(Received for publication, August 23, 1994)
From the
We studied the interactions of phosphorothioate
oligodeoxynucleotides and heparin-binding growth factors. By means of a
gel mobility shift assay, we demonstrated that phosphodiester and
phosphorothioate homopolymers bound to basic fibroblast growth factor
(bFGF). Binding of a probe phosphodiester oligodeoxynucleotide could
also be shown for other proteins of the FGF family, including acidic
fibroblast growth factor (aFGF), Kaposi's growth factor (FGF-4)
as well as for the bFGF-related vascular endothelial growth factor,
VEGF. No binding to epidermal growth factor (EGF) was observed. In
addition, using a radioreceptor assay, we have shown that
phosphorothioate homopolymers of cytidine and thymidine blocked binding
of not only I-bFGF, but also of
I-PDGF to
NIH 3T3 cells, whereas phosphodiester oligodeoxynucleotides were
ineffective. The extent of blockade of binding was dependent on the
chain length of the phosphorothioate oligodeoxynucleotide. Furthermore,
we have examined the effects of 18-mer phosphorothioate
oligodeoxynucleotides of different sequences on
I-bFGF
binding to low and high affinity sites on both NIH 3T3 fibroblasts and
DU-145 prostate cancer cells. Despite the fact that we have observed
inhibition of bFGF binding by the 18-mer phosphorothioate
oligodeoxynucleotides for both the high and low affinity classes of
bFGF receptor, the inhibition was sequence-selective only for the high
affinity receptors. We have also demonstrated that phosphorothioate
homopolymers of cytidine and thymidine release bFGF bound to low
affinity receptors in extracellular matrix (ECM). Finally, the most
potent phosphorothioate oligodeoxynucleotides used in these experiments (e.g. SdC28) were inhibitors of bFGF-induced DNA synthesis in
NIH 3T3 cells.
Phosphorothioate oligodeoxynucleotides are isoelectronic
congeners of phosphodiester oligodeoxynucleotides that retain the
property of aqueous solubility and Watson-Crick base pair
hybridization, but which are also nuclease-resistant (Stein et
al., 1988). These materials have found wide application as both in vitro and in vivo sequence-specific, or antisense,
inhibitors of gene expression (for a review, see Stein and
Cheng(1993)). However, it has been recognized for some years that these
compounds may have non-sequence-specific effects on cellular function.
These may result, at least in part, from their ability to bind to
cellular proteins. For example, phosphorothioate oligodeoxynucleotides
appear to bind non-sequence specifically to rsCD4 (Yakubov et
al., 1993), gp120 (Stein et al., 1993), and to protein
kinase C 1,
,
, and
isoforms. Other polyanions,
including pentosan polysulfate (Wellstein et al., 1991) and
suramin (Stein, 1993), can also bind to these proteins. Furthermore,
these latter polyanions also bind to heparin-binding growth factors,
including bFGF (
)(Moscatelli and Quarto, 1989) and other
growth factors as well (Coffey et al., 1987). We hypothesized
that oligodeoxynucleotides, which are also polyanions, might, similar
to suramin and pentosan polysulfate, interact with bFGF and other
heparin-binding proteins. In this report, we demonstrate direct binding
of a phosphodiester probe oligodeoxynucleotide to bFGF by means of a
mobility shift assay in denaturing polyacrylamide gels. We also
demonstrate that phosphorothioate oligodeoxynucleotides can block the
binding of human
I-labeled bFGF to both low and high
affinity receptors on the surface of NIH 3T3 and DU-145 cells. We
further show that phosphorothioate oligodeoxynucleotides, similar to
heparin, can remove
I-labeled bFGF from its low affinity
binding sites on subendothelial extracellular matrix. In addition, on
the basis of our data, we suggest that the ability of phosphorothioate
oligodeoxynucleotides to block the binding of
I-labeled
bFGF to its cell surface receptors may be sequence-selective.
In addition to phosphorothioate
homopolymers of cytidine and thymidine, we also used five 18-heteromer
oligodeoxynucleotides of different sequences to block binding of bFGF
to its cell surface receptors. Three oligodeoxynucleotides (1, 2, and
3) were complementary to codons 2-7 of either the rat or mouse
c-myb mRNA. In addition, one of the oligodeoxynucleotides (No.
