF promoter activity by wt p53 in SKLMS-1 cells. These data indicated that wt p53 can suppress angiogenesis in human soft-tissue sarcomas by transcriptional repression of VEGF expression.


    INTRODUCTION
 Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The growth and metastasis of tumors depend on the development of an adequate blood supply via angiogenesis. VEGF3 is a primary regulator of physiological angiogenesis, such as embryonic and reproductive manifestations, and it is a major mediator of pathological angiogenesis, such as tumor-associated neovascularization, proliferative retinopathy, and rheumatoid arthritis (1 , 2) . VEGF is a secreted, glycosylated dimeric polypeptide with a molecular mass of 34,000–45,000 daltons that can be expressed in four isoforms, i.e., VEGF121, VEGF165, VEGF189, and VEGF206, which are generated by alternative gene splicing (3) .

VEGF expression can be regulated by several cytokines, including transforming growth factor {alpha}, transforming growth factor ß, platelet-derived growth factor (2) , and insulin-like growth factor 1 (4) . In addition, the expression and function of VEGF in human tumors may be controlled by oncogenes and tumor suppressor genes. Mutant H-ras and K-ras oncogenes and v-src and v-raf induce VEGF expression (5) . Hypoxia induces VEGF expression through c-Src activation (6) and may involve transcriptional activation by hypoxia-inducible factor 1 and mRNA stabilization (7) ; v-src also induces expression of hypoxia-inducible factor 1 and transcription of the VEGF gene (8) . In renal cell carcinoma, the von Hippel-Lindau tumor suppressor inhibits VEGF expression by blocking protein kinase C (9) , interacting with Sp1 to repress VEGF transcription (10) , and modulating the stability of VEGF mRNA (11) .

The p53 tumor suppressor may also regulate VEGF expression. Mutant p53 potentiates protein kinase C induction of VEGF expression (12) . In a tumorigenicity model using human fibroblast cells, the loss of wt p53 and transfection of activated ras led to increased VEGF expression (13) . Transient transfection of wt p53 and of v-src has been found to exert opposing effects on human VEGF gene promoter activity in human glioblastoma and transformed human fetal kidney cells (14) . However, because wt p53 did not repress hypoxia-induced transcription of the VEGF gene in stably transfected human colorectal carcinoma and human hepatoblastoma cells with an inducible p53-estrogen-receptor fusion protein expression system (15) , it was proposed that there is a need for caution in interpreting p53 function on the basis of cells transiently overexpressing this protein (15) . Nevertheless, mutant p53 has been correlated with increased VEGF expression in human lung cancer (16) , bladder cancer (17) , and colorectal cancer (18) by immunohistochemical staining of surgical specimens. It has been reported that dysfunction of the p53/MDM-2 pathway can be found in more than two-thirds of angiosarcomas; thus, mutant p53 influenced the development of angiosarcoma not only by affecting growth and apoptosis control but also by up-regulating VEGF (19) .

In a recent study (20) , we used an adenovirus-mediated wt p53 gene-delivery system to investigate the therapeutic efficacy of restoring p53 function in human soft-tissue sarcoma. We found that introduction of the wt p53 gene can retard the growth of sarcoma in SCID mice. We know that the inhibition of cell cycle progression contributed to the antitumor effect of wt p53 in soft-tissue sarcoma (21) . Because several recent reports had demonstrated that p53 can inhibit tumor angiogenesis in other tumor types (14 , 22 , 23) , we hypothesized that the antitumor effect of wt p53 expression in sarcomas was caused not only by enhanced cell cycle control but also by inhibition of angiogenesis.


    MATERIALS AND METHODS
 Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
Cultured in DMEM/F12 with 10% fetal bovine serum (complete culture medium; Life Technologies, Grand Island, NY) in 5% CO2 and 95% air at 37°C were: SKLMS-1 human leiomyosarcoma cells, which have a p53 point mutation at codon 245 (American Type Culture Collection, Rockville, MD); SYNb-1 human synovial sarcoma cells, which contain wt p53 (established in our laboratory from the primary tumor of a patient); and SYNb-2 cells, which have a p53 point mutation at codon 135 (established in our laboratory from the metastatic tumors in the patient from whom SYNb-1 cells were obtained; Ref. 24 ).

