Holo-APP and G-protein-mediated signaling are required for sAPPα-induced activation of the Akt survival pathway.

Accumulating evidence indicates that loss of physiologic amyloid precursor protein (APP) function leads to reduced neuronal plasticity, diminished synaptic signaling and enhanced susceptibility of neurons to cellular stress during brain aging. Here we investigated the neuroprotective function of the soluble APP ectodomain sAPPα (soluble APPα), which is generated by cleavage of APP by α-secretase along the non-amyloidogenic pathway. Recombinant sAPPα protected primary hippocampal neurons and SH-SY5Y neuroblastoma cells from cell death induced by trophic factor deprivation. We show that this protective effect is abrogated in neurons from APP-knockout animals and APP-depleted SH-SY5Y cells, but not in APP-like protein 1- and 2- (APLP1 and APLP2) depleted cells, indicating that expression of membrane-bound holo-APP is required for sAPPα-dependent neuroprotection. Trophic factor deprivation diminished the activity of the Akt survival pathway. Strikingly, both recombinant sAPPα and the APP-E1 domain were able to stimulate Akt activity in wild-type (wt) fibroblasts, SH-SY5Y cells and neurons, but failed to rescue in APP-deficient neurons or fibroblasts. The ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) inhibitor GI254023X exacerbated neuron death in organotypic (hippocampal) slice cultures of wt mice subjected to trophic factor and glucose deprivation. This cell death-enhancing effect of GI254023X could be completely rescued by applying exogenous sAPPα. Interestingly, sAPPα-dependent Akt induction was unaffected in neurons of APP-ΔCT15 mice that lack the C-terminal YENPTY motif of the APP intracellular region. In contrast, sAPPα-dependent rescue of Akt activation was completely abolished in APP mutant cells lacking the G-protein interaction motif located in the APP C-terminus and by blocking G-protein-dependent signaling with pertussis toxin. Collectively, our data provide new mechanistic insights into the physiologic role of APP in antagonizing neurotoxic stress: they suggest that cell surface APP mediates sAPPα-induced neuroprotection via G-protein-coupled activation of the Akt pathway.

Antisense anti IGF-I cellular therapy of malignant tumours: immune response in cancer patients.

The treatment of cancer by antisense anti-IGF-I cellular therapy inducing immune response has evoked interest among many promising strategies. Here, we reported some results obtained from patients with cancer, mainly glioblastoma treated by this strategy, which was also extended to patients with colon carcinoma, ovary cystadenocarcinoma and prostate adenocarcinoma. It was shown that, in the phase I of clinical trial, patients vaccinated with their own tumour cells treated by antisense IGF-I presented a slight increase of temperature. Their peripheral blood lymphocytes showed a shift in the percentage of CD8 effector cells as judged by expression of cell surface markers CD8+ CD28+. Particularly, in two treated patients with glioblastoma, the survival time was 19 and 24 months respectively in comparison to the range of 12 to 15 months observed in the case of classical treatment such as surgery, radiation or chemotherapy. These results, although preliminary, gave indication that the reported strategy could deserve consideration owing to its safety. Furthermore, the increase in the percentage of peripheral blood monomorphonucleated cells (PBMNCs) with effector phenotype, i.e., CD8+ CD28+ in vaccinated patients might explain their prolonged survival time.

Proteomic and phosphoproteomic analysis of cellular responses in medaka fish (Oryzias latipes) following oral gavage with microcystin-LR.

