Transcription Factor Decoy Research Articles

An oligonucleotide decoy for nuclear factor-kappa B inhibits tumor necrosis factor-alpha-induced human umbilical cord vein endothelial cell tissue factor expression in vitro.

Abnormal tissue factor (TF) expression on vascular endothelial cells may account for thrombotic events associated with cardiovascular disease. The transcription factor nuclear factor-kappa B (NF-kappa B) activation plays a key role in endothelial cell injury and TF expression. Disruption of NF-kappa B activation in endothelial cells may inhibit TF expression and be protective in thrombosis. The purpose of the study was to determine whether NF-kappa B transcription factor decoy (TFD) could block TF expression. NF-kappa B TFD was transferred into cultured human umbilical cord vein endothelial cells (HUVEC) by liposomes, and the transfection efficiency was detected by flow cytometry. The effect of NF-kappa B TFD on TF mRNA levels was determined by reverse transcription-polymerase chain reaction. The expression of surface TF antigen was analyzed by flow cytometry. TF activity was studied by measuring enzymatic activation of factor X by the TF-activated factor VII complex. The results suggested that NF-kappa B decoy could be successfully transferred into HUVEC by liposome. The NF-kappa B TFD competed with the endogenous kappa B site sequence in the TF promoter for binding to transcription factor NF-kappa B in tumor necrosis factor-alpha-stimulated HUVEC, which could block the tumor necrosis factor-alpha-induced increase in TF mRNA levels, the upregulation of surface TF antigen and TF activity. This study demonstrated that NF-kappa B decoy could block HUVEC TF gene expression. Targeted genetic disruption of endothelial TF expression by NF-kappa B decoy may provide a possible therapeutic method for cardiovascular and thrombosis disease.

Peptide nucleic acid-DNA decoy chimeras targeting NF-κB transcription factors: Induction of apoptosis in human primary osteoclasts

Peptide nucleic acids (PNAs) are DNA mimics constituted by a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units. PNAs hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, forming Watson-Crick double helices and are resistant to both nucleases and proteases. While applications of PNAs as antisense and antigene molecules have been described, PNA/DNA and PNA/PNA hybrids are not useful for transcription factor decoy (TFD) pharmacotherapy. By contrast, PNA-DNA-PNA (PDP) chimeras, constituted of sequential PNA, DNA and PNA stretches, are potent decoy molecules in vitro. Interestingly, PDP-based decoys a) are more soluble than PNAs, b) are more resistant than synthetic oligonucleotides to enzymatic activity present in cellular extracts and serum and c) can be delivered with liposomes. In the present study we demonstrated that double-stranded PNA-DNA-PNA chimeras targeting NF-kappaB transcription factors induce apoptosis of human primary osteoclasts. Our data suggest that PDP-based induction of osteoclast apoptosis could be a therapeutic approach for disorders in which bone resorption is inappropriately excessive.

New trends in the development of transcription factor decoy (TFD) pharmacotherapy.

Many recent published observations firmly demonstrate that one of the ways to study and artificially modulate gene expression at the transcriptional level is offered by the "transcription factor decoy" (TFD) strategy. This experimental approach is based on the competition for trans-acting factors between endogenous cis-elements present within regulatory regions of target genes and exogenously added DNA sequences (the DNA-based drug) mimicking the specific cis-elements. The objective of this molecular intervention is to cause a decrease of the interactions of trans-factors with the target genomic cis-elements, leading to alteration of transcription. The characterisation of the biological activity of the designed decoy molecules is routinely assessed by molecular technologies, such as electrophoretic mobility gel shift assay (EMSA), competitive DNase I footprinting, in vitro transcription. New advances in this field employ biospecific interaction analysis (BIA) based on surface plasmon resonance (SPR) and biosensor technology. With respect to the design of the decoy biomolecules, in addition to double-stranded DNA/DNA hybrids, cross-linking between two DNA molecules either via photocrosslinking or by the introduction of a covalently linked, non-nucleotide bridge has been reported. Furthermore, RNA decoys have been described able to bind transcription factors via aptameric interactions. In addition, circular decoys assuming a dumbbell configuration or single-stranded decoys with intramolecular palindromic sequences have also been described. Decoy molecules were also produced by polymerase-chain reaction (PCR). More recently, peptide nucleic acids-DNA chimeras have been shown to exhibit decoy activity and high level of stability. This variety of decoy biomolecules facilitate the establishment of suitable delivery approaches, including pressure-mediated transfer, electrically enhanced transfer, biolistic bombardment, cationic liposomes, hemagglutinating virus of Japan (HVJ)-liposomes, microsphere-aided delivery, nano-particles, peptide-mediated delivery, steroid mediated gene transfer, and red-blood cells.

