Dual Blockade of Cyclic AMP Response Element- (CRE) and AP-1-directed Transcription by CRE-transcription Factor Decoy Oligonucleotide

Abstract

Alteration of gene transcription by inhibition of specific transcriptional regulatory proteins has important therapeutic potential. Synthetic double-stranded phosphorothioate oligonucleotides with high affinity for a target transcription factor can be introduced into cells as decoy cis-elements to bind the factors and alter gene expression. The CRE (cyclic AMP response element)-transcription factor complex is a pleiotropic activator that participates in the induction of a wide variety of cellular and viral genes. Because the CRE cis-element, TGACGTCA, is palindromic, a synthetic single-stranded oligonucleotide composed of the CRE sequence self-hybridizes to form a duplex/hairpin. Herein we report that the CRE-palindromic oligonucleotide can penetrate into cells, compete with CRE enhancers for binding transcription factors, and specifically interfere with CREand AP-1-directed transcription in vivo. These oligonucleotides restrained tumor cell proliferation, without affecting the growth of noncancerous cells. This decoy oligonucleotide approach offers great promise as a tool for defining cellular regulatory processes and treating cancer and other diseases.Eukaryotic transcription is regulated by the interplay of various protein factors at promoters (1, 2). It has been shown that prokaryotic repressors can function as negative regulators of eukaryotic promoters (3,4). This observation suggests that displacement of activating proteins might provide a general strategy for gene-specific repression in eukaryotes. Several approaches have been undertaken to control eukaryotic gene expression through such displacement.In one approach, trans-dominant mutants are generated that interfere with the function of transactivators. Mutants are generated that retain the ability to bind to cis-regulatory DNA sequences but that have dysfunctional transcriptional activation domains. These mutant transcription factors compete with their functional, wild-type counterparts for binding to the enhancer sequences and prevent the activation or repression of the target gene. Although this strategy has been successful, in vitro (5-8), the generation of such mutants is not always possible. The transcription factor must be well characterized such that the activation domain(s) is identified and can be mutated. Also, even with sufficient knowledge to generate such mutants, difficult gene therapy procedures would be required to express these proteins in vivo.Promoter competition strategy has also been utilized whereby plasmids containing cis-acting elements in common with the targeted gene are introduced in high copy number into cells (9). At high copy number, a majority of the transcription factors can be competitively bound away from the native enhancer sequences with gene expression accordingly regulated. Because these plasmids must be stably maintained at high copy number in target cells, a requirement that is difficult to achieve in vivo, this approach has also been limiting.Another alternative is to employ oligonucleotides to form triple heli...

CREB-Mediated Bcl-2 Protein Expression after Ischemic Preconditioning

“…As an important control, we show that the CRE decoy oligonucleotide did not effect CREB phosphorylation after preconditioning ischemia. The CRE decoy oligonucleotide blocked CREB binding to the bcl-2 CRE and the expression of the Bcl-2 protein after preconditioning, consistent with its hypothesized biological effect (Hara et al, 2003;Park et al, 1999).…”

“…Some cells were incubated with a CRE decoy oligonucleotide based on the bcl-2 CRE sequence or control oligonucleotides, similar to those previously described by Park et al (1999) (all 0.5 mmol/L) (CRE decoy oligonucleotide 5 0 -TGACGTCAGAGAGCGCTCTCTGACGTCA-3 0 ; Control CRE mis-match sequence 5 0 -TAGCTGCAGA-GAGCGCTCTCTGCAGCTA-3 0 ; (all linkages phosphorothioate protected) (Invitrogen Carlsbad, CA, USA or Sigma-Genosys, The Woodlands, TX, USA). Oligonucleotides were heated to 951C and then slowly cooled to room temperature before incubation with the cells.…”

“…To investigate the role of CREB in ischemic preconditioning, we used a CRE decoy oligonucleotide to block the binding of CREB to the bcl-2 CRE, as described by Park et al (1999). Cells were incubated with a hairpin loop of DNA containing the bcl-2 CRE, which differs from the consensus CRE site by one base (TGACGTTA versus TGACGTCA).…”

On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids

“…6 Cationic lipids provide a particularly powerful tool for the introduction of polynucleic acids into cells. For example, cationic lipid-nucleic acid complexes or 'lipoplexes' can promote the intracellular delivery of plasmid DNA, 6 RNA, 7 antisense oligonucleotides, 8 transcription factor decoy oligonucleotides, 9 ribozymes 10 and transcription factors. 11 Cationic lipid in complexes with plasmids or other polynucleic acids performs at least three functions.…”

Specific covalent binding of a NF‐κB decoy hairpin oligonucleotide targeted to the p50 subunit and induction of apoptosis

“…Such decoys are e⁄cient and speci¢c inhibitors of transcription. For instance, decoy oligodeoxyribonucleotides (ODNs) of the transcription factors E2F, NF-UB, CREB or AP-1 have been shown to e⁄ciently cure vascular proliferation [1], myocardial infarction [2], or to prevent tumor growth and invasiveness [3,4]. Initially shown to be implicated in host defense and in£ammatory response [5], NF-UB is associated with induction of apoptosis in some cells but it is also involved in protection against apoptosis in Epstein^Barr virus (EBV)-transformed B cells [6].…”