Inhibition of RNA Interference and Modulation of Transposable Element Expression by Cell Death in Drosophila

ABSTRACT Due to the limitations of effective treatments, avian influenza A H5N1 virus is the most lethal influenza virus strain that causes severe acute lung injury (ALI). To develop effective drugs ameliorating H5N1-induced ALI, we explore an RNA interference (RNAi) screening method to monitor changes in cell death induced by H5N1 infection. We performed RNAi screening on 19,424 genes in A549 lung epithelial cells and examined cell death induced by H5N1 infection. These screens identified 1,137 host genes for which knockdown altered cell viability by over 20%. DrugBank searches of these 1,137 host genes identified 146 validated druggable target genes with 372 drug candidates. We obtained 104 commercially available drugs with 65 validated target genes and examined their improvement of cell viability following H5N1 infection. We identified 28 drugs that could significantly recover cell viability following H5N1 infection and tested 10 in an H5N1-induced-ALI mouse model. The neurological drug ifenprodil and the anticancer drug flavopiridol markedly decreased leukocyte infiltration and lung injury scores in infected mouse lungs, significantly ameliorated edema in infected mouse lung tissues, and significantly improved the survival of H5N1-infected mice. Ifenprodil is an antagonist of the N-methyl-d-aspartate (NMDA) receptor, which is linked to inflammation and lung injury. Flavopiridol is an inhibitor of cyclin-dependent kinase 4 (CDK4), which is linked to leukocyte migration and lung injury. These results suggest that ifenprodil and flavopiridol represent novel remedies against potential H5N1 epidemics in addition to their proven indications. Furthermore, our strategy for identifying repurposable drugs could be a general approach for other diseases. IMPORTANCE Drug repurposing is a quick and economical strategy for developing new therapies with approved drugs. H5N1 is a highly pathogenic avian influenza virus subtype that can cause severe acute lung injury (ALI) and a high mortality rate due to limited treatments. The use of RNA interference (RNAi) is a reliable approach to identify essential genes in diseases. In most genomewide RNAi screenings, virus replication is the readout of interference. Since H5N1 virus infection could induce significant cell death and the percentage of cell death is associated with virus lethality, we designed a genomewide RNAi screening method to identify repurposable drugs against H5N1 virus with cell death as the readout. We discovered that the neurological drug ifenprodil and the anticancer drug flavopiridol could effectively ameliorate murine ALI after influenza A H5N1 virus infection, suggesting that they might be novel remedies for H5N1 virus-induced ALI in addition to the traditional indications.

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666. Lentiviral RNA Interference of CHOP Protects Dopaminergic Cells Against Human Alpha-Synuclein-Induced Cell Death

Molecular Therapy ◽

10.1016/s1525-0016(16)40069-9 ◽

2008 ◽

Vol 16 ◽

pp. S248-S249

Keyword(s):

Cell Death ◽

Rna Interference ◽

Alpha Synuclein ◽

Dopaminergic Cells

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Multiple insertions of COIN , a novel maize Foldback transposable element, in the Conring gene cause a spontaneous progressive cell death phenotype

The Plant Journal ◽

10.1111/tpj.14945 ◽

2020 ◽

Vol 104 (3) ◽

pp. 581-595

Author(s):

Saet‐Byul Kim ◽

Shailesh Karre ◽

Qingyu Wu ◽

Minkyu Park ◽

Emily Meyers ◽

...

Keyword(s):

Cell Death ◽

Transposable Element ◽

Cell Death Phenotype

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Lentiviral (HIV)-based RNA interference screen in human B-cell receptor regulatory networks reveals MCL1-induced oncogenic pathways

Blood ◽

10.1182/blood-2007-09-110601 ◽

2008 ◽

Vol 111 (3) ◽

pp. 1665-1676 ◽

Cited By ~ 11

Author(s):

Antonio Ruiz-Vela ◽

Mohit Aggarwal ◽

Paloma de la Cueva ◽

Cezary Treda ◽

Beatriz Herreros ◽

...

