scholarly journals Stress Kinase Signaling Regulates Androgen Receptor Phosphorylation, Transcription, and Localization

2006 ◽  
Vol 20 (3) ◽  
pp. 503-515 ◽  
Author(s):  
Daniel Gioeli ◽  
Ben E. Black ◽  
Vicki Gordon ◽  
Adam Spencer ◽  
Cristina T. Kesler ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Nuclear Export ◽  
Small Interfering Rna ◽  
Molecular Mechanisms ◽  
Specific Antigen ◽  
Mrna Levels ◽  
Kinase Signaling ◽  
Stress Kinase ◽  
Interfering Rna ◽  
Pharmacologic Inhibition

Abstract Activation of signal transduction kinase cascades is known to alter androgen receptor (AR) activity, but the molecular mechanisms are still poorly defined. Here we show that stress kinase signaling regulates Ser 650 phosphorylation and AR nuclear export. In LNCaP prostate cancer cells, activation of either MAPK kinase (MKK) 4:c-Jun N-terminal kinase (JNK) or MKK6:p38 signaling pathways increased Ser 650 phosphorylation, whereas pharmacologic inhibition of JNK or p38 signaling led to a reduction of AR Ser 650 phosphorylation. Both p38α and JNK1 phosphorylated Ser 650 in vitro. Small interfering RNA-mediated knockdown of either MKK4 or MKK6 increased endogenous prostate-specific antigen (PSA) transcript levels, and this increase was blocked by either bicalutamide or AR small interfering RNA. Stress kinase inhibition of PSA transcription is, therefore, dependent on the AR. Similar experiments involving either activation or inhibition of MAPK/ERK kinase:ERK signaling had little effect on Ser 650 phosphorylation or PSA mRNA levels. Ser 650 is proximal to the DNA binding domain that contains a nuclear export signal. Mutation of Ser 650 to alanine reduced nuclear export of the AR, whereas mutation of Ser 650 to the phosphomimetic amino acid aspartate restored AR nuclear export. Pharmacologic inhibition of stress kinase signaling reduced wild-type AR nuclear export equivalent to the S650A mutant without affecting nuclear export of the S650D mutant. Our data suggest that stress kinase signaling and nuclear export regulate AR transcriptional activity.

scholarly journals Optimization Procedure for Small Interfering RNA Transfection in a 384-Well Format

2007 ◽  
Vol 12 (4) ◽  
pp. 546-559 ◽  
Author(s):  
Jason Borawski ◽  
Alicia Lindeman ◽  
Frank Buxton ◽  
Mark Labow ◽  
L. Alex Gaither
Keyword(s):  
High Throughput Screening ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Dna Analysis ◽  
Lentiviral Vector ◽  
Mrna Levels ◽  
Sirna Transfection ◽  
Rna Transfection ◽  
Well Assay ◽  
Interfering Rna

High-throughput screening of RNAi libraries has become an essential part of functional analysis in academic and industrial settings. The transition of a cell-based RNAi assay into a 384-well format requires several optimization steps to ensure the phenotype being screened is appropriately measured and that the signal-to-background ratio is above a certain quantifiable threshold. Methods currently used to assess small interfering RNA (siRNA) efficacy after transfection, including quantitative PCR or branch DNA analysis, face several technical limitations preventing the accurate measurement of mRNA levels in a 384-well format. To overcome these difficulties, the authors developed an approach using a viral-based transfection system that measures siRNA efficacy in a standardized 384-well assay. This method allows measurement of siRNA activity in a phenotypically neutral manner by quantifying the knockdown of an exogenous luciferase gene delivered by a lentiviral vector. In this assay, the efficacy of a luciferase siRNA is compared to a negative control siRNA across many distinct assay parameters including cell type, cell number, lipid type, lipid volume, time of the assay, and concentration of siRNA. Once the siRNA transfection is optimized as a 384-well luciferase knockdown, the biologically relevant phenotypic analysis can proceed using the best siRNA transfection conditions. This approach provides a key technology for 384-well assay development when direct measurement of mRNA knockdown is not possible. It also allows for direct comparison of siRNA activity across cell lines from almost any mammalian species. Defining optimal conditions for siRNA delivery into mammalian cells will greatly increase the speed and quality of large-scale siRNA screening campaigns. ( Journal of Biomolecular Screening 2007:546-559)

