Oncogene (1999) 18, 6719 ± 6724
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Nuclear 24 kD ®broblast growth factor (FGF)-2 confers metastatic
properties on rat bladder carcinoma cells
M Okada-Ban*,1, G Moens1, JP Thiery1 and J Jouanneau1
1
UMR144 CNRS, Institut Curie, Section de recherche, 26 rue d'Ulm, 75248 Paris, Cedex 05, France
The tumorigenic and metastatic properties of rat bladder
carcinoma NBT-II cells transfected with a cDNA
encoding the 24 kD nuclear isoform of human ®broblast
growth factor-2 (FGF-2) were analysed and compared
with those cells producing the 18 kD cytoplasmic isoform
FGF-2. In transfected clones, 24 kD FGF-2 was found in
the nucleus, and no FGF-2 was secreted. RT ± PCR
analysis showed no upregulation of FGF-2-speci®c
receptor FGFR2c expression in these proliferating
transfected cells. A shorter latency period for in vivo
tumor formation and abundant spontaneous lung metastases were only seen if nuclear FGF-2-producing cells
were injected subcutaneously into nude mice. Intravenous
injection of 24 kD FGF-2-producing cells led to
extensive experimental lung metastases whereas injection
of control NBT-II cells or 18 kD FGF-2-producing cells
did not. As FGF-2-producing cells have no speci®c FGF2 receptors, our results suggest that the 24 kD FGF-2
has nuclear targets, and activates metastatic property of
carcinoma cells via a mechanism other than the
conventional FGF receptor-mediated signaling pathway.
Keywords: FGF-2; nuclear localization; tumor progression; lung metastasis
Introduction
Fibroblast growth factor-2 (FGF-2) is a multifunctional protein with well studied mitogenic and
angiogenic properties (Basilico and Moscatelli, 1992;
Burgess and Maciag, 1989; Fernig and Gallagher, 1994;
Mason, 1994). Several FGF-2 isoforms have been
identi®ed; higher molecular weight (21, 22, 24 and
34 kD) and lower molecular weight (18 kD) FGF-2
isoforms are produced from di€erent initiation codons
(Florkiewicz and Sommer, 1989; Prats et al., 1989;
Arnaud et al., 1999). Only the higher molecular weight
isoforms with a nuclear localization signal (NLS) are
translocated to the nucleus (Bugler et al., 1991), and
there have been several studies of the nuclear
translocation of the FGF-2 protein (Bouche et al.,
1987; Florkiewicz et al., 1991; Renco et al., 1990;
Tessler and Neufeld, 1990). Nuclear translocation of
peptide growth factors has been reported in various
types of cell (Levine and Prystowsky, 1995), but such
translocation is not necessarily linked to the functions
of nuclear growth factors. The functional di€erences
between nuclear and cytoplasmic FGF-2 isoforms are
*Correspondence: M Okada-Ban
Received 29 April 1999; revised 21 June 1999; accepted 14 July 1999
unclear. The NLS-dependent nuclear translocation of
high molecular weight FGF-2 suggests that this
isoform of FGF-2 has an unknown biological role
di€erent from that of cytoplasmic FGF-2. Several
studies investigated the possible functions of the
various FGF-2 isoforms. Endogenous selective expression of the high molecular weight isoform of FGF-2
induces immortalization in endothelial ABAE cells
(Couderc et al., 1991), morphological changes and
growth in pancreatic acinar AR4-2J cells (Estival et al.,
1993) and NIH3T3 cells (Bikfalvi et al., 1995; Quarto
et al., 1991) in low-serum or serum-free medium, and
binucleation of cardiac myocytes (Pasumarthi et al.,
1995). Cells with a transformed phenotype are
potentially tumorigenic and the role of FGF-2 in
tumorigenicity is thought to be associated with cellular
transformation (Basilico and Moscatelli, 1992). The
expression of all FGF-2 isoforms stimulates endothelial
HEC-1-B (Coltrini et al., 1995) and neural cell
proliferation (Joy et al., 1997), and in vivo tumorigenicity in ABAE (Couderc et al., 1991) and NIH3T3
cells (Quarto et al., 1991). However, no one particular
isoform of FGF-2 can be considered to be fully
responsible for tumorigenicity.
