Decoy Molecules Research Articles

CARD games in apoptosis and immunity.

A bewildering array of proteins containing the caspase recruitment domain (CARD) have now been identified. Previously, CARD-CARD interactions have been shown to be involved in the assembly of protein complexes that promote caspase processing and activation in the context of apoptosis. However, as the family of CARD-containing proteins has grown, it has become apparent that the majority of these proteins do not recruit caspases or promote caspase activation. Instead, many participate in NF-kappaB signalling pathways associated with innate or adaptive immune responses. Here, we suggest a simplified classification of the CARD proteins based upon their domain structures and discuss the divergent roles of these proteins in the context of host defence.

Design and characterization of decoy oligonucleotides containing locked nucleic acids.

Transfection of cis-element double-stranded oligonucleotides, referred to as decoy ODNs, has been reported to be a powerful tool that provides a new class of antigene strategies for gene therapy. However, one of the major limitations of the decoy approach is the rapid degradation of phosphodiester oligonucleotides by intracellular nucleases. To date, several DNA analogs have been employed to overcome this issue, but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this paper we have investigated the use of conformationally restricted nucleotides in the design of decoy molecules for nuclear transcription factor kappaB (NF-kappaB). Starting from a synthetic double-stranded oligonucleotide, containing the kappaB consensus binding sequence, we designed a panel of decoy molecules modified to various extents and at various positions with locked nucleic acids (LNAs). Our results indicate that the addition of terminal LNA bases, outside the kappaB sequence, to generate LNA-DNA-LNA co-polymers was sufficient to confer appreciable protection towards nuclease digestion, without interfering with transcription factor binding. Conversely, insertion of LNA substitutions in the context of the kappaB-binding site resulted in further increased stability, but caused a loss of affinity of NF-kappaB for the target sequence. However, our results also indicate that this latter effect was apparently dependent not only on the extent but also on strand positioning of the internal LNA substitutions. This observation is of great importance since it provides evidence for the possibility of tuning DNA-LNA duplexes with internal LNAs into decoy agents with improved features in terms of biological stability and inhibitory effect.

Osteoblastic Differentiation Induced by Transcription Factor Decoy against Estrogen Receptor α Gene

Osteoblasts have been shown to express both isoforms of estrogen receptor (ERα and ERβ). As a tool for the study of endogenous regulation of these genes the decoy strategy was employed. Human MG-63 osteoblast-like cells were transfected with a DNA decoy molecule containing a putative negative cis-element (DNA-102) located in the C distal promoter of ERα gene. Using real-time quantitative RT-PCR, we found that the DNA-102, but not scrambled DNA, produced a 36-fold increase in the level of total ERα mRNA and a 12-fold increase in the level of mRNA for the F isoform that is transcribed from the upstream F promoter, which is predominantly used in osteoblasts. This effect appears to be controlled by estrogen since 17-β-estradiol downregulated the mRNA increase. Notably, the same decoy was able to induce a 6-fold increase in ERβ mRNA transcription, indicating the coregulation of the ERα and ERβ expression. An increase in OPN but not in BMP4 expression was also observed. In addition, in decoy-treated cells, the cell growth decreased together with an increase in alkaline phosphatase activity. These findings indicated that DNA-102 decoy was able to induce a more differentiated osteoblastic phenotype. The augmentation of ERα and ERβ expression by the decoy approach may offer a further possibility for patient response to estrogenic therapy in the treatment of diseases related to estrogen deficiency.

Modulation of gene expression in human osteoblasts by targeting a distal promoter region of human estrogen receptor-alpha gene.

Estrogen receptor (ER) alpha is expressed during osteoblast differentiation; however, both its functional role in bone metabolism and its involvement in osteoporotic pathogenesis caused by estrogen deficiency are not well understood. Loss of ER alpha gene expression could be one of the mechanisms leading to osteoporosis. Therefore, we investigated a possible modulation of ER alpha gene expression in a human osteoblastic cell line and in four primary osteoblast cultures by using a decoy strategy. Double stranded DNA molecules, mimicking a regulatory region of the ER alpha gene promoter (DNA-102) and acting as a 'silencer' in breast cancer cells, were introduced into osteoblasts as 'decoy' cis-elements to bind and functionally inactivate a putative negative transcription factor, and thus to induce ER alpha gene expression. We found that the DNA-102 molecule was able to specifically bind osteoblast nuclear proteins. Before decoy treatment, absence or variable low levels of ER alpha RNAs in the different cultures were detected. When the cells were transfected with the DNA-102 decoy, an increase in expression of ER alpha and osteoblastic markers, such as osteopontin, was observed, indicating a more differentiated osteoblastic phenotype both in the cell line and in primary cultures. These results showed that the DNA-102 sequence competes with endogenous specific negative transcription factors that may be critical for a decrease in or lack of ER alpha gene transcription. Therefore, osteoblastic transfection with the DNA-102 decoy molecule may be considered a tempting model in a putative therapeutic approach for those pathologies, such as osteoporosis, in which the decrease or loss of ER alpha expression plays a critical role in bone function.

