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

Guvakova, Marina A.; Yakubov, L. A.; Vlodavsky, Israël; Tonkinson, John L.; Stein, C. A.

doi:10.1074/jbc.270.6.2620

Cited by 310 publications

(179 citation statements)

References 32 publications

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“…Substitution of the phosphodiester backbone of native DNA with phosphorothioate moieties markedly augments resistance to nuclease digestion. 35,36 Phosphorothioates, however, have also been found to bind with high affinity to a large number of proteins 37,38 presumably the result of alterations in the presentation of negative charges. Without appropriate controls, sequence-independent effects caused by phosphorothioate chemistry can be mistakenly attributed to a true antisense effect.…”

Section: Discussionmentioning

confidence: 99%

Vascular Smooth Muscle Cell Proliferation and Regrowth after Mechanical Injury in Vitro Are. Egr-1/NGFI-A-Dependent

Santiago

Atkins

Khachigian

1999

The American Journal of Pathology

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Smooth muscle cell (SMC) proliferation is a key event in renarrowing of blood vessels after balloon angioplasty. Mechanical injury imparted to the arterial wall in experimental models induces the expression of the immediate-early gene , egr-1. Egr-1 binds to and activates expression from the proximal promoters of multiple genes whose products can , in turn , influence the vascular response to injury. Here , we used antisense strategies in vitro to inhibit rat vascular SMC proliferation by directly targeting Egr-1. A series of phosphorothioate antisense oligonucleotides of 15 base length and complementary to various theoretically accessible regions within Egr-1 mRNA were synthesized and assessed for their ability to selectively inhibit SMC proliferation in an Egr-1-dependent manner. Western blot analysis revealed that two oligonucleotides , AS2 and E11 , inhibited Egr-1 synthesis in cells exposed to serum without affecting levels of the zinc finger protein Sp1. AS2 and E11 inhibited seruminducible [ 3 H]thymidine incorporation into DNA, as well as serum stimulation of total cell numbers. Sizematched phosphorothioate oligonucleotides with random , scrambled , sense or mismatch sequences failed to inhibit. Antisense Egr-1 inhibition was nontoxic and reversible. These oligonucleotides also inhibited SMC regrowth after mechanical injury in vitro. Egr-1 thus plays a key regulatory role in SMC proliferation and repair following injury. (Am J Pathol 1999, 155:897-905)

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Section: Discussionmentioning

confidence: 99%

Vascular Smooth Muscle Cell Proliferation and Regrowth after Mechanical Injury in Vitro Are. Egr-1/NGFI-A-Dependent

Santiago

Atkins

Khachigian

1999

The American Journal of Pathology

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“…3' exonucleases are thought to be more active than 5' exonucleases, so an amine group was added at the 3'-end, this modi®cation having been previously shown to increase the stability of classical phosphodiester antisense ODNs to levels similar to those for phosphothioate ODNs and the stability of the ODNstarget complex. In addition, these 3'-modi®ed ODNs are not toxic and do not compete with FGF1 and 2 for their binding sites (heparin-like activity), in contrast to phosphorothioate ODNs (Fennwald and Rando, 1995;Gukova et al, 1995). We used lipofectin (GIBCO/BRL, Cergy Pontoise, France) as a cationic lipid to deliver the ODNs, because this method results in high levels of uptake and stability of phosphodiester ODNs in the intracellular compartment without a ecting their ®nal nuclear location (Clarenc et al, 1993;Lezoulac'h et al, 1995) after endocytosis and release from the endocytic compartment.…”

Section: Oligonucleotides and Oligonucleotide Treatment Of Cellsmentioning

confidence: 99%

Both FGF1 and Bcl-x synthesis are necessary for the reduction of apoptosis in retinal pigmented epithelial cells by FGF2: role of the extracellular signal-regulated kinase 2

Bryckaert

Guillonneau²,

Hecquet³

et al. 1999

Oncogene

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Retinal pigmented epithelial (RPE) cells are of central importance in the maintenance of neural retinal function. Changes in the RPE cells associated with repair activities have been described as metaplasia, while RPE cell apoptosis is responsible for the development of a variety of retinal degenerations. We investigated the regulation of the anti-apoptotic properties of the ®broblast growth factors (FGF) 2 in serum-free cultures of RPE cells. In the absence of serum, con¯uent stationary RPE cells died by apoptosis via a caspase 3-dependent pathway. The addition of FGF2 greatly reduced apoptosis over a 7-day culture period. We demonstrated the involvement of an autocrine loop involving endogenous FGF1 in the mechanisms that govern FGF2-induced resistance to apoptosis by showing: (1) higher levels of apoptosis in cells treated with antisense FGF1 oligonucleotide or after neutralization of excreted FGF1; (2) the long-term activation of FGFR1 and of ERK2, (3) the inhibition of FGFR1 and ERK2 activation and an increase in apoptosis if excreted FGF1 was neutralized. FGF2 also increased the de novo synthesis and the production of Bcl-xl before the onset of apoptosis. Both inhibition of ERK2 activation, which decreased Bcl-xl synthesis, and downregulation of Bcl-x by antisense oligonucleotide treatment inhibited the survival-promoting activity of FGF2. Thus, FGF2-induced cell survival is a progressive adaptive phenomenon involving ERK2 activation by excreted FGF1 and ERK2-dependent Bcl-x production.

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“…However, preclinical and clinical studies showed that PS ASOs may have nonspecific effects, either due to nonsequence specific binding or activation of nonspecific mechanisms. In fact, it has been demonstrated that PS ASOs are able to bind to heparin-binding proteins, such as bFGF, PDGF, VEGF and their receptors (Fennewald and Rando, 1995;Guvakova et al, 1995). Under certain circumstances, PS ASOs are able to activate SP1 transcription factor and their degradation products can affect cell proliferation and differentiation (Perez et al, 1994).…”

Section: Antisense Therapy: Rationale and Limitationsmentioning

confidence: 99%

The future of antisense therapy: combination with anticancer treatments

Biroccio¹,

Leonetti²,

Zupi³

2003

Oncogene

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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

Cited by 310 publications

References 32 publications

Vascular Smooth Muscle Cell Proliferation and Regrowth after Mechanical Injury in Vitro Are. Egr-1/NGFI-A-Dependent

Vascular Smooth Muscle Cell Proliferation and Regrowth after Mechanical Injury in Vitro Are. Egr-1/NGFI-A-Dependent

Both FGF1 and Bcl-x synthesis are necessary for the reduction of apoptosis in retinal pigmented epithelial cells by FGF2: role of the extracellular signal-regulated kinase 2

The future of antisense therapy: combination with anticancer treatments

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