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Tumor Biology |
Research Institute of Molecular Pathology, A-1030 Vienna, Austria
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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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Whereas the involvement of VEGF in tumor angiogenesis has been demonstrated repeatedly in various murine tumor models and human tumor xenograft experiments (4) , experimental evidence for a direct involvement of FGFs in the regulation of tumor angiogenesis is rather circumstantial. Expression of FGF1 and FGF2 is often increased in tumors and correlates with the degree of cancer malignancy (11) , and high concentrations of FGF2 have been found in both the serum and urine of cancer patients (12) . However, a direct demonstration of an involvement of FGFs in tumor angiogenesis is hampered by the highly pleiotropic activities of the factors; FGFs can activate FGFRs expressed on a number of different target cells, including tumor cells, endothelial cells, and fibroblasts. For example, tumorigenicity of melanoma cells was efficiently repressed by interference with FGF2 or FGFR1 function (13) . However, melanoma cells are known to depend on FGF2 for their proliferation, and it was not possible to functionally separate inhibition of tumor angiogenesis from repression of tumor cell proliferation. Similarly, s.c. tumors induced by 3T3 fibroblasts expressing a constitutively secreted form of FGF2 were repressed in their growth by neutralizing antibodies against FGF2 (14) . Again, although tumor vascularization was impaired, the transforming and angiogenic activity of FGF2 could not be distinguished.
The onset of tumor angiogenesis (the angiogenic switch) has been functionally defined by Folkman et al. (15) in a transgenic mouse model of multistage tumorigenesis. In these mice, SV40 T antigen is expressed under the control of the rat insulin promoter, resulting in the development of ß-cell tumors in the pancreatic islets of Langerhans (Rip1Tag2; Ref. 16 ). Several tumor stages can be distinguished, including normal islets, ß-cell hyperplasia, adenoma, and carcinoma (17 , 18) . Notably, hyperplastic islets can be classified into two types: (a) nonangiogenic islets that do not affect cocultured endothelial cells; and (b) angiogenic islets that are able to induce proliferation, migration, and tube formation of endothelial cells (15) . Based on these experiments, it has been proposed that soluble factors are involved in the onset of tumor angiogenesis. Normal islets of Langerhans already express VEGF-A, and its expression and secretion are moderately up-regulated in ß tumor cells (19) . Similarly, FGF1 is expressed in normal islets of Langerhans and throughout ß-cell tumorigenesis. Notably, ß tumor cells gain the capability of exporting FGF1, although it does not carry a signal sequence for secretion (20) . A similar switch to the export of signal-less FGF2 has been reported during fibrosarcoma development in transgenic mice (21) , and it has been speculated that the export of these angiogenic FGFs may be involved in the onset of tumor angiogenesis.
Previously, soluble receptors have been used efficiently to address the role of growth factors during tumor neovascularization. For example, inhibition of VEGF activity by soluble VEGF receptors [sFlt or soluble Flk (sFLK)] has been instrumental in demonstrating that VEGF is required for angiogenesis (22, 23, 24, 25) . Similarly, experiments with soluble Tie-2 receptor have shown that angiopoietins contribute to tumor growth (26) . In this report, we have used a soluble form of FGFR2 IIIb (sFGFR) to interfere with FGF function during tumor angiogenesis. Previously, this soluble receptor has been reported to impair several developmental processes, causing early embryonic lethality when constitutively expressed in transgenic mice (27) . To express high levels of sFGFR in adult animals, we generated a recombinant adenovirus encoding for sFGFR. Expression of sFGFR dramatically repressed the induction and maintenance of tumor angiogenesis and tumor growth in allograft transplantation experiments and in Rip1Tag2 transgenic mice.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
HUVECs and mouse microvascular endothelial cells (1G11; Ref.