326 MICROINJECTIONS OF SMALL INTERFERING RNA AND COMPLEMENTARY RNA TO ELUCIDATE THE INVOLVEMENT OF ENDOGENOUS PHOSPHOLIPASE C ISOFORMS IN BOVINE OOCYTE ACTIVATION DURING FERTILIZATION

2010 ◽  
Vol 22 (1) ◽  
pp. 319
Author(s):  
B. R. Sessions ◽  
A. H. Bayles ◽  
J. Collier ◽  
K. Perry ◽  
L. S. Whitaker ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Small Interfering Rna ◽  
Calcium Oscillations ◽  
Activation Process ◽  
Mrna Levels ◽  
Post Injection ◽  
Oocyte Activation ◽  
Bovine Oocyte ◽  
Interfering Rna ◽  
Bovine Oocytes

Phospholipase C (PLC) isoforms stimulate the hydrolysis of phosphatidyl inositol (4,5)-bisphosphate (PIP2) to produce diacylglycerol (DAG) and 1,4,5 inositol trisphosphate (IP3), with IP3 regulating the release of calcium (Ca2+) from the endoplasmic reticulum. This release of calcium is essential for oocyte activation, and a sperm-specific PLC isoform, PLCγ;, has been proposed as the primary agent that initiates the activation process. However, the oocyte contains many endogenous PLC isoforms (PLC β, γ, and δ) that could also be involved in regulating or initiating these calcium oscillations downstream of other initiating events. In order to better elucidate the involvement of endogenous PLC isoforms as well as the specific role of the sperm-specific form, small interfering RNA (siRNA) directed against the specific bovine PLC isoforms (PLCζ;, PLCγ1, PLCγ2, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3) were microinjected into bovine oocytes, and the subsequent effects on PLC mRNA levels and bovine fertilization were evaluated. Real-time PCR (qPCR) was used to quantify the levels of PLC message present in bovine oocytes at the time of injection (15 h post-maturation) and 6, 10, and 14 h post-injection. The qPCR results indicated a near-complete knockdown of mRNA levels in bovine oocytes 10 h post-injection for the isotypes PLCγ1, PLCγ2, PLCδ3, PLCδ4, PLCβ1, PLCβ3, but only partial knockdown of PLCS 1 mRNA. Oocytes microinjected with PLC siRNA were also fertilized and cultured in vitro according to our standard laboratory procedures (Reed et al. 1996 Theriogenology 45, 439-449). The oocytes microinjected with PLCζ;, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3 siRNA resulted in cleavage rates similar to the negative control siRNA, non-injected, and sham-injected treatment groups, whereas bovine oocytes microinjected with PLCγ1 and PLCγ2 siRNA had significantly lower cleavage rates compared with the controls. Additionally, complementary cRNA for each specific PLC isoform was microinjected into bovine oocytes to ascertain each isoform’s ability to induce parthenogenetic activation. Development was observed in oocytes microinjected with a variety of cRNAs, and the activating effects of the cRNA were negligible if the oocytes were microinjected with the corresponding siRNA before microinjection with cRNA. Interestingly, siRNA specific for PLCζ; failed to reduce cleavage when treated bovine oocytes were fertilized. These data illustrate the potential involvement of multiple endogenous PLC isoforms and not just the sperm-specific PLCζ; isoform in bovine oocyte activation during fertilization.

scholarly journals Molecular mechanisms of Dicer: endonuclease and enzymatic activity

2017 ◽  
Vol 474 (10) ◽  
pp. 1603-1618 ◽  
Author(s):  
Min-Sun Song ◽  
John J. Rossi
Keyword(s):  
Enzymatic Activity ◽  
Small Rna ◽  
Small Rnas ◽  
Small Interfering Rna ◽  
Molecular Mechanisms ◽  
Cellular Processes ◽  
Interfering Rna ◽  
Exogenous Substrates

