Dr. Christian Stein

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Dr. Christian Stein forscht an der Anwendung von Oligonukleotiden als therapeutische Wirkstoffe, insbesondere zu deren Wechselwirkung mit Proteinen und Zellen sowie zur Verbesserung ihrer Aufnahme in Zielzellen. Seine Expertise umfasst die molekulare Charakterisierung von Antisense-Oligonukleotiden und deren Mechanismen bei der Behandlung von Virusinfektionen und Krebserkrankungen. Daneben leitet er Projekte zur Anwendung von Virtual Reality in der Therapie neurologischer Erkrankungen.

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Dr. Christian Stein

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• EntangleVR: Quantum Architect

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  Förderer: Investitionsbank Berlin (IBB) Zeitraum: 05/2026 - 04/2027 Projektleitung: Dr. Christian Stein

• EXGAVINE: Bewegungsspiele in der Virtuellen Realität als Therapieform neurologischer Erkrankungen

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  Förderer: Bundesministerium für Forschung, Technologie und Raumfahrt Zeitraum: 10/2018 - 03/2022 Projektleitung: Dr. Christian Stein, Kathleen Waak M.A.

• Viral Theatres

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  Förderer: Volkswagen Stiftung Zeitraum: 03/2021 - 03/2023 Projektleitung: Dr. Christian Stein, Dr. Ramona Mosse

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• Antisense Oligonucleotides as Therapeutic Agents—Is the Bullet Really Magical?

  1993

  Science · 1317 Zitationen · DOI

  Because of the specificity of Watson-Crick base pairing, attempts are now being made to use oligodeoxynucleotides (oligos) in the therapy of human disease. However, for a successful outcome, the oligo must meet at least six criteria: (i) the oligos can be synthesized easily and in bulk; (ii) the oligos must be stable in vivo; (iii) the oligos must be able to enter the target cell; (iv) the oligos must be retained by the target cell; (v) the oligos must be able to interact with their cellular targets; and (vi) the oligos should not interact in a non-sequence-specific manner with other macromolecules. Phosphorothioate oligos are examples of oligos that are being considered for clinical therapeutic trials and meet some, but not all, of these criteria. The potential use of phosphorothioate oligos as inhibitors of viral replication is highlighted.