31
) were cultured in DMEM supplemented with 20% FCS (Life
Technologies, Inc., Gaithersburg, MD), 2 mM glutamine, 40
µg/ml bovine brain extract, 80 units/ml heparin, and antibiotics. The
medium for bovine capillary endothelial cells was supplemented with
10% FCS and FGF2 (2.5 ng/ml). The ß tumor cell line ßHC13T
originated from a hyperplastic ß tumor cell line derived from
Rip1Tag2 transgenic mice (ßHC13; Ref. 32
) by
transplantation into immunodeficient mice and subsequent culture from
the allografted tumors in DMEM supplemented with 4.6 grams/liter (w/v)
glucose, 10% FCS, 2 mM glutamine, and antibiotics.
Immunoblotting.
HUVECs were infected with adenoviruses (2000 PPC) and starved for
48 h in serum-free medium. Lysates and conditioned media were
prepared, and proteins were resolved by 7.5% SDS-PAGE and transferred
to nylon membranes. Membranes were probed with 1 µg/ml anti-mouse
IgG1 antibody specific for mouse immunoglobulin heavy chain (Pierce,
Rockford, IL). Immunostained proteins were visualized using the
enhanced chemiluminescence detection system (Amersham, Buckinghamshire,
United Kingdom) according to the manufacturer’s recommendations. Serum
samples from Rip1Tag2 mice (500 µl serum/mouse) were precleared by
incubation with protein G-agarose beads. Unbound proteins were then
adsorbed to heparin-Sepharose, and bound proteins were analyzed by
immunoblotting as described above.
DNA Synthesis Assay.
HUVECs were plated in triplicate in 24-well dishes (40,000 cells/well)
and infected with adenoviruses (10,000 PPC) in reduced serum medium for
2–3 h. Cells were starved for 24 h in 5% FCS medium and then
stimulated overnight with recombinant FGF1 (provided by Dr. T. Maciag;
Maine Medical Center Research Institute, South Portland, ME) and
heparin, FGF2 (Promega, Madison, WI), or VEGF (R&D Systems,
Minneapolis, MN). After stimulation, 1 µCi of
[3
H]thymidine was added per well, and
incorporation of thymidine was determined by scintillation counting as
described previously (33)
.
Collagen Gel Assay.
Angiogenic tumor stages were isolated from Rip1Tag2 transgenic mice as
described previously (19)
. HUVECs were infected with
adenoviruses (10,000 PPC) and allowed to recover for 48 h in
complete medium. Cells were then trypsinized, resuspended in 10% FCS
RPMI 1640, and cocultured with tumor stages in collagen matrix
as described previously (15)
. After 2–3 days, the
response of endothelial cells to the angiogenic tumor stages was
scored. The experiment was performed three times with approximately
40–50 islets each.
Allograft Transplantation Experiments.
ßHC13T cells (1 x 106 cells in
200 µl of PBS) were injected s.c. together with 1 x 1010 particles of the respective
adenoviruses in both flanks of MF1 nu/nu mice. After 4 days,
5 x 109 particles of each
adenovirus in 100 µl of PBS were injected intratumorally. Tumor
diameter was determined daily, and tumor volume was calculated based on
the spheric shape of the tumors.
Injection of Adenoviruses in Rip1Tag2 Mice.
Particles (1 x 1010) of each
adenovirus in 200 µl of HEPES-buffered saline (HBS) were
injected i.v. in the tail vein of Rip1Tag2 mice. Mice were sacrificed,
and tumor volume was determined based on the spheric shape of the
tumors.
Histology and Immunohistochemistry.