The enzyme Dicer is best known for its role as a riboendonuclease in the small RNA pathway. In this canonical role, Dicer is a critical regulator of the biogenesis of microRNA and small interfering RNA, as well as a growing number of additional small RNAs derived from various sources. Emerging evidence demonstrates that Dicer's endonuclease role extends beyond the generation of small RNAs; it is also involved in processing additional endogenous and exogenous substrates, and is becoming increasingly implicated in regulating a variety of other cellular processes, outside of its endonuclease function. This review will describe the canonical and newly identified functions of Dicer.

scholarly journals Role of UBC9 in the Regulation of the Adipogenic Program in 3T3-L1 Adipocytes

Endocrinology ◽  
2010 ◽  
Vol 151 (11) ◽  
pp. 5255-5266 ◽  
Author(s):  
Angelo Cignarelli ◽  
Mariangela Melchiorre ◽  
Alessandro Peschechera ◽  
Antonella Conserva ◽  
Lucia Adelaide Renna ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Small Interfering Rna ◽  
Protein Modification ◽  
Covalent Attachment ◽  
Mrna Levels ◽  
Protein Levels ◽  
Mrna Induction ◽  
Induction Of Differentiation ◽  
Interfering Rna

The small ubiquitin-like modifier-conjugating enzyme UBC9, involved in protein modification through covalent attachment of small ubiquitin-like modifier and other less defined mechanisms, has emerged as a key regulator of cell proliferation and differentiation. To explore the role of UBC9 in adipocyte differentiation, the UBC9 protein levels were examined in differentiating 3T3-L1 cells. UBC9 mRNA and protein levels were increased 2.5-fold at d 2 and then gradually declined to basal levels at d 8 of differentiation. In addition, UBC9 was expressed predominantly in the nucleus of preadipocytes but shifted to cytoplasmic compartments after d 4, after induction of differentiation. UBC9 knockdown was then achieved in differentiating 3T3-L1 preadipocytes using a specific small interfering RNA. Oil-Red-O staining demonstrated accumulation of large triglyceride droplets in approximately 90% of control cells, whereas lipid droplets were smaller and evident in only 30% of cells treated with the UBC9-specific small interfering RNA. CCAAT/enhancer-binding protein (C/EBP)-δ, peroxisome proliferator-activated receptor-γ, and C/EBPα mRNA levels were increased severalfold 2–6 d after induction of differentiation in control cells, whereas the expression of these transcription factors was significantly lower in the presence of UBC9 gene silencing. Adenovirus-mediated overexpression of a catalytically inactive mutant UBC9 protein in 3T3-L1 cells resulted in no changes in expression of adipogenic transcription factors and conversion to mature adipocytes as compared with control. In conclusion, UBC9 appears to play an important role in adipogenesis. The temporal profile of UBC9 induction and its ability to affect C/EBPδ mRNA induction support a role for this protein during early adipogenesis.

Knockdown of Pathologic Immuoglobulin Light Chain Protein by Small Interfering RNA Molecules.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4407-4407
Author(s):  
Jonathan E Phipps ◽  
James S Foster ◽  
Daniel P Kestler ◽  
Robert Donnell ◽  
Alan Solomon ◽  
...  
Keyword(s):  
Cell Line ◽  
Plasma Cell ◽  
Light Chain ◽  
Small Interfering Rna ◽  
Mrna Levels ◽  
Light Chain Deposition Disease ◽  
Rna Molecules ◽  
Plasma Cell Dyscrasias ◽  
Rpmi 8226 ◽  
Interfering Rna