Estrogen, heat shock proteins, and NFkappaB in human vascular endothelium.

We hypothesized that estrogen would increase HSP72 in human coronary artery endothelial cells (HCAEC), and that these would be more sensitive to estrogen than our previous observations in myocytes. HCAEC were treated with 17beta-estradiol or tamoxifen, ranging from physiological to pharmacological(1 nM to 10 micromol/L) for either 24 hours (early) or 7 days (chronic). HSP expression was assessed by Western blots. Both early and chronic 17beta-estradiol and tamoxifen increased HSP72. Electromobility shift assays (EMSA) showed activation of HSF-1 with early, but not chronic, 17beta-estradiol. 17beta-Estradiol activated NFkappaB within 10 minutes, and the ER-alpha selective inhibitor, ICI 182 780, abolished this effect. Transcription factor decoys containing the heat shock element blocked HSP72 induction. Estrogen pretreatment decreased lactate dehydrogenase release with hypoxia. This protective effect persisted despite blockade of HSF-1 by decoys. However, an NF-kappaB decoy prevented the increase in HSP72 and abolished the estrogen-associated protection during hypoxia. 17beta-Estradiol upregulates HSP72 early and chronically via different mechanisms in HCAEC, and provides cytoprotection during hypoxia, independent of HSP72 induction. NF-kappaB mediates the early increase in HSP72, suggesting that estrogen activates NF-kappaB via a nongenomic, receptor-dependent mechanism, and this leads to activation of HSF-1. Activation of NF-kappaB was critical for estrogen-associated protection. Further studies are needed to elucidate the involved signaling pathways. We hypothesized that estrogen would increase HSP72 in human coronary artery endothelial cells (HCAEC). Both early and chronic treatment increased HSP72. EMSA showed activation of HSF-1 with early, but not chronic, 17beta-estradiol. Transcription factor decoys blocked estrogen-related HSP72 induction. Estrogen decreased LDH release with hypoxia. An NF-kappaB decoy blocked the HSP72 increase and estrogen-associated protection.

Differential inhibition of HSP72 and HSP25 produces profound impairment of cellular integrity.

To test a putative cause and effect relationship between heat-shock protein (HSP) expression and response to renal cell injury, HSP72 and HSP25 were differentially inhibited in LLC-PK1 cells by means of transcription factor decoy and short interference RNA (siRNA). Cellular injury was assessed by solubilization of NaK ATPase (S-NaK). An exonuclease-resistant, ethylene glycol-bridged, circular oligonucleotide decoy for heat-shock transcription factor (HSF)-1, based on the sequence of the porcine heat-shock element, was constructed and validated. It was found that under all experimental conditions, cells had comparable ATP levels; that decoy of unligated or scrambled sequence was ineffective; that HSP72 mRNA and HSP72/HSP25 proteins were significantly reduced in decoy-treated cells; and that the dampened response to HSF activation in decoy-treated, injured cells was accompanied by a substantially amplified loss of cellular integrity (S-NaK was 85% compared with baseline levels). Specific inhibition of HSP72 that used siRNA directed against an inducible porcine HSP72 gene resulted in complete ablation of injury-induced HSP72. Isolated inhibition of HSP72 was also associated with marked NaK ATPase detachment from the cytoskeleton (S-NaK was 135% compared with baseline levels). These studies suggest that an HSF-1 decoy effectively dampens the HSP72/HSP25 response to injury in renal cells; that HSP72 siRNA ablates injury-induced induction of HSP72; and that dampening of the HSP72/HSP25 response and ablation of the HSP72 response are both associated with impaired restitution of cellular polarity.

