Hagit Ashush

RNAi-Mediated Silencing of Nuclear Factor Erythroid-2–Related Factor 2 Gene Expression in Non–Small Cell Lung Cancer Inhibits Tumor Growth and Increases Efficacy of Chemotherapy

Nuclear factor erythroid-2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates the expression of electrophile and xenobiotic detoxification enzymes and efflux proteins, which confer cytoprotection against oxidative stress and apoptosis in normal cells. Loss of function mutations in the Nrf2 inhibitor, Kelch-like ECH-associated protein (Keap1), results in constitutive activation of Nrf2 function in non-small cell lung cancer. In this study, we show that constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by up-regulation of glutathione, thioredoxin, and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. RNAi-mediated reduction of Nrf2 expression in lung cancer cells induces generation of reactive oxygen species, suppresses tumor growth, and results in increased sensitivity to chemotherapeutic drug-induced cell death in vitro and in vivo. Inhibiting Nrf2 expression using naked siRNA duplexes in combination with carboplatin significantly inhibits tumor growth in a subcutaneous model of lung cancer. Thus, targeting Nrf2 activity in lung cancers, particularly those with Keap1 mutations, could be a promising strategy to inhibit tumor growth and circumvent chemoresistance.

siRNA Targeted to p53 Attenuates Ischemic and Cisplatin-Induced Acute Kidney Injury

Proximal tubule cells (PTCs), which are the primary site of kidney injury associated with ischemia or nephrotoxicity, are the site of oligonucleotide reabsorption within the kidney. We exploited this property to test the efficacy of siRNA targeted to p53, a pivotal protein in the apoptotic pathway, to prevent kidney injury. Naked synthetic siRNA to p53 injected intravenously 4 h after ischemic injury maximally protected both PTCs and kidney function. PTCs were the primary site for siRNA uptake within the kidney and body. Following glomerular filtration, endocytic uptake of Cy3-siRNA by PTCs was rapid and extensive, and significantly reduced ischemia-induced p53 upregulation. The duration of the siRNA effect in PTCs was 24 to 48 h, determined by levels of p53 mRNA and protein expression. Both Cy3 fluorescence and in situ hybridization of siRNA corroborated a short t(1/2) for siRNA. The extent of renoprotection, decrease in cellular p53 and attenuation of p53-mediated apoptosis by siRNA were dose- and time-dependent. Analysis of renal histology and apoptosis revealed improved injury scores in both cortical and corticomedullary regions. siRNA to p53 was also effective in a model of cisplatin-induced kidney injury. Taken together, these data indicate that rapid delivery of siRNA to proximal tubule cells follows intravenous administration. Targeting siRNA to p53 leads to a dose-dependent attenuation of apoptotic signaling, suggesting potential therapeutic benefit for ischemic and nephrotoxic kidney injury.

Rtp801, a suppressor of mTOR signaling, is an essential mediator of cigarette smoke-induced pulmonary injury and emphysema

Rtp801 (also known as Redd1, and encoded by Ddit4), a stress-related protein triggered by adverse environmental conditions, inhibits mammalian target of rapamycin (mTOR) by stabilizing the TSC1-TSC2 inhibitory complex and enhances oxidative stress–dependent cell death. We postulated that Rtp801 acts as a potential amplifying switch in the development of cigarette smoke–induced lung injury, leading to emphysema. Rtp801 mRNA and protein were overexpressed in human emphysematous lungs and in lungs of mice exposed to cigarette smoke. The regulation of Rtp801 expression by cigarette smoke may rely on oxidative stress–dependent activation of the CCAAT response element in its promoter. We also found that Rtp801 was necessary and sufficient for nuclear factor-κB (NF-κB) activation in cultured cells and, when forcefully expressed in mouse lungs, it promoted NF-κB activation, alveolar inflammation, oxidative stress and apoptosis of alveolar septal cells. In contrast, Rtp801 knockout mice were markedly protected against acute cigarette smoke–induced lung injury, partly via increased mTOR signaling, and, when exposed chronically to cigarette smoke, against emphysema. Our data support the notion that Rtp801 may represent a major molecular sensor and mediator of cigarette smoke–induced lung injury.

Ocular neuroprotection by siRNA targeting caspase-2.