In the rat bladder carcinoma NBT-II cell experimental model, the production of low molecular weight
(18 kD) FGF-2 induces angiogenic tumors, but does
not result in high level of tumorigenicity (Jouanneau et
al., 1997). In contrast we show herein that production
of the 24 kD FGF-2 alone results in NBT-II cells
becoming tumorigenic and lung metastatic. Thus,
regulation of the lung metastasis of 24 kD FGF-2producing cells may be controlled by FGF-2 in the
nucleus, with the possible involvement of speci®c
nuclear targets.
Results
Expression of 24 kD FGF-2
NBT-II cells were transfected with an expression vector
encoding the 24 kD FGF-2. This plasmid was designed
to produce exclusively the 24 kD FGF-2, and the
production of other isoforms of FGF-2 was prevented
by introducing point mutations into each of the
corresponding start codons. G418-resistant clones
were selected for the production of 24 kD FGF-2,
and FGF-2 mRNA was detected by Northern blot
analysis (data not shown). Most, but not all, of the
transfected cells were epithelial-like in morphology,
similar to parental NBT-II cells and 18 kD FGF-2producing bGF22 cells (Jouanneau et al., 1997). Three
clones, NLS20 (epithelial), NLS35 (epithelial) and
NLS42 (®broblast-like) were chosen for further study.
Nuclear 24 kD FGF-2 and metastasis
M Okada-Ban et al
6720
The presence of the FGF-2 protein in nuclei was
con®rmed by immuno¯uorescence staining using a
monoclonal antibody (Figure 1). Control NBT-II cells
did not react with the antibody (Figure 1A) and 18 kD
FGF-2-producing bGF22 cells only gave a signal in the
cytoplasm (Figure 1B). A strong signal was detected in
the nucleus of NLS20 (Figure 1C) and NLS35 (Figure
1D) clones with epithelial morphology and in
®broblast-like NLS42 cells (Figure 1E). The intensity
of FGF-2 immunostaining was higher in the nuclei of
subcon¯uent cells than in those of con¯uent cells (data
not shown).
FGF-2 protein was recovered from nuclear extracts
of subcon¯uent NLS cells by incubation with heparinsepharose gel, and was analysed by Western blotting.
The 24 kD FGF-2 was present in the nuclear fraction
of transfected cells (Figure 2, upper panel, lanes 1, 2
and 3), and a faint lower molecular weight band was
also visible in the nuclear fraction. A very small
amount of 24 kD FGF-2 protein was detected in the
cytoplasmic fraction (Figure 2, lower panel) whereas
18 kD FGF-2 was mostly cytoplasmic (Figure 2, lower
panel, lane 4). Parental NBT-II cells did not synthesize
endogenous FGF-2 (Figure 2, lane 5). Similar amounts
of 24 kD FGF-2 protein were produced in the
morphologically di€erent NLS cells, from 30 ± 100 ng
per 107 cells, as estimated by semiquantitative
determinations of the FGF-2 signal intensity from
Western blots.
or parental NBT-II cells (data not shown), either in
normal (10%) or low (1%) serum concentration
condition in cell culture.
Metastatic potential of 24 kD FGF-2-producing cells
It has been shown that 18 kD FGF-2-producing cells
are no more tumorigenic than control NBT-II cells in
nude mice, with a latency period for in vivo tumor
induction of more than 6 weeks (Jouanneau et al.,
1997). If NLS cells were injected subcutaneously into
nude mice, the tumor latency period decreased for
NLS42 and NLS20 cells, but not for NLS35 cells
(Figure 3, panel A, solid lines). NLS cells did not grow
any better in vitro than 18 kD FGF-2-producing cells
In each experiment involving the injection of 24 kD
FGF-2-producing cells subcutaneously into nude mice,
a large proportion of the lungs of the mice contained
foci of metastasized tumor cells (Figure 3, panel B).
Control untransfected NBT-II cells and 18 kD FGF-2transfected cells did not cause the development of such
foci. Macroscopic tumors were found in the lungs only
if 24 kD FGF-2-producing cells were injected into mice
(data not shown) when the primary tumor reached a
volume of approximately 2000 mm3. Both the highly
tumorigenic clones (NLS20/epithelial, NLS42/fibroblast-like) and the less tumorigenic clone (NLS35/
epithelial) gave lung metastases at a frequency of 10 ±
24% (Figure 3, panel B).