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.

Gene therapy with an E2F transcription factor decoy inhibits cell cycle progression in rat anti-Thy 1 glomerulonephritis

Mesangial cell (MC) proliferation is a central feature of many glomerular diseases. Various growth factors and cytokines are known to trigger MC proliferation both in vitro and in vivo. Regardless of the initial stimulus, proliferation is ultimately dependent upon the coordinated activation of cell cycle regulatory genes whose transcription is tightly controlled in mammalian cells. The transcription factor E2F plays an important role in the transactivation of the cell cycle regulatory genes proliferating-cell nuclear antigen (PCNA) and cdk2 kinase. To test whether or not E2F inhibition would blunt glomerular cell cycling in vivo, we treated rats with anti-Thy 1 antibody to induce glomerular injury, and that infused hemagglutinating virus of Japan (HVJ)-liposomes containing synthetic double stranded oligonucleotides (ODN) with high affinity for E2F (E2F decoy) directly into one kidney. First, we confirmed that with HVJ-liposome method fluorescence isothiocynate (FITC)-labeled ODN could be efficiently introduced into rat glomerular cells via renal artery. E2F decoy ODN treatment specifically inhibited mRNA expression of PCNA and cdk2 kinase in kidneys injured with anti-Thy 1 antibody as assessed by RT-PCR. This was associated with a significant decrease in number of glomerular cells in S phase as assessed by 5'-bromo-2'-deoxy-uridine labeling method, and attenuation of glomerular injury assessed histologically. The evidence suggests that intra-renal delivery of E2F decoy ODN by HVJ-liposome method prevents the induction of cell cycle regulatory gene expression and MC proliferation. These data also demonstrate the feasibility and the potential benefit of in vivo gene therapy as a novel strategy in the treatment of glomerular diseases.

Estrogen and regulation of heat shock protein expression in female cardiomyocytes: cross-talk with NFкB signaling

Estrogen is associated with increased heat shock protein (HSP)72 and protection during hypoxia-reoxygenation in cardiomyocytes from adult male rats, as previously reported. We have also reported that female rats have more cardiac HSP72 than males. We hypothesized that, despite higher endogenous estrogen levels and higher baseline HSP72, 17β-estradiol treatment would still result in increased HSP72 and protection during hypoxia-reoxygenation in cardiomyocytes from females. Methods/Results. – Cardiac cells isolated from adult female rats were treated for 12 hr with 17β-estradiol (0.1, 10, or 50 μM), tamoxifen, (10 or 25 μM; estrogen receptor agonist/antagonist), geldanamycin (2, 5, or 10 μg/ml; inactivates HSP90, preventing interaction with HSF1), or vehicle. Western blot analyses revealed that treatment with 17β-estradiol (10 or 50 μM), tamoxifen (25 μM), and geldanamycin (all doses) resulted in significant increases in HSP72. Electromobility shift assays revealed activation of HSF1 by 2 to 3 hr, and NFкB activation by 15 min. HSP72 induction via HSF1 activation was confirmed using transcription factor decoys containing the heat shock element, which prevented the estrogen-related HSP72 induction. Estrogen pretreatment resulted in decreased LDH release during 24 hr hypoxia. This protective effect persisted despite decoy-mediated blockade of nuclear HSF1 binding. However, transfection with an NFкB decoy not only prevented an estrogen-associated increase in HSP72, but also abolished the estrogenrelated protection during hypoxia. Conclusions. – Despite higher endogenous estrogen, 17β-estradiol and the selective estrogen receptor modulator, tamoxifen, upregulate HSP72 in cardiomyocytes from adult females, and provide cytoprotection during hypoxia, independent of HSP induction. NFкB activation is necessary for the increase in HSP72, suggesting that estrogen treatment activates NFкB, with subsequent HSF1 activation. NFкB activation is critical for estrogen-associated HSP induction, and protection during hypoxia in female cardiocytes. Treatment with 17β-estradiol and tamoxifen may provide a novel means of protecting both male and female cardiac myocytes against hypoxia-induced damage. Further studies are needed to define the cross-talk between HSF1 and NFкB signaling pathways.

