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Tumor Biology |
Department of Hematology and Oncology, Istituto Superiore di Sanità, 00161 Rome [A. C., F. F., E. M., L. B., C. P.]; Department of Experimental Oncology, Immunotherapy and Gene Therapy Unit, Istituto Nazionale Tumori, Milan 20133 [M. P., M. P. C.]; and Department of Experimental Medicine and Pathology, University La Sapienza, 00100 Rome [A. S.], Italy
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here, we have studied whether HOXB7, in addition to basic fibroblast
growth factor, may induce other genes directly or indirectly related to
neoangiogenesis and tumor invasion. Parental,
ß-galactosidase-transduced, and HOXB7-transduced SkBr3 cell
lines were examined for the expression of several growth factors and
growth factor receptors involved in the proliferative and angiogenic
processes. Vascular endothelial growth factor, melanoma
growth-stimulatory activity/growth-related oncogenene 
,
interleukin-8, and angiopoietin-2 were up-regulated by HOXB7
transduction. The exception was angiopoietin-1 expression that was
abrogated. Additional analyses included the expression levels of
enzymes such as matrix metalloprotease (MMP)-2 and MMP-9 and
heparanase, capable of proteolytic degradation of extracellular matrix
and basement membranes. Results showed an induction of only MMP-9.
The functional implication of such a finding was tested using an in vitro coculture assay in a three-dimensional matrix. A delay of differentiation with persistent nests of proliferating cells was found in endothelial cells cocultured with HOXB7-transduced SkBr3 cells. Tumorigenicity of these cells has been evaluated in vivo. Xenograft into athymic nude mice showed that SkBr3/HOXB7 cells developed tumors in mice, either irradiated or not, whereas parental SkBr3 cells did not show any tumor take unless mice were sublethally irradiated. Comparison of tumor nodules for vascularization by CD-31 and CD-34 immunostaining revealed an increased number of blood vessels in tumors expressing HOXB7. Together, the results indicate HOXB7 as a key factor up-regulating a variety of proangiogenic stimuli. Thus, HOXB7 gene or protein is a target to aim at to inhibit tumor-associated neoangiogenesis, considering the number and the redundancy of proangiogenic molecules that should be targeted one by one to theoretically achieve the same effect.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and VEGF are the most potent inducers of
angiogenesis; however, their precise role in the coordinate regulation
of this process remains veiled (4)
. Products of HOX genes are transcription factors responsible for tissue remodeling. Besides their function in embryonic development, inappropriate HOX gene expression has been associated with different neoplasias (5 , 6) . HOXB7-enforced expression in hematopoietic progenitor/stem cells, purified from adult human peripheral blood, markedly modulated the proliferative/differentiative program of this population inducing a prolonged proliferation of a discrete population of blast cells and granulo-monocytic oriented cells (7) . These results suggested a potential preleukemic immortalization step attributable to HOXB7 overexpression. This oncogenic potential has been already demonstrated by in vitro and in vivo transformation assays for several murine Hox genes (8) . Moreover, HOXB7 has been shown to be constitutively expressed in both melanoma primary lesions and cell lines (5) . Investigating downstream genes targeted by HOXB7, we identified bFGF as its main target. Indeed, HOXB7 binds and transactivates bFGF, whereas HOXB7 inhibition reduces bFGF expression and cell proliferation in melanoma cell lines. The screening of several tumor cell lines indicated SkBr3 from a breast adenocarcinoma (9) as the sole negative sample for the expression of both HOXB7 and bFGF genes. Retroviral-mediated transduction of the sole HOXB7 cDNA into SkBr3 cells resulted in transcription of both HOXB7 and bFGF and in several phenotypic changes including increased cell growth rate, independence from serum withdrawal, and ability to form colonies in semisolid medium (10) .
HOX genes likely regulate vasculogenesis and perhaps angiogenesis. Indeed, HOXD3 has been linked to the mechanism converting endothelial cells from a resting to angiogenic/invasive state (11) , whereas HOXB3 is required for the subsequent capillary morphogenesis of the new vascular sprouts (12) .