143Ala temperature-sensitive mutant p53-transfected SKLMS-1 cells designated SKAla-1, SKAla-2, and SKAla-3, respectively (21) were cultured in complete culture medium in 5% CO2 and 95% air at 37°C. These cells express wt p53 at 32°C and mutant p53 at 38°C. MluE cells (American Type Culture Collection, Rockville, MD) were cultured in complete culture medium containing 75 µg/ml endothelial cell growth supplement (Sigma, Saint Louis, MO) in 5% CO2 and 95% air at 37°C. Cells were passaged by treatment with 0.25% trypsin and 1 mM EDTA (Life Technologies) in PBS.

Preparation of CM.
Sarcoma-cell CM was prepared as described previously (25) . Briefly, cultures of the various sarcoma cell lines were rinsed twice with serum-free DMEM/F12 and cultured in 10 ml of serum-free DMEM/F12. One day later, the culture medium was removed by aspiration, and 10 ml of fresh serum-free DMEM/F12 medium was added to each culture. Sarcoma-cell CM was collected after a 72-h incubation, and 25 mM HEPES buffer (pH 7.4), 0.5 mg/ml leupeptin, 0.7 mg/ml pepstatin, 0.8 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 0.02% NaN3, and 0.1% BSA (Intergen, Purchase, NY) were added. Sarcoma-cell CM was clarified by centrifugation. CM was frozen and stored at -80°C until use.

Angiogenesis Assays in Vivo.
Angiogenesis was assessed in vivo as described previously (26) . Briefly, CM from SKLMS-1 cells, SKAla-1, -2, and -3 cells cultured at 32°C and 38°C; and SYNb-1 and SYNb-2 cells cultured at 37°C were concentrated with Centricon concentrator (Amicon, INC., Beverly, MA). Then 380 µl of Matrigel (Becton Dickinson, Bedford, MA) and 0.1 µg of basic fibroblast growth factor (R&D Systems, Minneapolis, MN) were mixed with 50 µl of the concentrated CM. A total of 0.5 ml of this modified Matrigel was injected into each SCID mouse. There were 10 treatment groups with five mice in each group. The animals were cared for in accordance with institutional and NIH guidelines. One week after the initial injection, the Matrigel plug was removed and bisected. One half of the Matrigel plug was used to measure hemoglobin using the Drakin method (Drakin reagent kit 525; SIGMA). The other half of the Matrigel plug was fixed in 4% formaldehyde and stained with H&E to permit determination of the gel for infiltrating vessels (27) .

Analysis of Microvessel Density in Tumor Sections.
For analysis of the microvessel density in SYNb-1 and SYNb-2 human synovial sarcoma samples, the paraffin-embedded blocks were stained with rabbit antihuman von Willebrand factor polyclonal antibody (DAKO, Carpinteria, CA) by the ABC staining system (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Tumor microvessel counts were analyzed also in paraffin-embedded tumor tissue blocks from SCID mice with SKLMS-1 tumors treated or not treated with Ad5p53, which is an E1A-deleted, replication-deficient adenovirus expressing a CMV promoter-driven wt p53 cDNA (20) . The microvessel density grade (on a scale of 1–4+) and microvessel counts were studied according to the method of Weidner (28 , 29) .

Endothelial Cell Growth Assay.
Endothelial cell growth was assayed in 96-well plate, which were precoated with 0.5% gelatin solution for 30 min. Five hundred MluE cells were loaded into each well along with 200 µl of CM from SKLMS-1 cells, SYNb-1 and -2 cells, and SKAla-1, -2, and -3 cells cultured at 32°C and 38°C and supplemented with 1% fetal bovine serum. Cell growth assays were terminated after 3 and 7 days of growth. Cell number was evaluated using crystal violet staining as described previously (30) . The absorbance was measured using a microplate reader set to 590 nm.