Cells from lung metastases derived from NLS20
tumors (NLS20-S5P) and NLS42 tumors (NLS42S32P) were grown in culture and used to inoculate
nude mice. These lung-derived cells have enhanced
tumorigenic potential, as the lag time for tumor
induction was 50% shorter than that of the parental
NLS cells, and also their lung metastatic potential was
increased (Figure 3, panel B). However, morphological
appearance in vitro and biochemical properties such as
24 kD FGF-2 production and its nuclear localization
(Figure 1F) were similar for cells derived from lung
metastases and parental NLS cells.
We then tested the lung-colonizing capacity in vivo
of NLS cells and lung-derived tumor cells, by
intravenous injection into either the tail vein or the
ocular sinus of nude mice. A large number of small foci
were observed in mouse lungs 2 weeks after the
injection of NLS cells or their metastatic derivatives,
but no metastatic foci were observed 2 weeks after
injection of control NBT-II cells or 18 kD FGF-2producing cells (data not shown).
Figure 1 Detection of FGF-2 protein by immuno¯uorescence.
Control NBT-II cells (A), 18 kD FGF-2-producing bGF22 cells
(B), and 24 kD FGF-2-producing cells (C) NLS20; (D) NLS35;
(E) NLS42 and (F) NLS20-S5P) were ®xed and stained. The
immunostaining was clearly associated with the nuclei of 24 kD
FGF-2-transfected cells. (Bars=10 mm)
Figure 2 Puri®cation of FGF-2 protein. FGF-2 proteins were
puri®ed from nuclear (upper, labeled as Nuclei) and cytoplasmic
fractions (lower, labeled as Cytoplasm) of cells. Lane 1: NLS42;
lane 2: NLS35; lane 3: NLS20; lane 4: bGF22 and lane 5: control
NBT-II cells
Tumorigenic properties of 24 kD FGF-2-producing cells
Nuclear 24 kD FGF-2 and metastasis
M Okada-Ban et al
Expression of FGF-2-speci®c receptors in 24 kD
FGF-2-producing cells
We investigated whether the biological properties of
NLS cells were mediated by an autocrine or paracrine
action of 24 kD FGF-2, by studying the expression of
FGF-2-speci®c receptors, by RT ± PCR. Proliferating
parental NBT-II cells do not have speci®c receptors for
FGF-2 (Savagner et al., 1994) because they only
express the FGFR2b spliced variant of FGFR2, which
does not bind FGF-2. In NLS cells (NLS20, NLS35,
NLS42, NLS20-S5P and NLS42-S32P), the FGF-2
receptor spliced variant, FGFR2c, was not detected by
PCR, and only FGFR2b was detected, as in parental
NBT-II cells and 18 kD FGF-2-producing cells (data
not shown).
Discussion
Production and distribution of the 24 kD FGF-2 protein
Western blot analysis of the FGF-2 produced in NLS
cells showed that a major band was present at the
expected molecular weight (24 kD) in the nuclear
fraction, consistent with nuclear immunostaining
results for these cells. A lower molecular weight band
was also detected in the nuclear fraction, but it is
unlikely to correspond to the low molecular weight
FGF-2 isoform (18 kD) because the NLS cells were
designed to produce 24 kD FGF-2 exclusively, and no
endogenous 18 kD or 24 kD FGF-2 is produced by
untransfected NBT-II. A loss of FGF-2 protein during
cell fractionation has been reported (Gualandris et al.,
1993; Pintucci et al., 1996), suggesting that the lower
molecular weight band may be generated by proteolysis
of 24 kD FGF-2, during or after puri®cation.
Heterogeneous immunostaining and the more intense
FGF-2 signal in subcon¯uent NLS cells than in
con¯uent cells suggest the cell-cycle and/or celldensity-dependent nuclear accumulation of endogenous 24 kD FGF-2 in NLS cells. Such endogenous cell
cycle dependent FGF-2 nuclear accumulation has been
observed in astrocytes (Hill and Logan, 1992).