Because tumor cells elaborate growth factors that act on the surrounding endothelial cells to induce new blood vessel formation, we studied whether HOXB7, besides bFGF activation, could affect neoangiogenesis through a direct or indirect regulation of growth factors and growth factor receptors.
Our data indicate that HOXB7-enforced expression in SkBr3 cells increased the transcription of the analyzed growth factor genes but not that of the corresponding receptors. The proangiogenic molecules produced by transduced SkBr3 cells are not used or sequestered by the tumor and remain available to the endothelial cells. Accordingly, in vitro and in vivo evaluation of the angiogenic activity showed an increased and persistent number of newly formed capillaries and an increased tumorigenicity, respectively, of HOXB7/SkBr3 cells. The possible role of HOXB7 as a key activator of tumor-associated neoangiogenesis and its targeting for tumor gene therapy are discussed.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RNA Analysis.
RT-PCR analysis and RNase protection were performed according to
standard procedures (5
, 10
, 13)
. The sequences of primers, the PCR
products, and the annealing conditions are reported in Table 1
. All of the amplified fragments were hybridized to a 30-base
end-labeled oligonucleotide according to Southern blot standard
procedures.
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was a 250-bp
BamHI. Fragments were cloned in pCRII vector (Invitrogen BV,
Groningen, the Netherlands). The ß-actin insert was a 93-bp
RsaI fragment, always labeled in the presence of cold GTP to
obtain a low specific activity. The expression levels were analyzed by
the Image Quant software (Molecular Dynamics, Sunnyvale, CA).
Antisense Experiments.
Antisense inhibition analysis was carried out as described previously
(5)
. Briefly, cells (2 x 103
) of each sample were incubated in triplicate
in 96-well plates in 0.2 ml of culture medium supplemented with 10%
fetal bovine serum, heat-inactivated for 30 min at 65°C to destroy
nucleases, in the presence or absence of antisense and scrambled
phosphorothioate oligomers. The antisense oligomers were complementary
to the translation start site. Oligomers at concentrations of 30
µM were added 24 h after cell plating, and
proliferation was tested on day 7. Antisense inhibition was calculated
relative to that of scrambled-treated cells, the inhibition of which
never exceeded 20% of the proliferation rate shown by their untreated
counterparts. The specificity of the inhibitory effect was ensured by a
series of controls: (a) the sense, scrambled, and antisense
sequences were tested against sequences from the European Molecular
Biology Laboratory database to demonstrate the absence of toxicity and
the specificity of inhibition; and (b) the abrogation of the
specific messages only in the antisense-treated cells was confirmed by
RT-PCR, whereas unrelated control mRNAs were not modified. For RT-PCR
analysis, on the last day of culture, cells from the third well of each
sample were lysed for total RNA extraction by a modification of the
guanidine-thiocyanate CsCl gradient made in a microultracentrifuge
(TL-100; Beckman Inst., Palo Alto, CA) in the presence of rRNA as
carrier.
ELISA.
Intracellular and secreted protein (bFGF, VEGF, IL-8, and GRO) were
quantified by ELISA kits from R&D Systems (Minneapolis, MN). Different
concentrations of cells ranging from 105/ml to
106/ml were cultured for 24 h and 48 h,
and their cell lysates and culture media were collected and stored at
-80°C until use. To prepare cell lysates, cells were washed in PBS
and resuspended in 10 mM Tris-HCl (pH 7.4), containing 1
M NaCl, in the presence of protease inhibitors. The cells
were disrupted by three cycles of freeze-thawing, and the homogenate
was centrifuged at 30,000 x g for 20 min.
Western Blot.
The expression of MMP-9 was evaluated in cell lysates by Western blot
according to standard procedures. Because the latent forms of
metalloproteases are activated by treatment in vitro with
proteinases like trypsin, to avoid artifactual results tumor cells were
detached from plastic by scraping. Anti-MMP-9 monoclonal
antibody (Oncogene Science Research, Boston, MA), which
recognizes both the latent and active forms of the protein, was used at
1:400 dilution.