Endothelial Cell Migration Assay.
Endothelial cell migration was assayed in 24-well, 6.5-mm-internal-diameter Transwell cluster plates (8.0-µm pores; Costar, Cambridge, MA) using MluE cells and serum-free sarcoma-cell CM (the same CM as used for the endothelial cell growth assay) as described previously (25) . Briefly, 2000 MluE cells were suspended in 0.1 ml of serum-free DMEM/F12 with 0.1% BSA and loaded into the 0.1-mg/ml gelatin-coated upper chamber of a Transwell cluster plate. The 600 µl of CM from the different sarcoma cell lines or CM neutralized with anti-VEGF polyclonal antibody (1500 pg/ml, mixed and stored at 4°C overnight; Santa Cruz) was placed in the lower chamber of the Transwell. DMEM/F12 plus 0.1% BSA was used as a negative control. The assay was allowed to proceed for 5.5 h at 37°C, after which the Transwell filters were fixed in methanol and stained with Giemsa. Five random fields were counted in five fields (x20 objective lens and x10 ocular lens), and the cell numbers were averaged and expressed as the number of migrated cells per high-power field.

Quantitation of VEGF in CM.
VEGF concentrations in CM were quantitated using a VEGF ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Northern Blot Analysis.
Total RNA was extracted using RNAzol B reagent (Biotecx Laboratories, Inc, Houston, TX) and separated on 1% agarose gel. Northern blot analysis was performed using Hybond-N nylon membrane (Amersham, Arlington Heights, IL) in an aqueous hybridization solution as described previously (31) . Hybridization probes were isolated VEGF cDNA or GAPDH cDNA fragments radiolabeled with a random-primed labeling kit (Life Technologies) and purified with QIAquick Nucleotide Remove Kit (QIAGEN Inc., Valencia, CA). Blots were washed at high stringency (0.5x SSC-0.1% SDS at 68°C) and exposed to Kodak BioMax film (Eastman Kodak Co., Rochester, NY) at -70°C. Densitometric quantitation was performed using an AlphaImager 2000 V4.0 (Alpha Innotech Corp., San Leandro, CA).

Construction of VEGF Promoter-Luciferase Plasmids.
The upstream regulatory sequences and promoter region of the human VEGF gene (-2362 to +956 relative to the transcription start site) were fused to the luciferase reporter gene in pGL3-basic vector (Promega, Madison, WI) to generate pVp-ecor. Deletion mutant constructs were generated by restriction digestion of pVp-ecor with SpeI (at -1810), PstI (at -794), ApaI (at -135), SmaI (at -88), EcoO1091 (at -68), NlaI (at -48), and SacII (at +688), followed by ligation. The deletion mutant constructs generated were pVp-spe, pVp-pst, pVp-apa, pVp-sma, pVp-eco, pVp-nla, and pVp-sac. The pVp-apa was digested with Age I (at +94) and NlaI (at -48) and generated pVp-nla'. The pRL-CMV plasmid (Promega E2261) was used as an internal control. pRL-CMV contains CMV immediate-early enhancer/promoter elements to provide high-level expression of Renilla luciferase in cotransfected mammalian cells. Expression of Renilla luciferase is detected using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). All of the DNA constructs were purified using QIAGEN columns (QIAGEN Inc.) and confirmed by restriction-enzyme-digestion analysis.

Transient Transfections and Luciferase and Renilla Luciferase Assays.
SKLMS-1 and SKAla-2 cells were cultured in 6-well plates. When cells were 50–70% confluent, 10 µg of promoter-reporter plasmid DNA and 1 µg of pRL/CMV (internal control) per well were transfected into the cells by using the ProFection Mammalian Transfection System-DEAE-Dextran (Promega). pGL3-basic plasmid served as vector control. pRL-CMV was used to normalize the transfection efficiency. Forty-eight h after transfection, the cells were rinsed with PBS and harvested. Luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega E1910) with a luminometer (Promega). Luciferase activity was normalized by the internal control. Assays were performed in three independent experiments, with duplicate transfection in each experiment.


    RESULTS
 Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
wt p53-inhibited Tumor Neovascularization and Tumor Cell-induced Angiogenesis in Vivo.
To determine whether wt p53 may inhibit angiogenesis in vivo, we used the Ad5p53 gene transfer to restore wt p53 expression in SKLMS-1 xenografts and examined the microvessel counts in tumor sections. Tumors treated with Ad5 p53 had significantly lower microvessel counts than did untreated or empty-Ad-vector (Ad5C4)-treated controls (Table 1)Citation . To further confirm that wt p53 gene restoration inhibited angiogenesis in leiomyosarcoma, in vivo Matrigel plug assays were performed and the results demonstrated that CM from cell lines containing wt p53 was associated with marked inhibition of neovascularizations, as measured by hemoglobin content (Fig. 1A)Citation and examination of the gel for infiltrating vessels (Fig. 1B)Citation . These observations suggest that wt p53 inhibits angiogenesis of human leiomyosarcoma in vivo.


