scholarly journals Amplification and attenuation of noisy expression by export processes

2021 ◽  
Author(s):  
Madeline Smith ◽  
Mohammad Soltani ◽  
Rahul Kulkarni ◽  
Abhyudai Singh
Keyword(s):  
Retention Time ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Mrna Level ◽  
Low Frequency ◽  
Random Variable ◽  
Arbitrary Distribution ◽  
Mrna Levels ◽  
Nuclear Retention ◽  
Poisson Arrivals

Inside mammalian cells, single genes are known to be transcribed in stochastic bursts leading to the synthesis of nuclear RNAs that are subsequently exported to the cytoplasm to create mRNAs. We systematically characterize the role of export processes in shaping the extent of random fluctuations (i.e. noise) in the mRNA level of a given gene. Using the method of Partitioning of Poisson arrivals, we derive an exact analytical expression for the noise in mRNA level assuming that the nuclear retention time of each RNA is an independent and identically distributed random variable following an arbitrary distribution. These results confirm recent experimental/theoretical findings that decreasing the nuclear export rate buffers the noise in mRNA level, and counterintuitively, decreasing the noise in the nuclear retention time enhances the noise in the mRNA level. Next, we further generalize the model to consider a dynamic extrinsic disturbance that affects the nuclear-to-cytoplasm export. Our results show that noise in the mRNA level varies non-monotonically with the disturbance timescale. More specifically, high- and low-frequency external disturbances have little impact on the mRNA noise level, while noise is amplified at intermediate frequencies. In summary, our results systematically uncover how the coupling of bursty transcription with nuclear export can both attenuate or amplify noise in mRNA levels depending on the nuclear retention time distribution and the presence of extrinsic fluctuations.

First-in-class, first-in-human phase I trial of KPT-330, a selective inhibitor of nuclear export (SINE) in patients (pts) with advanced solid tumors.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 2505-2505 ◽  
Author(s):  
Albiruni R A Razak ◽  
Morten Mau Soerensen ◽  
Amit Mahipal ◽  
Sharon Shacham ◽  
Cindy Y. F. Yau ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mrna Levels ◽  
Tumor Cell Death ◽  
Nuclear Retention ◽  
Minor Response ◽  
Mrna Induction ◽  
Ecog Ps ◽  
Tumor Biopsies ◽  
Dose Levels

2505 Background: In cancers, the majority of tumor suppressor proteins (TSP) are transported out of the nucleus exclusively by Exportin 1 (XPO1/CRM1), rendering these TSPs non-functional. KPT-330 is a potent inhibitor of XPO1, and forces the nuclear retention and activation of > 10 TSPs resulting in tumor cell death in vitro, in murine preclinical models and in dogs with spontaneous lymphomas. Methods: KPT-330 was administered orally for 10 doses in a 28-day cycle. Detailed pharmacokinetic (PK) and pharmacodynamic (PDn) analyses and serial tumor biopsies were performed. Response evaluation was done every 2 cycles (RECIST 1.1). All pts entering the study had documented progressive disease. Results: 23 pts (10 males; median age 62 yrs; ECOG PS 0/1: 5/18) received KPT-330 across 6 dose levels (3 to 30 mg/m2). There has been no dose limiting toxicity. Nine drug related grade 3/4 adverse events (AEs) post cycle 1 were reported in 6 pts (neutropenia, thrombocytopenia, hyponatremia, increased ALT, fatigue, vomiting [n=2], nausea [n=2]). The most common grade 1/2 AEs were nausea (78%), fatigue (74%) and anorexia (74%). PK analysis demonstrated a fairly proportional increase in Cmax and AUC with increasing dose, with no accumulation and without affecting half-life or clearance of KPT-330. At 30 mg/m2, AUC0-last. (4375 ng*h/mL) was comparable to the anti tumor exposure observed in mice and dogs. Tmax (~3 hrs) and T1/2 (6-7 hrs) were consistent across doses. Significant increase (2-20x) in XPO1 mRNA levels (PDn marker) in circulating leukocytes was observed at all doses, with higher doses demonstrating higher levels of XPO1 mRNA induction. Analysis of tumor biopsies confirmed nuclear localization of TSPs (e.g. p53, FOXO3A, IκB) and apoptosis of cancer cells following KPT-330 administration. RECIST response was evaluable in 13 pts. Stable disease (SD) was noted in 9 pts, with 3 (colon, endocervical & endometrial stromal tumors) remaining with SD at 6+ months (dose levels 3 & 6 mg/m2), as well as one minor response (colon). Conclusions: KPT-330 treatment is generally well tolerated, with favorable PK and PDn properties. Preliminary signals of clinical antitumor activity were observed. Clinical trial information: NCT01607905.

