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Results for “Small interfering RNA”

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PubMed2006

Serotonin stimulates GnRH secretion through the c-Src-PLC gamma1 pathway in GT1-7 hypothalamic cells.

Serotonin is a neurotransmitter that alters the hypothalamic-pituitary-adrenal axis. To date, however, the molecular mechanisms underlying the role of serotonin in hormone secretion have remained largely unclear. In this study, we report that serotonin activates phospholipase C (PLC) gamma1 in an Src-dependent manner in hypothalamic GT1-7 cells, and that pretreatment with either 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazole [3, 4-d] pyrimidine, an Src-kinase inhibitor, or U73122, a PLC inhibitor, attenuates the serotonin-induced increase in calcium levels. Also, PLC gamma1 binds to c-Src through the Src-homology (SH) 223 domain upon serotonin treatment. Moreover, calcium increase is alleviated in the cells transientlyexpressing SH223 domain-deleted PLC gamma1 or lipase inactive mutant PLC gamma1, as compared with cells transfected with wild-type PLC gamma1. Furthermore, the inhibition of the activities of either PLC or Src results in a significant diminution of the serotonin-induced release of gonadotropin-releasing hormone (GnRH). In addition, the results of our small-interfering RNA experiment confirm that endogenous PLC gamma1 is a prerequisite for serotonin-mediated signaling pathways. Taken together, our findings demonstrate that serotonin stimulates the release of GnRH through the Src-PLC gamma1 pathway, via the modulation of intracellular calcium levels.

Animals↗

PubMed2006

DMRT-1 expression during NEC8 human embryonic carcinoma cell differentiation.

To elucidate the relationship between dsx and mab-3 related transcription factor 1 (Dmrt-1) and differentiation, alteration in mRNA levels during differentiation of NEC8 human embryonic carcinoma cells was investigated. After stimulation with 50 nM phorbol 12-myristate 13-acetate (PMA), the cells differentiated into cells with mesenchymal characteristics and upregulated Dmrt-1 mRNA, possibly through the protein kinase C/mitogen-activated protein kinase/activated protein-1 signaling pathway. Conversely, knockdown of Dmrt-1 by small interfering RNA resulted in cell morphology that was different from that after PMA treatment. These results indicated that Dmrt-1 expression was apparently associated with the differentiation of NEC8, and this cell line may be a helpful in vitro tool to clarify the role of Dmrt-1 in the differentiation process.

Adaptor Proteins, Signal Transducing↗

PubMed2003

An overactivated ATR/CHK1 pathway is responsible for the prolonged G2 accumulation in irradiated AT cells.

Induction of checkpoint responses in G1, S, and G2 phases of the cell cycle after exposure of cells to ionizing radiation (IR) is essential for maintaining genomic integrity. Ataxia telangiectasia mutated (ATM) plays a key role in initiating this response in all three phases of the cell cycle. However, cells lacking functional ATM exhibit a prolonged G2 arrest after IR, suggesting regulation by an ATM-independent checkpoint response. The mechanism for this ataxia telangiectasia (AT)-independent G2-checkpoint response remains unknown. We report here that the G2 checkpoint in irradiated human AT cells derives from an overactivation of the ATR/CHK1 pathway. Chk1 small interfering RNA abolishes the IR-induced prolonged G2 checkpoint and radiosensitizes AT cells to killing. These results link the activation of ATR/CHK1 with the prolonged G2 arrest in AT cells and show that activation of this G2 checkpoint contributes to the survival of AT cells.

Alkaloids↗

PubMed2004

A small C-terminal sequence of Aurora B is responsible for localization and function.

Aurora B, a protein kinase required in mitosis, localizes to inner centromeres at metaphase and the spindle midzone in anaphase and is required for proper chromosome segregation and cytokinesis. Aurora A, a paralogue of Aurora B, localizes instead to centrosomes and spindle microtubules. Except for distinct N termini, Aurora B and Aurora A have highly similar sequences. We have combined small interfering RNA (siRNA) ablation of Aurora B with overexpression of truncation mutants to investigate the role of Aurora B sequence in its function. Reintroduction of Aurora B during siRNA treatment restored its localization and function. This permitted a restoration of function test to determine the sequence requirements for Aurora B targeting and function. Using this rescue protocol, neither N-terminal truncation of Aurora B unique sequence nor substitution with Aurora A N-terminal sequence affected Aurora B localization or function. Truncation of unique Aurora B C-terminal sequence from terminal residue 344 to residue 333 was without effect, but truncation to 326 abolished localization and function. Deletion of residues 326-333 completely abolished localization and blocked cells at prometaphase, establishing this sequence as critical to Aurora B function. Our findings thus establish a small sequence as essential for the distinct localization and function of Aurora B.