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Table 1 Microvessel counts assayed by immunohistochemistry with anti-F8RA/vWF in leiomyosarcoma tumor sections from differently treated SCID mice

Each sample was examined at low power (x10 objective lens and x10 ocular lens) to identify the area with the highest number of microvessels. The microvessels were then carefully counted in this area a x200 field (x20 objective lens and 10x ocular lens, 0.74 mm2 per field). The range of microvessel counts in the tumor samples was 8–50; x200.

 


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Fig. 1. Inhibition of wt p53 on angiogenesis in vivo. Angiogenesis assay using an in vivo Matrigel plug assay was performed as described in the "Materials and Methods" section. In A, hemoglobin assay was performed using the Drakin method and normalized by the weight of Matrigel. Data represent the mean hemoglobin value from 10 mice. SYNb-1/-2, CM from SYNb-1 and SYNb-2 cells cultured at 37°C. *, P < 0.05; **, P < 0.01; bar, SD. B, histology of recovered Matrigel plugs. Representative fields showed marked inhibition of angiogenesis by CM from SKAla-2 cells grown at 32°C expressing wt p53 relative to CM from SKAla-2 cells grown at 38°C with mutant p53. There is no significant difference in angiogenesis by CM from SKLMS-1 grown at 32°C and 38°C with mutant p53. Similarly, there is dramatic inhibition of angiogenesis by CM from SYNb-1 cells harboring wt p53 compared with CM from SYNb-2 cells harboring mutant p53. (x20 objective lens and x10 ocular lens).

 
Analysis of microvessel density and microvessel count in human specimens from a primary synovial sarcoma containing wt p53 (SYNb-1) and the autologous metastatic tumor containing mutant p53 (SYNb-2) demonstrated lower microvessel density and microvessel count in the primary tumor (microvessel density, 1+; microvessel count, 14 ± 3.2 at x200) than in the metastatic tumor (microvessel density, 2+; microvessel count, 60 ± 6.5 at x200). In addition, in vivo angiogenesis assay using Matrigel plug assays showed that CM from SYNb-1 cells significantly decreased neovascularization in SCID mice compared with CM from SYNb-2 cells (Fig. 1)Citation . These data supported the hypothesis that wt p53 can reduce angiogenesis in human synovial sarcoma in vivo.

wt p53-suppressed Angiogenic Ability of Sarcoma Cells in Vitro.
To determine whether the inhibition of angiogenesis by wt p53 in vivo was the result of reduced endothelial cell growth or migration, we used CM from SKLMS-1, SKAla, SYNb-1, and SYNb-2 cells with wt p53 or mutant p53 to treat MluE cells, and compared the effects of the various CM on endothelial cell growth and migration. After 3 days of culture, no significant differences were seen in endothelial cell growth among MluE cells treated with CM from SKAla-1, SKAla-2, and SKAla-3 cells growing at 32°C and 38°C, and SYNB-1 and SYNb-2 cells (data not shown). However, after 7 days of culture, the growth of MluE cells treated with CM from SKAla cells cultured at 32°C was reduced to 61% (SKAla-1), 57.6% (SKAla-2), and 43.3% (SKAla-3) of the growth of MluE cells treated with CM from SKAla cells cultured at 38°C (Fig. 2A)Citation . Endothelial cell migration was reduced 68.4%, 69.8%, and 36% in MluE cells treated with CM from SKAla-1, SKAla-2, and SKAla-3, respectively, growing at 32°C compared with migration in cells treated with CM from cells growing at 38°C (Fig. 2B)Citation . The endothelial growth-stimulating activity of the CM from SYNb-1 was 57.4% that of the CM from SYNb-2 cells (Fig. 2A)Citation , and the endothelial migration-stimulating activity of the CM from SYNb-1 was only 37.3% of that of the CM from SYNb-2 cells (Fig. 2B)Citation . Together, these data indicated that wt p53 can inhibit the angiogenic response of sarcoma cells in vitro as indicated by reduced endothelial cell growth and migration, both of which can contribute to reduced microvessel counts in vivo.