Molecular interactions with nuclear factor κB (NF‐κB) transcription factors of a PNA–DNA chimera mimicking NF‐κB binding sites

The decoy approach against nuclear factor κB (NF‐κB) is a useful tool to alter NF‐κB dependent gene expression using synthetic oligonucleotides (ODNs) carrying NF‐κB specific cis‐elements. Unfortunately, ODNs are not stable and need to be be extensively modified to be used in vivo or ex vivo. We have previously evaluated the possible use of peptide nucleic acids (PNAs) as decoy molecules. The backbone of PNAs is composed of N‐(2‐aminoethyl)glycine units, rendering these molecules resistant to both nucleases and proteases. We found that the binding of NF‐κB transcription factors to PNAs was either very low (binding to PNA–PNA hybrids) or exhibited low stability (binding to PNA–DNA hybrids). The main consideration of the present paper was to determine whether PNA–DNA chimeras mimicking NF‐κB binding sites are capable of stable interactions with proteins belonging to the NF‐κB family. Molecular modeling was employed for the design of PNA–DNA chimeras; prediction of molecular interactions between chimeras and NF‐κB nuclear proteins were investigated by molecular dynamics simulations, and interactions between PNA–DNA chimeras and NF‐κB proteins were studied by gel shifts. We found significant differences between the structure of duplex NF‐κB PNA–DNA chimera and duplex NF‐κB DNA–DNA. However, it was found that these differences do not prevent the duplex PNA–DNA chimera from binding to NF‐κB transcription factors, being able to suppress the molecular interactions between HIV‐1 LTR and p50, p52 and nuclear factors from B‐lymphoid cells. Therefore, these results demonstrate that the designed NF‐κB DNA–PNA chimeras could be used for a decoy approach in gene therapy.

Biospecific interaction analysis (BIA) as a tool for the design and development of gene transcription modifiers.

The applications of surface plasmon resonance (SPR) and biosensor technology for biospecific interaction analysis (BIA) of molecular interactions between transcription modifiers and target biomolecules is here described for the identification of possible candidates for drug research and development in antitumor and antiviral therapy. SPR-based BIA offers many advantages with respect to most of the other available methodologies to study biomolecular interactions. It should be underlined that (a) most commercially available biosensors are fully automated instruments; (b) no labelling is required; (c) a large variety of activated sensor chips are commercially available allowing the immobilization of either proteins or target DNA or RNA; (d) the amount of both ligand and analyte needed to obtain informative results is low; (e) the assay is rapid and (f) the sensor chip could be re-used many times, leading to low running costs, with the only limitation of verifying the stability of the immobilized ligand. Approaches employing SPR-based BIA were described for the development of (a) triple-helix forming oligonucleotides (TFO) and peptide nucleic acids (PNAs), (b) DNA-binding drugs, (c) decoy molecules and (d) PNAs able to perform strand invasion. All these biomolecules are of great interest for the development of transcription modifiers. Since alteration of the expression of transcription factors is involved in tumor cell growth and metastasis, SPR-based BIA appears to be a methodology of great impact in the design and development of anti-cancer agents.

Aberrant expression of caspase cascade regulatory genes in adult T-cell leukaemia: survivin is an important determinant for prognosis.