• 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

  1995

  Journal of Biological Chemistry · 330 Zitationen · DOI

  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 125I-bFGF, but also of 125I-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 125I-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. 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 125I-bFGF, but also of 125I-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 125I-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. INTRODUCTIONPhosphorothioate 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 β1, α, δ, and ε 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 ( 1The abbreviations used are: bFGFbasic fibroblast growth factoraFGFacidic fibroblast growth factorFGF-4fibroblast growth factor-4PDGFplatelet-derived growth factorVEGFvascular endothelial growth factorEGFepidermal growth factorSdC2828-mer phosphorothioate homopolymer of cytidineECMextracellular matrixBSAbovine serum albuminDMEMDulbecco's modified Eagle's mediumDPBSDulbecco's phosphate-buffered salinePAGEpolyacrylamide gel electrophoresis. )(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 125I-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 125I-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 125I-labeled bFGF to its cell surface receptors may be sequence-selective.MATERIALS AND METHODSReagents125I-Bolton-Hunter-labeled bFGF (human, recombinant), 125I-Bolton-Hunter-labeled PDGF, and 125I-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, Mr = 14,000, was obtained from Hepar Industries (Franklin, OH).Synthesis of OligodeoxynucleotidesPhosphodiester 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′-GSTSGCCGGGGTCTCSCSGSGSGSC-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-32P-OdT18 or -32P-OdT12 (RClNH32P-OdT18 or RClNH32P-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 32P of [γ-32P]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 32P by reaction with triphenylphosphine/dipyridyl disulfide. The final product was stored at −70°C.Modification of bFGF and Other Heparin-binding Growth Factors by ClRNH32P-OdT18 or ClRNH32P-OdT12This 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 ClRNH32P-OdT18 or ClRNH32P-OdT12. ( 2ClRNH32P-OdT12 is dodecathymidylate phosphodiester oligodeoxynucleotide derivative with an alkylator moiety (Fig. ZI) coupled to the 5′ radioactive phosphate through a phosphoroamide some a (e.g. phosphorothioate of the binding of the to bFGF was also as in the 1 of a containing 0.1 M 2% and was and was The were and to were The was and were by NIH 3T3 and the human prostate were obtained from NIH 3T3 were and in modified Eagle's containing serum and DU-145 were and in containing serum sodium, and For binding were at 1 well in or 1 well in and were used for experiments 1 to of endothelial were from as et al., were in of with and at in purified bFGF was other the phase of cell growth et al., et al., of 125I-bFGF to of were used for all were with phosphate-buffered and incubated in with at for 1 h the of binding For the binding were incubated at in containing 125I-bFGF, and the concentrations of the of the the was and 125I-bFGF was by one of bound bFGF, the were with and were with 1 M in other were with and with M in pH to remove 125I-bFGF bound to the low affinity binding the were with in 0.1 M sodium pH The binding to high affinity sites 1987). binding was in the of of The of in the and was determined in a 1, of with endothelial were from to with M sodium phosphate and and at an of were as that was in the growth and the were for addition of bFGF et al., et al., 1987). The subendothelial was by the cell with containing and by in The of cellular and to the of the et al., et al., of was incubated (3 with 125I-bFGF in containing bFGF was and the was incubated with concentrations of either or the oligodeoxynucleotide at for The were and in a to the of The was incubated (3 with 1 and the was in a The of 125I-bFGF was from the et al., on the of of the bound 125I-bFGF was with release could be by the to its release was from the to the specifically was performed to five similar 3T3 were at a concentration of well in were at the was and were with containing The was with with bFGF, and the appropriate concentration of phosphorothioate oligodeoxynucleotides and were for h with The of was on glass using a liquid of bFGF by the bFGF and the were incubated in 0.1 M for 1 and the reaction were to in a a was observed on the to the product of modification of the protein by the of the as oligodeoxynucleotide of not containing an moiety not with bFGF, in this gel were on the No in the modification of bFGF by could be in different including 0.1 M in M 0.1 M or in examined the concentration of the modification of bFGF by ClRNH32P-OdT18 (Fig. These are also in the concentration of oligodeoxynucleotide is as a of gel as determined by The of bFGF with the probe oligodeoxynucleotide the of the in is not and the fact that it is that the concentration of bFGF modification be by the However, at low concentrations of the concentration of the modification is and the to an of concentrations the of modification is also and the at = These that are at least binding with different for on the surface of the bFGF of bFGF by the oligodeoxynucleotide bFGF (3 μg/ml) was incubated in 0.1 M with ClRNH32P-OdT18 at the concentration for at The containing the was to polyacrylamide gel electrophoresis. The concentration of ClRNH32P-OdT18 was as 2, and ClRNH32P-OdT18 the of the gel and is not concentration of modification of bFGF by concentrations of The gel in 1 were by is a of oligodeoxynucleotide concentration The was of the in The of the in The of the with the were to this binding we used a gel to the binding of ClRNH32P-OdT18 to gel is shown in 3, and are the other bFGF a at the appropriate after The the of the gel at the for a bFGF of bFGF by the oligodeoxynucleotide ClRNH32P-OdT18 with gel performed by bFGF (3 μg/ml) was incubated in 0.1 M with ClRNH32P-OdT18 as in the The concentration of ClRNH32P-OdT18 was 1, and probe is at the of the are which at the of bFGF = The the of the gel at the for a bFGF of the for a of Oligodeoxynucleotide Binding to have examined the ability of other polyanions, including the phosphodiester