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Fig. 2. Wt p53 inhibited angiogenic ability of sarcoma cells in vitro. A, endothelial cell growth assay. MluE cells (500/well) were cultured in CM from different sarcoma cell lines at 37°C for 7 days. B, endothelial cell migration assay. MluE cells (2,000/well) were cultured with CM from different sarcoma cell lines in Transwell Cluster plates at 37°C for 5.5 h. SYNb-1/-2, CM from SYNb-1 and SYNb-2 cells cultured at 37°C. The assays were performed as described in "Materials and Methods" and done in triplicate. Bars, SD; *, P < 0.05; **, P < 0.01.

 
wt p53-suppressed VEGF Expression.
Endothelial cell growth and migration require the support of such angiogenic factors as VEGF, which is the most important growth factor in angiogenesis. We sought to determine whether p53 inhibits endothelial cell growth and migration in human soft-tissue sarcoma by inhibiting VEGF expression. We used ELISA to analyze VEGF protein expression in CM of SKAla-1, SKAla-2, and SKAla-3 cell lines growing at 32°C and 38°C. We found that wt p53 inhibited VEGF protein expression in these human leiomyosarcoma cells (Table 2)Citation . Similarly, VEGF protein levels were lower in the CM of SYNb-1 cells, which contain wt p53, than in the CM of SYNb-2 cells, which contain mutant p53 (Table 2)Citation . These data supported the notion that the inhibition of angiogenesis in human leiomyosarcoma and synovial sarcoma cells by wt p53 was attributable to suppression of VEGF expression.


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Table 2 Quantitation of VEGF level in CM of sarcoma cells by ELISAa

 
We next sought to determine whether wt p53 inhibited VEGF secretion by inhibiting VEGF mRNA expression. SKAla-1, -2, and -3 cell lines were cultured at 32°C (wt p53) or 38°C (mutant p53) for 5 days. Cells were then harvested, and total RNA was extracted for Northern blot analysis using a VEGF probe. We found that SKAla cells grown at 32°C expressed dramatically less VEGF mRNA than did cells grown at 38°C (Fig. 3)Citation . Furthermore, levels of VEGF mRNA in SYNb-1 cells were lower than the levels of VEGF mRNA in SYNb-2 cells. These data indicated that wt p53 can inhibit VEGF expression in soft-tissue sarcoma cells at the mRNA level.



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Fig. 3. Effects of wt and mutant p53 on VEGF mRNA expression in human sarcoma cells. Thirty µg of total RNA from SKLMS-1, SKAla-1, -2, and -3 cells, and 20 µg of total RNA from SYNb-1 and -2 cells were analyzed by Northern blot. Northern blot analysis of GAPDH was used as a loading control.

 
To determine whether the reduction in VEGF expression by wt p53 contributed to the decreased angiogenic ability of sarcoma cells, we used anti-VEGF polyclonal antibody to neutralize VEGF in CM. The endothelial cell migration-stimulating activity of CM from cells expressing wt p53 and those expressing mutant p53 was significantly reduced after anti-VEGF neutralizing antibody was added to the CM (Fig. 4)Citation . It is notable that the reduction of endothelial cell migration by anti-VEGF antibody was more dramatic in cells expressing mutant p53 than in cells expressing wt p53. These data suggested that wt p53 inhibition of VEGF expression contributed to the reduction of the angiogenic property of human soft-tissue sarcoma cells in vitro.



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Fig. 4. Endothelial cell migration in response to CM neutralized with anti-VEGF antibody. Rabbit antihuman VEGF polyclonal antibody (Ab+) or nonspecific rabbit IgG (Ab-) was added into CM from different sarcoma cells in 1500 pg/ml CM, was mixed, then was stored at 4°C overnight. SYNb-1/-2, CM from SYNb-1 and SYNb-2 cells cultured at 37°C. The assay was performed as described in "Materials and Methods" and done in triplicate. Bars, SD.