Derangement of either apoptosis or cell division is known to play an important role in tumorigenesis. Fas-mediated apoptosis on normal and leukaemic T cells is finely tuned by inhibitory proteins, such as FAP-1, FLIP and survivin, and defective caspase isoform which can attenuate the function of its intact caspase as a decoy molecule. However, complex involvement of such inhibitors in tumour biology relating to apoptotic pathology remains unclear in the neoplasms. We report the aberrant expression of FAP-1, FLIP and survivin mRNAs on leukaemic T cells from adult T-cell leukaemia (ATL) patients. Among these inhibitors, only survivin was aberrantly expressed in all ATL cases, but not in any normal peripheral blood mononuclear cells (PBMCs). Furthermore, survivin mRNA expression level was characteristic in each subtype of ATL and represented an important determinant for ATL prognosis. However, the apoptotic effector of casp-8, which is essential in Fas-mediated signal transduction, was dominant in defective casp-8 rather than intact casp-8 in ATL cells, suggesting a favourable biological situation for escape from apoptosis. Taken together, ATL cells probably possess many different regulatory mechanisms in order to attenuate Fas-mediated signalling and subsequently expand their populations under escape from apoptosis. Among these inhibitors, survivin is a useful bio-marker to assess tumour biology and may be a potential new target for apoptosis-based selective therapy in neoplasms as the expression is a general feature of neoplasia, but not normal tissues.

Modulation of estrogen receptor gene transcription in breast cancer cells by liposome delivered decoy molecules

It is well known that breast carcinomas without estrogen receptor (ER) have a poor prognosis and do not respond to antiestrogenic therapy. In analyzing the question of the lack of ER gene expression, we have considered the possibility to modify the ER gene expression by transfecting ER-negative breast cancer cells with a polymerase chain reaction product mimicking a putative negative regulatory region (−3258/−3157) inside the P3 ER gene promoter. Here we have demonstrated the efficacy of the selected sequence used as a decoy molecule in restoring the ER gene transcription. When this DNA was complexed and delivered by cationic liposomes (PC:DOTAP) a significant increase in the decoy effect was obtained. Breast cancer cells receiving the combination treatment responded substantially better to reactivation of quiescent ER gene than cells that had received DNA with calcium phosphate. This information may be useful for a series of in vitro transfections and also for in vivo application of the decoy strategy that is a potential therapeutic tool to control disease-related genes such as ER gene in breast cancer.

Death and decoy receptors and p53-mediated apoptosis.

Recently, several tumor necrosis factor receptor 1 (TNF-R1) and Fas-related death receptors have been discovered and include DR3, DR4, DR5 and DR6. These receptors contain an extracellular region containing varying numbers of cysteine-rich domains and an intracellular region that contains the death domain. The death receptors are activated in a ligand-dependent or independent manner and transduce apoptotic signals via their respective intracellular death domains. In addition to death receptors, several decoy molecules have also been identified and include DcR1/TRID, DcR2/TRUNDD, DcR3 and osteoprotegrin (OPG). The decoy molecules do not transduce apoptotic signals but rather compete with the death receptors for ligand binding and thereby inhibit ligand-induced apoptosis. Recent evidence suggests that p53 upregulates the expression of death receptors Fas and DR5, and thus, may mediate apoptosis in part via Fas and/or DR5. However, p53 also regulates the expression of TRAIL decoy receptors DcR1/TRID and DR2/TRUNDD. Although the significance of p53-dependent regulation of decoy receptors remains unclear, evidence suggests that DcR1/TRUNDD appears to inhibit 53-mediated apoptosis. It is, therefore, possible that p53 may blunt its DR5-dependent apoptotic effects by controlling the levels of decoy receptors.

Prostate cancer: molecular biology of early progression to androgen independence.

To improve the therapy for prostate cancer, it will be necessary to address the problems of progression to androgen independence and the process of metastatic spread of tumour. The complexity of the latter condition is likely to mitigate against the immediate development of relevant therapeutic approaches. However, the basis of androgen independence appears to be a problem of simpler dimensions and more amenable to treatment with current therapeutic technology. Since early tumour progression can be detected by an incomplete prostate-specific antigen (PSA) response to androgen withdrawal therapy, a study of the molecular biology of PSA gene regulation may well provide insight into new methods for preventing or delaying this problem. Mounting evidence suggests that ligand-independent activation of the androgen receptor may be one underlying mechanism of androgen independence. In the absence of androgen, a compensatory increase in the activity of cAMP-dependent protein kinase (PKA) enhances the ability of the androgen receptor to bind to the response elements regulating PSA gene expression. The activation of the androgen receptor through up-regulation of the PKA signal transduction pathway involves the amino-terminus of the androgen receptor, the function of which may be altered either by modifications such as phosphorylation, or through interactions with co-regulators or other proteins. Of therapeutic interest is the fact that this effect can be counteracted experimentally by the anti-androgen, bicalutamide, and clinically by several other similar agents. We speculate that the inhibition of PKA-activated androgen receptor might also be accomplished by decoy molecules that can bind to the relevant activated site on the amino-terminus or competitively interact with proteins recruited by the PKA pathway that are responsible for activating the receptor in the absence of androgen. Such molecules might include small mimetic substances or agents that can gain access to the nucleus of the cell.