scholarly journals Identification of Three Functions of the Adenovirus E4orf6 Protein That Mediate p53 Degradation by the E4orf6-E1B55K Complex

2001 ◽  
Vol 75 (2) ◽  
pp. 699-709 ◽  
Author(s):  
Emmanuelle Querido ◽  
Megan R. Morisson ◽  
Huan Chu-Pham-Dang ◽  
Sarah W.-L. Thirlwell ◽  
Dominique Boivin ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Export Signal ◽  
Direct Interaction ◽  
26S Proteasome ◽  
Productive Infection ◽  
Nuclear Retention ◽  
Hpv E6 ◽  
Export Signal

ABSTRACT Complexes containing adenovirus E4orf6 and E1B55K proteins play critical roles in productive infection. Both proteins interact directly with the cellular tumor suppressor p53, and in combination they promote its rapid degradation. To examine the mechanism of this process, degradation of exogenously expressed p53 was analyzed in p53-null human cells infected with adenovirus vectors encoding E4orf6 and/or E1B55K. Coexpression of E4orf6 and E1B55K greatly reduced both the level and the half-life of wild-type p53. No effect was observed with the p53-related p73 proteins, which did not appear to interact with E4orf6 or E1B55K. Mutant forms of p53 were not degraded if they could not efficiently bind E1B55K, suggesting that direct interaction between p53 and E1B55K may be required. Degradation of p53 was independent of both MDM2 and p19ARF, regulators of p53 stability in mammalian cells, but required an extended region of E4orf6 from residues 44 to 274, which appeared to possess three separate biological functions. First, residues 39 to 107 were necessary to interact with E1B55K. Second, an overlapping region from about residues 44 to 218 corresponded to the ability of E4orf6 to form complexes with cellular proteins of 19 and 14 kDa. Third, the nuclear retention signal/amphipathic arginine-rich α-helical region from residues 239 to 253 was required. Interestingly, neither the E4orf6 nuclear localization signal nor the nuclear export signal was essential. These results suggested that if nuclear-cytoplasmic shuttling is involved in this process, it must involve another export signal. Degradation was significantly blocked by the 26S proteasome inhibitor MG132, but unlike the HPV E6 protein, E4orf6 and E1B55K were unable to induce p53 degradation in vitro in reticulocyte lysates. Thus, this study implies that the E4orf6-E1B55K complex may direct p53 for degradation by a novel mechanism.

scholarly journals Nuclear Export Through Nuclear Envelope Remodeling inSaccharomyces cerevisiae

2017 ◽  
Author(s):  
Baojin Ding ◽  
Anne M. Mirza ◽  
James Ashley ◽  
Vivian Budnik ◽  
Mary Munson
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Budding Yeast ◽  
Mrna Export ◽  
Low Frequency ◽  
Nuclear Pore ◽  
Wild Type ◽  
Export Pathway ◽  
Wild Type Yeast