 
wt p53-inhibited VEGF Expression at the Transcription Level by Targeting a Specific cis Element in the VEGF Promoter.
To further understand the mechanisms by which wt p53 inhibited VEGF expression, we tested whether introduction of exogenous wt p53 could suppress VEGF expression at the transcription level in soft-tissue sarcoma cells. We first used the 2.4-kb VEGF promoter-luciferase construct (Fig. 5A)Citation to analyze the effect of wt or mutant p53 on VEGF promoter activity. SKAla-2 cells grown at 32°C (wt p53) or 38°C (mutant p53) were transiently transfected with VEGF promoter-luciferase constructs, and VEGF promoter activity was assayed 48 h later. As shown in Fig. 5BCitation , wt p53 markedly inhibited VEGF promoter activity. In contrast, SKLMS-1 cells containing mutant p53 had no inhibitory effect on VEGF promoter activity grown at 32°C.



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Fig. 5. Effect of wt and mutant p53 on VEGF promoter activity in the SKLMS-1 and SKAla-2 cell lines. In A, the promoter of VEGF including the 5'-untranslated region (3318 bp, -2362 to +956 relative to transcription initiation site) was fused to the luciferase reporter gene in pGL3-basic to generate pVp-ecor. In B, luciferase assay was performed as described in "Materials and Methods." Values are expressed as the percentage of the relative luciferase activity (100%) in lysate from SKLMS-1 cells (cultured at 32°C) cotransfected with VEGF promoter constructs and internal control plasmid. Bars, SD, derived from three independent experiments.

 
To localize the promoter region responsible for wt p53-mediated suppression of VEGF promoter activity, we generated a series of deletion constructs of the VEGF promoter that were composed of various lengths of 5' promoter sequences fused to the luciferase gene (Fig. 6A)Citation . The VEGF promoter-luciferase constructs and the pGL3-basic vector (vector control) were transiently transfected into SKLMS-1 and SKAla-2 cells, using pRL/CMV plasmid as internal control of transfection efficiency. The promoter activities of the various 5' deletion VEGF promoter-luciferase constructs, including pVp-ecor, pVp-spe, pVp-pst, pVp-apa, and pVp-nla, were similar between SKLMS-1 and SKAla-2 cells grown at 38°C when both cell lines expressed mutant p53 (Fig. 6B)Citation . Interestingly, when SKAla-2 cells were grown at 32°C expressing wt p53, wt p53 inhibited the activity of VEGF promoter in pVp-ecor-, pVp-spe-, pVp-pst-, and pVp-apa-transfected cells. However, the inhibitory effect of the wt p53 was abolished when pVp-nla was used. These results suggested that pVp-apa is sufficient to confer p53-induced inhibition, and the promoter sequence -135 to -48 might contain the p53-responsive element. In contrast, SKLMS-1 cells grown at 32°C and expressing mutant p53 showed no inhibitory effects on the promoter activities of any of the 5' deletion VEGF promoter constructs. As expected, the promoterless luciferase vector pGL3-basic had no promoter activity and could not be repressed by p53. These data suggested that wt p53 repression of the VEGF promoter activity is mediated by a cis-acting element located in the -135 to -48 region of the VEGF promoter.



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Fig. 6. Localization of the VEGF promoter response element mediating the inhibitory effect of p53 on VEGF transcription. In A, deletion mutant constructs were generated by restriction digestion of pVp-ecor with SpeI (at -1810), PstI (at -794), ApaI (at -135), NlaI (at -48), and SacII (at +688), followed by ligation. The deletion mutant constructs generated were pVp-spe, pVp-pst, pVp-apa, pVp-nla, and pVp-sac. In B, VEGF promoter-luciferase plasmids containing various lengths of the VEGF promoter were transiently transfected into SKLMS-1 and SKAla-2 cells, and luciferase activity was measured as described in "Materials and Methods." Values are expressed as the percentage of the relative luciferase activity (100%) in lysate from SKLMS-1 cells (cultured at 32°C) cotransfected with VEGF promoter constructs and internal control plasmid. Each value indicates the mean of the relative luciferase activity measured in three independent experiments. Bars, SD.