In vitro stability of polymerase chain reaction-generated DNA fragments in serum and cell extracts

The potential use of polymerase chain reaction (PCR)-generated DNA fragments (PCR-DNAs) as pharmaceutical agents has previously been suggested, with the demonstration of the in vitro cellular internalization and biologic activity of PCR-DNA decoy molecules targeted to human estrogen receptor gene. In order to provide information on the stability of these double-stranded DNA molecules, the nuclease resistance of PCR-DNAs of different sizes was studied in different conditions and experiments. Simulating in vitro and in vivo transfection protocol, we demonstrated that PCR-DNAs exhibited good stability toward fetal bovine serum (FBS) and adult human serum nuclease digestion. In addition, when the protective activity of liposome-based formulations toward nuclease digestion was tested, it was shown that the stability of PCR-DNAs could be further increased (up to 7 days) when a liposome-mediated delivery system was employed.

CD38 binding to human myeloid cells is mediated by mouse and human CD31.

Soluble forms of membrane receptors are emerging candidates as physiological regulators of leukocyte trafficking. In the present study, we found that the soluble form of the CD38 antigen (sCD38) bears a binding domain of low affinity for a cellular receptor on U937 cells. Cross-linking and peptide-mapping studies confirmed the physical association and the identification of the U937 receptor as a 130 kDa protein. The binding of sCD38 to the receptor was differentially inhibited by several monoclonal antibodies against the CD31 cell-adhesion molecule. Thus the interaction was analysed through direct association of soluble and membrane CD38 with soluble recombinant murine CD31 with three N-terminal and with all six extracellular Ig domains. Cross-linking experiments on U937 intact cells, and ligand blot assays of the immunoprecipitated CD38 molecule, indicated that (i) the recognized epitope is determined by the tertiary structure of the molecule, and that (ii) the binding domain involved resides in the ectocellular portion of the CD31 molecule, more precisely in the first three N-terminal domains. A comparative functional activity between murine and human CD31 was also explored. The data presented suggest that (i) human CD31 bears a highly functional similarity with its murine counterpart, as it is a receptor in myeloid cells with more than one ligand (the alphavbeta3 integrin and the CD38 molecule), and that (ii) the activity of sCD38 as decoy molecule for CD31 may play an important role in cell-cell interactions in physiological and pathological conditions.

Interleukin-4 ameliorates experimental glomerulonephritis and up-regulates glomerular gene expression of IL-1 decoy receptor

Monocytes/macrophages and pro-inflammatory cytokines such as interleukin (IL)-1 are important in the pathogenesis of acute glomerulonephritis. The aim of this study was to examine whether IL-4, a cytokine with anti-inflammatory activity, could modulate glomerular inflammation and reduce injury in vivo. Treatment with recombinant rat IL-4 in a model of anti-glomerular basement membrane (GBM) antibody mediated glomerulonephritis in rats reduced glomerular injury. Albuminuria was less (73% less at day 4) and a lower proportion of glomeruli had capillary thrombi (79% less at day 4). In IL-4 treated rats, there was a moderate reduction in the number of macrophages in the glomeruli and also suppression of pro-inflammatory activities of the macrophages. Northern blot analysis of glomerular RNA showed that treatment with IL-4 up-regulated mRNA levels of type II IL-1 receptor (IL-1RTII). IL-1RTII, also known as IL-1 decoy receptor, may act as a decoy molecule to inhibit the effect of IL-1 beta. To our knowledge, this is the first demonstration of (i) recombinant IL-4 reducing glomerular inflammation in vivo and (ii) a treatment that increases IL-1RTII expression in association with reduction of tissue injury in vivo.

The type II "receptor" as a decoy target for interleukin 1 in polymorphonuclear leukocytes: characterization of induction by dexamethasone and ligand binding properties of the released decoy receptor.