ABSTRACTIn eukaryotes, subsets of exported mRNAs are organized into large ribonucleoprotein (megaRNP) granules. How megaRNPs exit the nucleus is unclear, as their diameters are much larger than the nuclear pore complex (NPC) central channel. We previously identified a non-canonical nuclear export mechanism inDrosophila(Speese et al.,Cell2012) and mammals (Ding et al., in preparation), in which megaRNPs exit the nucleus by budding across nuclear envelope (NE) membranes. Here, we present evidence for a similar pathway in the nucleus of the budding yeast S.cerevisiae, which contain morphologically similar granules bearing mRNAs. Wild-type yeast displayed these granules at very low frequency, but this frequency was dramatically increased when the non-essential NPC protein Nup116 was deleted. These granules were not artifacts of defective NPCs; a mutation in the exportinXPO1(CRM1), in which NPCs are normal, induced similar megaRNP upregulation. We hypothesize that a non-canonical nuclear export pathway, analogous to those observed inDrosophilaand in mammalian cells, exists in yeast, and that this pathway is upregulated for use when NPCs or nuclear export are impaired.SUMMARYDing et al., describe a non-canonical mRNA export pathway in budding yeast similar to that observed inDrosophila. This pathway appears upregulated when the NPC is impaired, nuclear envelope integrity is disrupted, or the export factor Xpo1 (CRM1) is defective.

scholarly journals Short-term Exposure to 50-Hz Electromagnetic Field and Alterations in NQO1 and NQO2 Expression in MCF-7 Cells

2016 ◽  
Vol 4 (4) ◽  
pp. 548-550 ◽  
Author(s):  
Hamideh Mahmoudinasab ◽  
Mostafa Saadat
Keyword(s):  
Mrna Level ◽  
Rna Extraction ◽  
Low Frequency ◽  
Published Data ◽  
Mrna Levels ◽  
Quinone Oxidoreductase ◽  
Antioxidant Genes ◽  
Post Exposure ◽  
Short Term Exposure ◽  
Mcf 7

AIM: Extremely low-frequency electromagnetic fields (ELF-EMFs) have some genotoxic effects and it may alter the mRNA levels of antioxidant genes. The NAD(P)H: quinone oxidoreductase-1 (NQO1) and NQO2 are ubiquitously expressed. Considering that there is no published data on the effect(s) of ELF-EMF (50-Hz) exposure and expression levels of NQO1 and NQO2 in the human MCF-7 cells, the present study was carried out.METHODS: The ELF-EMF (0.25 and 0.50 mT) exposure patterns were: 5 min field-on/5 min filed-off, 15 min field-on/15 min field-off, and 30 min field-on continuously. In all exposure conditions, total exposure time were 30 minutes. The RNA extraction was done at two times; immediately post exposure and two hours post exposure. The effect of ELF-EMF on gene expression was assessed by real-time PCR.RESULTS: The NQO1 mRNA level (at 0h) decreased in the cells exposed to 5 min field-on/5 min filed-off condition at 0.25 mT EMF when compared with the unexposed cells. The NQO2 mRNA level (at 0h and 2h) increased in the cells exposed to 5 min field-on/5 min filed-off condition at 0.50 mT EMF when compared with the unexposed cells.CONCLUSIONS: Alterations in the NQO1 and NQO2 mRNA levels seem at the "5 min field-on/5 min field-off" condition.

scholarly journals eEF1A Is a Novel Component of the Mammalian Nuclear Protein Export Machinery

2008 ◽  
Vol 19 (12) ◽  
pp. 5296-5308 ◽  
Author(s):  
Mireille Khacho ◽  
Karim Mekhail ◽  
Karine Pilon-Larose ◽  
Arnim Pause ◽  
Jocelyn Côté ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Export Signal ◽  
Point Mutations ◽  
Nuclear Retention ◽  
Von Hippel Lindau ◽  
Trna Species