3; antisense rat c-myb) was a chimeric
phosphorothioate/diester (two phosphorothioate linkages at the 5` and
five phosphorothioate linkages at the 3` terminus). One
oligodeoxynucleotide (No. 4) was sense rat c-myb, and the
other (No. 5) was a scrambled version of the rat antisense c-myb oligodeoxynucleotide. The sequences are: 1,
5`-GTGCCGGGGTCTCCGGGC-3` (antisense rat c-myb, all
phosphorothioate); 2, 5`-GTGTCGGGGTCTCCGGGC-3` (antisense mouse
c-myb, all phosphorothioate); 3,
5`-GT
GCCGGGGTCTC
C
G
G
G
C-3`
(chimeric phosphorothioate/diester); 4, 5`-GCCCGGAGACCCCGGCAC-3` (sense
rat c-myb, all phosphorothioate); 5, 5`-CGCCGTCGCGGCGGTTGG-3`
(scrambled rat c-myb, all phosphorothioate).
We examined the concentration dependence of
the modification of bFGF by ClRNHP-OdT18 (Fig. 1).
These results are also depicted in Fig. 2(top), where
the concentration of alkylating oligodeoxynucleotide is plotted as a
function of gel band intensity, as determined by laser-scanner
densitometry. The association of bFGF with the alkylating probe
oligodeoxynucleotide exhibits approximate saturation binding. Fig. 2(bottom) depicts the double-reciprocal replot of
the data in Fig. 2(top). This plot is not linear, and
the fact that it is composite implies that the concentration dependence
of bFGF modification cannot be simply described by the Michaelis-Menton
equation. However, at low concentrations of ClRNH
P-OdT18,
the concentration dependence of the modification is approximately
linear and intersects the minus abscissa corresponding to an apparent K
value of 0.18 µM. At higher
concentrations (0.6-5.0 µM), the dependence of
modification is also linear and intersects the minus abscissa at
apparent K
= 1.1 µM. These
data imply, therefore, that there are at least two binding sites, with
different affinities for ClRNH
P-OdT18, on the surface of
the bFGF protein.
Figure 1:
Modification of bFGF by the alkylating
oligodeoxynucleotide ClRNHP-OdT18. bFGF (3 µg/ml) was
incubated in 0.1 M Tris-HCl (pH 7.5) with
ClRNH
P-OdT18 at the concentration given below for 45 min
at 37 °C. The mixture containing the bFGF
oligodeoxynucleotide
complex was then subjected to 12.5% polyacrylamide gel electrophoresis.
The concentration of ClRNH
P-OdT18 was as follows (lanes 1-9, respectively): 0.02, 0.04, 0.07, 0.15, 0.3,
0.6, 1.25, 2, and 5 µM. Unreacted
ClRNH
P-OdT18 ran off the end of the gel and is not
seen.
Figure 2:
Top, concentration dependence of
modification of bFGF by differing concentrations of
ClRNHP-OdT18. The gel bands in Fig. 1were
quantitated by laser scanning densitometry. Shown is a plot of band
intensity versus modifying oligodeoxynucleotide concentration
(micromolar). The curve fit was graphically approximated. Bottom, double-reciprocal plot of the data in top panel. The
two apparent double-reciprocal plot of the data in top panel. The two
apparent K
values (intersection of the
lines with the negative abscissa) were graphically
approximated.
In order to detect this putative second binding
site, we used a higher resolution 6% SDS-polyacrylamide gel to better
define the binding of ClRNHP-OdT18 to bFGF. This gel is
shown in Fig. 3, and two bands are clearly visible. On the other
hand, unmodified bFGF gave a single sharp band at the appropriate
migration rate after Coomassie Blue staining. The slower migrating band
near the top of the gel migrates at the position expected for a bFGF
dimer.
Figure 3:
Modification of bFGF by the alkylating
oligodeoxynucleotide ClRNHP-OdT18 with gel electrophoresis
performed by 6% SDS-PAGE. bFGF (3 µg/ml) was incubated in 0.1 M Tris-HCl (pH 7.5) with ClRNH
P-OdT18 as described in
the text. The concentration of ClRNH
P-OdT18 (lanes
1-5, respectively) was 0.01, 0.04, 0.2, 1, and 5
µM. Unreacted probe is seen at the bottom of the
gel. Two bands are clearly seen which migrate at the approximate
position of bFGF (M
= 17,000). The band
near the top of the gel migrates at the position expected for
a bFGF dimer.
We have,
in greater detail, examined the ability of a 15-mer phosphorothioate
homopolymer of thymidine, SdT15, to inhibit binding of the modifying,
radiolabeled oligodeoxynucleotide to bFGF. We have previously used this
method to determine the values of K for
competitors of modifying oligodeoxynucleotide binding to rsCD4. The
value of K
may be calculated from Equation 1 from
Cheng and Prusoff(1973): K
=
IC
(1+
[ClRNH
P-OdT12]/K
).
