Transcription Factor Decoy Approach Research Articles

γ-Hydroxymethyl PNAs: Synthesis, interaction with DNA and inhibition of protein/DNA interactions

The ability of PNA to interact with DNA double stranded has been recently investigated. In a decoy approach these interactions are of great importance as may lead to inhibition of interactions of DNA sequences to specific transcription factors and may be employed as a strategy for the inhibition of gene transcription alternative to the antisense strategy (targeting transcription factors mRNAs) and the transcription factor decoy approach (targeting transcription factors). We explored the ability of PNA and PNAs with modified monomers to bind to DNA and to interfere in the formation of DNA/transcription factor complex. We report a procedure for the synthesis of Fmoc–γ-hydroxymetyl PNA, the synthesis and CD analysis of PNA oligomers containing the modified monomer in different positions and EMSA assays to test the: (a) binding to double stranded DNA and (b) inhibition of DNA–protein interactions.

Transcription Inhibition Using Oligonucleotide-Modified Gold Nanoparticles

The capture of T7 RNA polymerase using double-stranded promoter DNA on the surface of gold nanoparticles has been demonstrated. The competitive binding and inhibition of T7 RNA polymerase due to specific interactions on the nanoparticle surface represents a transcription factor decoy approach in a model system. The efficiency of inhibition was determined for various nanoparticle sizes, surface coverage, and linker length for double-stranded promoter DNA on gold nanoparticles. The experiments provide a basis for determining the accessibility of binding sites on nanoparticle surfaces for applications involving cell targeting or the use of nanoparticles as binding agents in solution.

“In vivo” local transfection of a cis element decoy mimicking an estrogen receptor alpha gene promoter region induces apoptosis of osteoclasts following application of orthodontic forces to rat teeth

We have recently reported that it is possible to induce apoptosis of human primary osteoclasts (OCs) following a transcription factor decoy (TFD) approach specifically designed for increasing the expression of the human estrogen receptor alpha (ERα) gene [1]. ERα expression was increased by interfering with the activity of a negative transcription factor and by removing it with a powerful TFD oligonucleotide (ODN, named RA4-3′) mimicking a region of the distal promoter C of ERα gene. The RA4-3′ mediated induction of OCs apoptosis was also associated with increase of the levels of caspase 3 and Fas receptor [1]. From the theoretical point of view, our published study confirmed a role of ERα on osteoclastic cell function. From the practical point of view, we proposed this TFD approach for the therapy of diseases caused by OCs activation. In order to demonstrate the efficacy of our strategy “in vivo”, we employed an experimental model system constituted by rats subjected to orthodontic forces inducing alveolar bone resorption during orthodontic tooth movement [2]. This system is very useful for

Therapeutic Relevance of Targeting Nuclear Factor kappaB with Transcription Factor Decoy Molecules

Transcription factors belonging to the NF-kappaB superfamily are involved in several human pathologies, as well as in biological processes facilitating the onset of diseases. Among well established functions of NF-kappa B factors is the promotion of osteoclast differentiation in osteopenic diseases, the enhancement of inflammatory processes in cystic fibrosis, the involvement in asthma and pulmonary diseases associated to dust or smoke. In consideration of these roles of NF-kappaB, targeting of these transcription factors could be of great interest. A very promising approach to alter NFkappaB regulated gene expression is the transcription factor decoy (TFD) strategy. The TFD approach employs double stranded oligodeoxyribonucleic acids mimicking the NF-kappaB binding sites or bioactive analogues; therefore, treatment of cells with these decoy molecules causes a binding of NF-kappaB factors to them and not to the target promoter sequences, leading to a strong inhibition of NF-kappaB dependent biological functions. Decoy molecules targeting NFkappaB factors were found in vitro inducers of apoptosis, and strong inhibitors of cell cycle progression, and TNF-alpha induced gene expression in several experimental cell systems. In vivo, decoy molecules targeting NF-kappaB factors were employed for prolonged survival of renal allografts, regression of atopic dermatitis, and cardiac protective effects. Therefore, the design and development of novel molecules able to target NF-kappaB, including modified oligonucleotides, LNA (locked nucleic acids) and peptide nucleic acids (PNA) based transcription factors decoys are of great interest. In this respect, TFD activity of double stranded PNA-DNA-PNA chimeras has been demonstrated to be useful to inhibit NF-kappaB dependent functions.

