Establishment of an Isogenic Human Colon Tumor Model forNQO1 Gene Expression: Application to Investigate the Role of DT-Diaphorase in Bioreductive Drug Activation In Vitro and In Vivo

2000 ◽  
Vol 58 (5) ◽  
pp. 1146-1155 ◽  
Author(s):  
Swee Y. Sharp ◽  
Lloyd R. Kelland ◽  
Melanie R. Valenti ◽  
Lisa A. Brunton ◽  
Steve Hobbs ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Model ◽  
Human Colon ◽  
Colon Tumor ◽  
Dt Diaphorase ◽  
Drug Activation

scholarly journals PRMT5 Is Required for Bovine Leukemia Virus Infection In Vivo and Regulates BLV Gene Expression, Syncytium Formation, and Glycosylation In Vitro

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 650 ◽  
Author(s):  
Wlaa Assi ◽  
Tomoya Hirose ◽  
Satoshi Wada ◽  
Ryosuke Matsuura ◽  
Shin-nosuke Takeshima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Small Molecule ◽  
Bovine Leukemia Virus ◽  
Proviral Load ◽  
Leukemia Virus ◽  
Syncytium Formation ◽  
High Proviral Load

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle and is closely related to human T-cell leukemia viruses. We investigated the role of a new host protein, PRMT5, in BLV infection. We found that PRMT5 is overexpressed only in BLV-infected cattle with a high proviral load, but not in those with a low proviral load. Furthermore, this upregulation continued to the lymphoma stage. PRMT5 expression was upregulated in response to experimental BLV infection; moreover, PRMT5 upregulation began in an early stage of BLV infection rather than after a long period of proviral latency. Second, siRNA-mediated PRMT5 knockdown enhanced BLV gene expression at the transcript and protein levels. Additionally, a selective small-molecule inhibitor of PRMT5 (CMP5) enhanced BLV gene expression. Interestingly, CMP5 treatment, but not siRNA knockdown, altered the gp51 glycosylation pattern and increased the molecular weight of gp51, thereby decreasing BLV-induced syncytium formation. This was supported by the observation that CMP5 treatment enhanced the formation of the complex type of N-glycan more than the high mannose type. In conclusion, PRMT5 overexpression is related to the development of BLV infection with a high proviral load and lymphoma stage and PRMT5 inhibition enhances BLV gene expression. This is the first study to investigate the role of PRMT5 in BLV infection in vivo and in vitro and to reveal a novel function for a small-molecule compound in BLV-gp51 glycosylation processing.

Biologic consequences of androgen receptor (AR) activity in MDV3100-resistant LNCaP cells and dependence on AR full length.

2014 ◽  
Vol 32 (4_suppl) ◽  
pp. 74-74
Author(s):  
Yoshiaki Yamamoto ◽  
Yohann Loriot ◽  
Eliana Beraldi ◽  
Tianyuan Zhou ◽  
Youngsoo Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Androgen Receptor ◽  
Cell Growth ◽  
Functional Role ◽  
Full Length ◽  
Lncap Cells ◽  
Exon 1