Retinal ganglion cell (RGC) loss after optic nerve damage is a hallmark of certain human ophthalmic diseases including ischemic optic neuropathy (ION) and glaucoma. In a rat model of optic nerve transection, in which 80% of RGCs are eliminated within 14 days, caspase-2 was found to be expressed and cleaved (activated) predominantly in RGC. Inhibition of caspase-2 expression by a chemically modified synthetic short interfering ribonucleic acid (siRNA) delivered by intravitreal administration significantly enhanced RGC survival over a period of at least 30 days. This exogenously delivered siRNA could be found in RGC and other types of retinal cells, persisted inside the retina for at least 1 month and mediated sequence-specific RNA interference without inducing an interferon response. Our results indicate that RGC apoptosis induced by optic nerve injury involves activation of caspase-2, and that synthetic siRNAs designed to inhibit expression of caspase-2 represent potential neuroprotective agents for intervention in human diseases involving RGC loss.

ADHESION MOLECULES INVOLVED IN THE INTERACTIONS BETWEEN EARLY T CELLS AND MESENCHYMAL BONE MARROW STROMAL CELLS

We previously reported that among the various thymic lymphocyte subpopulations, the immature T cells preferentially adhere to mesenchymal bone marrow stroma. In the present study we examined the interactions between phenotypically defined populations of early T cells and stromal cell lines. The immature T cells segregated into two subpopulations according to their adhesive capacity. Whereas the majority of the adherent CD4-CD8- T cells were devoid of CD3/TCRalphabeta, most of the nonadherent CD4-CD8- T cells expressed this receptor complex. The adhesion of T cells to bone marrow stroma almost entirely was accounted for by CD49d and CD90, whereas that of adherent CD4-CD8- cells also was dependent on CD44, CD62L, and CD117 receptor. Blocking antibody combinations failed to reduce the adherence of these early T cells to less than 50% that of the control. On the other hand, the adhesion of unselected thymocytes to the stroma was reduced by 80%, using the same blocking antibodies. Therefore, the participation of additional molecules in the adhesion of early T cells to mesenchymal stroma is implicated. Comparison between the interaction of T cells with bone marrow mesenchymal or with thymus-derived epithelial stroma indicated that T cells utilize a selected set of adhesion molecules under each situation. Although CD49d and CD90 participated in both cases, CD11a, CD18, and CD2 receptors played a dominant role in the adhesion of T cells to thymic epithelium only. This study may point to a role of mesenchymal stroma in the regulation of early T-cell lymphopoiesis in the bone marrow.

Delayed Intrathecal Delivery of RhoA siRNA to the Contused Spinal Cord Inhibits Allodynia, Preserves White Matter, and Increases Serotonergic Fiber Growth

RhoA is a key regulator of the actin cytoskeleton that is upregulated after spinal cord injury (SCI). We analyzed different methods for siRNA delivery and developed siRNAs targeting RhoA (siRhoA) for SCI treatment. Cy 3.5-labeled siRNA delivered at the time of SCI yielded fluorescence in several cell types in the injury site. Intraspinal injections of chemically stabilized siRhoA into the spinal cord of injured rats reduced RhoA protein levels after 1 week and improved hindlimb walking over 6 weeks. To explore a less invasive route, we tested intrathecal injection of Cy 3.5-labeled siRNA via lumbar puncture 1 day after SCI, which resulted in robust uptake in the T9-T10 injury site. Lumbar injection of siRhoA 1 day after SCI reduced RhoA mRNA and protein levels 3 days after injection. Although siRhoA treatment did not yield significant improvement in locomotion, it decreased tactile hypersensitivity significantly compared to controls. Histological analysis at 8 weeks showed significant improvement in white matter sparing with siRhoA compared to control siRNA. siRhoA treatment also resulted in less accumulation of ED1+macrophages, increased PKC-γ immunoreactivity in the corticospinal tract rostral to the injury site, and increased serotonergic fiber growth 12 mm caudal to the contusion site. The ability of siRhoA to preserve white matter and promote serotonergic axonal regrowth caudal to the injury site is likely to suppress allodynia. This provides justification for considering clinical development of RhoA inhibitors to treat SCI sub-acutely to reduce allodynia, which occurs frequently in SCI patients.