Role of nuclear FGF-2 in tumorigenicity
This study indicates that the presence of 24 kD FGF-2
in nuclei may result in tumorigenic properties with no
obvious correlation with cell morphology. Both
epithelial (NLS20) and ®broblast-like (NLS42) cells
24 kD FGF-2-producing clones were more tumorigenic
in vivo than the epithelial 18 kD FGF-2-producing cells
(Figure 3, panel A). Another epithelial cell type,
NLS35 was no more tumorigenic than control NBTII cells, suggesting that there is no direct correlation
between high level of tumorigenicity and 24 kD FGF-2
production in the NBT-II cell system.
Parental NBT-II cells have no speci®c FGF-2
receptors on their surface (Savagner et al., 1994), and
there is no detectable upregulation of expression of the
FGF-2-speci®c receptor splice variant, FGFR2c, in
NLS cells. The 24 kD FGF-2 was not secreted by the
NLS cells. Thus the high level of tumorigenicity of
NLS cells does not result from the autocrine action of
FGF-2.
Figure 3 Tumor growth and lung metastasis frequency. The
tumor volume is the mean for experiments with ®ve animals (A).
Solid lines indicate growth curves for NLS cells and dotted lines
show the results of control experiments, as indicated in the inset.
Subcutaneous injection of transfected NBT-II cells resulted in
spontaneous lung metastasis with various frequencies (B).
Metastasis frequencies are given as percentages calculated from
numbers of mice with invaded lungs and total numbers of mice
tested, at the time which the tumor reached approximately
2000 mm3. Numbers of mice tested were as follows; NBT-II B43:
185; bGF22: 35; NLS20: 10; NLS35: 10; NSL42: 26; NLS20-S5P:
10; NLS42-S32P: 10
Tumor angiogenesis is not critical in NLS cell-derived
tumor
Histological analysis and von Willebrand Factor
staining were carried out on frozen sections of tumor
developed with 24 kD FGF-2-producing cells and
control NBT-II cells. The two types of tumor had
similar vascularization patterns (data not shown), very
unlike that of highly angiogenic 18 kD FGF-2producing cell-derived tumors (Jouanneau et al.,
1997). As 18 kD FGF-2-producing cells secrete
detectable amounts of biologically active 18 kD FGF-
6721
Nuclear 24 kD FGF-2 and metastasis
M Okada-Ban et al
6722
2, extensive tumor vascularization is attributed to
angiogenic activity of the 18 kD FGF-2. Angiogenesis
is essential for tumor growth, but rapidly growing NLS
tumors were less vascularized than tumors obtained
with 18 kD FGF-2-producing cells. Thus, the 24 kD
FGF-2 does not act as a tumor angiogenic factor, but
rather as a signaling molecule, increasing in vivo
tumorigenicity.
Role of nuclear FGF-2 in cancer cell metastasis
Lung metastasis of NBT-II-derived carcinoma cells is
speci®cally associated with the production of the FGF2 24 kD isoform, because 18 kD FGF-2-producing
cells did not give rise to metastatic foci in the lung
when primary tumors reached the same size. Micrometastases of NBT-II carcinoma cells and of transfected NBT-II cells, producing FGF-1 (Jouanneau et
al., 1994), FGF-4 (Jouanneau et al., 1995), TGFa
(Jouanneau, unpublished data) or HGF/SF (Bellusci et
al., 1994) are commonly found in the lymph nodes, but
never in the lungs or in other organs (Jouanneau et al.,
1994).
The lung colonizing capacity of NLS cells was
assayed in experimental metastasis by intravenous
injection in nude mice. We found that 24 kD FGF-2producing cells survived in the bloodstream and
colonized the lungs; in the same experimental
conditions, parental NBT-II cells and 18 kD FGF-2
producing cells did not give rise to lung foci.
NLS cells derived from lung metastatic foci were
more tumorigenic and lung metastatic than their
parental cells, even though their morphological and
biochemical characteristics were similar. The greater
tumorigenicity and metastatic properties of such lungderived cells may be due to in vivo selection. The
tumorigenicity and lung metastatic potentials of 24 kD
FGF-2-producing cells are not closely correlated
because, for example, NLS35 cells were no more
tumorigenic than control NBT-II cells but did produce
numerous lung metastatic foci in nude mice.