For
the competitor SdT15, we found (Fig. 4, A and B) that the value of IC = 0.12
µM. However, the determination of K
is complicated by the difficulty in accurately determining a
value of K
. This is because the value of the low
affinity K
and the high affinity K
of the modifying oligodeoxynucleotide binding to bFGF are quite
close, and there are significant errors in the determination of each.
Thus, we have used an average value (0.5 µM) for the value
of K
. The value of K
for
SdT15, as determined by the Cheng-Prusoff equation, is 60 nM.
Figure 4:
Competition by SdT15 for binding of
ClRNHP-OdT12 to bFGF. Top, SdT15 was used as a
competitor of ClNH
P-OdT12 (0.5 µM) binding to
bFGF as described in the text (6% PAGE). Bands represent bFGF modified
by the oligodeoxynucleotide. The concentration of SdT15 was (lanes
1-9, respectively) 0, 0.02, 0.04, 0.08, 0.16, 0.31, 0.625,
1.25, and 2.5 µM. Bottom, determination of the
value of K
for SdT15 by the Cheng-Prusoff
equation (Equation 1). The data from the top panel was
quantitated by laser scanner densitometry. Shown is a plot (r
= 0.96) of normalized counts/min versus the log of the SdT15 concentration (micromolar). The
IC
was 0.12 µM.
Figure 5:
Modification of growth factors by
ClRNHP-OdT18 or -OdT12. The growth factor was incubated
with either 2.5 µM ClNH
P-OdT18 (lanes
1-5) or 2.5 µM ClNH
P-OdT12 (lanes 6-10). Lanes 1 and 6, EGF (3
µg/ml); lanes 2 and 7, bFGF (10 µg/ml); lanes 3 and 8, aFGF (10 µg/ml); lanes 4 and 9, FGF4 (10 µg/ml); lanes 5 and 10, VEGF (25 µg/ml). Members of the FGF family migrate at
the position labeled 1 on the left. VEGF migrates at
the position labeled 2. No binding of either probe
oligodeoxynucleotide to EGF was observed. In contrast, bFGF, aFGF,
FGF-4, and VEGF all underwent modification. Other slower migrating
bands, seen particularly in lanes 2 and 7 (bFGF
lanes) probably represent protein multimers. The bands designated by
the arrow represent modification of albumin, which is added to
the reagent by the manufacturer.
Figure 6:
Effects of phosphorothioate and
phosphodiester homopolymers of cytidine and thymidine on total binding
of I-bFGF to NIH 3T3 cells. Cells were plated in 6-well
plates at 1.0
10
cells per well. For binding, test
cells were incubated at 4 °C in serum-free medium containing 0.1%
BSA, 0.05 ng/ml
I-bFGF, and the indicated concentrations
of oligodeoxynucleotides. After 2 h, medium was removed and cells were
washed 3 times with Dulbecco's phosphate-buffered saline and
lysed with 1 M NaOH. Incorporated radioactivity was determined
by
counting. Error bars are the S.E. of three
independent experiments.
, SdC28;
, SdT28;
,
OdC25;
, OdT28.
Furthermore, by use
of the same binding assay, we demonstrated that the inhibition of bFGF
binding correlated, in general, directly with the oligodeoxynucleotide
chain length for homopolymers of cytidine and thymidine; i.e. the longer the oligodeoxynucleotide (3, 15, or 28 bases), the
greater its inhibitory activity at any given concentration (Fig. 7). We also examined the ability of phosphorothioate
oligodeoxynucleotides to affect the binding of another heparin-binding
growth factor to cells. In a binding assay using I-labeled PDGF, we demonstrated that phosphorothioate
homopolymers of cytidine of different chain length inhibit PDGF binding
to fibroblasts. Again, the inhibition is chain length-dependent, with
SdC3 and SdC28 being least and most potent, respectively (Fig. 8A). On the other hand, these same
phosphorothioate oligodeoxynucleotides did not affect binding of EGF to
cell surface receptors of 3T3 cells (Fig. 8B).
Figure 7:
Chain length dependent effects of
phosphorothioate homopolymers of cytidine (A) and thymidine (B) on binding of I-bFGF to NIH 3T3 cells. Cells
were plated in 6-well plates at 1.0
10
cells per
well. For binding, test cells were incubated at 4 °C in serum-free
medium, containing 0.1% BSA, 0.05 ng/ml
I-bFGF, and the
indicated concentrations of oligodeoxynucleotides. After 2 h, medium
was removed and cells were washed three times with Dulbecco's
phosphate-buffered saline and lysed with 1 M NaOH.