Keyword(s):

Cell Death ◽

Rna Interference ◽

Programmed Cell Death ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

B Cell Lymphomas ◽

Clonal Deletion ◽

A Genome ◽

Human B Cell

AbstractAberrant inhibition of B-cell receptor (BCR)-induced programmed cell death pathways is frequently associated with the development of human auto-reactive B-cell lymphomas. Here, we integrated loss-of-function, genomic, and bioinformatics approaches for the identification of oncogenic mechanisms linked to the inhibition of BCR-induced clonal deletion pathways in human B-cell lymphomas. Lentiviral (HIV)-based RNA interference screen identified MCL1 as a key survival molecule linked to BCR signaling. Loss of MCL1 by RNA interference rendered human B-cell lymphomas sensitive to BCR-induced programmed cell death. Conversely, MCL1 overexpression blocked programmed cell death on BCR stimulation. To get insight into the mechanisms of MCL1-induced survival and transformation, we screened 41 000 human genes in a genome-wide gene expression profile analysis of MCL1-overexpressing B-cell lymphomas. Bioinformatic gene network reconstruction illustrated reprogramming of relevant oncoproteins within β-catenin–T-cell factor signaling pathways induced by enforced MCL1 expression. Overall, our findings not only illustrate MCL1 as an aberrantly expressed reprogramming oncoprotein in follicular lymphomas but also highlight MCL1 as key therapeutic target.

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The knockdown of Ha-GRIM-19 by RNA interference induced programmed cell death

Amino Acids ◽

10.1007/s00726-010-0824-8 ◽

2010 ◽

Vol 42 (4) ◽

pp. 1297-1307 ◽

Cited By ~ 10

Author(s):

Du-Juan Dong ◽

Peng-Cheng Liu ◽

Jin-Xing Wang ◽

Xiao-Fan Zhao

Keyword(s):

Cell Death ◽

Rna Interference ◽

Programmed Cell Death

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p400 Is Required for E1A to Promote Apoptosis

Journal of Biological Chemistry ◽

10.1074/jbc.m414564200 ◽

2005 ◽

Vol 280 (23) ◽

pp. 21915-21923 ◽

Cited By ~ 38

Author(s):

Andrew V. Samuelson ◽

Masako Narita ◽

Ho-Man Chan ◽

Jianping Jin ◽

Elisa de Stanchina ◽

...

Keyword(s):

Cell Death ◽

Rna Interference ◽

Chromatin Remodeling ◽

Retinoblastoma Protein ◽

Cellular Proteins ◽

Creb Binding Protein ◽

Adenovirus E1a ◽

Chromatin Remodeling Complex ◽

Cellular Machinery ◽

Promote Cell Death

The adenovirus E1A oncoprotein promotes proliferation and transformation by binding cellular proteins, including members of the retinoblastoma protein family, the p300/CREB-binding protein transcriptional coactivators, and the p400-TRRAP chromatin-remodeling complex. E1A also promotes apoptosis, in part, by engaging the ARF-p53 tumor suppressor pathway. We show that E1A induces ARF and p53 and promotes apoptosis in normal fibroblasts by physically associating with the retinoblastoma protein and a p400-TRRAP complex and that its interaction with p300 is largely dispensable for these effects. We further show that E1A increases p400 expression and, conversely, that suppression of p400 using stable RNA interference reduces the levels of ARF, p53, and apoptosis in E1A-expressing cells. Therefore, whereas E1A inactivates the retinoblastoma protein, it requires p400 to efficiently promote cell death. These results identify p400 as a regulator of the ARF-p53 pathway and a component of the cellular machinery that couples proliferation to cell death.

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RNA Interference-Mediated Silencing of Ornithine Decarboxylase and Spermidine Synthase Genes in Trypanosoma brucei Provides Insight into Regulation of Polyamine Biosynthesis

Eukaryotic Cell ◽

10.1128/ec.00047-09 ◽

2009 ◽

Vol 8 (5) ◽

pp. 747-755 ◽

Cited By ~ 36

Author(s):

Yanjing Xiao ◽

Diane E. McCloskey ◽

Margaret A. Phillips

Keyword(s):

Cell Death ◽

Rna Interference ◽

Trypanosoma Brucei ◽

Ornithine Decarboxylase ◽

Cellular Response ◽

Sleeping Sickness ◽

Spermidine Synthase ◽

African Sleeping Sickness ◽

Polyamine Biosynthesis ◽

Insight Into

ABSTRACT Polyamine biosynthesis is a drug target for the treatment of African sleeping sickness; however, mechanisms regulating the pathway in Trypanosoma brucei are not well understood. Recently, we showed that RNA interference (RNAi)-mediated gene silencing or the inhibition of S-adenosylmethionine decarboxylase (AdoMetDC) led to the upregulation of the AdoMetDC activator, prozyme, and ornithine decarboxylase (ODC) proteins. To determine if this regulatory response is specific to AdoMetDC, we studied the effects of the RNAi-induced silencing of the spermidine synthase (SpdSyn) and ODC genes in bloodstream form T. brucei. The knockdown of either gene product led to the depletion of the polyamine and trypanothione pools and to cell death. Decarboxylated AdoMet levels were elevated, while AdoMet was not affected. There was no significant effect on the protein levels of other polyamine pathway enzymes. The treatment of parasites with the ODC inhibitor α-difluoromethylornithine gave similar results to those observed for ODC knockdown. Thus, the cellular response to the loss of AdoMetDC activity is distinctive, suggesting that AdoMetDC activity controls the expression levels of the other spermidine biosynthetic enzymes. RNAi-mediated cell death occurred more rapidly for ODC than for SpdSyn. Further, the ODC RNAi cells were rescued by putrescine, but not spermidine, suggesting that the depletion of both putrescine and spermidine is more detrimental than the depletion of spermidine alone. This finding may contribute to the effectiveness of ODC as a target for the treatment of African sleeping sickness, thus providing important insight into the mechanism of action of a key antitrypanosomal agent.