Chronic and subchronic toxicity resulting from exposure to microcystins (MCs) receives increasing attention due to the risk of bioaccumulation of these toxins by aquatic animals, including fish. The mechanisms of action of MCs that target the liver, involve modifications of protein phosphorylation resulting from phosphatases 1 and 2A inhibition. Therefore, studying phosphoprotein modifications by using a specific phosphoprotein stain Pro-Q Diamond in fish liver contaminated with MC-leucine-arginine (MC-LR), the most toxic MC, should help dissecting disturbed signaling and metabolic networks. We have recently used this technology to identify several proteins that are modulated either in expression or phosphorylation in the liver of medaka following short-term exposure to MC-LR by balneation. In the present study, we have decided to use an alternative way of introducing the toxin into fish; that is by gavage (force-feeding). This was first achieved using tritiated MC-LR and allowed us to quantify the quantity of toxin incorporated into fish and to demonstrate that the toxin is mainly accumulated in liver. Afterwards a proteomics study limited to liver cytosolic proteins of contaminated animals showed that several proteins were up or down regulated either in quantity or in phosphorylation or both. Some of them had been previously detected as modified in balneation experiments but new molecules were identified as involved in signal transduction pathways activated by the toxin. In addition, in the conditions used (5 microg toxin/g body weight) anatomopathological studies supported a process of apoptonecrosis established after 24h, which was suggested to proceed by the evolution of some of the proteins after 2h contamination.

siRNA relieves chronic neuropathic pain.

Double stranded, short interfering RNAs (siRNA) of 21-22 nt length initiate a sequence-specific, post-trancriptional gene silencing in animals and plants known as RNA interference (RNAi). Here we show that RNAi can block a pathophysiological pain response and provide relief from neuropathic pain in a rat disease model by down regulating an endogenous, neuronally expressed gene. Rats, intrathecally infused with a 21 nt siRNA perfectly complementary to the pain-related cation-channel P2X3, showed diminished pain responses compared to missense (MS) siRNA-treated and untreated controls in models of both agonist-evoked pain and chronic neuropathic pain. This form of delivery caused no adverse effects in any of the animals receiving P2X3 siRNA, MS siRNA or vehicle. Molecular analysis of tissues revealed that P2X3 mRNA expressed in dorsal root ganglia, and P2X3 protein translocated into the dorsal horn of the spinal cord, were significantly diminished. These observations open a path toward use of siRNA as a genetic tool for drug target validation in the mammalian central nervous system, as well as for proof of concept studies and as therapeutic agents in man.

Human glioma cells transformed by IGF-I triple helix technology show immune and apoptotic characteristics determining cell selection for gene therapy of glioblastoma.

AIMS: Insulin-like growth factor type I (IGF-I) antisense cellular gene therapy of tumours is based on the following data: rat glioma or hepatoma cells transfected with the vector encoding IGF-I antisense cDNA lose their tumorigenicity and induce a tumour specific immune response involving CD8(+) T cells. Recently, using the IGF-I triple helix approach in studies of tumorigenicity, major histocompatibility complex class I (MHC-I) antigens were demonstrated in rat glioma transfected cells. This study used comparative IGF-I antisense and triple helix technologies in human primary glioma cells to determine the triple helix strategy that would be most appropriate for the treatment of glioblastoma. METHODS: The cells were transfected using the IGF-I triple helix expression vector, pMT-AG, derived from the pMT-EP vector. pMT-AG contains a cassette comprising a 23 bp DNA fragment transcribing a third RNA strand, which forms a triple helix structure within a target region of the human IGF-I gene. Using pMT-EP, vectors encoding MHC-I or B7 antisense cDNA were also constructed. RESULTS: IGF-I triple helix transfected glioma cells are characterised by immune and apoptotic phenomena that appear to be related. The expression of MHC-I and B7 in transfected cells (analysed by flow cytometry) was accompanied by programmed cell death (detected by dUTP fluorescein terminal transferase labelling of nicked DNA and electron microscopic techniques). Cotransfection of these cells with MHC-I and B7 antisense vectors suppressed the expression of MHC-I and B7, and was associated with a pronounced decrease in apoptosis. CONCLUSION: When designing an IGF-I triple helix strategy for the treatment of human glioblastoma, the transfected tumour cells should have the following characteristics: the absence of IGF-I, the presence of both MHC-I and B7 molecules, and signs of apoptosis.

The spherulites(TM): a promising carrier for oligonucleotide delivery.