The metastatic potential of NLS cells cannot be
predicted from cellular morphology, because all the
NLS clones tested gave similar level of lung
metastasis despite being morphologically di€erent.
These di€erences may be due to di€erence in
metalloproteinase (MMP) production in the cells.
However, using zymography we found that the
gelatinase activities present in secretions from NBTII cell transfected with FGF-1 (®broblast-like
morphology) and from NLS cells were similar
(unpublished date). Nuclear FGF-2 probably stimulates or initiates metastatic signaling the mechanism
of which is unknown.
Impact of endogenous nuclear FGF-2 production
We have clearly demonstrated the importance of the
24 kD nuclear isoform of FGF-2, which confers lung
metastatic properties on NBT-II carcinoma cells and
also, in some but not all cases, increases their
tumorigenicity. The biological activities of most
growth factors are thought to be expressed via binding
to their receptors, but our experimental system
excludes such an autocrine/paracrine mechanism for
FGF-2.
The lung metastatic properties of carcinoma cell are
clearly associated with the endogenous production of
nuclear FGF-2; cytoplasmic FGF-2 production had no
e€ect. Our results provide evidence for a novel FGF-2
biological activity that may be involved in the
induction of metastatic and tumorigenic potential in
carcinoma cells, because this nuclear growth factor
may stimulate novel signaling pathways or transcriptional activities (Nakanishi et al., 1992; Bonnet et al.,
1996). Molecular mechanisms for tumor metastasis are,
in many cases, attributed to a limited number of
putative metastatic markers (Sidransky, 1997). FGF-2
has not previously been considered to be such a
marker, but our study indicates that nuclear FGF-2 is
a potential metastasis inducer in carcinoma cells.
Screening for possible nuclear targets of FGF-2 and
its investigation of functions in tumor metastasis
induction are underway. Our experimental system
provides an in vivo model for the study of FGF-2induced tumor progression and metastasis, independent
of the conventional FGF-2 receptor-mediated signaling
pathway.
Materials and methods
Cells
NBT-II cells, which were originally isolated from a
chemically-induced rat bladder carcinoma (Toshima et al.,
1971), were provided by Professor M Mareel (University of
Gent, Belgium). Cells from NBT-II clone 19 were used
(Jouanneau et al., 1994). Cells were routinely cultured in
monolayers in Dulbecco's modi®ed Eagle's minimal essential
medium with 10% heat-inactivated fetal calf serum with 1%
glutamine and antibiotics (complete medium). The cells were
grown at 378C in a humidi®ed atmosphere containing 6%
CO2 and were routinely cultured after trypsin treatment
(0.05% trypsin and 0.02% ethylenediamine tetraacetic acid
w/v). NBT-II B43 cells, which express b-galactosidase, were
used as controls (Jouanneau et al., 1994).
Expression plasmid for nuclear FGF-2 and transfection
The pRFE-neo biscistonic expression vector, a gift from Drs
B Bugler and H Prats (INSERM U397, Toulouse, France),
contains the neomycin resistance (NeoR) gene and a human
FGF-2 cDNA under control of the CMV promoter such that
it produces only the 24 kD FGF-2. NBT-II cells were
transfected by the calcium-phosphate coprecipitation method
(Graham and Van der Eb, 1973) with 10 mg of the pRFE-neo
expression plasmid. The parental cells were seeded in 10 cmdiameter plastic culture dishes with 0.86106 cells/dish, 24 ±
36 h before transfection. Transfected cells were incubated for
24 h, treated with trypsin and cultured at 1 : 4 dilution in
culture medium containing the neomycin analog G418
(400 mg/ml) (selective medium). After 2 ± 3 weeks, G418resistant colonies were picked and ampli®ed using the
selective medium for the ®rst two passages. Selected clones
were grown in complete medium from the 3rd passage, and
24 kD FGF-2-producing clones were renamed as NLS cells.