oligodeoxynucleotides and to with the probe oligodeoxynucleotide for binding to of these polyanions are of binding to bFGF of the probe oligodeoxynucleotide of probe = Furthermore, a (Stein, to be to bind to heparin-binding growth factors and to block their binding to cell surface also is a of the binding of to ( and in examined the ability of a phosphorothioate homopolymer of thymidine, to binding of the oligodeoxynucleotide to We have used this method to the of for of oligodeoxynucleotide binding to The of may be from 1 from and = the we found (Fig. 4, and that the of = However, the of is by the in a of is the of the low affinity and the high affinity of the oligodeoxynucleotide binding to bFGF are and are in the of we have used an for the of The of for as determined by the is by for binding of to was used as a of binding to bFGF as in the bFGF modified by the oligodeoxynucleotide. The concentration of was and of the of by the The from the was by is a = of the of the concentration The was of to Other Heparin-binding Growth was performed in a gel and is shown in can be not only bFGF, but acidic FGF4 and VEGF can bind to either of the probe oligodeoxynucleotides or concentration of = In to the with these growth factors, is at the of of EGF, which to only be that the of aFGF, and VEGF all has been demonstrated et al., that albumin can bind phosphodiester oligodeoxynucleotides with low The modification of the albumin in these by can be at the in of growth factors by ClRNH32P-OdT18 or The growth factor was incubated with either or 1 and EGF (3 and bFGF (10 and aFGF (10 and FGF4 (10 and VEGF (25 of the FGF at the 1 on the VEGF at the No binding of either probe oligodeoxynucleotide to EGF was observed. In bFGF, aFGF, and VEGF all Other in and protein The by the modification of which is to the reagent by the of Growth Binding to by to oligodeoxynucleotides can interact with growth factors and can their binding to cell surface we examined the binding of basic FGF to 3T3 fibroblasts in the of different phosphodiester and phosphorothioate homopolymers of cytidine and thymidine and were for their ability to 125I-labeled bFGF binding (Fig. a of oligodeoxynucleotide concentrations and we observed inhibition of bFGF binding by phosphodiester However, phosphorothioate homopolymers of thymidine as well as cytidine of of phosphorothioate and phosphodiester homopolymers of cytidine and thymidine on binding of 125I-bFGF to NIH 3T3 cells. were in at For were incubated at in containing 125I-bFGF, and the concentrations of was and were with phosphate-buffered and with 1 M was determined by are the of by of the binding assay, we demonstrated that the inhibition of bFGF binding in with the oligodeoxynucleotide chain length for homopolymers of cytidine and the the oligodeoxynucleotide or the its at concentration (Fig. We also examined the ability of phosphorothioate oligodeoxynucleotides to the binding of heparin-binding growth factor to cells. In a binding assay using 125I-labeled PDGF, we demonstrated that phosphorothioate homopolymers of cytidine of different chain length binding to the inhibition is chain with and least and most (Fig. the other these phosphorothioate oligodeoxynucleotides not binding of EGF to cell surface receptors of 3T3 (Fig. length dependent effects of phosphorothioate homopolymers of cytidine and thymidine on binding of 125I-bFGF to NIH 3T3 cells. were in at For were incubated at in containing 125I-bFGF, and the concentrations of was and were with phosphate-buffered and with 1 M was determined by are the of of phosphorothioate homopolymers of cytidine on binding of 125I-PDGF and 125I-EGF to NIH 3T3 cells. were in at For were incubated at in containing or 0.1 125I-EGF and the concentrations of the was and were with phosphate-buffered and with 1 M was determined by is the of which by of bFGF Binding to and by Oligodeoxynucleotide bFGF binding to low and high affinity we used method of 18-mer phosphorothioate oligodeoxynucleotides the binding of bFGF to the low affinity receptors of fibroblasts with similar (Fig. However, in oligodeoxynucleotide were found the ability to high affinity binding of bFGF was determined (Fig. In these the concentrations of oligodeoxynucleotides were (Fig. the oligodeoxynucleotide concentration the high affinity binding of bFGF was by in the of both the antisense c-myb rat and mouse 1 and In binding was only by by the scrambled phosphorothioate oligodeoxynucleotide (No. and by only in the of chimeric phosphorothioate/diester oligodeoxynucleotide (No. Furthermore, we to of the sense oligodeoxynucleotide (No. 4) on high affinity binding at the concentrations were high and low affinity binding of bFGF to the prostate cancer cell DU-145 were (Fig. and of 18-mer phosphorothioate oligodeoxynucleotides on binding of to low and high affinity receptors on cells. were in at For binding were incubated at in containing 125I-bFGF, and the concentrations of was and bFGF bound to low and high affinity receptors was determined as and are the of Oligodeoxynucleotide are and oligodeoxynucleotide oligodeoxynucleotide oligodeoxynucleotide 3; oligodeoxynucleotide oligodeoxynucleotide of 18-mer oligodeoxynucleotides on binding of 125I-bFGF to low and high affinity receptors on prostate cancer cells. were in at For were incubated at in containing 125I-bFGF, and the concentrations of was and bFGF bound to low and high affinity receptors was determined as and are the of Oligodeoxynucleotide are and oligodeoxynucleotide oligodeoxynucleotide oligodeoxynucleotide 3; oligodeoxynucleotide oligodeoxynucleotide are of of bFGF by Phosphorothioate on the of bFGF with the subendothelial have shown that bFGF to in the extracellular matrix and can be by and et al., In the was release of bFGF by a phosphodiester homopolymer containing to a concentration of In of extracellular matrix to phosphorothioate homopolymers of thymidine and cytidine in release of release was dependent on oligodeoxynucleotide chain for example, bound bFGF, whereas of the bound bFGF (Fig. was as as release at but with release at Phosphorothioate of cytidine were the thymidine congeners of the length release at for release of of the extracellular bFGF was obtained in the of a concentration of both and of bFGF by homopolymer