The cytoplasmic translation factor eEF1A has been implicated in the nuclear export of tRNA species in lower eukaryotes. Here we demonstrate that eEF1A plays a central role in nuclear export of proteins in mammalian cells. TD-NEM (transcription-dependent nuclear export motif), a newly characterized nuclear export signal, mediates efficient nuclear export of several proteins including the von Hippel-Lindau (VHL) tumor suppressor and the poly(A)-binding protein (PABP1) in a manner that is dependent on ongoing RNA polymerase II (RNA PolII)-dependent transcription. eEF1A interacts specifically with TD-NEM of VHL and PABP1 and disrupting this interaction, by point mutations of key TD-NEM residues or treatment with actinomycin D, an inhibitor of RNA PolII-dependent transcription, prevents assembly and nuclear export. siRNA-induced knockdown or antibody-mediated depletion of eEF1A prevents in vivo and in vitro nuclear export of TD-NEM–containing proteins. Nuclear retention experiments and inhibition of the Exportin-5 pathway suggest that eEF1A stimulates nuclear export of proteins from the cytoplasmic side of the nuclear envelope, without entering the nucleus. Together, these data identify a role for eEF1A, a cytoplasmic mediator of tRNA export in yeast, in the nuclear export of proteins in mammalian cells. These results also provide a link between the translational apparatus and subcellular trafficking machinery demonstrating that these two central pathways in basic metabolism can act cooperatively.

HIF1A is Overexpressed in Medulloblastoma and its Inhibition Reduces Proliferation and Increases EPAS1 and ATG16L1 Methylation

2018 ◽  
Vol 18 (3) ◽  
pp. 287-294 ◽  
Author(s):  
Gustavo Alencastro Veiga Cruzeiro ◽  
Maristella Bergamo dos Reis ◽  
Vanessa Silva Silveira ◽  
Regia Caroline Peixoto Lira ◽  
Carlos Gilberto Carlotti Jr ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Cellular Proliferation ◽  
Mrna Level ◽  
Relevant Information ◽  
Protein Stabilization ◽  
Mrna Levels ◽  
Medulloblastoma Cell Line ◽  
Pediatric Medulloblastoma ◽  
Fresh Frozen

Background: Genetic and epigenetic modifications are closely related to tumor initiation and progression and can provide guidance for understanding tumor functioning, potentially leading to the discovery of new therapies. Studies have associated hypoxia-related genes to tumor progression and chemo/radioresistance in brain tumors. Information on the expression profile of hypoxiarelated genes in pediatric medulloblastoma, although scarce, may reveal relevant information that could support treatment decisions. Objective: Our study focused on evaluation the of CA9, CA12, HIF1A, EPAS1, SCL2A1 and VEGF genes in 41 pediatric fresh-frozen medulloblastoma sample. Additionally, we analyzed the effect of hypoxia and normoxia in the pediatric medulloblastoma cell-line UW402. Furthermore, we assessed the effects of HIF1A knockdown in cell-proliferation and methylation levels of genes related to hypoxia, apoptosis and autophagy. Method: qPCR was performed to evaluate mRNA levels, and Western blot to confirm HIF1A silencing in both patient samples and cell line. Pyrosequencing was performed to asses the methylation levels after HIF1A knockdown in the UW402 cell line. Results: A higher HIF1A mRNA level was observed in MB patients when compared to the cerebellum (non-tumor match). In UW402 MB cell-line, chemically induced hypoxic resulted in an increase of mRNA levels of HIF1A, VEGF, SCL2A1 and CA9 genes. Additionally, HIF1A knockdown induced a decrease in the expression of hypoxia related genes and a decrease of 30% in cell proliferation was also observed. Also, a significant increase in the methylation of ATG16L1 promoter and decrease in the methylation of EPAS1 promoter were observed after HIF1A knockdown. Conclusion: HIF1A knockdown in medulloblastoma cells lead to decreased cellular proliferation, suggesting that HIF1A can be a potential therapeutic target to be explored in the medulloblastoma. However, the mechanisms behind HIF1A protein stabilization and function are very complex and more data need to be generated to potentially use HIF1A as a therapeutical target.

scholarly journals Rheology of rounded mammalian cells over continuous high-frequencies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gotthold Fläschner ◽  
Cosmin I. Roman ◽  
Nico Strohmeyer ◽  
David Martinez-Martin ◽  
Daniel J. Müller
Keyword(s):  
High Frequency ◽  
Mammalian Cells ◽  
Mechanical Response ◽  
Cell Mass ◽  
Low Frequency ◽  
Mass Measurements ◽  
High Frequencies ◽  
Polymer Relaxation ◽  
Continuous Frequency ◽  
Rheological Experiments