In vivo antitumor efficacy of STAT3 blockade using a transcription factor decoy approach: implications for cancer therapy

The development of more effective prevention and treatment strategies for solid tumors is limited by an incomplete understanding of the critical growth pathways that are activated in carcinogenesis. Signal transducers and activators of transcription (STAT) proteins have been linked to transformation and tumor progression. Several approaches have been used to block STAT3 in cancer cells resulting in reduced proliferation and apoptosis. We tested the hypothesis that blocking STAT3 activation using a transcription factor decoy approach would decrease tumor growth and STAT3 target gene expression in vivo. In a xenograft model of squamous cell carcinoma of the head and neck (SCCHN), daily administration of the STAT3 decoy (25 microg) resulted in decreased tumor volumes, abrogation of STAT3 activation, and decreased expression of STAT3 target genes (VEGF, Bcl-xL, and cyclin D1) compared to treatment with a mutant control decoy. Blockade of STAT3 with the STAT3 decoy also induced apoptosis and decreased proliferation, an effect that was augmented when the STAT3 decoy was combined with cisplatin, both in vitro and in vivo. These results suggest that a transcription factor decoy approach may be used to target STAT3 in cancers that demonstrate increased STAT3 activation including SCCHN.

Peptide Nucleic Acids (PNA)-DNA Chimeras Targeting Transcription Factors as a Tool to Modify Gene Expression

Peptide nucleic acids (PNAs)-DNA chimeras have been recently described as DNA mimics constituted of a part of PNA and of a part of DNA. We have demonstrated that double stranded molecules based on PNA-DNA chimeras bind to transcription factors in a sequence-dependent manner. Accordingly, these molecules can be used for transcription factor decoy (TFD) pharmacotherapy. Effects of double stranded PNA-DNA chimeras targeting NF-kappaB and Sp1 were determined on in vitro cultured human cells and were found to be comparable to those observed using double-stranded DNA decoys. The TFD molecules based on PNA-DNA chimeras can be further engineered by addition of short peptides facilitating cell penetration and nuclear localization. Therefore, these engineered molecules could be of great interest for in vivo experiments for non-viral gene therapy of a variety of diseases, including neoplastic and viral diseases, for which the TFD approach has been already demonstrated as a very useful strategy.

Biological activity and delivery of peptide nucleic acids (PNA)-DNA chimeras for transcription factor decoy (TFD) pharmacotherapy.

Peptide nucleic acids (PNAs) are recently described DNA mimics, in which the sugar-phosphate backbone is replaced by N-(2-aminoethyl)glycine units. These molecules efficiently hybridize with complementary DNA, forming Watson-Crick double helices. In addition, the interest of PNAs and PNA-based analogs is related to the fact that they are resistant to DNases and proteinases. While applications of PNAs as antisense and antigéne molecules in non-viral gene therapy are well documented, their effects as potential transcription factor decoy (TFD) molecules is not demonstrated. PNA/PNA and PNA/DNA duplex are not suitable for TFD. In fact, PNA/PNA duplex does not recognize transcription factors, while, in the case of PNA/DNA hybrids containing nuclear factor binding sites, the interaction with transcription factors is unstable. By sharp contrast, double stranded molecules based on PNA-DNA chimeras exhibit strong TFD activity, display enzymatic stability in serum and cellular extracts and can be delivered to target cells after complexation with liposomes and microspheres. The TFD molecules based on PNA-DNA chimeras can be further engineered by addition of short peptides facilitating cell penetration and nuclear localization. Therefore, these engineered molecules could be of great interest for in vivo experiments on non-viral gene therapy of a variety of diseases, including neoplastic and viral diseases, for which the TFD approach has been already demonstrated as a very useful strategy.