Amino Acid Sequence↗

PubMed2005

Interferon regulatory factor 5 represses expression of the Epstein-Barr virus oncoprotein LMP1: braking of the IRF7/LMP1 regulatory circuit.

We have reported evidence for a positive regulatory circuit between interferon regulatory factor 7 (IRF7) and the Epstein-Barr virus (EBV) oncoprotein 1 (LMP1) (S. Ning, A. M. Hahn, and J. S. Pagano, J. Virol. 77:9359-9368, 2003). To explore a possible braking mechanism for this circuit, several type II EBV-infected cell lines that express different levels of LMP1 and IRF7 proteins and therefore are convenient for studying modulation of expression of LMP1 were analyzed. Endogenous levels of IRF7 and LMP1 were directly correlated. Transient expression of an IRF7 dominant-negative mutant decreased LMP1 levels. Endogenous IRF5 and IRF7 proteins were shown to physically associate in EBV-positive cells. Transient expression of IRF5 decreased activation of the LMP1 promoter by IRF7 in a dose-dependent manner. Finally, transfection of either an IRF5 dominant-negative construct or IRF5 small interfering RNA in these cells resulted in increases in endogenous levels of LMP1. These results indicate that IRF5 can downregulate IRF7's induction of expression of LMP1 most likely by interacting with IRF7 and provide a means of modulating a regulatory circuit between IRF7 and LMP1.

Antigens, Viral↗

PubMed2005

Escape from therapy-induced accelerated cellular senescence in p53-null lung cancer cells and in human lung cancers.

Accelerated cellular senescence (ACS) has been described for tumor cells treated with chemotherapy and radiation. Following exposure to genotoxins, tumor cells undergo terminal growth arrest and adopt morphologic and marker features suggestive of cellular senescence. ACS is elicited by a variety of chemotherapeutic agents in the p53-null, p16-deficient human non-small cell H1299 carcinoma cells. After 10 to 21 days, infrequent ACS cells (1 in 10(6)) can bypass replicative arrest and reenter cell cycle. These cells express senescence markers and resemble the parental cells in their transcription profile. We show that these escaped H1299 cells overexpress the cyclin-dependent kinase Cdc2/Cdk1. The escape from ACS can be disrupted by Cdc2/Cdk1 kinase inhibitors or by knockdown of Cdc2/Cdk1 with small interfering RNA and can be promoted by expression of exogenous Cdc2/Cdk1. We also present evidence that ACS occurs in vivo in human lung cancer following induction chemotherapy. Viable tumors following chemotherapy also overexpress Cdc2/Cdk1. We propose that ACS is a mechanism of in vivo tumor response and that mechanisms aberrantly up-regulate Cdc2/Cdk1 promotes escape from the senescence pathway may be involved in a subset of tumors and likely accounts for tumor recurrence/progression.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2006

Role of ABCC1 in export of sphingosine-1-phosphate from mast cells.

Mast cells play a pivotal role in inflammatory and immediate-type allergic reactions by secreting a variety of potent inflammatory mediators, including sphingosine-1-phosphate (S1P). However, it is not known how S1P is released from cells. Here, we report that S1P is exported from mast cells independently of their degranulation and demonstrate that it is mediated by ATP binding cassette (ABC) transporters. Constitutive and antigen-stimulated S1P release was inhibited by MK571, an inhibitor of ABCC1 (MRP1), but not by inhibitors of ABCB1 (MDR-1, P-glycoprotein). Moreover, down-regulation of ABCC1 with small interfering RNA, which decreased its cell surface expression, markedly reduced S1P export from both rat RBL-2H3 and human LAD2 mast cells. Transport of S1P by ABCC1 influenced migration of mast cells toward antigen but not degranulation. These findings have important implications for S1P functions in mast cell-mediated immune responses.

Animals↗

PubMed2005

Ataxia-telangiectasia-mutated (ATM) is a T-antigen kinase that controls SV40 viral replication in vivo.