 
Transcriptional Repression of VEGF Promoter Activity by wt p53 Required Sp1-binding Element.
It has been reported (32) that wt p53 expressed at high levels can repress transcription of promoters containing a TATA box in the absence of a classical p53- binding sites. The human VEGF promoter contains no TATA or CCAAT boxes or putative p53-binding site (33, 34, 35) . Thus, the repression of the VEGF promoter activity that we observed may be mediated by indirect mechanisms, e.g., other transcriptional factors targeted by wt p53. Four Sp1-binding sites have been identified in the region of -135 to +1 in the human VEGF promoter (33) . To examine whether Sp1 may mediate p53’s inhibitory effect on VEGF promoter activity in human leiomyosarcoma cells, we further deleted various Sp1 binding sites of the VEGF promoter (Fig. 7A)Citation . The sequential deletion of Sp1 binding sites resulted in the progressive reduction of VEGF promoter activity (Fig. 7A)Citation . Comparing the effect of wt p53 on VEGF promoter constructs containing one (pVp-eco), three (pVp-sma), and four (pVp-apa) Sp1-binding sites, we found that the reductions were 37, 59.6 and 72.3%, respectively, of VEGF promoter activity in cells containing mutant p53 with one, three, and four Sp1-binding sites (Fig. 7B)Citation . When all of the four Sp1 sites were deleted (pVp-nla), wt p53 completely lost its VEGF promoter repression activity (Fig. 7B)Citation . These results strongly suggested that the Sp1-binding elements in the -135 to -48 region are required for the inhibitory effect of wt p53 on VEGF promoter activity in human leiomyosarcoma cells.



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Fig. 7. Effect of 5' deletion in the cis-regulatory Sp1-binding element on the inhibitory effects of p53 on VEGF promoter activity. In A, the deletion mutant constructs generated were pVp-apa with four Sp1 binding sites, pVp-sma with three Sp1 binding sites, pVp-eco with one Sp1 binding sites, pVp-nla without Sp1-binding sites, and pVp-nla' without Sp1 binding sites and deletion of most 5'-UTR. In B, luciferase assays were performed as described in "Materials and Methods." Values are expressed as the percentage of the relative luciferase activity (100%) in lysate from SKLMS-1 cells (cultured at 32°C) cotransfected with VEGF promoter constructs and internal control plasmid, and each value represents the mean of the relative luciferase activity measured in three independent experiments. Bars, SD.

 

    DISCUSSION
 Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we investigated the role of p53 in the regulation of angiogenesis in human soft-tissue sarcoma cells using SKLMS-1 human leiomyosarcoma cells stably transfected with a temperature-sensitive mutant p53 gene construct and two human synovial sarcoma cell lines expressing wt p53 and mutant p53, respectively. We found that the restoration of wt p53 function in SKLMS-1 cells and expression of wt p53 in synovial sarcoma cells significantly suppressed angiogenesis in vivo and in vitro, as well as decreased expression of both VEGF mRNA and VEGF protein. We also found that wt p53 inhibited VEGF expression by suppression of VEGF promoter activity in human-soft-tissue sarcoma cells, and that an 87-bp region in the VEGF promoter containing Sp1-binding sites was required for wt p53 repression.

The tumor suppressor function of p53 may involve transcriptional activation of important cellular genes, such as p21; regulation of cell cycle control; and regulation of apoptosis (36) . Recently, p53 was found to be involved in the regulation of angiogenesis inhibitors and stimulators, such as thrombospondin and VEGF (13) . Previous reports (37) indicated that tumor growth could be restricted to a diameter of approximately 0.4 cm if capillaries were physically prevented from reaching the tumor implant or were inhibited from undergoing angiogenesis by a tumor suppressor. Our observations in this study suggested that the inhibition of angiogenesis by suppressing the VEGF expression may be another important mechanism by which restoration of wt p53 suppresses tumor growth in human soft-tissue sarcoma bearing mutant p53.

Tumor-derived expression of VEGF has been reported to be a critical factor in tumor expansion and vascular function (38) . In addition, in human colon cancer, cell lines bearing mutant p53, transient restoration of wt p53 function by adenovirus-mediated gene transfer has been found to down-regulate VEGF expression (22) . However, to the best of our knowledge, the precise mechanism of p53-induced inhibition of VEGF expression is not yet completely known. Our results here showed that the induction of wt p53 caused the suppression of VEGF mRNA and protein expression in human leiomyosarcoma and synovial sarcoma cells. Additional work with the 2.4-kb VEGF promoter-driven luciferase reporter gene showed that VEGF promoter activity was strongly repressed in SKAla-2 cells grown at 32°C expressing wt p53 compared with cells grown at 38°C expressing mutant p53. These data suggested that the restoration of wt p53 repressed VEGF expression at the transcriptional level in human leiomyosarcoma cells.