Thrombospondin-1 mediates distal tubule hypertrophy induced by glycated albumin.

Diabetic nephropathy is characterized by early hypertrophy in both glomerular and tubuloepithelial elements. However, no studies to date have established a direct causal link between hyperglycaemia and renal hypertrophy. Our previous studies have found that high glucose does not induce cellular hypertrophy or expression of TGF-beta1 (transforming growth factor-beta1) in distal renal tubule cells [Yang, Guh, Yang, Lai, Tsai, Hung, Chang and Chuang (1998) J. Am. Soc. Nephrol. 9, 182-193]. In the present study, we used AGEs (advanced glycation end-products) to mimic long-term hyperglycaemia. Similar to glucose, AGEs did not induce TGF-beta1 mRNA in distal renal tubule cells [MDCK (Madin-Darby canine kidney) cells]; however, TGF-beta1 bioactivity was increased significantly. This result indicated post-translational regulation. Since TSP-1 (thrombospondin-1) has been demonstrated to activate latent TGF-beta1 in a variety of systems, the following experiments were performed. We found that AGEs dose-dependently increased both intracellular and extracellular levels of TSP-1. Purified TSP-1, like AGEs, increased the cellular protein content. Furthermore, anti-TSP-1 neutralizing antibodies attenuated the AGE-induced increase in TGF-beta1 bioactivity and hypertrophy. Thus TSP-1 might mediate AGE-induced distal renal tubule hypertrophy. In addition, we observed several putative transcription factor binding sites in the TSP-1 promoter, including those for AP-1 (activator protein-1), CREB (cAMP response element binding protein), NF-kappaB (nuclear factor-kappaB), SRF (serum response factor) and HSF (heat-shock factor), by sequence mapping. We used an enhancer assay to screen possible transcription factors involved. We showed that AP-1 and CREB were specifically induced by AGEs; furthermore, TFD (transcription factor decoy) for AP-1 could attenuate the AGE-induced increases in TSP-1 levels and cellular hypertrophy. Thus regulation of TSP-1 might be critical for hyperglycaemic distal tubule hypertrophy. Furthermore, TSP-1 TFD might be a potential approach to ameliorate diabetic renal hypertrophy.

Transcription factor decoy oligonucleotides modified with locked nucleic acids: an in vitro study to reconcile biostability with binding affinity

Double-stranded oligonucleotides (ODNs) containing the consensus binding sequence of a transcription factor provide a rationally designed tool to manipulate gene expression at the transcriptional level by the decoy approach. However, modifications introduced into oligonucleotides to increase stability quite often do not guarantee that transcription factor affinity and/or specificity of recognition are retained. We have previously evaluated the use of locked nucleic acids (LNA) in the design of decoy molecules for the transcription factor kappaB. Oligo nucleotides containing LNA substitutions displayed high resistance to exo- and endonucleolytic degradation, with LNA-DNA mix-mers being more stable than LNA-DNA-LNA gap-mers. However, insertion of internal LNA bases resulted in a loss of affinity for the transcription factor. This latter effect apparently depended on positioning of the internal LNA substitutions. Indeed, here we demonstrate that intra- and inter-strand positioning of internal LNAs has to be carefully considered to maintain affinity and achieve high stability, respectively. Unfortunately, our data also indicate that LNA positioning is not the only parameter affecting transcription factor binding, the interference in part being dependent on the intrinsic conformational properties of this nucleotide analog. To circumvent this problem, the successful use of an alpha-L-ribo- configured LNA is demonstrated, indicating LNA-DNA-alpha-L-LNA molecules as promising new decoy agents.