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RNA Silencing of Aurora Kinase Genes Sensitizes Human Leukemia Cells to the Ara-C-Induced Cell Death Via Caspase-Independent Pathway.

Blood ◽

10.1182/blood.v106.11.2468.2468 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2468-2468

Author(s):

Yoo-Hong Min ◽

Jin Koo Lee ◽

Ju In Eom ◽

In Hae Park ◽

Sun Young Park ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Rna Interference ◽

Acute Leukemia ◽

Caspase 3 ◽

Aurora Kinase ◽

Leukemia Cells ◽

Cell Cycle Distribution ◽

U937 Cells ◽

U937 Leukemia Cells

Abstract Aurora kinases play critical roles in chromosome segregation and cell division. Furthermore, Aurora A and B kinases are both frequently amplified and overexpressed in a various kinds of human cancers including leukemia disorders. To address its possibility as a therapeutic target for leukemia, we employed the RNA interference technique to knockdown Aurora kinase gene expression and analyzed its effect on the cell cycle distribution and the chemotherapy-induced cell death in leukemia cells. The expression of Aurora kinase A (AURKA) and Aurora kinase B (AURKB) was observed in all the leukemia cell lines evaluated and 15 out of 18 cultured leukemic cell specimens obtained from untreated patients with acute myeloid leukemia (AML). An arrest in the G(2)/M phase 24 h after specific knockdown of AURKA was shown in U937 leukemia cells. Following the silencing of AURKA, we observed a striking increase in the proportion of cells in the S phase, tetraploid state, and showing multi-nucleated morphologic changes. Cell death was observed in 17.0 ± 0.9% and 20.1 ± 2.5% of U937 cells after AURKA and AURKB silencing, respectively. The proportion of cells in the subG(0)/G(1) phase was 23% and 11% with AURKA and AURKB silencing, respectively. Although the disruption of mitochondrial membrane potential was observed after AURKA or AURKB silencing in U937 cells, the cleavage of caspase-3, -8, -9, and PARP was not observed in the apoptotic cells. Pretreatment of U937 cells with the caspase-3 inhibitor, DEVD-CHO, did not abrogate the AURKA or AURKB silencing-induced cell death. The U937 leukemia cells are resistant to Ara-C-induced cell death. However, we observed a striking synergistic enhancement of the cytotoxicity of Ara-C (10 μM), an important antimetabolic chemotherapeutic agent used for acute leukemia, by the RNA interference-mediated knockdown of AURKA and AURKB through a caspase-independent manner in U937 cells. Collectively, we demonstrate that the specific inhibition of either AURKA or AURKB can potentially regulate the cell cycle distribution of leukemia cells and induce synergistic interaction with Ara-C for inducing the cell death through the mechanism of mitotic catastrophe. These results indicate that Aurora kinase inhibition provides a novel approach for the treatment of human acute leukemia.

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Image-Based RNA Interference Screening Identifies Microenvironmental Signals Supporting Primary Acute Lymphoblastic Leukemia Cell Survival.

Blood ◽

10.1182/blood.v120.21.2348.2348 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2348-2348

Author(s):