The mesenchyme expresses T cell receptor mRNAs: Relevance to cell growth control

The mesenchyme plays a crucial regulatory role in organ formation and maintenance. However, comprehensive molecular characterization of these cells is lacking. We found unexpectedly that primary mesenchyme, as well as mesenchymal cell clones, express T cell receptor (TCR)αβ mRNAs, lacking the variable region. Immunological and genetic evidence support the expression of a corresponding TCRβ protein. Additionally, mRNAs encoding TCR complex components including CD3 and ζ chain are present. A relatively higher expression of the mesenchymal TCRβ mRNA by cultured mesenchymal cell clones correlates with fast growth, whereas poorly expressing cells are slow growers and are contact inhibited. The clones that express relatively higher amount of the TCR mRNA exhibit an increased capacity to form tumors in nude mice. However, the expression of this mRNA in the mesenchyme is not per se leading to tumorigenesis, as demonstrated by primary mesenchyme that does not form tumors in mice while expressing moderate amounts of the TCR transcripts. The expression of mesencymal TCRβ was confined to the G2/M phases of the cell cycle in the MBA-13 mesenchymal cell line. This cell cycle dependent expression, considered together with the correlation between growth properties and the level of TCR expression by cell clones, implies association of mesenchymal TCR with cell growth control.

siRNA-Mediated Knockdown of the mTOR Inhibitor RTP801 Promotes Retinal Ganglion Cell Survival and Axon Elongation by Direct and Indirect Mechanisms

PURPOSE To investigate, using in vivo and in vitro models, retinal ganglion cell (RGC) neuroprotective and axon regenerative effects and underlying mechanisms of siRTP801, a translatable small-interfering RNA (siRNA) targeting the mTOR negative regulator RTP801. METHODS Adult rats underwent optic nerve (ON) crush (ONC) followed by intravitreal siRTP801 or control siRNA (siEGFP) every 8 days, with Brn3a+ RGC survival, GFAP+ reactive gliosis, and GAP43+ regenerating axons analyzed immunohistochemically 24 days after injury. Retinal cultures, prepared from uninjured animals or 5 days after ONC to activate retinal glia, were treated with siRTP801/controls in the presence/absence of rapamycin and subsequently assessed for RGC survival and neurite outgrowth, RTP801 expression, glial responses, and mTOR activity. Conditioned medium was analyzed for neurotrophin titers by ELISA. RESULTS Intravitreal siRTP801 enabled 82% RGC survival compared to 45% with siEGFP 24 days after ONC, correlated with greater GAP43+ axon regeneration at 400 to 1200 μm beyond the ONC site, and potentiated the reactive GFAP+ Müller glial response. In culture, siRTP801 had a direct RGC neuroprotective effect, but required GFAP+ activated glia to stimulate neurite elongation. The siRTP801-induced neuroprotection was significantly reduced, but not abolished, by rapamycin. The siRTP801 potentiated the production and release of neurotrophins NGF, NT-3, and BDNF, and prevented downregulation of RGC mTOR activity. CONCLUSIONS The RTP801 knockdown promoted RGC survival and axon elongation after ONC, without increasing de novo regenerative sprouting. The neuroprotection was predominantly direct, with mTORC1-dependent and -independent components. Enhanced neurite/axon elongation by siRTP801 required the presence of activated retinal glia and was mediated by potentiated secretion of neurotrophic factors.

Therapeutic ultrasound induces apoptosis in p53 positive and p53 negative malignant hemopoietic cell lines

Abstract None of the modalities currently employed to induce apoptosis involves the use of ultrasound energy. We have demonstrated (Cancer Res. 60: 1014, 2000) that selected physical parameters utilized in therapeutic ultrasound (ULS) application resulting in cavitation process induce an apoptotic cell death. The aim of the present study was to investigate ULS application with rational selected physical parameters to optimize ultrasound cavitation- induced cell death and to determine whether apoptosis is involved. High intensity focussed ULS sonication was delivered with an induction of transient cavitation and intensity of 54 W/cm 2 to malignant T and B lymphocyte-derived cell lines expressing p53 as Raji, Jurkat and NALM-6 as well as myeloid leukemia cell lines p53 negative HL-60, K562, U937. Here we present evidence that much of the cell damage surviving ULS exposure appears to occur through an apoptotic mechanism. Morphological alterations of apoptotic cells involve nuclear fragmentation and apoptotic body formation. Positive identification of apoptotic cells was based also on the detection of nuclear DNA strand break and changes in the surface of treated cells undergoing apaptosis expressed by the breakup of phosphatidylserine from the inner to the outer side of the membrane layer. In summary, after ULS exposure malignant hemopoietic cell lines can be induced to undergo apoptosis by p53-dependent and p53-independent apoptotic pathways.