74 Background: While recent reports link androgen receptor (AR) variants (AR-Vs) to castration resistant prostate cancer (CRPC), the biological significance of AR-Vs in AR-regulated cell survival and proliferation, independent of AR full length (AR-FL), remains controversial. To define the functional role of AR-FL and AR-Vs in MDV3100-resistant (MDV-R), we designed antisense oligonucleotide (ASO) targeting exon 1 and exon 8 in AR to knockdown AR-FL alone or in combination with AR-Vs and examined these effects in MDV-R LNCaP-derived cells in vitro and in vivo. Methods: We generated by selection MDV-R LNCaP-derived sub-lines that uniformly expressed high levels of both AR-FL and AR-V7 compared to CRPC LNCaP xenografts. Cell growth rates, protein and gene expression were analyzed using crystal violet assay, western blotting and real-time PCR, respectively. Exon 1 and 8 AR-ASO were evaluated in MDV-R49F CRPC LNCaP xenografts. Results: AR-V7 was transiently transfected in MDV-R49F cells and differential knockdown of AR-V7 and/or AR-FL by exon 1 versus exon 8 AR-ASO was used to evaluate relative biologic contributions of AR-FL versus AR-V7 in MDV-R LNCaP AR-V7 overexpressing cells. Exon 1 and 8 AR-ASO treatment in these cells similarly decreased prostate-specific antigen (PSA) expression and induced apoptosis as measured by caspase-3 and PARP cleavage and cell growth inhibition. To further define the functional role of AR-Vs in MDV-R LNCaP cells, we used a CE3 siRNA that specifically silenced AR-V7, but not AR-FL in MDV-R LNCaP cells. AR-V7 knockdown did not decrease PSA levels, did not induce apoptosis, and did not inhibit cell growth. In MDV-R LNCaP cells, exon 1 and 8 ASO similarly suppressed cell growth and AR-regulated gene expression in vitro and in vivo. Conclusions: These results indicate that the AR remains an important driver of MDV3100 resistance and, the biologic consequences mainly driven by AR-FL in MDV-R LNCaP models.

Role of the snake venom toxin jararhagin in proinflammatory pathogenesis: In vitro and in vivo gene expression analysis of the effects of the toxin

2005 ◽  
Vol 441 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Paul Gallagher ◽  
Yongde Bao ◽  
Solange M.T. Serrano ◽  
Gavin D. Laing ◽  
R. David G. Theakston ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Snake Venom ◽  
Gene Expression Analysis ◽  
Venom Toxin ◽  
Snake Venom Toxin

HEPATOCYTE TOLL-LIKE RECEPTOR 2 EXPRESSION IN VIVO AND IN VITRO: ROLE OF CYTOKINES IN INDUCTION OF RAT TLR2 GENE EXPRESSION BY LIPOPOLYSACCHARIDE

Shock ◽  
2000 ◽  
Vol 14 (3) ◽  
pp. 361-365 ◽  
Author(s):  
Shubing Liu ◽  
Neil A. Salyapongse ◽  
David A. Geller ◽  
Yoram Vodovotz ◽  
Timothy R. Billiar
Keyword(s):  
Gene Expression ◽  
Toll Like Receptor ◽  
Tlr2 Gene ◽  
Toll Like Receptor 2

scholarly journals Epigenetic factors in atherogenesis: microRNA

2016 ◽  
Vol 62 (2) ◽  
pp. 134-140
Author(s):  
A.V. Smirnova ◽  
V.N. Sukhorukov ◽  
V.P. Karagodin ◽  
A.N. Orekhov
Keyword(s):  
Gene Expression ◽  
Antisense Oligonucleotides ◽  
Noncoding Rna ◽  
Cell Cycle Progression ◽  
Rna Sequences ◽  
Cycle Progression ◽  
Atherosclerosis Progression

MicroRNAs (miRNAs) are small (~22 nucleotides in length) noncoding RNA sequences regulating gene expression at posttranscriptional level. MicroRNAs bind complementarily to certain mRNA and cause gene silencing. The involvement of miRNAs in the regulation of lipid metabolism, inflammatory response, cell cycle progression and proliferation, oxidative stress, platelet activation, endothelial and vascular smooth muscle cells (VSMC) function, angiogenesis and plaque formation and rapture indicates important roles in the initiation and progression of atherosclerosis. The key role of microRNAs in pathophysiology of cardiovascular diseases (CVDs), including atherosclerosis, was demonstrated in recent studies. Creating antisense oligonucleotides is a novel technique for selective changes in gene expression both in vitro and in vivo. In this review, we draw attention to the role of miRNAs in atherosclerosis progression, using miRNA as the potential biomarkers and targets in the CVDs, as well as possible application of antisense oligonucleotides

scholarly journals ASXL2 Is Recurrently Mutated in t(8;21) AML and Regulates Hematopoietic Development

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1051-1051
Author(s):  
Vikas Madan ◽  
Lin Han ◽  
Norimichi Hattori ◽  
Anand Mayakonda ◽  
Qiao-Yang Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Research Funding ◽  
Hematopoietic Differentiation ◽  
Wild Type ◽  
Flow Cytometric ◽  
Deficient Mice