phosphorothioate oligodeoxynucleotides of different chain of were incubated (3 with 125I-bFGF The was and incubated (3 with concentrations of and is as the of 125I-bFGF of of 125I-bFGF in the of oligodeoxynucleotides not of the is the of and the standard not other we examined the ability of 18-heteromer oligodeoxynucleotides 1, 2, and 4) to release extracellular bFGF was by of these a concentration of 1 the of bFGF was for the antisense c-myb mouse compound and for the antisense and sense rat c-myb a similar the release for and was and of bFGF-induced by Phosphorothioate used bFGF as a of fibroblast We the effects of phosphorothioate oligodeoxynucleotides on the ability of the to These oligodeoxynucleotides phosphorothioate homopolymers of cytidine and and oligodeoxynucleotides 1, 2, and as The addition of compounds 1 and 2, as well as in a The most effects were observed a of concentrations In the sense was to the extent (Fig. In with as cell and were of phosphorothioate oligodeoxynucleotides on by NIH 3T3 cells. were for h in containing bFGF only or phosphorothioate oligodeoxynucleotides, as was a are of fibroblast growth factors of a of at least effects are through a of high affinity receptors = but also interact with affinity receptors = et al., Despite the of a both aFGF and bFGF have also been in the extracellular matrix by and 1989) and endothelial et al., 1987). the of bFGF in of the rat et al., et al., 1988), and human et al., that may as a for bFGF et al., that bFGF specifically to and in the and cell as by its by heparin, or but not by or et al., et al., 1989). These may bFGF from can also be by the = et al., release from the the growth may at is to suggest that may to the et al., By a gel mobility shift assay, et have demonstrated that aFGF can with phosphodiester In this we have demonstrated that both phosphorothioate and phosphodiester oligodeoxynucleotides are of binding to In the of phosphorothioate oligodeoxynucleotides this binding may 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 the bFGF-induced in in 3T3 ability of oligodeoxynucleotides to bind to growth factors is not to These compounds can also interact with aFGF, VEGF, and not with the basis of these data, it is not to that oligodeoxynucleotides may be to interact with heparin-binding growth factors. of oligodeoxynucleotides is of that of other polyanions, including suramin (Stein, et al., et al., et al., 1991) and pentosan polysulfate (Wellstein et al., et al., is a and a to bFGF and VEGF, PDGF, and to other proteins as only some of which are binding (Stein et al., least in to these suramin has and effects and is in cancer (Stein et al., et al., The in the of phosphorothioate oligodeoxynucleotides to that of suramin that the may also have sequence-selective and is in the addition to growth factors, are other proteins to which phosphorothioate oligodeoxynucleotides, suramin and pentosan polysulfate, can the studied of these is rsCD4 (Yakubov et al., In this binding sites have been to both the and of the In the bFGF are basic that are on its surface et al., the basis of a et al., et al., it has been that the of a bound be by the of in addition to the chain of binding also to and the chain of as well as the of and may be this was in demonstrated that for the binding of to bFGF et al., it is that phosphorothioate oligodeoxynucleotides also bind to bFGF at or the heparin-binding However, the and heparin-binding sites of bFGF appear to be et al., as shown by the fact that that the binding of bFGF to its not block it has been that as suramin phosphorothioate oligodeoxynucleotides as to binding to bFGF, either to the binding similar to and other et al., a in the bFGF et al., to our the for the binding of some of the to bFGF may be as low as Furthermore, some in a can block the binding of bFGF to high affinity receptors with an = 1 et al., However, of this by with phosphorothioate oligodeoxynucleotides of their to is that the ability of phosphorothioate oligodeoxynucleotides to block the binding of 125I-bFGF to cell surface receptors is at least has to experiments in which phosphorothioate oligodeoxynucleotides are to complementary on mRNA. In some experiments et al., it is that the antisense and the may ability to block the binding of heparin-binding growth factors to their cell surface receptors. determined to be a direct of Watson-Crick base pair may be to the non-sequence-specific effects of phosphorothioate oligodeoxynucleotides may at similar concentrations as the the may not be suggest that the the and the its the it is that observed effects have a non-sequence-specific is by our on the effects of phosphorothioate oligodeoxynucleotides on in 3T3 it be to that antisense oligodeoxynucleotide not interact the concentrations with the protein product of the (Stein and the we also that phosphorothioate homopolymers of thymidine and cytidine are of has been demonstrated that containing as as of bFGF of release of bFGF was by a et al., was or release of bFGF by and of the other was by of heparin, but was not by containing et al., In the for the phosphorothioate release of bFGF and for inhibition of were ( and suggest that the oligodeoxynucleotides their effects by of their the effects of are to a of the bFGF and In this phosphorothioate oligodeoxynucleotides to the of and may release bFGF from its in phosphorothioate oligodeoxynucleotides may bFGF that is from and cell and its in and These are also INTRODUCTIONPhosphorothioate 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 β1, α, δ, and ε 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 ( 1The abbreviations used are: bFGFbasic fibroblast growth factoraFGFacidic fibroblast growth factorFGF-4fibroblast growth factor-4PDGFplatelet-derived growth factorVEGFvascular endothelial growth factorEGFepidermal growth factorSdC2828-mer phosphorothioate homopolymer of cytidineECMextracellular matrixBSAbovine serum albuminDMEMDulbecco's modified Eagle's mediumDPBSDulbecco's phosphate-buffered salinePAGEpolyacrylamide gel electrophoresis. )(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 125I-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 125I-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 125I-labeled bFGF to its cell surface receptors may be