Tumors and pancreata were fixed overnight in 4% paraformaldehyde in
PBS and immersed in 30% sucrose/PBS for 12 h. Finally, they were
embedded in OCT (Tissue Tek, Torrance, CA) and snap frozen in liquid
nitrogen. For BrdUrd labeling, 2 h before sacrifice, mice were
injected i.p. with 100 µg of BrdUrd (Sigma, St. Louis, MO) per gram
of body weight. Sections (10-µm thick) were cut, mounted on
silane-coated slides, and immunostained as described previously
(34)
. For detection of apoptosis, the TUNEL technique was
used as described previously (34)
. Vessel density was
determined by CD31 (PECAM-1) immunostaining. Specific antibody staining
was visualized using the ABC-Vector horseradish peroxidase kit
according to the manufacturer’s recommendations (Vector Laboratories,
Burlington, CA). Antibodies were antimouse CD31 antibody (1:50;
PharMingen, San Diego, CA) and anti-BrdUrd antibody (1:50; Zymed, San
Francisco, CA). Proliferating and apoptotic cells were counted in at
least 10 comparable fields per section (magnification, x200), and
blood vessels were counted in at least 10 comparable fields per section
(magnification, x100).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. To
assess the capability of sFGFR to block FGF-induced endothelial cell
proliferation, HUVECs were infected with AdsFGFR, AdFlt, or a control
virus expressing eGFP (AdeGFP) at 10,000 PPC, starved in 5% FCS serum
medium, and then stimulated with FGF1 (10 ng/ml) and heparin (10
µg/ml), FGF2 (5 ng/ml), or VEGF (5 ng/ml). The incorporation of
radiolabeled thymidine was determined as a measurement for the
induction of DNA synthesis and thus proliferation. FGF1/heparin-induced
DNA synthesis was completely inhibited by AdsFGFR but not by AdeGFP
(Fig. 1B)
. In contrast, only a mild inhibitory effect of
AdsFGFR was observed with cells that were treated with FGF2, indicating
that sFGFR interfered predominantly with FGF1 function, interfered to a
lesser extent with FGF2 function, and did not interfere at all
with VEGF function (Fig. 1B)
The strong specificity of sFGFR
for FGF1 reflects the higher binding affinity of FGFR2 IIIb for FGF1 in
comparison to FGF2 (27)
. Conversely, AdsFlt was able to
block endothelial cell proliferation induced by VEGF but not that
induced by FGF1/heparin or FGF2 (Fig. 1B)
. Similar results
were obtained with mouse microvascular endothelial cells (1G11) and
bovine capillary endothelial cells (data not shown).
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The Onset of Tumor Angiogenesis.
Having established that sFGFR is a valid tool to interfere with FGF
function in vitro, we next wanted to determine whether sFGFR
inhibits tumor angiogenesis in vivo. For this purpose, we
used the Rip1Tag2 transgenic mouse model of ß-cell carcinogenesis
(see "Introduction"). In these mice, the switch to the angiogenic
state can be visualized by coculturing hyperplastic islets of
Langerhans with endothelial cells in a three-dimensional collagen gel
matrix (15)
. In the presence of an angiogenic hyperplastic
islet, endothelial cells proliferate and migrate toward the tumor
biopsy. To assess whether sFGFR is able to interfere with this
angiogenic response, HUVECs were infected with either AdeGFP or AdsFGFR
before coculturing them with angiogenic islets isolated from Rip1Tag2
transgenic mice. Three classes of endothelial cell response could be
distinguished: (a) a strong response characterized by
chemotactic migration of endothelial cells toward the islet and
survival of many endothelial cells in the vicinity of the islet;
(b) a weak response characterized by the migration and
survival of only a subset of endothelial cells; and (c) no
response characterized by failure of the endothelial cells to migrate
toward the angiogenic islet and by their subsequent death (Fig. 2A)
. With noninfected or
AdeGFP-infected endothelial cells, 60–80% of the samples showed a
strong response toward the islets, and only a minority of cases
exhibited a weak response or no response (Fig. 2B)
, also
indicating that most of the selected islets were indeed angiogenic. In
contrast, with endothelial cells expressing sFGFR, 60–70% of the
samples did not exhibit any reaction toward the angiogenic islets (Fig. 2B)
. In control experiments, infected endothelial cells were
cultured in a collagen gel in the absence of any tumor biopsy. These
experiments did not reveal any significant difference between cells
infected with AdsFGFR or AdeGFP, indicating that the inhibitory effect
of AdsFGFR was due to the inhibition of FGFs released by the tumor
biopsies (data not shown). These results indicate that FGFs are
required for endothelial cell migration, proliferation, and survival,
which are hallmarks of ongoing tumor angiogenesis, as visualized in
this collagen gel assay.