Incorporated radioactivity was determined by
counting. Error
bars are the S.E. of three independent experiments. A:
, SdC3;
, SdC15;
, SdC28. B:
, SdT3;
, SdT15;
, SdT28.
Figure 8:
Effects of phosphorothioate homopolymers
of cytidine on binding of I-PDGF (A) and
I-EGF (B) to NIH 3T3 cells. Cells were plated in
6-well plates at 1.0
10
cells per well. For
binding, test cells were incubated at 4 °C in serum-free medium,
containing 0.1% BSA, 0.5 ng/ml
I-PDGF, or 0.1 ng/ml
I-EGF and the indicated concentrations of
oligodeoxynucleotides. After 2 h, the medium was removed and cells were
washed three times with Dulbecco's phosphate-buffered saline and
lysed with 1 M NaOH. Incorporated radioactivity was determined
by
counting. Each point is the mean of duplicate
measurements, which varied by less than 10%. &cjs2113;, SdC3;
&cjs2110;, SdC15;
, SdC28.
Figure 9:
Effects of 18-mer phosphorothioate
oligodeoxynucleotides on binding of I-bFGF to low (A) and high (B) affinity receptors on NIH3T3 cells.
Cells were plated in 24-well plates at 1.0
10
cells
per well. For binding test, cells were incubated at 4 °C in
serum-free medium, containing 0.1% BSA, 0.6 ng/ml
I-bFGF,
and the indicated concentrations of oligodeoxynucleotides. After 2 h,
medium was removed, and bFGF bound to low and high affinity receptors
was determined as described above under ``Materials and
Methods.'' Error bars are the S.E. of three independent
experiments. Oligodeoxynucleotide numbers are defined under
``Materials and Methods.'' Closed stars,
oligodeoxynucleotide 1; circles, oligodeoxynucleotide 2; diamonds, oligodeoxynucleotide 3; open triangles,
oligodeoxynucleotide 4; open diamonds, oligodeoxynucleotide
5.
Figure 10:
Effects
of 18-mer oligodeoxynucleotides on binding of I-bFGF to
low (A) and high (B) affinity receptors on DU 145
prostate cancer cells. Cells were plated in 24-well plates at 1.0
10
cells per well. For binding, test cells were
incubated at 4 °C in serum-free medium, containing 0.1% BSA, 0.6
ng/ml
I-bFGF, and the indicated concentrations of
oligodeoxynucleotides. After 2 h, medium was removed, and bFGF bound to
low and high affinity receptors was determined as described under
``Materials and Methods.'' Error bars are the S.E.
of three independent experiments. Oligodeoxynucleotide numbers are
defined under ``Materials and Methods.'' Closed
stars, oligodeoxynucleotide 1; circles,
oligodeoxynucleotide 2; diamonds, oligodeoxynucleotide 3; open triangles, oligodeoxynucleotide 4; open
diamonds, oligodeoxynucleotide 5. Error bars are S.E. of
three independent experiments.
Figure 11:
Release of ECM-bound bFGF by homopolymer
phosphorothioate oligodeoxynucleotides of different chain length.
ECM-coated wells of four-well plates were incubated (3 h, 24 °C)
with I-bFGF (2.5
10
cpm/well). The
ECM was washed four times and incubated (3 h, 24 °C) with
increasing concentrations of heparin (closed circles), SdC28 (closed triangles), SdT28 (closed squares), SdT15 (open triangles), SdT18 (crosses), SdT5 (open
circles), and OdT28 (open squares). Released
radioactivity is expressed as the percent of total ECM-bound
I-bFGF (1
10
cpm/well; 34 pg of
bFGF/well). Release of
I-bFGF in the absence of
oligodeoxynucleotides did not exceed 14% of the total ECM-bound bFGF.
Each data point is the average of triplicate wells, and the standard
deviation did not exceed ±7%.
In other experiments, we examined the ability of three 18-heteromer oligodeoxynucleotides (Nos. 1, 2, and 4) to release extracellular matrix-bound bFGF. ECM-bound bFGF was efficiently released by each of these oligodeoxynucleotides. At a concentration of 1 µM, the amount of bFGF released was 50% for the antisense c-myb mouse compound and about 40% for the antisense and sense rat c-myb compounds. At a similar concentration, the release for SdC18 and SdT18 was 50% and 38%, respectively.