Abstract Abstract 4407 Introduction A major contributing factor to the morbidity and mortality of patients with plasma cell dyscrasias (i.e., light chain deposition disease, light chain amyloidosis (AL), etc.) is the overproduction and deposition of immunoglobulin light chain (LC) protein. Accumulation of insoluble fibrillar or amorphous aggregates within vital organs, particularly the kidneys, contributes to progressive loss of function and failure. Recent reports have shown that reducing levels of precursor LC proteins by 50% correlated with improved prognosis in patients with AL. Current therapies to suppress the production of monoclonal free LC rely on conventional or high dose myeloablative chemotherapeutic intervention. These therapies carry inherent risk and may not be appropriate in every situation, thus there is a need to employ alternative or adjuvant therapeutic approaches. To this end, we have investigated the ability of small interfering RNA molecules to decrease production of LC mRNA and protein secretion in both a synthetic and naturally occurring myeloma cell line. We report here that exposure of these lines to small interfering RNA (siRNA) oligonucleotides inhibited LC production in a specific and non-cytotoxic manner. Methods SP2/O mouse myeloma cells were stably transfected with a construct encoding a human λ6 LC protein (Wil) and designated SP2/O-1 for this study. The human λ2 producing cell line RPMI 8226 was purchased from ATCC. For both lines, LC mRNA was sequenced and used to design siRNA molecules targeting the V-, J-, or C-region of the LC. Cells were cultured for 1-3 d with sequence-specific siRNA, sham siRNA, or with media alone after which time LC mRNA levels were assessed by real-time PCR, and cellular or secreted LC protein concentrations were measured using flow cytometry or ELISA, respectively. Results Treatment with siRNA was well tolerated and no significant reduction in cell viability was observed in either the SP2/O-1 or RPMI 8226 cells at any of the timepoints assayed. Exposure of either SP2/O-1 or 8226 cells to siRNAs targeting the LC resulted in reductions of mRNA as compared to sham siRNA-treated controls. Flow cytometric analysis of 8226 cells immunostained with anti-LC antibodies at 48 h post transfection, indicated that ∼63% and 83% of the total cell number contained reduced amounts of cytoplasmic LC following treatment with V-, or C- region-specific siRNA, respectively. The 8226 LC protein was also significantly reduced by 20-60% in culture supernatants over the 72h period in cells exposed to experimental siRNAs as compared with sham-treated cells. Similarly, treating the SP2/O-1 cells with siRNAs led to significant (20-52%; P < 0.05) reductions in secreted LC Wil over 72 h as compared to sham-siRNA treatments. Conclusions We demonstrate here that siRNA provides a viable option for preventing the synthesis and thus secretion of pathologic LC protein using two cell lines, and that this reduction is carried out in a specific and non-cytotoxic manner. These results provide support for the potential use of siRNA to decrease the secretion of pathologic LC proteins in patients diagnosed with plasma cell dyscrasias. Our future goal will be to evaluate the action of siRNAs on LC production in vivo using a murine model of the disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Molecular mechanisms that funnel RNA precursors into endogenous small-interfering RNA and microRNA biogenesis pathways in Drosophila

RNA ◽  
2010 ◽  
Vol 16 (3) ◽  
pp. 506-515 ◽  
Author(s):  
K. Miyoshi ◽  
T. Miyoshi ◽  
J. V. Hartig ◽  
H. Siomi ◽  
M. C. Siomi
Keyword(s):  
Small Interfering Rna ◽  
Molecular Mechanisms ◽  
Microrna Biogenesis ◽  
Interfering Rna

scholarly journals Sirtuin 1 Is Required for Antagonist-Induced Transcriptional Repression of Androgen-Responsive Genes by the Androgen Receptor

2007 ◽  
Vol 21 (8) ◽  
pp. 1807-1821 ◽  
Author(s):  
Yan Dai ◽  
Duyen Ngo ◽  
Lora W. Forman ◽  
David C. Qin ◽  
Johanna Jacob ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Transcriptional Repression ◽  
Molecular Mechanisms ◽  
Specific Antigen ◽  
Sirtuin 1 ◽  
Therapeutic Modality ◽  
Class Iii ◽  
Androgen Ablation ◽  
Androgen Antagonists