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.
These observations allowed us to assess the contribution of FGF1 to
tumor growth in a setting where FGFs did not directly affect tumor cell
proliferation but rather stimulated cells of the stromal compartment,
for example, endothelial cells.
|
. Consistent with the
inability of sFGFR to interfere with this process, an intermediate
delay in tumor onset was observed with the combination of the two
adenoviruses where half the dose of AdsFlt was applied together with
AdsFGFR (Fig. 4A)
. Four days after transplantation of the
tumor cells, 5 x 109 particles of
the respective adenoviruses were injected intratumorally, based on a
previous report demonstrating that intratumoral injection of AdsFlt
maintained high levels of sFlt expression (23)
. One mouse
per group was sacrificed to monitor tumor state, and, subsequently,
tumor size was determined daily. AdeGFP-injected tumors started to grow
exponentially, whereas AdsFGFR treatment resulted in a significant
repression of tumor outgrowth over the following 6 days (Fig. 4B)
. At this later time point, AdsFlt-injected tumors
started to grow at rates that were comparable with those of
AdeGFP-injected controls. In contrast, combined expression of sFGFR and
sFlt exhibited a strong synergistic effect in blocking tumor growth
(Fig. 4B)
, which was also demonstrated by the dramatic
decrease in final tumor weight at the end of the experiment (Fig. 4C
; P < 0.005). Histopathological
analyses of the latter tumors revealed a high degree of necrosis, and
the residual tumor cells were found to cuff around major vessels (Fig. 4D)
, a situation that is reminiscent of tumors that have
been treated with antiangiogenic compounds (37)
.
Quantitation of tumor cell proliferation (BrdUrd incorporation),
apoptosis (TUNEL staining), and blood vessel density (CD31 staining)
revealed that the mitotic index of tumor cells was not affected by the
expression of sFGFR or sFlt, whereas the extent of apoptosis was
marginally increased in tumors of AdsFGFR-infected mice (Table 1)
. In contrast, vessel density, a
measure of the extent of tumor vascularization, was decreased by
approximately 50% in tumors of AdsFGFR- and AdsFlt-infected mice
(Table 1)
. Tumors of mice that had been treated with both AdsFGFR and
AdsFlt were too small to be included in this statistical analysis.
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Tumorigenesis in Rip1Tag2 Transgenic Mice.
To assess the contribution of FGFs to tumor progression and tumor
angiogenesis in vivo, Rip1Tag2 mice were injected i.v. once
a week with 1 x 1010 particles of
AdeGFP, AdsFlt, AdsFGFR, or a combination of AdsFGFR and AdsFlt.
Titration experiments in unrelated control mice revealed that in this
experimental setting, serum levels of soluble receptors were highest 6
days after i.v. injection, and approximately 70% were still detectable
10 days after injection (data not shown). Tumor progression in Rip1Tag2
transgenic mice is highly reproducible, i.e., all mice
develop ß-cell tumors with similar incidence and kinetics and die
around 14 weeks. Hence, injections were applied between 8 and 12 weeks
of age, a time period during which angiogenesis was known to be highly
active in ß-cell tumorigenesis. No apparent effects on the general
health of the animals was observed during the course of the
experiments. At 12 weeks of age, the mice were sacrificed, and tumor
volumes were determined. In AdsFGFR-infected mice, tumor volumes were
dramatically reduced as compared with AdeGFP-infected control mice
(P < 0.005; Fig. 5A
). Tumor volumes were also
significantly reduced in AdsFlt-infected mice, however, they were
reduced to a lesser degree as compared with AdsFGFR-infected mice
(P < 0.01; Fig. 5A
). A
synergistic effect between AdsFGFR and AdsFlt was not apparent in these
experiments, rather the reduction of the AdsFGFR dose by half in the
combination treatment resulted in an intermediate response (Fig. 5A)
. These results indicate that in this experimental
setting, sFGFR is more efficient than sFlt in repressing tumor growth.