Concentric multilamellar microvesicles, named spherulites(TM), were evaluated as an oligonucleotide carrier. Up to 80% oligonucleotide was encapsulated in these vesicles. The study was carried out on two different spherulite(TM) formulations. The spherulite(TM) size and stability characteristics are presented. Delivery of encapsulated oligonucleotide was performed on a rat hepatocarcinoma and on a lymphoblastoid T cell line, both expressing the luciferase gene. We showed that spherulites(TM) were able to transfect both adherent and suspension cell lines and deliver the oligonucleotide to the nucleus. Moreover, 48-62% luciferase inhibition was obtained in the rat hepatocarcinoma cell line when the antisense oligonucleotide targeted to the luciferase coding region was encapsulated at 500 nM concentration in spherulites(TM) of different compositions.

Alterations in tumorigenicity of embryonal carcinoma cells by IGF-I triple-helix induced changes in immunogenicity and apoptosis.

IGF-I antisense gene therapy has been applied successfully to animal models of glioma, hepatoma and teratocarcinoma. The antisense strategy has shown that tumor cells transfected with vectors encoding IGF-I antisense RNA lose tumorigenicity, become immunogenic and are associated with tumor specific immune response involving CD8+ lymphocytes. An IGF-I triple helix approach to gene therapy for glioma was recently described. The approach we have taken is to establish parameters of change using the IGF-I triple helix strategy. PCC-3 embryonal carcinoma cells derived from murine teratocarcinoma which express IGF-I were used as a model. The cells were transfected with vector which encodes an oligoribonucleotide that forms RNA-IGF-I DNA triple-helix structure. The triple-helix stops the production of IGF-I. Cells transfected in this manner underwent changes in phenotype and an increase in MHC-I and B-7 cell surface molecules. They also showed enhancement in the production of apoptotic cells (60-70%). The "triple helix" transfected cells lost the ability to induce tumor when injected subcutaneously in syngeneic 129 Sv mice. When co-transfected in vitro with expression vectors encoding both MHC-I and B-7 cDNA in antisense orientation, the "triple-helix" transfected cells were down-regulated in expression of MHC-I and B-7 and the number of apoptotic cells was significantly decreased. Injection of the doubly co-transfected cells into 129 Sv mice was associated with induction of teratocarcinoma. Comparison between antisense and triple-helix transfected cells strategies showed similar immunogenic and apoptotic changes. The findings suggest that triple-helix technology may offer a new clinical approach to treatement of tumors expressing IGF-I.

Recognition of hairpin-containing single-stranded DNA by oligonucleotides containing internal acridine derivatives.

Oligodeoxynucleotides with an internal intercalating agent have been targeted to single-stranded sequences containing hairpin structures. The oligonucleotide binds to nonadjacent single-stranded sequences on both sides of the hairpin structure in such a way as to form a three-way junction. The acridine derivative is inserted at a position that allows it to interact with the three-way junction. The melting temperature (Tm) of complexes formed between the hairpin-containing target and oligonucleotides containing one internal acridine derivative was higher than that obtained with the same target and an unmodified oligonucleotide (DeltaTm = +13 degrees C). The internal acridine provided the oligonucleotide with a higher affinity than covalent attachment to the 5' end. Oligonucleotides could also be designed to recognize a hairpin-containing single-stranded nucleic acid by formation of Watson-Crick hydrogen bonds with a single-stranded part and Hoogsteen hydrogen bonds with the stem of the hairpin. An internal acridine derivative was introduced at the junction between the two domains, the double helix domain with Watson-Crick base pairs and the triple helix domain involving Hoogsteen base triplets in the major groove of the hairpin stem. Oligonucleotides with an internal acridine or an acridine at their 5' end have similar binding affinities for the stem-loop-containing target. The bis-modified oligonucleotide containing two acridines, one at the 5' end and one at an internal site, did not exhibit a higher affinity than the oligonucleotides with only one intercalating agent. The design of oligonucleotides with an internal intercalating agent might be of interest to control gene expression through recognition of secondary structures in single-stranded targets.

Inhibition of gene expression by anti-sense C-5 propyne oligonucleotides detected by a reporter enzyme.