One question that arises is: which cis DNA element is responsible for the wt p53 inhibitory effect on VEGF promoter activity. The VEGF promoter has been reported not to possess a TATA box nor a p53 DNA-binding sequence (14 , 33) . Therefore, other, as yet unknown, cis elements and trans protein factors must be responsible for repression of VEGF transcription expression by p53. Using a series of 5' deletion constructs of the VEGF promoter, we have found that an 87-bp VEGF promoter element (bp -135 to -48) contains the cis element responsible for repression of VEGF promoter activity by wt p53. The Sp1 protein is a well-known transcription factor that is involved in the control of transcription of many important genes (39) . Transcriptional repression of some other genes by p53 has shown that Sp1 protein was prevented from binding to the promoter region by a p53-Sp1 protein complex (40, 41, 42) . It was reported that p53 can form a heterocomplex with Sp1 and inhibit Sp1 activity in the human erythroleukemia cell line TF-1 (41) . The region between -135 and - 48 in human VEGF promoter is known to be GC rich and to contain four Sp1-binding sites (33) . Our finding that wt p53 lost repression effect on VEGF promoter activity when the four Sp1 binding sites were deleted indicated that transcriptional repression of VEGF promoter activity by wt p53 requires the Sp1 elements in human leiomyosarcoma cells. To our knowledge, this is the first evidence that Sp1-binding sites are required for p53-mediated transcriptional repression of VEGF expression. Currently, we are further investigating the interactions between p53 and Sp1 in human soft-tissue sarcoma cells that may bring new insights for developing novel molecular tools to inhibit VEGF in cancer.

VEGF contains a very long (1038 bp) 5'-UTR that also contains putative binding sites for various transcription factors (33) . It was reported that interleukin 6-induced VEGF expression is mediated not only by the cis DNA element at the promoter region but also by a specific motif located in the 5'-UTR of VEGF mRNA (43) . We also tested whether the 5'-UTR of VEGF is involved in the inhibition by wt p53 of VEGF promoter activity and found that, in human leiomyosarcoma cells, the wt p53 inhibition of VEGF promoter activity does not change significantly with most 5'-UTR deletions (pVp-nla') compared with this inhibition without 5'-UTR deletion (pVp-nla). The role of 5'-UTR in the inhibitory effect of wt p53 on VEGF expression in human soft-tissue sarcoma cells will be further investigated. Additionally the impact of p53 on the stabilization of VEGF mRNA and its contribution to p53-mediated inhibition of VEGF expression will be investigated in our future study.

In summary, the results of this study demonstrated that the restoration of wt p53 in human leiomyosarcoma and synovial sarcoma containing mutant p53 markedly inhibited tumor angiogenesis in vivo and in vitro and that transcriptional repression of VEGF by wt p53 via Sp1-binding sites contributes to the p53-mediated inhibition of angiogenesis and growth of human soft-tissue sarcoma. Our data suggested that molecular-based therapies targeting mutant p53 and VEGF may lead to an effective new strategy for controlling the progression of this type of tumor.


    ACKNOWLEDGMENTS
 
We thank Dongtong Sun for technical assistant, Dr. Ann Killary for the use of microscope, and Stephanie Deming for editorial support.


    FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by Cancer Center Core Grant P30-16672 from the National Cancer Institute and National Cancer Institute, Department of Health and Human Services Grants CA 67802 (to R. E. P.), CA 60488 (to D. Y.), and CA74821 (to L. M. E.). Back

2 To whom requests for reprints should be addressed, at Department of Surgical Oncology, Box 106, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-6928; Fax: (713) 792-0722. Back

3 The abbreviations used are: VEGF, vascular endothelial growth factor; wt, wild-type; DMEM/F12, 1:1 mixture of DMEM and Ham’s F-12 nutrient mixture; MluE, murine lung endothelial (cell); CM, conditioned medium/media; CMV, cytomegalovirus; UTR, untranslated region; SCID, severe combined immunodeficient; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

Received 12/28/99. Accepted 5/ 2/00.


    REFERENCES
 Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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