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; Table 2
). Concomitantly, a modest increase in
the apoptotic index was observed. Proliferation rates remained
unchanged, indicating that interfering with FGF and VEGF function did
not directly affect ß tumor cell proliferation (Table 2)
. A
comparable decrease in vessel density has been reported in Rip1Tag2
mice treated with antiangiogenic agents (38
, 39)
,
suggesting that sFGFR and sFlt repressed the formation of new blood
vessels (angiogenesis) rather than eliminating preexisting vessels.
Histological examination of AdeGFP-infected mice showed that when these
viruses were applied i.v., they predominantly infected the parenchymal
cells of the liver and, to a lesser degree, the spleen (data not
shown). Immunoblotting analysis of blood serum samples from
adenovirus-infected mice revealed detectable levels of sFGFR and sFlt
(Fig. 5B)
, suggesting that the soluble receptors were
produced mainly by hepatic and splenic cells and delivered via the
blood stream to the tumor sites in the pancreas. It should be noted
that serum levels of the soluble receptors as determined by
immunoblotting did not significantly correlate with the degree of tumor
repression, indicating that the levels of soluble receptors were not
rate-limiting in these experiments.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The onset of tumor angiogenesis can be nicely observed in a collagen
gel assay in which hyperplastic islets of Langerhans isolated from
Rip1Tag2 mice are cocultured with endothelial cells (15)
.
Angiogenic islets readily induce endothelial cell proliferation,
chemotactic migration, and survival. Besides VEGF-A, angiogenic islets
express FGF1, FGF7, and FGF8, among which FGF1 is the only FGF with
angiogenic activity (36)
. The fact that sFGFR has high
specific affinity for FGF1 (Fig. 1)
and the fact that expression of
sFGFR efficiently inhibits the angiogenic response in the collagen gel
assay indicate that FGF1 is one of the factors required for the
transition from a nonangiogenic to an angiogenic tumor stage. Moreover,
despite the lack of a signal sequence for secretion, FGF1 is
efficiently exported by ß tumor cell lines (20)
, raising
the possibility that FGF1 secretion by ß tumor cells contributes to
the onset of tumor angiogenesis. The requirement for VEGF in this
experimental setting has been demonstrated recently (42)
. Because
VEGF-A exhibits a synergistic effect with FGF1 and FGF2 under
these experimental conditions, it is conceivable that both FGF1 and
VEGF-A are required for the switch to the angiogenic phenotype during
Rip1Tag2 tumorigenesis (7
, 8
, 19
, 20)
.
In allograft transplantation experiments, coinjection of AdsFlt with ß tumor cells resulted in an initial delay of tumor formation; however, at later time points, tumor growth was only moderately affected. In contrast, expression of sFGFR did not inhibit the early phases of tumor formation but rather repressed tumor growth at later time points. Similar to sFlt, sFGFR mediated its inhibitory function by interfering with tumor angiogenesis, as demonstrated by a 2-fold reduction in vessel density as compared with control tumors. Finally, a combination of sFGFR and sFlt revealed a synergistic effect in the repression of tumor growth, indicating that FGF1 and VEGF-A cooperate in inducing tumor neovascularization. Tumor cell proliferation was not affected by any of the soluble receptors in vitro or in vivo, excluding the possibility that tumor repression was due to a direct inhibition of tumor cell proliferation.