Using a reporter plasmid containing the luciferase gene under the control of the insulin-like growth factor 1 (IGF-1) promoter region [including its 5' untranslated region (UTR)], we demonstrate that a 17-mer oligophosphorothioate containing C-5 propyne pyrimidines is able to inhibit luciferase gene expression in the nanomolar concentration range when the anti-sense oligonucleotide is targeted either to a coding sequence in the luciferase gene or to the 5' UTR of the gene for IGF-1. Inhibition was obtained independently of whether the plasmid and the anti-sense oligonucleotide were co-transfected or transfected separately into hepatocarcinoma cells. However, the efficiency of inhibition by the anti-sense oligonucleotides was 10-fold greater in the first case. The unmodified oligophosphorothioate targeted to the 5' UTR of IGF-1 did not inhibit luciferase gene expression at a 100-fold higher concentration unless its length was increased from 17 to 21 nt, in which case an inhibition of gene expression was obtained and an IC50 of 200 nM was observed.

Triple helix formation with purine-rich phosphorothioate-containing oligonucleotides covalently linked to an acridine derivative.

Purine-rich (GA)- and (GT)-containing oligophosphorothioates were investigated for their triplex-forming potential on a 23 bp DNA duplex target. In our system, GA-containing oligophosphorothioates (23mer GA-PS) were capable of triplex formation with binding affinities lower than (GA)-containing oligophosphodiesters (23mer GA-PO). The orientation of the third strand 23mers GA-PS and GA-PO was antiparallel to the purine strand of the duplex DNA target. In contrast, (GT)-containing oligophosphorothioates (23mer GT-PS) did not support triplex formation in either orientation, whereas the 23mer GT-PO oligophosphodiester demonstrated triplex formation in the antiparallel orientation. GA-PS oligonucleotides, in contrast to GT-PS oligonucleotides, were capable of self-association, but these self-associated structures exhibited lower stabilities than those formed with GA-PO oligonucleotides, suggesting that homoduplex formation (previously described for the 23mer GA-PO sequence by Noonberg et al.) could not fully account for the decrease in triplex stability when phosphorothioate linkages were used. The 23mer GA-PS oligonucleotide was covalently linked via its 5'-end to an acridine derivative (23mer Acr-GA-PS). In the presence of potassium cations, this conjugate demonstrated triplex formation with higher binding affinity than the unmodified 23mer GA-PS oligonucleotide and even than the 23mer GA-PO oligonucleotide. A (GA)-containing oligophosphodiester with two phosphorothioate linkages at both the 5'- and 3'-ends exhibited similar binding affinity to duplex DNA compared with the unmodified GA-PO oligophosphodiester. This capped oligonucleotide was more resistant to nucleases than the GA-PO oligomer and thus represents a good alternative for ex vivo applications of (GA)-containing, triplex-forming oligonucleotides, allowing a higher binding affinity for its duplex target without rapid cellular degradation.

Inhibition of replication initiation by triple helix-forming oligonucleotides.

Oligonucleotide-directed triple helix formation constitutes a new approach to block gene expression via transcription inhibition. In addition triple helices might inhibit replication. We have examined the capacity of triple helix-forming oligonucleotides to inhibit the initiation of replication on a single-stranded DNA template using T7 DNA polymerase (Sequenase). We show that triple helix formation at the primer initiation site efficiently inhibits DNA polymerization, by preventing binding of the polymerase. The effect is dependent on the distance between the 3'-end of the primer and the triple helix boundary. Inhibition becomes ineffective when this distance is greater than 3 nucleotides. The presence of three base-pairs outside the triple-helical region on the 3'-side of the primer is therefore sufficient to allow for initiation of DNA replication.

Triplex formation with alpha anomers of purine-rich and pyrimidine-rich oligodeoxynucleotides.

