Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential ...delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem-loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design.
Effective delivery of siRNA (small interfering RNA) into the cells requires the translocation of siRNA into the cytosol. One potential delivery strategy uses cell-delivery peptides that facilitate ...this step. In the present paper, we describe the characterization of an amphipathic peptide that mediates the uptake of non-covalently bound siRNA into cells and its subsequent release into the cytosol. Biophysical characterization of peptide and peptide/siRNA mixtures at neutral and lysosomal (acidic) pH suggested the formation of α-helical structure only in endosomes and lysosomes. Surprisingly, even though the peptide enhanced the uptake of siRNA into cells, no direct interaction between siRNA and peptide was observed at neutral pH by isothermal titration calorimetry. Importantly, we show that peptide-mediated siRNA uptake occurred through endocytosis and, by applying novel endosomal-escape assays and cell-fractionation techniques, we demonstrated a pH-dependent alteration in endosome and lysosome integrity and subsequent release of siRNA and other cargo into the cytosol. These results indicate a peptide-mediated siRNA delivery through a pH-dependent and conformation-specific interaction with cellular membranes and not with the cargo.
To determine the impact of tumor progression on the reversibility of Neu-induced tumorigenesis, we have used the tetracycline regulatory system to conditionally express activated Neu in the mammary ...epithelium of transgenic mice. When induced with doxycycline, bitransgenic MMTV-rtTA/TetO-NeuNT mice develop multiple invasive mammary carcinomas, essentially all of which regress to a clinically undetectable state following transgene deinduction. This demonstrates that Neu-initiated tumorigenesis is reversible. Strikingly, extensive lung metastases arising from Neu-induced mammary tumors also rapidly and fully regress following the abrogation of Neu expression. However, despite the near universal dependence of both primary tumors and metastases on
Neu transgene expression, most animals bearing fully regressed Neu-induced tumors ultimately develop recurrent tumors that have progressed to a Neu-independent state.
Aberrant activation of Wnt signaling is oncogenic and has been implicated in a variety of human cancers. We have developed a doxycycline-inducible Wnt1 transgenic mouse model to determine the ...dependence of established mammary adenocarcinomas on continued Wnt signaling. Using this model we show that targeted down-regulation of the Wnt pathway results in the rapid disappearance of essentially all Wnt-initiated invasive primary tumors as well as pulmonary metastases. Tumor regression does not require p53 and occurs even in highly aneuploid tumors. However, despite the dependence of primary mammary tumors and metastases on continued Wnt signaling and the dispensability of p53 for tumor regression, we find that a substantial fraction of tumors progress to a Wnt-independent state and that p53 suppresses this process. Specifically, loss of one p53 allele dramatically facilitates the progression of mammary tumors to a Wnt1-independent state both by impairing the regression of primary tumors following doxycycline withdrawal and by promoting the recurrence of fully regressed tumors in the absence of doxycycline. Thus, although p53 itself is dispensable for tumor regression, it nevertheless plays a critical role in the suppression of tumor recurrence. Our findings demonstrate that although even advanced stages of epithelial malignancy remain dependent upon continued Wnt signaling for maintenance and growth, loss of p53 facilitates tumor escape and the acquisition of oncogene independence.
Asthma is a chronic lung disease exhibiting airway obstruction, hyperresponsiveness, and inflammation, characterized by the infiltration of eosinophils into the airways and the underlying tissue. ...Prolonged eosinophilic inflammation depends on the balance between the cell's inherent tendency to undergo apoptosis and the local eosinophil-viability enhancing activity. TRAIL, a member of the TNF family, induces apoptosis in most transformed cells; however, its role in health and disease remains unknown. To test the hypothesis that Ag-induced inflammation is associated with TRAIL/TRAIL-R interactions, we used a segmental Ag challenge (SAC) model in ragweed-allergic asthmatics and nonasthmatic patients and analyzed bronchoalveolar lavage (BAL) material for 2 wk. In asthmatic patients, the level of TRAIL in BAL fluid dramatically increased 24 h after SAC, which significantly correlated with BAL eosinophil counts. Immunohistochemical analysis of bronchial biopsies from asthmatic patients demonstrated that TRAIL staining was increased in epithelial, airway smooth muscle, and vascular smooth muscle cells and throughout the interstitial tissue after SAC. This was confirmed by quantitative immunocytochemical image analysis of BAL eosinophils and alveolar macrophages, which demonstrated that expression levels of TRAIL and DcR2 increased, whereas expression levels of the TRAIL-Rs DR4 and DR5 decreased in asthmatic subjects after SAC. We also determined that TRAIL prolongs eosinophil survival ex vivo. These data provide the first in vivo evidence that TRAIL expression is increased in asthmatics following Ag provocation and suggest that modulation of TRAIL and TRAIL-R interactions may play a crucial role in promoting eosinophil survival in asthma.
There are many opportunities to use macromolecules, such as peptides and oligonucleotides, for intracellular applications. Despite this, general methods for delivering these molecules to the cytosol ...in a safe and efficient manner are not available. Efforts to develop a variety of intracellular drug delivery systems such as viral vectors, lipoplexes, nanoparticles, and amphiphilic peptides have been made, but various challenges such as delivery efficiency, toxicity, and controllability remain. A central challenge is the ability to selectively perturb, not destroy, the membrane to facilitate cargo introduction. Herein, we describe our efforts to design and characterize peptides that form pores inside membranes at acidic pH, so-called pH-switchable pore formation (PSPF) peptides, as a potential means for facilitating cargo translocation through membranes. Consistent with pore formation, these peptides exhibit low-pH-triggered selective release of ATP and miRNA, but not hemoglobin, from red blood cells. Consistent with these observations, biophysical studies (tryptophan fluorescence, circular dichroism, size-exclusion chromatography, analytical ultracentrifugation, and attenuated total reflectance Fourier transformed infrared spectroscopy) show that decreased pH destabilizes the PSPF peptides in aqueous systems while promoting their membrane insertion. Together, these results suggest that reduced pH drives insertion of PSPF peptides into membranes, leading to target-specific escape through a proposed pore formation mechanism.