The structurally related ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) protein kinases fulfill overlapping yet non-redundant functions as key regulators of cellular DNA damage responses. We recently showed that ATM phosphorylates the cyclic AMP response element-binding protein, CREB, following exposure to ionizing radiation (IR) and other DNA-damaging stimuli. Here, we show that a phospho-specific antibody recognizing the major ATM phosphorylation site in CREB cross-reacts with SV40 large tumor antigen (LTag), a multifunctional oncoprotein required for replication of the SV40 minichromosome. The relevant IR-induced phosphorylation site in LTag recognized by phospho-CREB antibody was mapped to Ser-120. IR strongly induced the phosphorylation of Ser-120 in an ATM-dependent manner in mouse embryo fibroblasts. Infection of African green monkey CV1 cells with SV40 resulted in the activation of ATM and phosphorylation of LTag and endogenous ATM substrates. Infection-induced LTag phosphorylation correlated with the onset of DNA replication, was ATM-dependent, and peaked when viral DNA levels reached their maximum. SV40 replication in CV1 cells required an intact LTag Ser-120 phosphorylation site and was inhibited following transfection with ATM small interfering RNA suggesting that ATM is required for optimal SV40 replication in primate cells. Our findings uncover a direct link between ATM and SV40 LTag that may have implications for understanding the replication cycle of oncogenic polyoma viruses.

Amino Acid Sequence↗

PubMed2006

Cross talk between PML and p53 during poliovirus infection: implications for antiviral defense.

PML nuclear bodies (NBs) are dynamic intranuclear structures harboring numerous transiently or permanently localized proteins. PML, the NBs' organizer, is directly induced by interferon, and its expression is critical for antiviral host defense. We describe herein the molecular events following poliovirus infection that lead to PML-dependent p53 activation and protection against virus infection. Poliovirus infection induces PML phosphorylation through the extracellular signal-regulated kinase pathway, increases PML SUMOylation, and induces its transfer from the nucleoplasm to the nuclear matrix. These events result in the recruitment of p53 to PML NBs, p53 phosphorylation on Ser15, and activation of p53 target genes leading to the induction of apoptosis. Moreover, the knock-down of p53 by small interfering RNA results in higher poliovirus replication, suggesting that p53 participates in antiviral defense. This effect, which requires the presence of PML, is transient since poliovirus targets p53 by inducing its degradation in a proteasome- and MDM2-dependent manner. Our results provide evidence of how poliovirus counteracts p53 antiviral activity by regulating PML and NBs, thus leading to p53 degradation.

Apoptosis↗

PubMed2004

Jab1/CSN5, a component of the COP9 signalosome, regulates transforming growth factor beta signaling by binding to Smad7 and promoting its degradation.

Smad7 inhibits responses mediated by transforming growth factor beta (TGF-beta) and acts in a negative-feedback loop to regulate the intensity or duration of the TGF-beta signal. However, the aberrant expression and continued presence of Smad7 may cause TGF-beta resistance. Here we report that Jab1/CSN5, which is a component of the COP9 signalosome complex, associates constitutively with Smad7 and that overexpression of Jab1/CSN5 causes the translocation of Smad7 from the nucleus to the cytoplasm, promoting its degradation. Overexpression of Jab1/CSN5 increases Smad2 phosphorylation and enhances TGF-beta-induced transcriptional activity. The inhibition of endogenous Jab1/CSN5 expression by small interfering RNA (siRNA) induces Smad7 expression. This study thus defines Jab1/CSN5 as an adapter that targets Smad7 for degradation, thus releasing Smad7-mediated suppression of TGF-beta signaling.

Animals↗

PubMed2004

Persistent c-FLIP(L) expression is necessary and sufficient to maintain resistance to tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in prostate cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in a variety of tumorigenic and transformed cell lines but not in many normal cells. Hence, TRAIL has the potential to be an ideal cancer therapeutic agent with minimal cytotoxicity. FLICE inhibitory protein (c-FLIP) is an important regulator of TRAIL-induced apoptosis. Here, we show that persistent expression of c-FLIP(Long) [c-FLIP(L)] is inversely correlated with the ability of TRAIL to induce apoptosis in prostate cancer cells. In contrast to TRAIL-sensitive cells, TRAIL-resistant LNCaP and PC3-TR (a TRAIL-resistant subpopulation of PC3) cells showed increased c-FLIP(L) mRNA levels and maintained steady protein expression of c-FLIP(L) after treatment with TRAIL. Ectopic expression of c-FLIP(L) in TRAIL-sensitive PC3 cells changed their phenotype from TRAIL sensitive to TRAIL resistant. Conversely, silencing of c-FLIP(L) expression by small interfering RNA in PC3-TR cells reversed their phenotype from TRAIL resistant to TRAIL sensitive. Therefore, persistent expression of c-FLIP(L) is necessary and sufficient to regulate sensitivity to TRAIL-mediated apoptosis in prostate cancer cells.

Antineoplastic Agents↗

PubMed2003

Regulated expression of ATF5 is required for the progression of neural progenitor cells to neurons.