Nuclease-resistant alpha anomers of pyrimidine-rich CT- and purine-rich GA- and GT-containing oligonucleotides were investigated for their triplex-forming potential and compared with their corresponding nuclease-sensitive beta anomers. Both 23mer CT-alpha and 23mer CT-beta had quite similar triplex binding affinities. Synthetic 23mer GT-alpha oligonucleotides were capable of triplex formation with binding affinities slightly lower than corresponding 23mer GT-beta oligonucleotides. The orientation of third strand GT-alpha binding was parallel to the purine strand of the duplex DNA target, whereas the orientation of third strand GT-beta binding was found to be antiparallel. Triplex formation with both GT oligonucleotides showed the typical dependence on magnesium and temperature. In contrast, 23mer GA-alpha oligonucleotides did not support triplex formation in either orientation under a variety of experimental conditions, whereas the corresponding 23mer GA-beta oligonucleotides demonstrated strong triplex formation in the antiparallel orientation. GA-alpha oligonucleotides covalently conjugated to acridine were similarly unable to demonstrate triplex formation. GA-alpha oligonucleotides, in contrast to GT-alpha oligonucleotides, were capable of self-association, detectable by gel retardation and UV spectroscopy, but competing self-association could not fully account for the lack of triplex formation. Thus for in vivo triplex gene regulation strategies using GT oligonucleotides the non-natural alpha anomer may be a feasible alternative to the natural beta anomer, allowing for a comparable degree of triplex formation without rapid cellular degradation. However, alpha anomeric inversion does not appear to be a feasible alternative in applications involving GA oligonucleotides.

Effect of competing self-structure on triplex formation with purine-rich oligodeoxynucleotides containing GA repeats.

Competition between triplex formation with double-stranded DNA and oligonucleotide self-association was investigated in 23mer GA and GT oligonucleotides containing d(GA)5 or d(GT)5 repeats. Whereas triplex formation with GT oligonucleotides was diminished when temperature increased from 4 to 37 degrees C, triplex formation with GA oligonucleotides was enhanced when temperature increased within the same range due to the presence of competing intermolecular GA oligonucleotide self-structure. This self-structure was determined to be a homoduplex stabilized by the internal GA repeats. UV spectroscopy of these homoduplexes demonstrated a single sharp transition with rapid kinetics (Tm = 38.5-43.5 degrees C over strand concentrations of 0.5-4 microM, respectively, with transition enthalpy, delta H = -89 +/- 7 kcal/mol) in 10 mM MgCl2, 100 mM NaCl, pH 7.0. Homoduplex formation was strongly stabilized by multivalent cations (spermine > Mg2+ = Ca2+) and destabilized by low concentrations of monovalent cations (K+ = Li+ = Na+) in the presence of divalent cations. However, unlike GA or GT oligonucleotide-containing triplexes, the homoduplex formed even in the absence of multivalent cations, stabilized by only moderate concentrations of monovalent cations (Li+ > Na+ > K+). Through the development of multiple equilibrium states and the resulting depletion of free oligonucleotide, it was found that the presence of competing self-structure could decrease triplex formation under a variety of experimental conditions.

Recognition and cleavage of single-stranded DNA containing hairpin structures by oligonucleotides forming both Watson-Crick and Hoogsteen hydrogen bonds.

A new approach is described to design antisense oligonucleotides targeted against single-stranded nucleic acids containing hairpin structures by use of both Watson-Crick and Hoogsteen hydrogen bond interactions for recognition. The oligonucleotide has two different domains, one allowing double helix formation involving Watson-Crick base pairs and the other one forming a triple helix involving Hoogsteen-type base triplets in the major groove of a hairpin stem. Spectroscopic and gel retardation experiments provided evidence for such Watson-Crick/Hoogsteen (WC/H) recognition of hairpin structures in single-stranded DNA. An antisense oligonucleotide designed to form only Watson-Crick base pairs was unable to disrupt the stable stem structure of the target under conditions where the oligonucleotide designed with the Watson-Crick/Hoogsteen interactions could bind efficiently to the hairpin-containing target. The addition of one nucleotide to the oligonucleotide at the junction between the double helix and triple helix regions in WC/H complexes had an effect on stability which was dependent on the relative orientation of the Watson-Crick and Hoogsteen domains in the target. An oligodeoxynucleotide-phenanthroline conjugate targeted against such a hairpin-containing DNA fragment induced specific cleavage in the double-stranded stem. This WC/H approach may be useful in designing artificial regulators of gene expression.

Photochemically and chemically activatable antisense oligonucleotides: comparison of their reactivities towards DNA and RNA targets.