CO 2 response for expression of ribulose-1,5-bisphosphate carboxylase/oxygenase genes is inhibited by AT-rich decoy in the cyanobacterium

The CO 2-regulatory function of the AT-rich element in the promoter for ribulose-1,5-bisphosphate carboxylase/oxygenase ( rbc) genes in the cyanobacterium Synechococcus sp. PCC7002 was analyzed using the transcription factor decoy approach. Double-stranded phosphorothioate AT-rich oligonucleotides with high affinity for a sequence-specific DNA-binding protein were successfully introduced into cyanobacterial cells in culture without any transfection reagent. The AT-rich decoy oligonucleotides interfered with CO 2 regulation of rbc expression by blocking the binding of the sequence-specific DNA-binding protein, indicating that the AT-rich element plays a critical role in CO 2 regulation for rbc genes. The decoy oligonucleotide approach to cyanobacteria provides a simple and excellent tool for investigating transcriptional regulation in vivo.

Targeted inhibition of Stat3 with a decoy oligonucleotide abrogates head and neck cancer cell growth.

The transcription factor signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a variety of cancers including squamous cell carcinoma of the head and neck (SCCHN). Previous investigations have demonstrated that activated Stat3 contributes to a loss of growth control and transformation. To investigate the therapeutic potential of blocking Stat3 in cancer cells, we developed a transcription factor decoy to selectively abrogate activated Stat3. The Stat3 decoy was composed of a 15-mer double-stranded oligonucleotide, which corresponded closely to the Stat3 response element within the c-fos promoter. The Stat3 decoy bound specifically to activated Stat3 and blocked binding of Stat3 to a radiolabeled Stat3 binding element. By contrast, a mutated version of the decoy that differed by only a single base pair did not bind the activated Stat3 protein. Treatment of head and neck cancer cells with the Stat3 decoy inhibited proliferation and Stat3-mediated gene expression, but did not decrease the proliferation of normal oral keratinocytes. Thus, disruption of activated Stat3 by using a transcription factor decoy approach may serve as a novel therapeutic strategy for cancers characterized by constitutive Stat3 activation.

Transcription factor decoy (TFD) in breast cancer research and treatment.

Synthetic oligonucleotides have recently been the object of many investigations aimed to develop sequence-selective compounds able to modulate, either positively or negatively, transcription of eukaryotic and viral genes. Alteration of transcription could be obtained by using synthetic oligonucleotides mimicking target sites of transcription factors (the transcription factor decoy -TFD- approach). This could lead to either inhibition or activation of gene expression, depending on the biological functions of the target transcription factors. Since several transcription factors are involved in tumor onset and progression, this issue is of great interest in order to design anti-tumor compounds. In addition to oligonucleotides, peptide nucleic acids (PNA) can be proposed for the modulation of gene expression. In this respect, double-stranded PNA-DNA chimeras have been shown to be capable to exhibit strong decoy activity. In the case of treatment of breast cancer cells, decoy oligonucleotides mimicking CRE binding sites, promoter region of estrogen receptor alpha gene, NF-kB binding sites have been used with promising results. Therefore, the transcription factor decoy approach could be object of further studies to develop protocols for the treatment of breast cancer. In the future, transcription factors regulating cell cycle, hormone-dependent differentiation, tumor invasion and metastasis are expected to be suitable targets for transcription factor decoy.

Transcription factor decoy to study the molecular mechanism of negative regulation of renin gene expression in the liver in vivo.

Renin is synthesized in high quantities in the juxtaglomerular cells of the kidney, but little or none is synthesized in the liver. Our previous in vitro and biochemical studies have demonstrated that tissue-specific expression of the mouse renin gene is regulated by the specific interaction between negative regulatory element (NRE) in the 5'-flanking region of the renin gene and NRE binding protein (NREB). In this study, we examined the hypothesis that this interaction between the NRE in the promoter region of the rat renin gene and the NREB in the liver contributes to the suppressed renin gene expression in this tissue in vivo. We used in vivo transfection of NRE transcription factor decoy (TFD) double-stranded oligonucleotide into the rat liver via portal vein infusion. A gel mobility shift assay showed that transfected NRE TFD blocked endogenous NREB binding with the rat renin gene. This resulted in enhanced hepatic renin mRNA expression, immunohistochemical detection of renin in the liver, and consequently, increased plasma renin concentration. Taken together, these results document the importance of NREB in the inhibition of renin gene expression in rat liver in vivo and suggest the possibility of in vivo renin gene modulation by the TFD approach.