Abstract Androgen antagonists or androgen deprivation is a primary therapeutic modality for the treatment of prostate cancer. Invariably, however, the disease becomes progressive and unresponsive to androgen ablation therapy (hormone refractory). The molecular mechanisms by which the androgen antagonists inhibit prostate cancer proliferation are not fully defined. In this report, we demonstrate that sirtuin 1 (SIRT1), a nicotinamide adenosine dinucleotide-dependent histone deacetylase (HDAC) linked to the regulation of longevity, is required for androgen antagonist-mediated transcriptional repression and growth suppression. Androgen antagonist-bound androgen receptor (AR) recruits SIRT1 and nuclear receptor corepressor to AR-responsive promoters and deacetylates histone H3 locally at the prostate-specific antigen promoter. Furthermore, SIRT1 down-regulation by small interfering RNA or by pharmacological means increased the sensitivity of androgen-responsive genes to androgen stimulation, enhanced the sensitivity of prostate cancer cell proliferative responses to androgens, and decreased the sensitivity of prostate cancer cells to androgen antagonists. In this study, we demonstrate the ligand-dependent recruitment of a class III HDAC into a corepressor transcriptional complex and a necessary functional role for a class III HDAC as a transcriptional corepressor in AR antagonist-induced transcriptional repression. Collectively, these findings identify SIRT1 as a corepressor of AR and elucidate a new molecular pathway relevant to prostate cancer growth and approaches to therapy.

scholarly journals Twist Modulates Human Trophoblastic Cell Invasion via Regulation of N-Cadherin

Endocrinology ◽  
2012 ◽  
Vol 153 (2) ◽  
pp. 925-936 ◽  
Author(s):  
York Hunt Ng ◽  
Hua Zhu ◽  
Peter C. K. Leung
Keyword(s):  
Cell Invasion ◽  
Small Interfering Rna ◽  
Molecular Mechanisms ◽  
Dependent Manner ◽  
Trophoblastic Cell ◽  
Protein Levels ◽  
Bewo Cells ◽  
Interfering Rna ◽  
Tgf Β1

The invasion of extravillous cytotrophoblasts (EVT) into the underlying maternal tissues and vasculature is a key step in human placentation. The molecular mechanisms involved in the development of the invasive phenotype of EVT include many that were first discovered for their role in cancer cell metastasis. Previous studies have demonstrated that N-cadherin and its regulatory transcription factor Twist play important roles in the onset and progression of cancers, but their roles in human trophoblastic cell invasion is not clear. The goal of the study was to examine the role of Twist and N-cadherin in human trophoblastic cell invasion. Twist and N-cadherin mRNA and protein levels were determined by RT-PCR and Western blotting in human placental tissues, highly invasive EVT, and poorly invasive JEG-3 and BeWo cells. Whether IL-1β and TGF-β1 regulate Twist mRNA and protein levels in the EVT was also examined. A small interfering RNA strategy was employed to determine the role of Twist and N-cadherin in HTR-8/SVneo cell invasion. Matrigel assays were used to assess cell invasion. Twist and N-cadherin were highly expressed in EVT but were poorly expressed in JEG-3 and BeWo cells. IL-1β and TGF-β1 differentially regulated Twist expression in EVT in a time- and concentration-dependent manner. Small interfering RNA specific for Twist decreased N-cadherin and reduced invasion of HTR-8/SVneo cells. Similarly, a reduction in N-cadherin decreased the invasive capacity of HTR-8/SVneo cells. Twist is an upstream regulator of N-cadherin-mediated invasion of human trophoblastic cells.

scholarly journals Leupaxin, a Novel Coactivator of the Androgen Receptor, Is Expressed in Prostate Cancer and Plays a Role in Adhesion and Invasion of Prostate Carcinoma Cells

2008 ◽  
Vol 22 (7) ◽  
pp. 1606-1621 ◽  
Author(s):  
Silke Kaulfuss ◽  
Michal Grzmil ◽  
Bernhard Hemmerlein ◽  
Paul Thelen ◽  
Stefan Schweyer ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Nuclear Export ◽  
Mutational Analysis ◽  
Specific Antigen ◽  
High Rate ◽  
Dependent Manner ◽  
Lncap Cells ◽  
Normal Prostate ◽  
Progression Marker

