©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorothioate Oligodeoxynucleotides Bind to Basic Fibroblast Growth Factor, Inhibit Its Binding to Cell Surface Receptors, and Remove It from Low Affinity Binding Sites on Extracellular Matrix (*)

(Received for publication, August 23, 1994)

Marina A. Guvakova (1) (2) Leonid A. Yakubov (1) (3) Israel Vlodavsky (4) John L. Tonkinson (1) C. A. Stein (1)(§)

From the  (1)Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, New York 10032, the (2)Institute of Cytology and Genetics, 10 Lavrentiev Prospekt, Novosibirsk 630090 Russia, the (3)Institute of Bioorganic Chemistry, 8 Lavrentiev Prospekt, Novosibirsk 630090 Russia, and the (4)Department of Oncology, Hadassah Hospital, Jerusalem, Israel 91120

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 beta1, alpha, , 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 (^1)(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.


MATERIALS AND METHODS

Reagents

I-Bolton-Hunter-labeled bFGF (human, recombinant), I-Bolton-Hunter-labeled PDGF, and I-EGF (murine) were obtained from NEN Research Products. Unlabeled EGF, VEGF, aFGF, bFGF, and FGF-4 were obtained from R and D Systems (Minneapolis, MN). Basic fibroblast growth factor (human, recombinant) was also obtained from Promega or as a generous gift from Takeda Chemical Industries (Osaka, Japan). Bovine serum albumin (BSA) was purchased from Sigma. Heparin sodium, M(r) = 14,000, was obtained from Hepar Industries (Franklin, OH).

Synthesis of Oligodeoxynucleotides

Phosphodiester oligodeoxynucleotides were synthesized by standard phosphoramidite chemistry on an Applied Biosystems 380B synthesizer. Phosphorothioate oligodeoxynucleotides were also synthesized by standard methods (Stein et al., 1988), and sulfurization was performed using the tetraethylthiuram disulfide/acetonitrile reagent (TETD; Applied Biosystems). Following cleavage from the controlled glass support, oligodeoxynucleotides were base-deblocked in ammonium hydroxide at 60 °C for 8 h and purified by reversed phase high performance liquid chromatography (0.1 M triethylammonium bicarbonate/acetonitrile, PRP-1 support). Oligomers were detritylated in 3% acetic acid and precipitated with 2% lithium perchlorate/acetone dissolved in sterile water and reprecipitated as the sodium salt from 1 M NaCl/ethanol. Oligodeoxynucleotide concentrations were determined by UV spectroscopy.

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`-G(S)T(S)GCCGGGGTCTC(S)C(S)G(S)G(S)G(S)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).

Synthesis of Alkylating, Radioactive Phosphodiester Oligodeoxynucleotide 5`-N-Methyl-N-(2-chloroethyl)aminobenzylamine-P-OdT18 or -P-OdT12 (RClNHP-OdT18 or RClNHP-OdT12)

This compound was synthesized by a modification of the method of Knorre et al.(1985). Briefly, after 5`-phosphorylation of OdT18 or OdT12 by Chemical Phosphorylation Reagent (Glen Research, Herndon, VA), a reaction exchanging the P of [-P]ATP was carried out using T4 polynucleotide kinase with ADP as the phosphate acceptor (Sambrook et al., 1989). Then, N-methyl-N-(2-chloroethyl)aminobenzylamine was coupled to the 5`-terminal P by reaction with triphenylphosphine/dipyridyl disulfide. The final product was stored at -70 °C.

Modification of bFGF and Other Heparin-binding Growth Factors by ClRNHP-OdT18 or ClRNHP-OdT12

This was accomplished by the method of Yakubov et al.(1993). bFGF (10 µg/ml), EGF (3 µg/ml), aFGF (10 µg/ml), FGF4 (10 µg/ml), or VEGF (25 µg/ml) was incubated in 0.1 M Tris-HCl, pH 7.6, containing the appropriate concentration of ClRNHP-OdT18 or ClRNHP-OdT12. (^2)In some experiments, a putative competitor (e.g. phosphorothioate oligodeoxynucleotide) of the binding of the modifying oligomer to bFGF was also added, as indicated in the figure legends. After 1 h, 0.5 volume of a buffer containing 50% glycerol, 0.1 M dithiothreitol, 2% SDS, and 0.0001% bromphenol blue was added and SDS-PAGE (12% acrylamide) was performed. The gels were dried and allowed to expose Kodak x-ray film until bands were visualized. The film was developed, and band densities were quantitated by laser scanning densitometry.

Cells

Contact-inhibited NIH 3T3 cells and the human prostate carcinoma line DU 145 were obtained from American Type Culture Collection. NIH 3T3 cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc.), containing 10% (v/v) calf serum (Life Technologies, Inc.) and 50 µg/ml gentamicin sulfate. DU-145 cells were grown and maintained in RPMI 1640 medium (Life Technologies, Inc.), containing 5% (v/v) fetal calf serum (Life Technologies, Inc.), 100 units/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate. For binding experiments, cells were plated at 1 times 10^6 cells per well in 6-well plates (Falcon) or 1 times 10^5 cells per well in 24-well plates (Corning) and were used for experiments 1 to 3 days later. Cultures of bovine corneal endothelial cells were established from steer eyes as described previously (Gospodarowicz et al., 1977). Stock cultures were maintained in DMEM (1 g of glucose/liter) supplemented with 10% newborn calf serum, 5% fetal calf serum, 50 units/ml penicillin, and 50 µg/ml streptomycin at 37 °C in 10% CO(2)-humidified incubators. Partially purified brain-derived bFGF (100 ng/ml) was added every other day during the phase of active cell growth (Gospodarowicz et al., 1977; Ishai-Michaeli et al., 1992).

Binding of I-bFGF to Cells

Confluent cultures of cells were used for all experiments. All cells were washed twice with Dulbecco's phosphate-buffered saline (DPBS) and then incubated in DMEM with 0.1% (w/v) BSA at 37 °C for 1 h before the initiation of binding experiments. For the binding test, cells were incubated at 4 °C in serum-free medium, containing 0.1% (w/v) BSA, 0.05 ng/ml (or 0.6 ng/ml) I-bFGF, and the desired concentrations of oligodeoxynucleotides. At the end of the incubation, the medium was removed, and cell-bound I-bFGF was analyzed by one of two procedures. 1) To determine total bound bFGF, the cells were washed three times with DPBS and were solubilized with 1 M NaOH; 2) in other experiments, cells were washed three times with DPBS and once with 2 M NaCl in 20 mM HEPES, pH 7.5, to remove I-bFGF bound to the low affinity binding sites; then the cells were extracted with 0.5% Triton X-100 in 0.1 M sodium phosphate, pH 8.1. The radioactivity thus released represented specific binding to high affinity sites (Moscatelli, 1987). Nonspecific binding was measured in the presence of 1000-fold molar excess of nonradioactive bFGF. The amount of radioactivity released in the washes and extracts was determined in a -counter (Multi-Prias 1, Packard Instruments).

Preparation of Dishes Coated with Extracellular Matrix (ECM)

Bovine corneal endothelial cells were dissociated from stock cultures (second to fifth passage) with 0.05% trypsin, 0.01 M sodium phosphate (pH 7.4), and 0.02% EDTA and plated into 4-well plates (Nunc; Roskilde, Denmark) at an initial density of 2 times 10^5 cells/ml. Cells were maintained as described above except that 5% dextran T-40 was included in the growth medium and the cells were maintained for 10-12 days without addition of bFGF (Ishai-Michaeli et al., 1992; Vlodavsky et al., 1987). The subendothelial ECM was exposed by dissolving (5 min, room temperature) the cell layer with PBS containing 0.5% Triton X-100 and 20 mM NH(4)OH, followed by four washes in PBS. The ECM remained intact, free of cellular debris, and firmly attached to the entire area of the tissue culture dish (Ishai-Michaeli et al., 1992; Vlodavsky et al., 1987).

Displacement of ECM-bound bFGF

ECM was incubated (3 h, 24 °C) with I-bFGF (2.5 times 10^4 cpm/0.25 ml/well) in PBS containing 0.02% gelatin. Unbound bFGF was washed away, and the ECM was incubated with increasing concentrations of either heparin or the desired oligodeoxynucleotide at room temperature for 3 h. The incubation media were collected and counted in a counter to determine the amount of released iodinated material. The remaining ECM was incubated (3 h, 37 °C) with 1 N NaOH, and the solubilized radioactivity was counted in a -counter. The percentage of released I-bFGF was calculated from the total ECM-associated radioactivity (Ishai-Michaeli et al., 1992). Depending on the batch of ECM, 8-15% of the bound I-bFGF was released during incubation with PBS alone. This ``spontaneous'' release could be markedly reduced by first heating (80 °C, 60 min) the ECM to inactivate its intrinsic proteolytic activity. Spontaneous release was subtracted from the experimental values to assess the specifically released I-bFGF. Each experiment was performed three to five times, yielding similar results.

[^3H]Thymidine Incorporation

NIH 3T3 cells were plated into 96-well plates at a concentration of 2 times 10^4 cells per well in culture medium. After 72 h, when cells were at confluence, the culture medium was removed and cells were washed once with DMEM containing 0.1% BSA. The medium was replaced with DMEM supplemented with 0.1% BSA, 10.0 ng/ml bFGF, and the appropriate concentration of phosphorothioate oligodeoxynucleotides (0.2, 2.0, 20.0 µM). Control wells contained medium plus 0.1% BSA and 10.0 ng/ml bFGF. After 18-20 h, cells were pulsed for 3 h with [^3H]thymidine (0.5 µCi/well). The amount of radioactivity incorporated into trichloroacetic acid-insoluble material was counted on glass filters, using a 1450 Microbeta liquid scintillation counter (Pharmacia Biotech Inc.).


RESULTS

Modification of bFGF by the Alkylating PO Oligodeoxynucleotide

When bFGF and the P-labeled alkylating, radiolabeled oligodeoxynucleotide, ClRNHP-OdT18, were incubated in 0.1 M Tris-HCl (pH 7.6) for 1 h, and the reaction products were then subjected to electrophoresis in a 12% SDS-polyacrylamide gel, a single band was observed on the exposed x-ray film. This band corresponded to the product of chemical modification of the protein by the alkylating group of the oligodeoxynucleotide, as described above. P-Labeled oligodeoxynucleotide of identical sequence not containing an alkylating moiety did not strongly associate with bFGF, and, in this case, no gel bands were seen on the x-ray film. No difference in the quantitative modification of bFGF by RClNHP-OdT18 could be detected in different buffers, including 0.1 M Tris-HCl ± 5 mM CaCl(2), in 0.05 M Tris-HCl ± 0.1 M NaCl, or in PBS.

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 ClRNHP-OdT18, the concentration dependence of the modification is approximately linear and intersects the minus abscissa corresponding to an apparent K(d) 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(d) = 1.1 µM. These data imply, therefore, that there are at least two binding sites, with different affinities for ClRNHP-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 ClRNHP-OdT18 at the concentration given below for 45 min at 37 °C. The mixture containing the bFGFbulletoligodeoxynucleotide complex was then subjected to 12.5% polyacrylamide gel electrophoresis. The concentration of ClRNHP-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 ClRNHP-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 ClRNHP-OdT18 as described in the text. The concentration of ClRNHP-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(r) = 17,000). The band near the top of the gel migrates at the position expected for a bFGF dimer.



Determination of the Competition Constant (K(c)) for SdT18, a Competitor of Modifying Oligodeoxynucleotide Binding to bFGF

We have examined the ability of several other polyanions, including the phosphodiester oligodeoxynucleotides OdC15, OdT18, and OdT28 (all 5 µM) to compete with the radiolabeled probe oligodeoxynucleotide for binding to bFGF. All of these polyanions are competitors of binding to bFGF of the modifying, radiolabeled probe oligodeoxynucleotide (concentration of probe = 2.5 µM). Furthermore, suramin, a polyanion long known (Stein, 1993) to be able to bind to heparin-binding growth factors and to block their binding to cell surface receptors, also is a competitor of the binding of RClNHP-OdT12 to bFGF. (^3)

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(c) for competitors of modifying oligodeoxynucleotide binding to rsCD4. The value of K(c) may be calculated from Equation 1 from Cheng and Prusoff(1973): K(c) = IC(1+ [ClRNHP-OdT12]/K(d)).

For the competitor SdT15, we found (Fig. 4, A and B) that the value of IC = 0.12 µM. However, the determination of K(c) is complicated by the difficulty in accurately determining a value of K(d). This is because the value of the low affinity K(d) and the high affinity K(d) 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(d). The value of K(c) 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 ClNHP-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 Kfor 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^2 = 0.96) of normalized counts/min versus the log of the SdT15 concentration (micromolar). The IC was 0.12 µM.



Binding of RClNHP-OdT18 to Other Heparin-binding Growth Factors

This experiment was performed in a 4-20% SDS-polyacrylamide gradient gel and is shown in Fig. 5. As can be seen, not only bFGF, but acidic FGF, FGF4 (K-FGF/hst), and VEGF can bind to either of the modifying probe oligodeoxynucleotides (RClNHP-OdT12 or -OdT18; concentration of each = 2.5 µM). In sharp contrast to the behavior with these growth factors, no band is present at the approximate place of migration of EGF, which binds to heparin only weakly. It should be noted that the commercially available preparations of aFGF, FGF4, and VEGF all contain albumin. It has previously been demonstrated (Vlassov et al., 1993) that albumin can bind phosphodiester oligodeoxynucleotides with low affinity. The modification of the albumin present in these preparations by RClNHP-OdT18 can be seen at the indicated position in Fig. 5.


Figure 5: Modification of growth factors by ClRNHP-OdT18 or -OdT12. The growth factor was incubated with either 2.5 µM ClNHP-OdT18 (lanes 1-5) or 2.5 µM ClNHP-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.



Inhibition of Growth Factor Binding to Cells by Homopolymeric Oligodeoxynucleotides

In order to determine whether oligodeoxynucleotides can interact with growth factors and can influence their binding to cell surface receptors, we examined the binding of basic FGF to 3T3 fibroblasts in the presense of different oligodeoxynucleotides. Both phosphodiester and phosphorothioate homopolymers of cytidine and thymidine (28- and 25-mers) were initially tested for their ability to affect I-labeled bFGF binding (Fig. 6). At a range of oligodeoxynucleotide concentrations between 2.0 and 10.0 µM, we observed slight (<20%) inhibition of bFGF binding by phosphodiester homopolymers. However, phosphorothioate homopolymers of thymidine as well as cytidine inhibited about 60-70% of total binding.


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 times 10^6 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. bullet, SdC28; , SdT28; box, OdC25; up triangle, 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 times 10^6 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: up triangle, SdC3; , SdC15; bullet, SdC28. B: up triangle, SdT3; , SdT15; bullet, 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 times 10^6 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.



Inhibition of bFGF Binding to Low and High Affinity Receptors by Oligodeoxynucleotide Heteromers

To distinguish bFGF binding to low and high affinity receptors, we used method of Moscatelli(1987). All tested 18-mer phosphorothioate oligodeoxynucleotides (Nos. 1-5) inhibited the binding of bFGF to the low affinity receptors of fibroblasts with similar efficiency (Fig. 9A). However, differences in oligodeoxynucleotide potency were found when the ability to inhibit high affinity binding of bFGF was determined (Fig. 9B). In these experiments, the concentrations of oligodeoxynucleotides employed were 0.2-20.0 µM (Fig. 9B). At the highest oligodeoxynucleotide concentration (20.0 µM), the high affinity binding of bFGF was inhibited by 60-70% in the presence of both the all-phosphorothioate antisense c-myb rat and mouse constructs (Nos. 1 and 2). In contrast, binding was only reduced by 20% by the scrambled phosphorothioate oligodeoxynucleotide (No. 5), and by only 10% in the presence of chimeric phosphorothioate/diester oligodeoxynucleotide (No. 3). Furthermore, we failed to identify any inhibitory activity of the sense sequence oligodeoxynucleotide (No. 4) on high affinity binding at the concentrations tested. Similar results were achieved when high and low affinity binding of bFGF to the prostate cancer cell line DU-145 were measured (Fig. 10, A and B).


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 times 10^5 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 times 10^5 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.



Release of Extracellular Matrix-bound bFGF by Phosphorothioate Oligodeoxynucleotides

Previous studies on the interaction of bFGF with the subendothelial ECM have shown that bFGF binds to heparan sulfate in the extracellular matrix and can be released by heparin and heparin-like molecules (Ishai-Michaeli et al., 1992). In the present studies, there was no release of ECM-bound bFGF by a phosphodiester homopolymer containing 28 thymidines (OdT28) up to a concentration of 20 µM. In contrast, exposure of extracellular matrix to phosphorothioate homopolymers of thymidine and cytidine resulted in efficient release of bFGF. This release was dependent on oligodeoxynucleotide chain length; for example, SdT5 (20 µM) released virtually no bound bFGF, whereas SdT28 (2 µM) released 50% of the bound bFGF (Fig. 11). SdT18 was almost as active as SdT28 (60-70% release at 5 µM), but activity diminished with SdT15 (50% release at 20 µM). Phosphorothioate polymers of cytidine were slightly more active than the thymidine congeners of the same length (50% release at 0.5 µM for SdC28). A maximal release of about 75% of the extracellular matrix-bound bFGF was obtained in the presence of a 5 µM concentration of both SdC18 and SdC28.


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 times 10^4 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 times 10^4 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.

Inhibition of bFGF-induced [^3H]Thymidine Incorporation by Phosphorothioate Oligodeoxynucleotides

We used bFGF as a stimulator of NIH3T3 fibroblast proliferation. We then analyzed the effects of several phosphorothioate oligodeoxynucleotides on the ability of the cells to incorporate [^3H]thymidine. These oligodeoxynucleotides included phosphorothioate homopolymers of cytidine SdC3 and SdC28 and oligodeoxynucleotides 1, 2, and 4 as given above. The addition of compounds 1 and 2, as well as SdC28, decreased [^3H]thymidine incorporation in a dose-dependent manner. The most significant effects were observed over a range of concentrations 2.0-20.0 µM. In contrast, neither the sense oligo (4) nor SdC3 was active to the same extent (Fig. 12). In parallel with decreased [^3H]thymidine incorporation, morphological changes such as cell rounding and clumping were observed.


Figure 12: Effects of phosphorothioate oligodeoxynucleotides on [^3H]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 [^3H]thymidine was measured following a 3-h pulse. Error bars are S.E. of three independent experiments.




DISCUSSION

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(d) = 2-20 times 10M), but they also interact with lower affinity receptors (K(d) = 2 times 10-2 times 10M (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(r) = 7.5 kDa (Kajio et al., 1992)). After release from the ECM, the soluble growth factorbulletGAG 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 [^3H]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 [^3H]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. (^4)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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Columbia University, College of Physicians and Surgeons, 630 W. 168 St., New York, NY 10032. Tel.: 212-305-3606; Fax: 212-305-8466; stein{at}cuccfa.ccc.columbia.edu.

(^1)
The abbreviations used are: bFGF, basic fibroblast growth factor; aFGF, acidic fibroblast growth factor; FGF-4, fibroblast growth factor-4; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; EGF, epidermal growth factor; SdC28, 28-mer phosphorothioate homopolymer of cytidine; ECM, extracellular matrix; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; DPBS, Dulbecco's phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.

(^2)
ClRNHP-OdT12 is dodecathymidylate phosphodiester oligodeoxynucleotide derivative with an alkylator moiety (Fig. ZI) coupled to the 5` radioactive phosphate through a phosphoroamide bond.


Figure ZI:


(^3)
M. Guvakova and C. A. Stein, unpublished results.

(^4)
M. Guvakova, I. Vlodavsky, and C. A. Stein, unpublished results.


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