Coculture of Tumor and Endothelial Cells: in Vitro
Capillary-like Structure and Cell Growth Assays.
HUVEC, isolated from umbilical cord vein according to standard
procedures (PromoCell, Heidelberg, Germany), were used at passage 2–6.
HUVEC were kept in culture on gelatin-coated plastic in M199 medium as
reported (14)
. For coculture experiments,
105 endothelial cells were plated in 6-well
plates coated with Matrigel Growth Factor Reduced (Collaborative
Research) and allowed to adhere. Inserts (0.4-µm pore transwell
inserts; Falcon; Becton Dickinson, Franklin Lakes, NJ)
containing 6 x 105 parental or
HOXB7-transduced SkBr3 cells were then applied on the plates containing
the HUVEC. In vitro angiogenesis was monitored after 24 h and 48 h, and plates were photographed with a Zeiss microscope.
For cell growth assay, 3 x 104
HUVEC were plated in 24-well plates coated with gelatin in M199 medium
containing 10% FCS and 10% newborn serum without factors. After
24 h, the medium was removed and replaced with M199 medium
containing 2.5% FCS without factors. After an additional 24 h,
transwell inserts containing 5 x 104 tumor cells in M199 with 2.5% FCS were
applied on the plates containing endothelial cells. For quantification
of cell growth, after 40 h of coculture, endothelial cells were
photographed with a Zeiss microscope and then trypsinized and counted.
Coculture experiments in both proliferative and differentiative
conditions of endothelial cells were performed also in the presence of
increasing amounts (0.5–2.5 µg/ml) of a blocking anti-VEGF antibody
(R&D Systems).
In Vivo Assay.
SkBr3 or SkBr3/HOXB7 cells in exponential growth phase were injected
s.c. at dose of 3 x 106 and
107 into adult athymic nude mice purchased from
Charles River (Calco, Italy) and maintained at the Istituto Nazionale
Tumori (Milan, Italy) under standard conditions according to
institutional guidelines. Tumor growths were monitored twice a week. At
different time points, tumors were excised and fixed for
histopathological examination.
Immunohistochemistry.
Tumor fragments were embedded in OCT compound (Miles Lab., Elkert, IN),
snap-frozen in liquid nitrogen, and stored at -80°C. Immunochemical
analysis using the peroxidase-antiperoxidase method was performed as
described (15)
. Briefly, 5-µm cryostat sections were
fixed in acetone and immunostained with rat antimouse monoclonal
antibodies CD31/PECAM-1 (Mec 13.3 hybridoma) and CD34 (RAM34;
PharMingen, San Diego, CA). Sections were preincubated with rabbit
serum and sequentially incubated with optimal dilutions of primary
antibodies, rabbit antirat IgG (Zymed Laboratories, Inc., San
Francisco, CA), and rat peroxidase antiperoxidase (Abbot Laboratories,
North Chicago, IL). Serial sections from the tumors were analyzed, and
the number of vessels was evaluated in the entire growing area at x400
magnification. The statistical analysis was performed by Student’s
t test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Parental, ß-gal-transduced, and HOXB7-transduced SkBr3 cells were
analyzed by RNase protection and/or RT-PCR to test whether enforced
expression of HOXB7 may affect genes, in addition to bFGF, having a
direct or indirect role in neoangiogenesis. The expression level of
VEGF, GRO, IL-8, Ang-1, Ang-2, and the corresponding receptor genes
was evaluated (Figs. 1–3
). All of the examined growth factors, except Ang-1 that was sharply
down-regulated, were increased in the HOXB7-transduced cells. A
3–10-fold increase of VEGF, Ang-2, GRO, and IL-8 was found in
SkBr3/HOXB7 cells.
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shows a clear increase of VEGF in the HOXB7-transduced
cells ranging from 1.5-fold for VEGF121 to 7- and
12-fold for VEGF165 and
VEGF189, respectively. To see whether such
pattern of transcripts might be regulated by HOXB7, we analyzed
melanoma cell lines that constitutively express HOXB7 (5)
.