Beat C Bornhauser ◽

Jeannette Boutter ◽

Peter Horvath ◽

Martin Stanulla ◽

Jean-Pierre Bourquin

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Rna Interference ◽

Cell Survival ◽

Candidate Genes ◽

Stromal Cells ◽

Leukemia Cell ◽

Leukemia Cells ◽

Down Regulation ◽

Stromal Support

Abstract Abstract 2348 Leukemia cells are critically dependent upon interactions with the microenvironment in the bone marrow and at extramedullary sanctuary sites, which is likely to provide a protective mechanism to escape chemotherapy. In vitro, co-culture of primary ALL cells on human bone marrow derived mesenchymal stromal cells (MSCs) provides survival cues allowing long-term cultures. In contrast, primary ALL cells rapidly undergo cell death when cultured without stromal support. We developed an automated microscopy-based approach to identify pro-survival signals by RNA interference of candidate genes in MSCs and subsequent evaluation of leukemia cell survival, enabling us to functionally profile primary leukemia cells. We took advantage of our leukemia xenograft system as a renewable source of well characterized samples derived from cases with very high risk (VHR) ALL, which were selected based on clinical resistance to chemotherapy. Based on gene expression and cell surface proteomic data that we had obtained from both cellular compartments, we generated a customized siRNA library for 110 candidate genes with a potential function in stromal support. Primary ALL cells were seeded on reversely transfected MSC cells and ALL cell viability was assessed with a fluorescent vital dye after 6 days. Image analysis and machine learning algorithms were developed for quantification of surviving ALL cells on top of MSC. Evaluating three VHR-ALL cases we observed a strong decrease of viability when interfering with the expression of 14 candidate genes in 2 out of 3 patients. Interestingly, in validation studies with 7 additional cases, the pattern of dependence on the genes tested were confirmed to be only partially overlapping between patients, indicating the existence of functional differences in distinct subsets. As a proof of concept, we could show that down-regulation of VCAM1 or the VEGF pathway in MSCs decreased ALL survival supporting earlier studies. Concomitantly, inhibitors of VEGF signalling recapitulated ALL cell viability decrease for patients that showed to be dependent on VEGFC expression in MSCs. One of the strongest effects on ALL survival was achieved by down-regulation of the membrane protein Basigin (CD147). Specifically, 13 out of 17 ALL cases were affected by the modulation of Basigin on MSC level. Basigin has been implicated in cell signalling, in interactions with extracellular matrix and serves as chaperone to different membrane carrier proteins. Among putative Basigin interactors we identified the heteromeric amino acid transporter SLC3A2 (CD98) to be required for ALL survival in the same set of ALL cases. The down-regulation of Basigin, SLC3A2 or both together in MSC cells induces an increase in ROS in ALL cells resulting in apoptotic cell death, which indicates that Basigin/SLC3A2 function is important for the integrity of ALL cell metabolism in this model of the leukemia niche. We are now investigating which metabolites are implicated in the mechanism of action. Taken together, we have established a robust platform for systematic functional investigation of primary ALL survival in a 2-D model of the microenvironment and obtained evidence for patient-specific dependence of leukemia cell survival on stromal support. Critical interactions between ALL cells and bone marrow stromal cells can be identified with this approach, which will be useful for unbiased higher throughput screening and combinatorial testing. This platform will also be of great interest for preclinical drug profiling on clinically relevant patients samples in the context of protective bone marrow signals. Disclosures: No relevant conflicts of interest to declare.

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Phosphorylation of the Ndc80 complex protein, HEC1, by Nek2 kinase modulates chromosome alignment and signaling of the spindle assembly checkpoint

Molecular Biology of the Cell ◽

10.1091/mbc.e11-01-0012 ◽

2011 ◽

Vol 22 (19) ◽

pp. 3584-3594 ◽

Cited By ~ 45

Author(s):

Randy Wei ◽

Bryan Ngo ◽

Guikai Wu ◽

Wen-Hwa Lee

Keyword(s):

Cell Death ◽

Rna Interference ◽

Molecular Mechanism ◽

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Distribution Characteristic ◽

Checkpoint Signaling ◽

Chromosome Alignment ◽

Ndc80 Complex ◽

Complex Protein

The spindle assemble checkpoint (SAC) is critical for accurate chromosome segregation. Hec1 contributes to chromosome segregation in part by mediating SAC signaling and chromosome alignment. However, the molecular mechanism by which Hec1 modulates checkpoint signaling and alignment remains poorly understood. We found that Hec1 serine 165 (S165) is preferentially phosphorylated at kinetochores. Phosphorylated Hec1 serine 165 (pS165) specifically localized to kinetochores of misaligned chromosomes, showing a spatiotemporal distribution characteristic of SAC molecules. Expressing an RNA interference (RNAi)-resistant S165A mutant in Hec1-depleted cells permitted normal progression to metaphase, but accelerated the metaphase-to-anaphase transition. The S165A cells were defective in Mad1 and Mad2 localization to kinetochores, regardless of attachment status. These cells often entered anaphase with lagging chromosomes and elicited increased segregation errors and cell death. In contrast, expressing S165E mutant in Hec1-depleted cells triggered defective chromosome alignment and severe mitotic arrest associated with increased Mad1/Mad2 signals at prometaphase kinetochores. A small portion of S165E cells eventually bypassed the SAC but showed severe segregation errors. Nek2 is the primary kinase responsible for kinetochore pS165, while PP1 phosphatase may dephosphorylate pS165 during SAC silencing. Taken together, these results suggest that modifications of Hec1 S165 serve as an important mechanism in modulating SAC signaling and chromosome alignment.

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