In Rip1Tag2 mice, i.v. injection of AdsFGFR dramatically repressed
tumor growth. Whereas tumor cell proliferation was not affected, vessel
density was modestly but significantly decreased (Table 2)
. In
particular, the number of small microcapillaries was reduced, whereas
larger vessels were less affected, suggesting that sFGFR repressed
tumor growth by inhibition of tumor angiogenesis (Fig. 6)
. Several
lines of evidence support the notion that ß tumor cell proliferation
or survival is not directly affected by interfering with FGF function:
(a) proliferation of ß tumor cells was not affected by the
expression of sFGFR in Rip1Tag2 transgenic mice (Table 2)
;
(b) tumor cell lines established from Rip1Tag2 transgenic
mice did not respond to exogenous FGFs and were not affected by the
expression of dominant-negative or soluble forms of FGFR (Fig. 3
; data
not shown); and (c) tumorigenesis was not affected in
Rip1Tag2 mice deficient for FGFR4, the FGF receptor that is
predominantly expressed by ß tumor cells (36)
.
In our experiments, sFGFR was more effective in the inhibition of tumor growth than sFlt, a treatment that has been demonstrated to efficiently repress physiological and tumor angiogenesis (22 , 23 , 25 , 40 , 41) . However, although both soluble receptors were detectable in the serum of infected mice, protein levels and half-life might vary significantly; therefore, a quantitative comparison between the two reagents is not possible. The differences observed between the allograft transplantation experiments and the experiments with Rip1Tag2 mice may be due to the fundamental differences between fast tumor formation of cultured tumor cell lines and the slow genesis of endogenously growing tumors.
From our results, we conclude that FGFs, together with VEGF, contribute to tumor angiogenesis and that inhibition of their activities represses tumor growth. During the multiple stages of tumor development, FGFs, in particular the angiogenic factors FGF1 and FGF2, exert highly pleiotropic functions. They can promote tumor cell proliferation in an autocrine fashion; they can stimulate the growth, survival, and migration of stromal cells, including fibroblasts and smooth muscle cells; and, as demonstrated in this study, they participate in the induction of tumor angiogenesis. Thus, therapeutic approaches that are based on the inhibition of FGF function may allow the simultaneous targeting of different cell types. Moreover, such treatment may potentiate therapeutic inhibition of VEGF function in cases in which both growth factors are expressed and act in a synergistic manner.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by the Austrian Industrial
Research Promotion Fund. 
2 To whom requests for reprints should be
addressed, at Research Institute of Molecular Pathology, Dr. Bohr-Gasse
7, A-1030 Vienna, Austria. Phone: 43-1-79730-840; Fax: 43-1-798-7153;
E-mail: christofori{at}nt.imp.univie.ac.at
3 The abbreviations used are: VEGF, vascular
endothelial growth factor; FGF, fibroblast growth factor; FGFR, FGF
receptor; sFGFR, soluble FGFR; sFlt, soluble Flt; HUVEC, human
umbilical vein endothelial cell; PPC, particles per cell; BrdUrd,
bromodeoxyuridine; eGFP, enhanced green fluorescent protein; TUNEL,
terminal deoxynucleotidyl transferase-mediated nick end labeling.
Received 7/ 5/00. Accepted 10/17/00.