In the A375 and 665/1 melanoma cells, antisense phosphorothioate
oligomers to HOXB7 specifically reduced VEGF165
and VEGF189 of 5- and 10-fold, respectively,
whereas VEGF121 remained the same (see Fig. 2
).
Growth Factor Receptor Analysis.
The analysis of the corresponding receptors included bFGF receptors
from 1 to 4, VEGF receptors Flt-1, KDR and Flt-4, IL-8 receptors and
Tie-1 and Tie-2/Tek. The expression of all of them remained
generally unchanged or slightly down-regulated (Fig. 3
and Ref. 10
). Parental SkBr3/ß-gal and SkBr3/HOXB7 cells
constitutively expressed bFGF R-2, -3, and -4 (10)
and
IL-8 high affinity receptors (Fig. 3)
, whereas they were negative for
bFGF receptor-1 (10)
and for Tie-2/Tek (Fig. 3)
. In
parental cells, the three VEGF receptors, Flt-1, KDR, and Flt-4, were
expressed from low to high level, respectively, whereas they were shut
off in the SkBr3/HOXB7 cells. IL-8R1 was down-regulated, whereas IL-8R2
was expressed at constant level (Fig. 3)
. Only Tie-1, which has no
known ligand, showed a 5–10-fold increase in transduced cells. Lack or
down-regulation of these receptors in the presence of their respective
ligands, being against any possible autocrine loop, may rather suggest
a strategy by which tumor cells avoid to sequester proangiogenic
factors making them available to the neovessels. Accordingly, VEGF did
not show any proliferative effect on SkBr3/HOXB7, and the addition of a
blocking antibody up to 72 h did not change the cell growth rate
(Fig. 4)
. The specificity of the antibody was confirmed by measuring a
reduction in the level of detectable VEGF from 1580 to 54 pg/ml in
culture supernatants as assayed by an ELISA.
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. This up-regulation was confirmed at protein level by
Western blot analysis as shown in Fig. 5B
and quantified by
densitometric analysis. MMP-9 protein was barely detectable in the
SkBr3 parental cells, whereas in the HOXB7-transduced cells we observed
a 38-fold increase of the mature Mr
84,000 enzyme and a 16-fold increase of the
Mr 68,000 form, attributable to
autocatalytic cleavage of the COOH terminus.
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and Ref. 10
). The secretion of the other proangiogenic
factors may likely activate the cell surface receptors present on
endothelial cells. ELISA assays detected production of VEGF, GRO,
and IL-8 (Fig. 6)
in supernatants of SkBr3/HOXB7. A 10-fold increase of
VEGF was found in the secreted fraction of the transduced
versus the parental cells (Fig. 6)
, according to
up-regulation of VEGF mRNA (Fig. 1)
. IL-8 was undetectable in parental
cells, whereas SkBr3/HOXB7 produced up to 
2 ng/ml of IL-8 that was
totally found in the extracellular secreted fraction. Finally, GRO
was found in the secreted fraction of the transduced cells,
up-regulated of 40-fold as compared with the original cell line (Fig. 6)
.
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. The experiment was made also by adding an anti-VEGF blocking
antibody. Fig. 7D
shows how the inhibition of the
SkBr3/HOXB7-secreted VEGF could modify endothelial cell morphology
reducing the differentiative delay we have observed. No clear
differences were found by adding the anti-VEGF antibody in HUVEC
cocultured with the parental cell line (data not shown). The growth
stimulatory activity of parental versus HOXB7/SKBr3 on
endothelial cells was compared in a cocultivation assay (Fig. 8)
. An increased number (3–5-fold) of HUVEC grown in the presence of
HOXB7/SKBr3-expressing cells was observed in four independent
experiments. Assays were repeated in the presence or absence of an
anti-VEGF blocking antibody. The proliferative rate of the endothelial
cells cocultured with SkBr3/HOXB7 cells and the anti-VEGF antibody was
48% of that obtained in its absence. The same assay performed with
HUVEC cocultured with parental SkBr3 showed a proliferation rate of
32% of that obtained with SkBr3/HOXB7 that was further reduced to 20%
in the presence of the blocking antibody (Fig. 8C)
.
