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Tumor Biology |
Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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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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VEGF expression can be regulated by several cytokines, including
transforming growth factor 
, 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.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 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)
and examination of the gel for infiltrating vessels (Fig. 1B)
. These observations suggest that wt p53
inhibits angiogenesis of human leiomyosarcoma in vivo.
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. 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)
. 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)
. The endothelial growth-stimulating activity of the CM
from SYNb-1 was 57.4% that of the CM from SYNb-2 cells (Fig. 2A)
, 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)
. 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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. 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)
. 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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. 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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. 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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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. 5B
, 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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. 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)
.
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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. The sequential deletion of Sp1 binding sites resulted in
the progressive reduction of VEGF promoter activity (Fig. 7A)
. 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)
. When all of the
four Sp1 sites were deleted (pVp-nla), wt p53 completely
lost its VEGF promoter repression activity (Fig. 7B)
. 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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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.). 
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.
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.
Received 12/28/99. Accepted 5/ 2/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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