Transcription factor decoy for AP-1 reduces mesangial cell proliferation and extracellular matrix production in vitro and in vivo.

Diabetic nephropathy is characterized by an expansion of glomerular mesangium, caused by mesangial cell proliferation and excessive accumulation of extracellular matrix (ECM) proteins, which eventually leads to glomerulosclerosis and renal failure. Activator protein-1 (AP-1), a transcription factor, is implicated in the transcriptional regulation of a wide range of genes participating in cell proliferation and ECM production. This investigation was undertaken to test the hypothesis that AP-1 plays an important role in ECM gene expression, and to develop a molecular therapeutic strategy based on decoy oligodeoxynucleotides (ODN). In this report, we show that transfection with AP-1 decoy ODN strongly inhibits high glucose- and angiotensin II-induced cell proliferation and expression of ECM genes in cultured mesangial cells in vitro. Administration of AP-1 decoy ODN into streptozotocin-induced diabetic rat kidney in vivo using the hemagglutinating virus of Japan (HVJ)-liposome method virtually abolished TGF-beta1 and plasminogen activator inhibitor-1 expression. Our results collectively indicate that AP-1 activation is crucial for mesangial cell proliferation and ECM production in response to high glucose and angiotensin II. Moreover, use of stable AP-1 decoy ODN combined with the highly effective HVJ-liposome method provides a novel potential molecular therapeutic strategy for the prevention of diabetic nephropathy.

Molecular therapy to inhibit NFkappaB activation by transcription factor decoy oligonucleotides.

Molecular therapy is emerging as a potential strategy for the treatment of various diseases for which few known effective therapies exist. One strategy for combating disease processes has been to target the transcriptional process. Two approaches have been used to accomplish this: the use of antisense complimentary to the mRNA of interest and the use of ribozymes, a unique class of RNA molecules that not only store information but also process catalytic activity. Ribozymes are known to catalytically cleave specific target RNA, leading to its degradation, whereas antisense molecules inhibit translation by binding to mRNA sequences on a stoichiometric basis. More recently, small interfering RNA has been shown to inhibit target gene expression. The application of oligonuclotide technology, such as antisense, to regulate the transcription of disease-related genes in vivo has important therapeutic potential. Transfection of cis-element double-stranded oligodeoxynucleotides has been reported as a powerful tool in a new class of anti-gene strategies for molecular therapy.

Transcription factor as molecular targets: is transcription factor decoy a novel drug?

Transcription factor as molecular targets: is transcription factor decoy a novel drug?

Decoy oligodeoxynucleotides targeting NF-kappaB transcription factors: induction of apoptosis in human primary osteoclasts

Proteins belonging to the nuclear factor kappaB (NF-kappaB) superfamily are involved in osteoclast formation, playing a very important role for both differentiation of osteoclast precursors and survival of mature osteoclasts. Several drugs used to fight bone loss in a variety of human pathologies, including osteoporosis, act by increasing the frequency of osteoclast apoptosis, since it was demonstrated that small changes in osteoclast apoptosis can result in large changes in bone formation. In this respect, targeting of NF-kappaB transcription factor could be of great interest. Among nonviral gene therapy strategies recently proposed to inhibit or even block NF-kappaB activity, the transcription factor decoy (TFD) should be taken in great consideration. The main issue of the present study was to examine the effects of decoy DNA/DNA molecules targeting NF-kappaB on apoptosis of human osteoclasts (OCs), with the aim to interfere with the pathway regulating osteoclast differentiation and programmed cell death. To this aim, we used a mixture of receptor activator of NF-kappaB ligand (RANKL), macrophage colony-stimulating factor (M-CSF) and parathyroid hormone (PTH) to prepare human OCs from peripheral blood cells. Then, transfection with the decoy molecules targeting NF-kappaB was performed. The results obtained demonstrate that in primary cells expressing typical osteoclast markers such as TRAP and MMP9, NF-kappaB decoy significantly stimulated apoptosis. Inhibition of IL-6 expression and induction of Caspase 3 were found in OCs treated with NF-kappaB DNA/DNA decoys. We consider these data as the basis for setting up experimental conditions allowing nonviral gene therapy of several bone disorders.