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. Although parental cells originated barely or not detectable tumors,
all of the mice that received SkBr3/HOXB7 injections showed tumors
growing at the injection site. To obtain tumors from the parental cell
line to be compared for vessels content with the HOXB7-transduced
nodules, recipient mice were irradiated with 400 rad the day before the
injection. The analysis of incipient SkBr3 tumors from irradiated mice
showed poor vascularization with most of the tissue necrotic. Vessels
were outside the tumor, and the presence of muscular walls was
suggestive of residual s.c. vessels (Fig. 9A)
. Immunohistology of SkBr3/HOXB7 tumor nodules showed
increased vascularization, as evaluated by anti-CD31 and anti-CD34
immunostaining. Vascular lacunas, unicellular and small vessels without
an organized wall, as well as muscular vessels, were present within the
tumor site and in the peritumoral area (Fig. 9B)
. In tumors,
beside the increased number of new vessels, HOXB7 expression seems to
directly or indirectly up-regulate CD31 expression to a bright
immunodetection (Fig. 9)
. The number of vessels were counted in the
entire growing areas, and the differences, evaluated by Student’s
t test, were statistically significant (Table 3)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have reported (5) the direct relationship between a homeogene, HOXB7, and bFGF in melanomas and in the SKBr3 breast carcinoma cell line. SKBr3 cells upon transduction of the sole HOXB7 gene, became bFGF-positive, increased the proliferative rate, and acquired independence from serum and the ability to form foci in semisolid agar (10) .
In this study, we show that several genes involved in the
proliferative and angiogenic processes are also affected by HOXB7
expression. Growth factors and growth factor receptors, known to have
partially overlapping but specific roles in controlling the growth of
new vessels, were analyzed. In particular in the HOXB7-transduced SkBr3
cells, besides the induction of bFGF, we found an increase of
3–10-fold of VEGF, GRO, IL-8, and Ang-2 at both mRNA (see Figs. 1
2
) and, when possible, protein levels (Fig. 6)
. On the contrary,
Ang-1 was totally abrogated as outlined by the use of primers that can
amplify all of the Ang-1 isoforms identified thus far
(22)
. This finding could appear in contrast to transgenic
studies (23)
where overexpression of Ang-1 was associated
to increased skin vascularization. In agreement with our results,
recent data showed that 90% of breast primary tumors were negative for
Ang-1 transcription. Moreover, enforced expression of Ang-1 in a breast
carcinoma cell line reduced tumor growth, suggesting an inhibitory
action in this cellular model (24)
. In the highly
disordered tumor vasculature, the reported stabilizing effect of Ang-1
might interfere with the intense sprouting of neovessels
(24)
. An interplay between VEGF and Angs in regulating
tumor angiogenesis has been hypothesized. High-grade malignant tumors,
showing a lot of immature tumor vessels, do not express Ang-1, but
Ang-2 (25)
, a natural antagonist of Ang-1, and VEGF.
Ang-1/Tie-2 interaction seems to stabilize microvessels, and a
down-regulation of Ang-1 may prime tumor vasculature for active
propagation, likely through cooperation between VEGF and Ang-2
(26)
. Also, the HOXB7-transduced HeLa cell line showed
VEGF and Ang-2 up-regulation paralleled by Ang-1 down-regulation (data
not shown), thus confirming a possible specific role of HOXB7 in
regulation of vasculogenesis during embryonic formation and in
neoangiogenesis during tumor progression when its expression is
deregulated.
Our results underscore the selective induction of
VEGF165 and VEGF189
isoforms by HOXB7. In fact, although expression of
VEGF121 remained substantially unchanged,
VEGF165 and 189
were expressed at a ratio of 1:7.5 and 1:12 in SkBr3 and SkBr3/HOXB7,
respectively (see Fig. 1
). The same pattern was observed in the A375
and 665/1 melanoma cell lines after treatment with antisense oligomers
targeting HOXB7. This treatment down-regulated selectively the
expression of VEGF189 and
VEGF165 (7-fold and 4-fold decrease,
respectively), but not of VEGF121 (Fig. 2)
.