Antibodies and immuno¯uorescence analysis
Anti FGF-2 monoclonal antibody was purchased from
Transduction Laboratories (USA). Cells were grown on thin
glass coverslips for 1 ± 3 days and ®xed in 4% paraformaldehyde in phosphate-bu€ered saline (PBS). Fixed cells were
permeabilized by incubation with PBS containing 0.1%
Triton X-100 for 20 min at room temperature, followed by
Nuclear 24 kD FGF-2 and metastasis
M Okada-Ban et al
washing and blocking with 5% normal serum in PBS. Cells
were incubated with FGF-2 antibody (1 : 25 in dilution in
PBS) overnight at 48C in a humid atmosphere, washed with
washing bu€er (PBS containing 0.25% Tween-20, w/v)
several times, and stained with ¯uorescent secondary antibody for 1 h at room temperature. They were then rinsed
with washing bu€er and mounted.
Isolation of nuclear fraction and detection of FGF-2 protein
Cell pellets were collected by trypsin treatment of subcon¯uent cells. They were washed thoroughly with PBS,
centrifuged, suspended in 100 mM Tris-HCl pH 7.5, 1 mM
ethylenediamine tetraacetic acid, 10 mM NaCl, 1 mM phenylmethyl-sulfonyl ¯uoride, 10 mg/ml leupeptin, 10 mg/ml pepstatin-A and homogenized with a glass homogenizer for 30
strokes. Cell nuclei were separated by centrifugation at
2000 g through a sucrose and were solubilized in PBS
containing 1% Nonidet P-40, 0.5% sodium deoxycholate,
0.1% sodium dodecyl sulfate, 1 mM phenyl-methyl-sulfonyl
¯uoride, 10 mg/ml leupeptin, 10 mg/ml pepstatin-A. They were
then centrifuged at 5000 g for 10 min to remove insoluble
substances. Nuclear fractions (soluble) were incubated with
heparin-sepharose (Pharmacia Biotech, Uppsala, Sweden) to
recover FGF-2 protein. Fixed proteins were solubilized with
reducing sample bu€er, and detected after SDS ± PAGE by
Western blot analysis with a peroxidase-linked secondary
antibody (antibody dilution 1 : 250). FGF-2 was determined
semiquantitatively by comparison of the signal with that of
control FGF-2 protein.
PCR-based detection of the FGF receptor
FGF receptors were detected by RT ± PCR, as described in a
previous report (Savagner et al., 1994). Brie¯y, total RNA
was extracted from NBT-II cells and transfected cells; cDNA
was obtained by reverse transcription used as a template for
PCR, using speci®c oligomeric DNA primers to distinguish
the alternative spliced variants of FGFR2, FGFR2b and
FGFR2c. Synthetic oligomeric DNA primers were purchased
from Genset (France), and were based on previously
determined sequences (Savagner et al., 1994).
In vivo experiments
Untransfected NBT-II cells or FGF-2-producing cells
(3.56106 cells) were injected subcutaneously into the ¯ank
of 6-week-old female Swiss nude mice (nu/nu, I€a Credo, Les
Oncins, France). Series of ®ve animals were used in each
experiment. Tumor growth was assessed twice a week and
spheroid volume was calculated from the measurements of
two perpendicular dimensions. When tumor volume reached
approximately 2000 mm3, tumors and lungs were aseptically
removed and fragments grown in culture, or treated with
Bouin ®xative solution for anatomical and pathological
analysis. Experimental metastasis was obtained by intravenous injection of 1.756106 cells into either the tail vein or the
ocular sinus. Lungs were removed 2 weeks after injection.
Abbreviations
FGF, ®broblast growth factor; FGFR, ®broblast growth
factor receptor
Acknowledgments
We would like to thank B Bugler and H Prats for the high
molecular weight 24 kD FGF-2 expression vector, Z Werb
for suggestions and discussions, Y Bourgeois for nude
mouse injections, D Morineau for photographs and IS
Coulter for proof reading. This work was supported by the
Centre National de la Recherche Scienti®que and the
Institut Curie, and by grants from the Association pour la
Recherche sur le Cancer (ARC-4045 and a postdoctoral
fellowship to M Okada-Ban), the Fondation pour la
Recherche MeÂdicale (postdoctoral fellowship to M Okada-Ban), the Ligue Nationale FrancËaise contre le Cancer
(National and Paris committees) and the Groupement des
Entreprises FrancËaises contre le Cancer (Ge¯uc).
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