Abstract In the present study, we demonstrate that leupaxin mRNA is overexpressed in prostate cancer (PCa) as compared with normal prostate tissue by using cDNA arrays and quantitative RT-PCR analyses. Moderate to strong expression of leupaxin protein was detected in approximately 22% of the PCa tissue sections analyzed, and leupaxin expression intensities were found to be significantly correlated with Gleason patterns/scores. In addition, different leupaxin expression levels were observed in PCa cell lines, and at the subcellular level, leupaxin was usually localized in focal adhesion sites. Furthermore, mutational analysis and transfection experiments of LNCaP cells using different green fluorescent protein-leupaxin constructs demonstrated that leupaxin contains functional nuclear export signals in its LD3 and LD4 motifs, thus shuttling between the cytoplasm and the nucleus. We could also demonstrate for the first time that leupaxin interacts with the androgen receptor in a ligand-dependent manner and serves as a transcriptional activator of this hormone receptor in PCa cells. Down-regulation of leupaxin expression using RNA interference in LNCaP cells resulted in a high rate of morphological changes, detachment, spontaneous apoptosis, and a reduction of prostate-specific antigen secretion. In contrast, knockdown of leupaxin expression in androgen-independent PC-3 and DU 145 cells induced a significant decrease of both the invasive capacity and motility. Our results therefore indicate that leupaxin could serve as a potential progression marker for a subset of PCa and may represent a novel coactivator of the androgen receptor. Leupaxin could function as a putative target for therapeutic interventions of a subset of advanced PCa.

scholarly journals RELA is Sufficient to Mediate Interleukin-1 (IL-1) Repression of Androgen Receptor Expression and Activity in LNCaP Disease Progression Model

2019 ◽  
Author(s):  
Shayna E. Thomas-Jardin ◽  
Haley Dahl ◽  
Mohammed S. Kanchwala ◽  
Freedom Ha ◽  
Joan Jacob ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Androgen Receptor ◽  
Disease Progression ◽  
Cell Lines ◽  
Pathway Analysis ◽  
Molecular Mechanisms ◽  
Interleukin 1 ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Lncap Cells

ABSTRACTBackgroundThe Androgen Receptor (AR) nuclear transcription factor is a therapeutic target for prostate cancer (PCa). Unfortunately, patients can develop resistance to AR-targeted therapies and progress to lethal disease, underscoring the importance of understanding the molecular mechanisms that underlie treatment resistance. Inflammation is implicated in PCa initiation and progression and we have previously reported that the inflammatory cytokine, interleukin-1 (IL-1), represses AR mRNA levels and activity in AR-positive (AR+) PCa cell lines concomitant with the upregulation of pro-survival biomolecules. Thus, we contend that IL-1 can select for AR-independent, treatment-resistant PCa cells.MethodsTo begin to explore how IL-1 signaling leads to the repression of AR mRNA levels, we performed comprehensive pathway analysis on our RNA sequencing data from IL-1-treated LNCaP PCa cells. Our pathway analysis predicted Nuclear Factor Kappa B (NF-κB) p65 subunit (RELA), a canonical IL-1 signal transducer, to be significantly active and potentially regulate many genes, including AR. We used siRNA to silence the NF-κB family of transcription factor subunits, RELA, RELB, c-REL, NFKB1, or NFKB2, in IL-1-treated LNCaP, C4-2, and C4-2B PCa cell lines. C4-2 and C4-2B cell lines are castration-resistant LNCaP sublines and represent progression towards metastatic PCa disease; and we have previously shown that IL-1 represses AR mRNA levels in C4-2 and C4-2B cells.ResultssiRNA revealed that RELA alone is sufficient to mediate IL-1 repression of AR mRNA and AR activity. Intriguingly, while LNCaP cells are more sensitive to IL-1-mediated repression of AR than C4-2 and C4-2B cells, RELA siRNA led to a more striking de-repression of AR mRNA levels and AR activity in C4-2 and C4-2B cells than in LNCaP cells.ConclusionsThese data indicate that there are RELA-independent mechanisms that regulate IL-1-mediated AR repression in LNCaP cells and suggest that the switch to RELA-dependent IL-1 repression of AR in C4-2 and C4-2B cells reflects changes in epigenetic and transcriptional programs that evolve during PCa disease progression.

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