Figure 12:
Effects of phosphorothioate
oligodeoxynucleotides on [H]thymidine
incorporation by NIH 3T3 cells. Cells were cultured for 18-20 h
in medium containing 10.0 ng/ml bFGF only (shaded column) or
medium plus 20 µM phosphorothioate oligodeoxynucleotides,
as indicated (black columns). Incorporated
[
H]thymidine was measured following a 3-h pulse. Error bars are S.E. of three independent
experiments.
The fibroblast growth factors consist of a family of at least
nine related polypeptides. Their biological effects are mediated
through a set of specific high affinity receptors (K = 2-20
10
M),
but they also interact with lower affinity receptors (K
= 2
10
-2
10
M (Roghani et al., 1994)).
Despite the lack of a signal peptide, both aFGF and bFGF have also been
identified in the extracellular matrix (ECM) deposited by cultured
myoblasts (Weiner and Swain, 1989) and endothelial cells (Vlodavsky et al., 1987). Immunohistochemical staining revealed the
presence of bFGF in basement membranes of the rat fetus (Gonzalez et al., 1990), bovine cornea (Folkman et al., 1988),
and human blood vessels (Cardon-Cardo et al., 1990),
suggesting that ECM may serve as a reservoir for bFGF (Vlodavsky et
al., 1991). It appears that bFGF binds specifically to heparan
sulfate and heparin-like molecules in the ECM and cell surface, as
indicated by its displacement by heparin, heparan sulfate, or heparan
sulfate-degrading enzymes, but not by unrelated glycosaminoglycans
(GAGs) or GAG-degrading enzymes (Ishai-Michaeli et al., 1992;
Bashkin et al., 1989). These GAGs may protect bFGF from
proteolytic cleavage. Protection can also be afforded by the polyanion
dextran sulfate (M
= 7.5 kDa (Kajio et
al., 1992)). After release from the ECM, the soluble growth
factor
GAG complex may then act at distant sites.
There is
evidence to suggest that FGFs may localize to the nucleus (Amalric et al., 1994). By a gel mobility shift assay, Maciag et
al.(1994) have demonstrated that aFGF can associate with
double-stranded random phosphodiester 89-mers. In this study, we have
demonstrated that both phosphorothioate and phosphodiester
oligodeoxynucleotides are capable of binding to bFGF. In the case of
phosphorothioate oligodeoxynucleotides only, this binding may promote
release of bFGF from its low affinity binding sites on extracellular
matrix. Furthermore, phosphorothioate oligodeoxynucleotides can block
the binding of bFGF to both low and high affinity cell surface
receptors and can abrogate the bFGF-induced increment in
[H]thymidine incorporation in 3T3 fibroblasts.
The ability of oligodeoxynucleotides to bind to growth factors is not limited to bFGF. These compounds can also interact with aFGF, FGF-4, VEGF, and PDGF (but not apparently with EGF). On the basis of these data, it is not unreasonable to speculate that oligodeoxynucleotides may be able to interact with many additional heparin-binding growth factors. This behavior of oligodeoxynucleotides is highly reminiscent of that of other polyanions, including suramin (Stein, 1993; Williams et al., 1984; Coffey et al., 1987; Bikfalvi et al., 1991) and pentosan polysulfate (Wellstein et al., 1991; Zugmaier et al., 1991). Suramin is a hexaanion and a polysulfonated naphthylurea. It directly binds to bFGF (Yayon and Klagsbrun, 1990), VEGF, PDGF, and to many other proteins as well, only some of which are heparin binding (Stein et al., 1993). At least in part due to these properties, suramin has significant antineoplastic and antiangiogenic effects and is currently being evaluated in clinical cancer trials (Stein et al., 1989; Eisenberger et al., 1993). The similarities in the behavior of phosphorothioate oligodeoxynucleotides to that of suramin suggests that the former may also have sequence-selective antiangiogenic and antineoplastic activities. This possibility is currently being evaluated in the laboratory.