An important milestone in brain development is the transition of neuroprogenitor cells to postmitotic neurons. We report that the bZIP transcription factor ATF5 plays a major regulatory role in this process. In developing brain ATF5 expression is high within ventricular zones containing neural stem and progenitor cells and is undetectable in postmitotic neurons. In attached clonal neurosphere cultures ATF5 is expressed by neural stem/progenitor cells and is undetectable in tau-positive neurons. In PC12 cell cultures nerve growth factor (NGF) dramatically downregulates endogenous ATF5 protein and transcripts, whereas exogenous ATF5 suppresses NGF-promoted neurite outgrowth. Such inhibition requires the repression of CRE sites. In contrast, loss of function conferred by dominant-negative ATF5 accelerates NGF-promoted neuritogenesis. Exogenous ATF5 also suppresses, and dominant-negative ATF5 and a small-interfering RNA targeted to ATF5 promote, neurogenesis by cultured nestin-positive telencephalic cells. These findings indicate that ATF5 blocks the differentiation of neuroprogenitor cells into neurons and must be downregulated to permit this process to occur.

Activating Transcription Factors↗

PubMed2006

Phorbol 12-myristate 13-acetate and serum synergize to promote rapamycin-insensitive cell proliferation via protein kinase C-eta.

Previously, we have shown that PKC-eta (protein kinase C-eta) positively regulates glioblastoma proliferation and confers resistance to irradiation-induced apoptosis. In this study, we investigated the efficacy of rapamycin in inhibiting cell proliferation in two glioblastoma cell lines U-251MG (PKC-eta expressing) and U-1242MG (PKC-eta deficient) following PKC-eta activation. In U-251MG cells, rapamycin (10 nM) treatment was less effective as an antiproliferative agent when cells were concurrently stimulated with 10% serum and phorbol 12-myristate 13-acetate (PMA, 100 nM), a potent activator of PKC isozymes. Rapamycin-insensitive growth was owing to PKC-eta, as U-1242MG and U-251MG cells infected with a kinase-dead form of PKC-eta (U-251kr) were susceptible to rapamycin-induced inhibition of cell proliferation. Furthermore, U-251MG cells transfected with PKC-eta antisense oligonucleotides were sensitive to rapamycin. PKC-eta-expressing cells stimulated with PMA maintained p70S6K phosphorylation on Thr389 and phosphorylation of rpS6 (ser235/36), suggesting p70S6K kinase activity was still intact. Inhibition of p70S6K expression with small interfering RNA oligonucleotides inhibited cell proliferation greater than 50% in the presence of a combination of PMA and serum. Additionally, p70S6K co-precipitated with PKC-eta, suggesting a physical interaction between PKC-eta and p70S6K regulates the observed phosphorylation. Taken together, these data demonstrate that rapamycin-insensitive glioblastoma proliferation involves PKC-eta signaling.

Blotting, Western↗

PubMed2003

Annexin II is required for apical transport in polarized epithelial cells.

The sorting of apical proteins comprises an initial recognition step in the trans Golgi network and a final partitioning of the apical pool of proteins into at least two different types of vesicular carriers. One criteria of these carriers is the association or non-association of the protein content with lipid rafts. We have previously characterized a population containing the raft-associated sucrase-isomaltase-carrying vesicles (SAVs) and another one, the non-raft-associated lactase-phlorizin hydrolase-carrrying vesicles (LAVs) that are targeted separately to the apical membrane. Here, we demonstrate biochemically and by employing confocal laser microscopy that the annexin II-S100A10 complex is a component of SAVs and is absent from LAVs. The unequivocal role of annexin II in the apical targeting of SI is clearly demonstrated when down-regulation of this protein by annexin II-specific small interfering RNA drastically decreases the apical delivery of SI in the epithelial cell line Madin-Darby canine kidney. The annexin II-S100A10 complex plays therefore a crucial role in routing SAVs to the apical membrane of epithelial cells.

Animals↗

PubMed2005

Negative regulation of monocyte adhesion to arterial elastic laminae by signal regulatory protein alpha and Src homology 2 domain-containing protein-tyrosine phosphatase-1.