Because the alternative splicing originated polypeptides with a
different secretion pattern and possibly different function,
VEGF189 and 165 may
influence tumor progression (27)
. Although
VEGF121 is a diffusible protein, the longer
VEGF189 isoform is tightly associated with the
ECM and can be released as a soluble and bioactive factor by heparin
and plasmin. VEGF165 shows an intermediate
behavior (28)
. The preferential induction of the longer,
membrane-bound isoforms by HOXB7 could be related to the role of the
HOX genes in ECM remodeling during development
(29)
. Extracellular proteolysis and degradation of the ECM
occurring during morphogenesis are thought to be involved in
invasiveness and metastases of tumor cells (30)
. In
agreement with these data, we found a strong increase of MMP-9 in the
HOXB7-transduced cells (see Fig. 5
).
Growth factor receptors have been studied to recognize whether the
induction of proliferation and angiogenesis is based on autocrine or
paracrine loops. The lack of a marked up-modulation of receptors for
angiogenic factors on neoplastic cells suggested that their ligands,
except bFGF, did not play autocrine activity. Such ligands, not
sequestered by tumor cells, remain free to activate cell surface
receptors on endothelial cells. This function was confirmed by the
experiments performed by adding an anti-VEGF blocking antibody in the
SkBr3/HOXB7 culture medium. Although the presence of this antibody did
not interfere with tumor cell proliferation (see Fig. 4
), persisting
capillary-like structures formed by endothelial cells grown in the
presence of HOXB7-producing cells, although still showing a morphology
different from SkBr3 parental coculture, did not last as long as in the
presence of the antibody (Fig. 7D)
. Endothelial cells are
known to express several members of the receptor tyrosine kinase
family, including receptors for VEGF and FGF and Tie-1 and Tie-2/Tek
(31
, 32)
. This may explain the induction of angiogenesis
and tumorigenesis in xenografted SkBr3/HOXB7 cells. The presence of
these endothelial receptors can well account for the induction of the
angiogenic phenotype that we observed. Among the receptors only Tie1
was found up-regulated in SkBr3/HOXB7 cells but, because its ligand is
unknown, no functional inference could be formulated at this moment.
To assess the actual function of the HOXB7-based microenvironment, in vitro and in vivo angiogenic assays were performed. In both cases, HOXB7 overexpression correlated with an enhanced angiogenic response, and our data indicate that this effect could be likely attributable to the induction of many different proangiogenic factors.
In this view, HOXB7 might represent a master gene to be targeted to inhibit tumor-associated angiogenesis. Its ability of activating many angiogenic molecules makes it an attractive upstream gene, the inhibition of which could be a highly effective antiangiogenic strategy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Italian Association for Cancer
Research (AIRC) and in part by Ministero della Sanità and The
Italy-USA Special Project on Therapy of Tumors. F. F. was supported by
a fellowship from Fondazione Italiana per la Ricerca sul
Cancro. 
2 To whom requests for reprints should be
addressed, at Istituto Superiore di Sanità, Hematology/Oncology
Department, Viale Regina Elena, 299-00161 Rome, Italy. Phone:
0039-06-49902411; Fax: 0039-06-49387087; E-mail: a.care{at}iss.it
3 The abbreviations used are: bFGF, basic
fibroblast growth factor; VEGF, vascular endothelial growth factor;
MGSA/GRO, melanoma growth-stimulatory activity/growth-related
oncogene ; HOX, homeobox; ß-gal, ß-galactosidase; RT-PCR,
reverse transcription-PCR; IL, interleukin; MMP, matrix
metalloprotease; HUVEC, human umbilical vein endothelial cell; Ang,
angiopoietin; ECM, extracellular matrix; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
Received 3/15/01. Accepted 6/21/01.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.. J. Exp. Med., 178: 151-161, 1993.This article has been cited by other articles:
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