Dodecadeoxyribonucleotides derivatized with 1,10-phenanthroline or psoralen were targeted to the point mutation (G<-->U) in codon 12 of the Ha-ras mRNA. DNA and RNA fragments, 27 nucleotides in length, and containing the complementary sequence of the 12mers, were used to compare the reactivity of the activatable dodecamers (cleavage of the target by the phenanthroline-12mer conjugates; photo-induced cross-linking of psoralen-12mer conjugates to the target). The reactivity of the RNA with the dodecamers was weaker than that of the DNA target. With psoralen-substituted oligonucleotides, it was possible to obtain complete discrimination between the mutated target (which contained a psoralen-reactive T(U) in the 12th codon) and the normal target (which contained G at the same position). When longer Ha-ras RNA fragments were used as targets (120 and 820 nucleotides), very little reactivity was observed. Part of the reactivity could be recovered by using 'helper' oligonucleotides that hybridized to adjacent sites on the substrate. A 'helper' chain length greater than 13 was required to improve the reactivity of dodecamers. However, the dodecanucleotides induced RNase H cleavage of the target RNA in the absence of 'helper' oligonucleotide. Therefore, in the absence of the RNase H enzyme, long oligonucleotides are needed to compete with the secondary structures of the mRNA. In contrast, formation of a ternary complex oligonucleotide-mRNA-RNase H led to RNAT cleavage with shorter oligonucleotides.

Recognition and cleavage of hairpin structures in nucleic acids by oligodeoxynucleotides.

The possibility of designing antisense oligodeoxynucleotides complementary to non-adjacent single-stranded sequences containing hairpin structures was studied using a DNA model system. The structure and stability of complexes formed by a 17mer oligonucleotide with DNA fragments containing hairpin structures was investigated by spectroscopic measurements (melting curves) and chemical reactions (osmium tetroxide reaction, copper-phenanthroline cleavage). A three-way junction was formed when the oligonucleotide was bound to both sides of the hairpin structure. When the complementary sequences of the two parts of the oligonucleotide were separated by a sequence which could not form a hairpin, the oligonucleotide exhibited a slightly weaker binding than to the hairpin-containing target. An oligodeoxynucleotide-phenanthroline conjugate was designed to form Watson-Crick base pairs with two single-stranded regions flanking a hairpin structure in a DNA fragment. In the presence of Cu2+ ions and a reducing agent, two main cleavage sites were observed at the end of the duplex structure formed by the oligonucleotide-phenanthroline conjugate with its target sequence. Competition experiments showed that both parts of the oligonucleotide must be bound in order to observe sequence-specific cleavage. Cleavage was still observed with target sequences which could not form a hairpin, provided the reaction was carried out at lower temperatures. These results show that sequence-specific recognition and modification (cleavage) can be achieved with antisense oligonucleotides which bind to non-adjacent sequences in a single-stranded nucleic acid.

Stability of triple helices containing RNA and DNA strands: experimental and molecular modeling studies.

UV-absorption spectrophotometry and molecular modeling have been used to study the influence of the chemical nature of sugars (ribose or deoxyribose) on triple helix stability. For the Pyrimidine.purine* Pyrimidine motif, all eight combinations were tested with each of the three strands composed of either DNA or RNA. The chemical nature of sugars has a dramatic influence on triple helix stability. For each double helix composition, a more stable triple helix was formed when the third strand was RNA rather than DNA. No stable triple helix was detected when the polypurine sequence was made of RNA with a third strand made of DNA. Energy minimization studies using the JUMNA program suggested that interactions between the 2'-hydroxyl group of the third strand and the phosphates of the polypurine strand play an important role in determining the relative stabilities of triple-helical structures in which the polypyrimidine third strand is oriented parallel to the polypurine sequence. These interactions are not allowed when the third strand adopts an antiparallel orientation with respect to the target polypurine sequence, as observed when the third strand contains G and A or G and T/U. We show by footprinting and gel retardation experiments that an oligoribonucleotide containing G and A or G and U fails to bind double helical DNA, while the corresponding DNA oligomers form stable triple-helical complexes.