The insulin receptor substrate-1 (IRS-1) is one of the major substrates of both the insulin and IGF-I receptors and is generally
localized in the cytosol/membrane fraction of the cell. We show here ...that a substantial fraction of IRS-1 is translocated
to the nucleus in mouse embryo fibroblasts (MEF) expressing the simian virus 40 T antigen. Nuclear translocation of IRS-1
occurs also in MEF stimulated with IGF-I or in MEF expressing the oncogene v-src. Nuclear translocation of IRS-1 can be demonstrated
by confocal microscopy, immunohistochemistry, or subcellular fractionation. An antibody to IRS-1 immunoprecipitates from nuclear
fractions (but not from cytosolic fractions) the upstream binding factor, which is a key regulator of RNA polymerase I activity
and ribosomal RNA (rRNA) synthesis. In agreement with this finding, in 32D murine hemopoietic cells, nuclear translocation
of IRS-1 correlates with a markedly increased rRNA synthesis. Our experiments suggest that nuclear IRS-1 may play a specialized
role in rRNA synthesis and/or processing.
Adenosine A2A receptors are predominantly localized on striatopallidal gamma-aminobutyric acid (GABA) neurons, where they are colocalized with dopamine D2 receptors and are involved in the regulation ...of movement. Adenosine A2A receptor antagonists have been evaluated as a novel treatment for Parkinson׳s disease and have demonstrated efficacy in a broad spectrum of pharmacological and toxicological rodent and primate models. Fewer studies have been performed to evaluate the efficacy of adenosine A2A receptor antagonists in genetic models of hypodopaminergic states. SCH 412348 is a potent and selective adenosine A2A receptor antagonist that shows efficacy in rodent and primate models of movement disorders. Here we evaluated the effects of SCH 412348 in the MitoPark mouse, a genetic model that displays a progressive loss of dopamine neurons. The dopamine cell loss is associated with a profound akinetic phenotype that is sensitive to levodopa (l-dopa). SCH 412348 (0.3–10mg/kg administered orally) dose dependently increased locomotor activity in the mice. Moreover, SCH 412348 retained its efficacy in the mice as motor impairment progressed (12–22 weeks of age), demonstrating that the compound was efficacious in mild to severe Parkinson׳s disease–like impairment in the mice. Additionally, SCH 412348 fully restored lost functionality in a measure of hind limb bradykinesia and partially restored functionality in a rotarod test. These findings provide further evidence of the anti-Parkinsonian effects of selective adenosine A2A receptor antagonists and predict that they will retain their efficacy in both mild and severe forms of motor impairment.
Adenosine A sub(2A) receptors are predominantly localized on striatopallidal gamma-aminobutyric acid (GABA) neurons, where they are colocalized with dopamine D sub(2) receptors and are involved in ...the regulation of movement. Adenosine A sub(2A) receptor antagonists have been evaluated as a novel treatment for Parkinson's disease and have demonstrated efficacy in a broad spectrum of pharmacological and toxicological rodent and primate models. Fewer studies have been performed to evaluate the efficacy of adenosine A sub(2A) receptor antagonists in genetic models of hypodopaminergic states. SCH 412348 is a potent and selective adenosine A sub(2A) receptor antagonist that shows efficacy in rodent and primate models of movement disorders. Here we evaluated the effects of SCH 412348 in the MitoPark mouse, a genetic model that displays a progressive loss of dopamine neurons. The dopamine cell loss is associated with a profound akinetic phenotype that is sensitive to levodopa (l-dopa). SCH 412348 (0.3-10 mg/kg administered orally) dose dependently increased locomotor activity in the mice. Moreover, SCH 412348 retained its efficacy in the mice as motor impairment progressed (12-22 weeks of age), demonstrating that the compound was efficacious in mild to severe Parkinson's disease-like impairment in the mice. Additionally, SCH 412348 fully restored lost functionality in a measure of hind limb bradykinesia and partially restored functionality in a rotarod test. These findings provide further evidence of the anti-Parkinsonian effects of selective adenosine A sub(2A) receptor antagonists and predict that they will retain their efficacy in both mild and severe forms of motor impairment.
The insulin receptor substrate-1 (IRS-1) is one of the major substrates of both the insulin and IGF-I receptors and is generally localized in the cytosol/membrane fraction of the cell. We show here ...that a substantial fraction of IRS-1 is translocated to the nucleus in mouse embryo fibroblasts (MEF) expressing the simian virus 40 T antigen. Nuclear translocation of IRS-1 occurs also in MEF stimulated with IGF-I or in MEF expressing the oncogene v-src. Nuclear translocation of IRS-1 can be demonstrated by confocal microscopy, immunohistochemistry, or subcellular fractionation. An antibody to IRS-1 immunoprecipitates from nuclear fractions (but not from cytosolic fractions) the upstream binding factor, which is a key regulator of RNA polymerase I activity and ribosomal RNA (rRNA) synthesis. In agreement with this finding, in 32D murine hemopoietic cells, nuclear translocation of IRS-1 correlates with a markedly increased rRNA synthesis. Our experiments suggest that nuclear IRS-1 may play a specialized role in rRNA synthesis and/or processing.
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