AbstractUnderstanding the viscoelastic properties of living cells and their relation to cell state and morphology remains challenging. Low-frequency mechanical perturbations have contributed considerably to the understanding, yet higher frequencies promise to elucidate the link between cellular and molecular properties, such as polymer relaxation and monomer reaction kinetics. Here, we introduce an assay, that uses an actuated microcantilever to confine a single, rounded cell on a second microcantilever, which measures the cell mechanical response across a continuous frequency range ≈ 1–40 kHz. Cell mass measurements and optical microscopy are co-implemented. The fast, high-frequency measurements are applied to rheologically monitor cellular stiffening. We find that the rheology of rounded HeLa cells obeys a cytoskeleton-dependent power-law, similar to spread cells. Cell size and viscoelasticity are uncorrelated, which contrasts an assumption based on the Laplace law. Together with the presented theory of mechanical de-embedding, our assay is generally applicable to other rheological experiments.

scholarly journals Polypurine Reverse-Hoogsteen Hairpins as a Tool for Exon Skipping at the Genomic Level in Mammalian Cells

2021 ◽  
Vol 22 (7) ◽  
pp. 3784
Author(s):  
Véronique Noé ◽  
Carlos J. Ciudad
Keyword(s):  
Dna Sequencing ◽  
Rare Diseases ◽  
Mammalian Cells ◽  
Mrna Level ◽  
Exon Skipping ◽  
Selective Medium ◽  
Rt Pcr ◽  
Exon 2 ◽  
Reading Frame ◽  
Additional Exon

Therapeutic strategies for rare diseases based on exon skipping are aimed at mediating the elimination of mutated exons and restoring the reading frame of the affected protein. We explored the capability of polypurine reverse-Hoogsteen hairpins (PPRHs) to cause exon skipping in NB6 cells carrying a duplication of exon 2 of the DHFR gene that causes a frameshift abolishing DHFR activity. Methods: Different editing PPRHs were designed and transfected in NB6 cells followed by incubation in a DHFR-selective medium lacking hypoxanthine and thymidine. Surviving colonies were analyzed by DNA sequencing, RT-PCR, Western blotting and DHFR enzymatic activity. Results: Transfection of editing PPRHs originated colonies in the DHFR-selective medium. DNA sequencing results proved that the DHFR sequence in all these colonies corresponded to the wildtype sequence with just one copy of exon 2. In the edited colonies, the skipping of the additional exon was confirmed at the mRNA level, the DHFR protein was restored, and it showed high levels of DHFR activity. Conclusions: Editing-PPRHs are able to cause exon skipping at the DNA level and could be applied as a possible therapeutic tool for rare diseases.

scholarly journals Role of ASM/Cer/TXNIP signaling module in the NLRP3 inflammasome activation

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Jianjun Jiang ◽  
Yining Shi ◽  
Jiyu Cao ◽  
Youjin Lu ◽  
Gengyun Sun ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Nlrp3 Inflammasome ◽  
Mrna Level ◽  
Scavenger Receptor ◽  
Nuclear Factor Κb ◽  
Inflammasome Activation ◽  
Mrna Levels ◽  
Irreversible Inhibitor ◽  
Nlrp3 Inflammasome Activation