• Differentiation of immortal cells inhibits telomerase activity.

  1995

  Proceedings of the National Academy of Sciences · 270 Zitationen · DOI

  Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, terminal differentiation was induced in leukemic cell lines by diverse agents. A pronounced downregulation of telomerase activity was produced as a consequence of the differentiated status. The differentiation-inducing agents did not directly inhibit telomerase activity, suggesting that the inhibition of telomerase activity is in response to induction of differentiation. The loss of telomerase activity was not due to the production of an inhibitor, since extracts from differentiated cells did not cause inhibition of telomerase activity. By using additional cell lineages including epithelial and embryonal stem cells, down-regulation of telomerase activity was found to be a general response to the induction of differentiation. These findings provide the first direct link between telomerase activity and terminal differentiation and may provide a model to study regulation of telomerase activity.

• Hochschule Düsseldorf

  EXGAVINE: Bewegungsspiele in der Virtuellen Realität als Therapieform neurologischer Erkrankungen

  other

• RetroBrain R&D UG

  EXGAVINE: Bewegungsspiele in der Virtuellen Realität als Therapieform neurologischer Erkrankungen

  company

• Universität Hamburg

  EXGAVINE: Bewegungsspiele in der Virtuellen Realität als Therapieform neurologischer Erkrankungen

  university