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B. Rousseau, F. Larrieu-Lahargue, S. Javerzat, F. Guilhem-Ducleon, F. Beermann, and A. Bikfalvi The tyrp1-Tag/tyrp1-FGFR1-DN Bigenic Mouse: A Model for Selective Inhibition of Tumor Development, Angiogenesis, and Invasion into the Neural Tissue by Blockade of Fibroblast Growth Factor Receptor Activity Cancer Res., April 1, 2004; 64(7): 2490 - 2495. [Abstract] [Full Text] [PDF]
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 P. Magnusson, C. Rolny, L. Jakobsson, C. Wikner, Y. Wu, D. J. Hicklin, and L. Claesson-Welsh Deregulation of Flk-1/vascular endothelial growth factor receptor-2 in fibroblast growth factor receptor-1-deficient vascular stem cell development J. Cell Sci., March 15, 2004; 117(8): 1513 - 1523. [Abstract] [Full Text] [PDF]
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 L. Zilberberg, S. Shinkaruk, O. Lequin, B. Rousseau, M. Hagedorn, F. Costa, D. Caronzolo, M. Balke, X. Canron, O. Convert, et al. Structure and Inhibitory Effects on Angiogenesis and Tumor Development of a New Vascular Endothelial Growth Inhibitor J. Biol. Chem., September 12, 2003; 278(37): 35564 - 35573. [Abstract] [Full Text] [PDF]
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R. Giavazzi, B. Sennino, D. Coltrini, A. Garofalo, R. Dossi, R. Ronca, M. P. M. Tosatti, and M. Presta Distinct Role of Fibroblast Growth Factor-2 and Vascular Endothelial Growth Factor on Tumor Growth and Angiogenesis Am. J. Pathol., June 1, 2003; 162(6): 1913 - 1926. [Abstract] [Full Text] [PDF]
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S. E. Woodman, A. W. Ashton, W. Schubert, H. Lee, T. M. Williams, F. A. Medina, J. B. Wyckoff, T. P. Combs, and M. P. Lisanti Caveolin-1 Knockout Mice Show an Impaired Angiogenic Response to Exogenous Stimuli Am. J. Pathol., June 1, 2003; 162(6): 2059 - 2068. [Abstract] [Full Text] [PDF]
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O. Straume and L. A. Akslen Importance of Vascular Phenotype by Basic Fibroblast Growth Factor, and Influence of the Angiogenic Factors Basic Fibroblast Growth Factor/Fibroblast Growth Factor Receptor-1 and Ephrin-A1/EphA2 on Melanoma Progression Am. J. Pathol., March 1, 2002; 160(3): 1009 - 1019. [Abstract] [Full Text] [PDF]
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H. Seno, M. Oshima, T.-o Ishikawa, H. Oshima, K. Takaku, T. Chiba, S. Narumiya, and M. M. Taketo Cyclooxygenase 2- and Prostaglandin E2 Receptor EP2-dependent Angiogenesis in Apc{Delta}716 Mouse Intestinal Polyps Cancer Res., January 1, 2002; 62(2): 506 - 511. [Abstract] [Full Text] [PDF]
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G. Gannon, S. J. Mandriota, L. Cui, D. Baetens, M. S. Pepper, and G. Christofori Overexpression of Vascular Endothelial Growth Factor-A165 Enhances Tumor Angiogenesis but not Metastasis during {beta}-Cell Carcinogenesis Cancer Res., January 1, 2002; 62(2): 603 - 608. [Abstract] [Full Text] [PDF]
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D. Leali, M. Belleri, C. Urbinati, D. Coltrini, P. Oreste, G. Zoppetti, D. Ribatti, M. Rusnati, and M. Presta Fibroblast Growth Factor-2 Antagonist Activity and Angiostatic Capacity of Sulfated Escherichia coli K5 Polysaccharide Derivatives J. Biol. Chem., October 5, 2001; 276(41): 37900 - 37908. [Abstract] [Full Text] [PDF]
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 M. Papetti and I. M. Herman Mechanisms of normal and tumor-derived angiogenesis Am J Physiol Cell Physiol, May 1, 2002; 282(5): C947 - C970. [Abstract] [Full Text] [PDF]
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), FGF2 (5 ng/ml; 
), or VEGF (5 ng/ml; 
), as
indicated. The induction of DNA synthesis is given as fold stimulation
by comparing thymidine incorporation in the absence of growth factor
and in the presence of the particular growth factors. The results
represent the averages of triplicate experiments.
) or infected with 10,000
PPC of AdeGFP (
). Cell numbers were
determined daily after adenoviral infection as indicated. Error
bars represent the SD on the average of triplicate
experiments.