In addition to growth factors, there are other proteins to which phosphorothioate oligodeoxynucleotides, suramin and pentosan polysulfate, can bind. Perhaps the best studied of these is rsCD4 (Yakubov et al., 1993). In this case, polyanion binding sites have been mapped to both the CDR2- and CDR3-like loops of the D1 (N-terminal) domain. In the bFGF molecule, there are 24 basic residues that are exposed on its surface (Zhang et al., 1991). On the basis of a crystallographic structural determination (Eriksson et al., 1991; Zhang et al., 1991), it has been proposed that the four sulfate oxygen atoms of a bound glycosaminoglycan would be hydrogen-bonded by the side chains of Asn-27, Arg-120, Lys-125, in addition to the main chain amide of 120. A secondary sulfate binding site also appears to exist, and the main chain amide of Leu-126, as well as the side chains of Lys-119 and Lys-129 may be involved. A study published while this work was in progress demonstrated that RNA competes for the binding of heparin to bFGF (Jellinek et al., 1993). Thus, it is likely that phosphorothioate oligodeoxynucleotides will also bind to bFGF at or near the heparin-binding site. However, the receptor-binding and heparin-binding sites of bFGF appear to be independent (Eriksson et al., 1991), as shown by the fact that neutralizing antibodies that inhibit the binding of bFGF to its receptor do not block heparin binding. Thus, it has been proposed that bulky polyanionic compounds, such as suramin (and perhaps phosphorothioate oligodeoxynucleotides as well) may, subsequent to binding to bFGF, either prevent access to the receptor binding region, or, similar to heparin and other polysulfated carbohydrates (Prestrelski et al., 1992), cause a conformational change in the bFGF molecule (Eriksson et al., 1991).
In contrast to
our results, the dissociation constants for the binding of some of the
oligoribonucleotide constructs to bFGF may be as low as 0.2
nM. Furthermore, some constructs, in a sequence-specific
manner, can block the binding of bFGF to high affinity receptors with
an IC = 1 µM (Jellinek et
al., 1993). However, molecules of this type inevitably suffer by
comparison with phosphorothioate oligodeoxynucleotides because of their
extreme sensitivity to nucleases.
It is significant that the ability
of phosphorothioate oligodeoxynucleotides to block the binding of I-bFGF to cell surface receptors is at least partially
sequence-selective. This observation has potentially important
applications to experiments in which sequence-specific phosphorothioate
oligodeoxynucleotides are targeted to complementary regions on mRNA. In
some experiments (Simons et al., 1992), it is possible that
the antisense construct and the controls may exhibit differential
ability to block the binding of heparin-binding growth factors to their
cell surface receptors. Thus, experimentally determined biological end
points thought to be a direct consequence of Watson-Crick base pair
hybridization may ultimately be due to apatameric effects. Indeed,
because the non-sequence-specific effects of phosphorothioate
oligodeoxynucleotides may occur at similar concentrations as the
sequence-specific effects, the two may not be easily separable. Our
data suggest that the longer the oligodeoxynucleotide, and the higher
its concentration, the more likely it is that observed biological
effects have a significant non-sequence-specific effect. This is
exemplified by our data on the effects of phosphorothioate
oligodeoxynucleotides on [
H]thymidine
incorporation in 3T3 fibroblasts. Thus, it would be advisable to ensure
that any mRNA-targeted antisense oligodeoxynucleotide does not directly
interact (at the concentrations employed) with the protein product of
the targeted mRNA (Stein and Krieg, 1994).
In the present study, we
also report that phosphorothioate homopolymers of thymidine and
cytidine are capable of efficiently releasing ECM-bound bFGF. It has
been previously demonstrated that heparin-derived oligosaccharides
containing as little as 4 sugar units exhibited 30-40% of bFGF
releasing activity of native heparin. A nearly maximal release of
ECM-bound bFGF was induced by a decasaccharide (Ishai-Michaeli et
al., 1992). There was little or no release of bFGF by unrelated
glycosaminoglycans and N-substituted species of heparin. On
the other hand, heparanase activity was efficiently inhibited by N-substituted species of heparin, but was not affected by
heparin-derived oligosaccharides containing <12 sugar units
(Ishai-Michaeli et al., 1992). In striking contrast, the
structural requirements for the phosphorothioate
oligodeoxynucleotideinduced release of ECM-bound bFGF and for
inhibition of heparanase activity were quite similar. ()These data suggest that while the oligodeoxynucleotides
exert their effects by virtue of their polyanionic character, the
effects of heparin are due to a more specific recognition of the bFGF
and heparanase molecules. In this respect, phosphorothioate
oligodeoxynucleotides again behave similarly to the polyanion suramin.
Consequently, while various species of heparin and heparin-like
molecules may release active bFGF from its storage in ECM,
phosphorothioate oligodeoxynucleotides may displace bFGF that is
inactive from ECM and cell surfaces and hence prevent its possible
utilization in tissue repair and neovascularization. These
possibilities are also currently under investigation.
Figure ZI: