(Received for publication, April 10, 1995; and in revised form, June 10, 1995)
From the
Oligonucleotides can be used to inhibit the binding of basic fibroblast growth factor to cells. Though standard phosphodiester oligonucleotides show a slight inhibition of binding, the oligonucleotides with phosphorothioate internucleoside linkages have inhibition levels equivalent to that of the polyanion heparin. Variations in sequence of the oligonucleotides does lead to differences in the inhibitory action of the oligonucleotides. This inhibition of basic fibroblast growth factor by phosphorothioate oligonucleotides may account for much of the published data on inhibition of various genes by proposed antisense oligonucleotides and needs to be taken into account when considering the mechanism of action of oligonucleotides in biological systems.
Oligonucleotides have been used extensively in the past several
years to inhibit gene expression. Compounds are most often designed as
antisense agents and, as such, have shown efficacy in cell culture and
in some animal models of disease. Efficacious oligonucleotides have
been used against viruses, including human cytomegalovirus, herpes
simplex virus, human immunodeficiency virus, and papillomavirus, and
against various cellular targets, including the oncogenes
c-myc, c-myb, and
c-abl(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) .
In addition investigators have used anti-c-myc/-myb antisense oligonucleotides to prevent restenosis in animal
models(13, 14, 15) . Several of these
compounds are progressing into human drug trials based on their
proposed antisense inhibition of viral or oncogene targets in disease.
Oligonucleotide therapeutics are targeted to human cytomegalovirus in
retinitis, papillomavirus in genital warts, human immunodeficiency
virus in AIDS, c-abl in myelogenous leukemia, and c-myc and c-myb in restenosis. At the same time, increased
numbers of unexplained, nonspecific effects have been reported
especially for the phosphorothioate (PS) ()oligonucleotides(16, 17, 18, 19, 20) .
Phosphorothioate oligonucleotides contain internucleoside linkages in
which one of the nonbridging oxygen atoms has been replaced by a sulfur
atom in order to enhance nuclease resistance.
Our own studies progressed from the initial use of oligonucleotides as anti-herpesvirus agents on Vero and MRC-5 cells(9) . The action of oligonucleotides against herpes simplex virus and human cytomegalovirus have been reported previously(3, 8, 9) , but the actual analysis of the data has been complicated by the ability of the oligonucleotides to operate on the exterior of the cell as inhibitors of viral adsorption. The binding of herpes simplex virus to the cell involves an initial attachment to cell surface heparan sulfate molecules and can be competed by the addition of heparin to the media(21) . Phosphorothioate oligonucleotides are particularly potent inhibitors of viral adsorption with effective concentrations in the nanomolar range(9, 22) . Because basic fibroblast growth factor (bFGF) binding, like herpes simplex virus adsorption, involves interaction with cell surface heparan sulfate, we investigated whether oligonucleotides would also inhibit the binding of bFGF to cells.
bFGF is a member of the heparin binding family of growth factors(23, 24) . It is a widely distributed growth factor found in all organs, solid tissues, tumors, and cultured cells examined(25) . bFGF binds to high affinity protein receptors in association with cellular matrix heparan sulfate. This association, which is competed by the presence of exogenously added heparin, is necessary for the high affinity and functional binding of bFGF. Heparin-induced dimerization of the growth factor/receptor is required for full activity of the growth factor (26) . Heparin and heparin subfragments can be used to inhibit the biological activity of FGF(27, 28) , although it may not be required for all of the FGF effects(29) . The bFGF receptors are members of the group of tyrosine kinase receptors capable of autophosphorylation and dimerization; it is this dimerization that results in an increase in receptor/ligand interaction and signal transduction(30, 31) . In this model of bFGF receptor activity, exogenously added heparin competes with cell surface heparan sulfate and inhibits bFGF activity through the disruption of the dimerization of the receptor. This is similar in concept to the disruption of ligand binding to receptors by the uncoupling of the receptor and the G protein complex for those receptors, which signal through G protein complex formation(32, 33) . FGF binding/dimerization and signaling results in a variety of cellular responses including myc, fos, collagenase, and platelet-derived growth factor receptor up-regulation, increases in intracellular pH, hydrolysis of polyphosphoinositol, and phosphorylation of cellular proteins. It induces growth in various cell types including astrocytes, glioma cells, fibroblasts, and, in synergy with hematopoietic growth factors, stem cells. In animal models it is most directly connected with angiogenesis and wound healing.
Our studies show that oligonucleotides, especially PS oligonucleotides, are able to inhibit bFGF binding to cells. These observations on bFGF and oligonucleotides, taken together, indicate that although the oligonucleotides studied to date may have therapeutic potential, their level of biologic activity may not be due to the original rational ``antisense'' design.
bFGF binding to cells is assayed by the incubation of radiolabeled bFGF with cell monolayers with subsequent wash conditions able to distinguish the low affinity heparan sulfate binding from the high affinity protein receptor-specific binding(29) . The bFGF bound to the heparan sulfate is removed with 2 M NaCl at neutral pH. This heparan sulfate binding is not saturated and is not competed away at 100-fold concentrations of unlabeled bFGF. It is however competed away by exogenous heparin or by the addition of other polyanions such as pentosan polysulfate and suramin. Phosphorothioate oligonucleotides can also act as appropriate polyanions and inhibit the low affinity heparan sulfate binding of bFGF (Fig. 1A) at submicromolar (0.6 µM) concentrations.
Figure 1:
Inhibition of
basic FGF binding to low and high affinity receptors. The binding of I-bFGF to Vero cell low (A) and high (B) affinity receptors was assayed as described under
``Experimental Procedures.'' For the competition with cold
FGF, 100 ng/ml unlabeled bFGF was added. Heparin was used at 100
µg/ml; the phosphorothioate oligonucleotide ODN-1 was used at 0.6
µM (7 µg/ml). Each value is the average of duplicate
samples of a typical experiment. All points were assayed in at least
two separate experiments.
The bFGF bound to the high affinity receptors is then removed from the cells with a subsequent low pH rinse. It is this high affinity binding that is specific for the bFGF receptor proteins. This high affinity receptor binding is effectively competed away with unlabeled bFGF (Fig. 1B). Heparan sulfate binding is necessary for the dimerization of the bFGF receptor; therefore, an inhibition of the bFGF low affinity heparan sulfate binding results in a change in the binding to the high affinity protein receptor. The binding constant has been reported to shift from 50 pM with heparan sulfate involvement to 175 pM without such involvement (37) . If the bFGF concentration is kept low, then only the high affinity receptor binding resulting from dimerization is detected; the concentration of bFGF is not sufficient for the receptor binding that occurs in the absence of dimerization. Both heparin and PS oligonucleotides inhibit high affinity binding under such conditions (Fig. 1B).
When a standard saturation curve is plotted, the difference in binding over an increasing range of bFGF concentrations is seen for both the high and low affinity receptors (Fig. 2). The binding to the high affinity receptors increases more slowly in the presence of the oligonucleotides or heparin, approaching a plateau only at higher concentrations. The concentrations are too low to saturate the binding in the presence of heparin or PS oligonucleotides. At higher bFGF concentrations the bFGF may bind in the presence of heparin, but this binding is not expected to result in signal transduction due to the inability of the receptors to dimerize. It has been shown that although cell surface heparan sulfate is not required for receptor specific binding, it does increase the affinity of basic bFGF for its receptor and facilitates dimerization and activity of FGF (37, 38
Figure 2:
Inhibition of bFGF binding by
phosphorothioate oligonucleotides and heparin: saturation curves. Low (A) and high (B) affinity-specific FGF binding were
assayed as described. For high affinity-specific binding, background
values obtained from competition with 100-fold excess of unlabeled bFGF
were subtracted before plotting. Assays contained I-bFGF
alone (
) or in the presence of 100 µg/ml heparin (
), 20
µM (78 µg/ml) phosphorothioate ODN-4 (
), or 0.6
µM (7 µg/ml) ODN-1
(
).
Because a variation in the effect of
different antisense oligonucleotides is seen when they are assayed for
efficacy in the various systems employed, we tested a variety of
different oligonucleotides to determine if this variability could be
attributed to differences in bFGF binding inhibition. A series of
competition experiments was performed in which the concentration of I-bFGF was kept constant while the dose of
oligonucleotide used in competition was varied. In this way the
relative binding affinities of the various oligonucleotides could be
compared. Representative results are shown (Fig. 3). Though
unlabeled bFGF competed for binding at only the high affinity
receptors, as expected, the oligonucleotides and heparin showed similar
competition at both the low and the high affinity binding sites.
Although the oligonucleotides varied in their ability to block bFGF
binding, we were unable to distinguish any sequence or structure that
was responsible. The concentration at which 50% of the high affinity
binding was inhibited (ID
) was calculated for several
representative oligonucleotides and in several conditions (Table 1). The PS oligonucleotides were consistently competitive
at lower concentrations than the phosphodiester or partial
phosphorothioate oligonucleotides, though the phosphodiester
oligonucleotides did have some effect at concentrations greater than 10
µM. A comparison of PS oligonucleotides of various lengths
(12-36 bases) showed no direct correlation of length to
ID
, but the exact sequence did influence bFGF binding
inhibition; two PS oligonucleotides (ODN-1 and ODN-3) of identical
length but different sequence both diminish bFGF binding, but the
ID
values differ by 10-fold. The oligonucleotides we used
that were composed of G's and T's were generally more
effective than mixed sequences, but no firm rules could be established
for predicting the relative binding inhibition of the
phosphorothioates. The oligonucleotides and heparin are both polymeric
anions, but the phosphorothioate modification of the oligonucleotide
backbone presumably causes a change in charge or secondary structure
that renders the oligonucleotides even more effective in their
inhibition of bFGF binding.
Figure 3:
Competitive inhibition of basic FGF
binding by phosphorothioate oligonucleotides and heparin: competitive
inhibition curves. Low (A) and high (B)
affinity-specific I-bFGF binding were assayed as
described. For high affinity-specific binding, background values
obtained from competition with 100-fold excess of unlabeled bFGF and
the highest concentration of each competitor were subtracted before
plotting. Assays contained 2.5 ng/ml
I-bFGF and the
indicated concentrations of unlabeled bFGF (
), heparin
(
), phosphorothioate ODN-1 (
), phosphorothioate ODN-3
(
), or phosphodiester ODN-2 (
).
In an effort to more closely mimic the
various antisense experiments, we also varied the time of addition and
incubation of the oligonucleotide. In the standard protocol, the
oligonucleotides were mixed with the bFGF prior to the addition to the
cells. If the oligonucleotide was preincubated with the cells, instead
of with the growth factor, the ID was only slightly higher (Table 1). The ID
was significantly higher if the
oligonucleotide was removed prior to the addition of the bFGF but was
still less than 5 µM. Although there is some variation in
the extent of bFGF inhibition depending on whether the growth factor or
the cells are preincubated with oligonucleotide, there is a significant
level of activity against bFGF under the conditions of most antisense
assays. It should be kept in mind that many cell lines synthesize and
secrete bFGF and that endogenously produced bFGF may have significant
effect on cell growth and gene expression even in the absence of
exogenously added growth factor. Under such conditions there is no
possibility of separating the presence of oligonucleotide and bFGF by
washing, and the full effect of the binding inhibition may be seen.
Inhibition of bFGF binding by phosphorothioate oligonucleotides may account for some of the experimental results attributed in the literature to antisense mechanisms. Antisense experiments often report results using concentrations of PS oligonucleotides in the range of 0.1-50 µM, which we would expect to influence bFGF binding. The 10-fold variation in the activity of the PS oligonucleotides is in the same range as the specific/nonspecific activity of antisense and control oligonucleotides reported for gene disruption experiments. This would be especially true when the assays are for those activities known to be influenced by bFGF, including myc and fos regulation, platelet-derived growth factor and bFGF receptor regulation, hematopoiesis, and cell growth, especially that of vascular smooth muscle cells. Other cellular factors known to interact with polymers include granulocyte macrophage-stimulating factor, interleukin-3, pleitrophin, and platelet factor 4(39) , as well the entire family of heparin binding growth factors. Any or all of these (and other factors) may be affected in their interaction with cells by polyanions such as PS oligonucleotides. Recently, it has been reported that at least some PS oligonucleotides exert their effect by inhibiting the extracellular binding of interferon gamma (40) or by inhibiting the autophosphorylation of the bcr-abl tyrosine kinase(41) . Interpretation of antisense data must be made in light of this effect of polyanions on extracellular binding. In particular, the inhibition of vascular smooth muscle cell growth by antisense oligonucleotides has progressed rapidly into the effective use of these molecules in animal models of restenosis. Although the proposed mechanism of action of these compounds has been as antisense inhibitors of myc or myb, the simpler explanation is that they are acting, like heparin, as growth factor inhibitors. Heparin and its low molecular weight subfragments have been shown to be potent vascular smooth muscle cell growth inhibitors in vitro(42) , are active in animal models of restenosis, and have been used in human clinical trials (43, 44, 45) . The interpretation of the results of these cell culture and animal trials of antisense therapeutics for restenosis and other indications should be made in light of the similarity in action between polyanions, heparin, and PS oligonucleotides on the action of growth factors.