Whereas the signaling function of the interleukin 1 (IL-1) receptor type I (IL-1R I) has been well documented, the type II "receptor" has been suggested to act as a decoy target for this cytokine. Since IL-1 may represent a key target of the immunomodulatory and antiinflammatory properties of glucocorticoids (GC), the aim of this study was to investigate the effects of dexamethasone (Dex) on IL-1R expression in human polymorphonuclear leukocytes (PMN), which express predominantly the type II molecule (IL-1R II). We found that Dex augments the levels of steady state transcripts encoding the IL-1R I and, most prominently, those of IL-1R II. Dex induced both transcripts via transcription-dependent mechanisms and by prolongation of the mRNAs half-lives. Inhibition of protein synthesis superinduced basal and Dex-augmented IL-1R II mRNA, whereas it completely inhibited the induction by Dex of IL-1R I transcripts. Induction of IL-1R II mRNA by Dex was associated with augmented membrane expression and release of the type II IL-1 binding molecule. This effect was mediated by the GC receptor. Other steroids (17 beta-estradiol, progesterone, and testosterone) were ineffective. The concentrations of IL-1 alpha and IL-1 receptor antagonist required to displace the binding of IL-1 beta to the soluble form of the decoy molecule induced by Dex from PMN were, respectively, 100 and 2 times higher compared with IL-1 beta. The induction by Dex of the type II receptor, a decoy molecule for IL-1, may contribute to the immunosuppressive and antiinflammatory activities of Dex.

The class II MHC and HIV-1 binding surfaces of CD4

C D4, the receptor for HIV entry into human lymphoid and monocytoid cells, has been the object of structural scrutiny since its molecular characterization by P. Maddon and coworkers eight years ago 1. It has been a goal of several laboratories to design effective CD4-based antiviral therapeutics, by building up an understanding of CD4 interaction with the HW gp120 envelope glycoprotein during viral entry and cytopathogenesis. CD4 is also a ligand for class II major histocompatibility (MHC) antigen, necessary for T cell recognition of antigen-presenting cells and activation of the cellular arm of the immune response. Understanding this interaction also has potential applications to transplantation and autoimmune disease therapy. These arenas of investigation are closely linked: antiviral agents that disrupt the CD4-gp120 interaction must spare interactions between CD4 and class II MHC, to avoid replacing virus-induced with antiviral-induced T cell impairment. CD4 has been a seductively attractive target as a therapeutic agent for AIDS: unlike the envelope glycoprotein itself, it is not a rapidly mutating molecule and hence is not a moving target for antiviral drug design. Attempts to capitalize on 'receptor decoy' strategies employing soluble CD4 (the truncated extracellular, and hence watersoluble, region of CD4 that contains the binding sites for both gp120 and class II MHC) have met with limited success for a variety of reasons. One initially perceived drawback of soluble CD4 (sCD4) unexpectedly did not emerge as a problem: it appears that sCD4 does not appreciably block helper T cell responses that depend on CD4 interaction with class II MHC. The chief obstacle to the therapeutic use of sCD4 is structural differences in the CD4-gp120 interaction among different isolates of HW-1, depending on the interaction of gp120 and gp41 in the viral envelope glycoprotein complex 2. Laboratory strains of HIV-1 are potently blocked by sCD4, while isolates obtained from HIV1-infected individuals after minimal passage in culture (i.e. most like the actual targets of sCD4 therapy) are not 3. The search for CD4based receptor decoy molecules that can bind gp120 of primary/ clinical isolates of HIV virions as effectively as authentic CD4 itself, perhaps including membraneanchored or polyvalent CD4 molecules, may produce a molecule that also efficiently binds class II MHC. If so, mutations of the molecules that can bind and disarm HIV virions without serving as ligands for class II MHC restricted antigen-presenting cells must also be sought. Several studies have addressed whether the class II MHC binding site on CD4 is separable from the gp120 binding site. These studies differ in the methods used to assess the ability of CD4 mutants to interact with class II MHC. Lamarre et al. 4 and Fleury et al. s employed mouse hybridoma cell lines conraining a murine class I MHC restricted T cell receptor (mTCR) in which human CD4 is stably expressed. Exposure of these cells to target cells expressing murine class I MHC and human class II MHC results in a greater than 20-fold enhancement in cell adhesion and IL-2 production, which depends