Gene therapy with transcription factor decoy oligonucleotides as a potential treatment for cardiovascular diseases.

Cardiovascular diseases including renal diseases are the leading causes of mortality and morbidity in developed countries. Most conventional therapy is inefficient and tends to treat the symptoms rather than the underlying causes of the disorder. Gene therapy based on oligonucleotides (ODN) offers a novel approach for the prevention and treatment of cardiovascular diseases. Gene transfer into somatic cells to interfere with the pathogenesis contributing to cardiovascular disease may provide such a novel approach for better prevention and treatment of cardiovascular disorders. The major development of gene transfer has importantly contributed to intense investigation of the potential of gene therapy in cardiovascular including renal medicine. The amazing advances in molecular biology have provided a dramatic improvement of the technology that is necessary to transfer target genes into somatic cells. Gene transfer methods have been surprisingly improved. In fact, some of them (retroviral vectors, adenoviral vectors or liposome based vectors, etc) have been used in the clinical trials already. Recent progress in molecular biology has provided new techniques to inhibit target gene expression. Especially, application of DNA technology such as an antisense strategy to regulate the transcription of disease-related genes in vivo has important therapeutic potential. Recently, transfection of cis-clement double-stranded ODN (= decoy) has been reported as a new powerful tool in a new class of anti-gene strategies for gene therapy. Transfection of double-stranded ODN corresponding to the cis sequence will result in attenuation of the authentic cis-trans interaction, leading to removal of trans-factors from the endogenous cis-elements with subsequent modulation of gene expression.

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.

NF-κB as a Therapeutic Target for Transcription Factor Decoy Strategy in Inflammatory Diseases

Nuclear factor (NF)-κB is a transcription factor associated with modulation of the immune response. NF-κB is a heterodimer consisting of subunits p50, p52 and p65 (Rel A) or Rel B or cRel (the most common NF-κB heterodimer consist of subunits p50 and p65) that belong to the Rel family of genes, which includes the v-rel oncogene, the c-rel protooncogene and dorsal, a Drosophia morphogen. In its quiesecent state, NF-κB is in the cytoplasm, bound to a family of inhibitory proteins that are collectively called IkB. Several molecules, including interleukin (IL)-1, lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, reactive oxygen intermediates and viral products activate NF-κB through phosphorylation and subsequent degradation of IkB. Activation by these agents leads to exposure of the nuclear localization signal of NF-κB and entry into the nucleus. Once in the nucleus, NF-κB transcriptionally activates a variety of genes involved in the inflammatory process, including intracellular adhesion molecule (ICAM)-1, vascular cellular adhesion molecule (VCAM)-1, endothelial leukocyte adhesion molecule (ELAM) and IL-8. The family of NF-κB transcription factor is a topic of interest in the biomedical community stemming from the role NF-κB plays in almost every aspect of cell regulation such as stress responses, immune cell activation, apoptosis, proliferation, differentiation and oncogenic transformation. Recently, we have developed a novel molecular strategy termed the decoy approach to inhibit NF-κB activity. Moreover, E2F decoy strategy has already been used in the clinical trials to prevent the vascular disease. Taken together, the decoy strategy is considered to have the potential of clinical application. Keywords: transcription factor, decoy, gene therapy, glomerulonephritis, rheumatoid arthritis, myocardial infarction, transplantation

Effect of Transcription Factor Decoy for NF-κB on the TNF-α Induced Cytokine and ICAM-1 Expression in Cultured HaCaT cells

Effect of Transcription Factor Decoy for NF-κB on the TNF-α Induced Cytokine and ICAM-1 Expression in Cultured HaCaT cells

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.