Abstract Background This study aimed to explore the effects of ceramide (Cer) on NLRP3 inflammasome activation and their underlying mechanisms. Methods Lipopolysaccharide (LPS)/adenosine triphosphate (ATP)-induced NLRP3 inflammasome activation in J774A.1 cells and THP-1 macrophages was used as an in vitro model of inflammation. Western blotting and real-time PCR (RT-PCR) were used to detect the protein and mRNA levels, respectively. IL-1β and IL-18 levels were measured by ELISA. ASM assay kit and immunofluorescence were used to detect ASM activity and Cer content. Results Imipramine, a well-known inhibitor of ASM, significantly inhibited LPS/ATP-induced activity of ASM and the consequent accumulation of Cer. Additionally, imipramine suppressed the LPS/ATP-induced expression of thioredoxin interacting protein (TXNIP), NLRP3, caspase-1, IL-1β, and IL-18 at the protein and mRNA level. Interestingly verapamil, a TXNIP inhibitor, suppressed LPS/ATP-induced activation of TXNIP/NLRP3 inflammasome but did not affect LPS/ATP-induced ASM activation and Cer formation. TXNIP siRNA and verapamil inhibited C2-Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome. In addition, the pretreatment of cells with sulfo-N-succinimidyl oleate (SSO), an irreversible inhibitor of the scavenger receptor CD36, blocked Cer-induced upregulation of nuclear factor-κB (NF-κB) activity, TXNIP expression, and NLRP3 inflammasome activation. Inhibition of NF-κB activation by SN50 prevented Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome but did not affect CD36 expression. Conclusion This study demonstrated that the ASM/Cer/TXNIP signaling pathway is involved in NLRP3 inflammasome activation. The results documented that the CD36-dependent NF-κB-TXNIP signaling pathway plays an essential role in the Cer-induced activation of NLRP3 inflammasomes in macrophages.

scholarly journals CTNI-18. FINAL RESULTS OF A PHASE 2 STUDY OF EFFICACY, SAFETY AND INTRATUMORAL PHARMACOKINETICS (PK) OF SELINEXOR MONOTHERAPY IN RECURRENT GLIOBLASTOMA (rGBM)

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii46-ii46
Author(s):  
Andrew Lassman ◽  
Patrick Wen ◽  
Martin van den Bent ◽  
Scott Plotkin ◽  
Annemiek Walenkamp ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Phase 1 ◽  
Progression Free Survival ◽  
Median Number ◽  
Recurrent Glioblastoma ◽  
Phase 2 ◽  
Nuclear Retention ◽  
Phase 2 Trial ◽  
Human Gbm

Abstract BACKGROUND Selinexor is an FDA-approved first-in-class, oral selective nuclear export inhibitor which forces nuclear retention of many tumor suppressor proteins. METHODS We conducted a phase 2 trial of selinexor monotherapy for adults with recurrent GBM including a surgical arm to explore intratumoral PK and 3 medical arms to optimize dosing. Prior treatment with radiotherapy and temozolomide was required; prior bevacizumab was exclusionary. The primary endpoint was 6-month progression-free survival (6mPFS) rate. RESULTS Selinexor administered ~2 hours pre-operatively yieleded average intratumoral concentration (136 nM, n=6) comparable to the in vitro IC50 (130 nM) from 7 primary human GBM cell lines. Among all 68 patients accrued to 3 medical arms (~85 mg BIW, n=24; 60 mg BIW, n=14; 80 mg QW, n=30), median age was 56 years (21–78). Median number of prior lines of therapies was 2 (1–7). At 80 mg QW, 28% patients were progression-free at the end of cycle 6; the 6mPFS was 17%; disese control rate by RANO was 37% (1 CR, 2 PRs, 7 SD) among 27 evaluable patients; responses were durable (median 11.1 months), and treatment lasted for 442, 547 and 1282 days in 3 responders, as of data lock, with one responder remaining on treatment off study; median overall survival was 10.2 months with 95% CI (7.0, 15.4). The ~85 mg BIW-schedule was abandoned due to poor tolerability. The related adverse events (all grades) in patients on ~85 mg BIW/60 mg BIW/80 mg QW were nausea (41.7%/64.3%/66.7%), fatigue (70.8%/71.4%/50.0%), neutropenia (29.2%/14.3%/33.3%), decreased appetite (45.8%/71.4%/26.7%), thrombocytopenia (66.7%/28.6%/23.3%) and weight loss (16.7%,/42.9%/6.7%). CONCLUSION Selinexor monotherapy demonstrated encouraging intratumoral penetration and efficacy, with durable disease control in rGBM. Monotherapy dose at 80 mg QW is recommended for further development in rGBM. A phase 1/2 study of combination therapy for newly diagnosed or rGBM has been initiated (NCT04421378).

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