Elastic laminae are extracellular matrix constituents that not only contribute to the stability and elasticity of arteries but also play a role in regulating arterial morphogenesis and pathogenesis. We demonstrate here that an important function of arterial elastic laminae is to prevent monocyte adhesion, which is mediated by the inhibitory receptor signal regulatory protein (SIRP) alpha and Src homology 2 domain-containing protein-tyrosine phosphatase (SHP)-1. In a matrix-based arterial reconstruction model in vivo, elastic laminae were resistant to leukocyte adhesion and transmigration compared with the collagen-dominant arterial adventitia. The density of leukocytes within the elastic lamina-dominant media was about 58-70-fold lower than that within the adventitia from 1 to 30 days. An in vitro assay confirmed the inhibitory effect of elastic laminae on monocyte adhesion. The exposure of monocytes to elastic laminae induced activation of SIRP alpha, which in turn activated SHP-1. Elastic lamina degradation peptides extracted from arterial specimens could also activate SIRP alpha and SHP-1. The knockdown of SIRP alpha and SHP-1 by specific small interfering RNA diminished the inhibitory effect of elastic laminae, resulting in a significant increase in monocyte adhesion. These observations suggest that SIRP alpha and SHP-1 potentially mediate the inhibitory effect of elastic laminae on monocyte adhesion.

Animals↗

PubMed2005

Syntabulin-mediated anterograde transport of mitochondria along neuronal processes.

In neurons, proper distribution of mitochondria in axons and at synapses is critical for neurotransmission, synaptic plasticity, and axonal outgrowth. However, mechanisms underlying mitochondrial trafficking throughout the long neuronal processes have remained elusive. Here, we report that syntabulin plays a critical role in mitochondrial trafficking in neurons. Syntabulin is a peripheral membrane-associated protein that targets to mitochondria through its carboxyl-terminal tail. Using real-time imaging in living cultured neurons, we demonstrate that a significant fraction of syntabulin colocalizes and co-migrates with mitochondria along neuronal processes. Knockdown of syntabulin expression with targeted small interfering RNA or interference with the syntabulin-kinesin-1 heavy chain interaction reduces mitochondrial density within axonal processes by impairing anterograde movement of mitochondria. These findings collectively suggest that syntabulin acts as a linker molecule that is capable of attaching mitochondrial organelles to the microtubule-based motor kinesin-1, and in turn, contributes to anterograde trafficking of mitochondria to neuronal processes.

Animals↗

PubMed2004

The p73 gene is an anti-tumoral target of the RARbeta/gamma-selective retinoid tazarotene.

Tazarotene, a member of the new class of acetylenic retinoids, has been shown to be effective in the treatment of several hyperproliferative skin diseases, including non-melanoma skin cancer. Its effectiveness is thought to rely on the ability to activate retinoic acid receptors beta and gamma and to induce a number of downstream anti-proliferative genes. Here, we show that the p53-related gene p73 is a target of tazarotene. Indeed, tazarotene modulates the expression of the p73 gene in immortalized keratinocyte cell lines by inducing the pro-apoptotic and anti-proliferative TAp73 isoforms and by repressing the anti-apoptotic and pro-proliferative DeltaNp73 isoforms. This occurs at the transcriptional level through a coordinated action on P1p73 and P2p73 promoters that control the expression of TA and DeltaN isoforms, respectively. The selective downregulation of DeltaNp73 expression by small interfering RNA led to an enhancement of tazarotene-induced bax activation and apoptosis, whereas the downregulation of both TA and DeltaN isoforms impairs tazarotene-mediated apoptosis. These results indicate the relevance of p73 gene products in tazarotene-induced growth inhibition and effectiveness in the treatment of skin tumors.

Animals↗

PubMed2006

A critical role of TRPM2 in neuronal cell death by hydrogen peroxide.

A brief exposure to hydrogen peroxide (H2O2) induces severe deterioration of primary cultured neurons in vitro. We have investigated a link between the H2O2-induced neuronal death and Ca2+-permeable TRPM2 channels regulated by ADP-ribose (ADPR). In cultured cerebral cortical neurons from fetal rat, TRPM2 proteins were detected at cell bodies and neurite extensions. Application of H2O2 to the cultured neurons elicited an increase in intracellular Ca2+ concentration ([Ca2+]i) caused by Ca2+ influx and the Ca2+-dependent neuronal death in a similar concentration range. Molecular cloning of TRPM2 cDNA from rat brain revealed several differences in amino acid sequences within the Nudix box region as compared with those of human and mouse TRPM2. ADPR-induced current responses, H2O2-induced Ca2+ influx, and H2O2-induced cell death were induced in human embryonic kidney cells heterologously expressing rat TRPM2. Treatment of cultured neurons with small interfering RNA against rat TRPM2,which efficiently suppressed immunoreactive TRPM2 content and the H2O2-induced Ca2+ influx,significantly inhibited H2O2-induced neuronal death. These results suggest that TRPM2 plays a pivotal role in H2O2-induced neuronal death as redox-sensitive Ca2+-permeable channels expressed in neurons.

Animals↗