Abstract Chromosomal translocation t(8;21) (q22;q22) leading to generation of oncogenic RUNX1-RUNX1T1 fusion is a cytogenetic abnormality observed in about 10% of acute myelogenous leukemia (AML). Studies in animal models and recent next generation sequencing approaches have suggested cooperativity of secondary genetic lesions with t(8;21) in inducing leukemogenesis. In this study, we used targeted and whole exome sequencing of 93 cases (including 30 with matched relapse samples) to profile the mutational landscape of t(8;21) AML at initial diagnosis and post-therapy relapse. We identified recurrent mutations of KIT, TET2, MGA, FLT3, NRAS, DHX15, ASXL1 and KMT2Dgenes in this subtype of AML. In addition, high frequency of truncating alterations in ASXL2 gene (19%) also occurred in our cohort. ASXL2 is a member of mammalian ASXL family involved in epigenetic regulation through recruitment of polycomb or trithorax complexes. Unlike its closely related homolog ASXL1, which is mutated in several hematological malignancies including AML, MDS, MPN and others; mutations of ASXL2 occur specifically in t(8;21) AML. We observed that lentiviral shRNA-mediated silencing of ASXL2 impaired in vitro differentiation of t(8;21) AML cell line, Kasumi-1, and enhanced its colony forming ability. Gene expression analysis uncovered dysregulated expression of several key hematopoiesis genes such as IKZF2, JAG1, TAL1 and ARID5B in ASXL2 knockdown Kasumi-1 cells. Further, to investigate implications of loss of ASXL2 in vivo, we examined hematopoiesis in Asxl2 deficient mice. We observed an age-dependent increase in white blood cell count in the peripheral blood of Asxl2 KO mice. Myeloid progenitors from Asxl2 deficient mice possessed higher re-plating ability and displayed altered differentiation potential in vitro. Flow cytometric analysis of >1 year old mice revealed increased proportion of Lin-Sca1+Kit+ (LSK) cells in the bone marrow of Asxl2 deficient mice, while the overall bone marrow cellularity was significantly reduced. In vivo 5-bromo-2'-deoxyuridine incorporation assay showed increased cycling of LSK cells in mice lacking Asxl2. Asxl2 deficiency also led to perturbed maturation of myeloid and erythroid precursors in the bone marrow, which resulted in altered proportions of mature myeloid populations in spleen and peripheral blood. Further, splenomegaly was observed in old ASXL2 KO mice and histological and flow cytometric examination of ASXL2 deficient spleens demonstrated increased extramedullary hematopoiesis and myeloproliferation compared with the wild-type controls. Surprisingly, loss of ASXL2 also led to impaired T cell development as indicated by severe block in maturation of CD4-CD8- double negative (DN) population in mice >1 year old. These findings established a critical role of Asxl2 in maintaining steady state hematopoiesis. To gain mechanistic insights into its role during hematopoietic differentiation, we investigated changes in histone marks and gene expression affected by loss of Asxl2. Whole transcriptome sequencing of LSK population revealed dysregulated expression of key myeloid-specific genes including Mpo, Ltf, Ngp Ctsg, Camp and Csf1rin cells lacking Asxl2 compared to wild-type control. Asxl2 deficiency also caused changes in histone modifications, specifically H3K27 trimethylation levels were decreased and H2AK119 ubiquitination levels were increased in Asxl2 KO bone marrow cells. Global changes in histone marks in control and Asxl2 deficient mice are being investigated using ChIP-Sequencing. Finally, to examine cooperativity between the loss of Asxl2 and RUNX1-RUNX1T1 in leukemogenesis, KO and wild-type fetal liver cells were transduced with retrovirus expressing AML1-ETO 9a oncogene and transplanted into irradiated recipient mice, the results of this ongoing study will be discussed. Overall, our sequencing studies have identified ASXL2 as a gene frequently altered in t(8;21) AML. Functional studies in mouse model reveal that loss of ASXL2 causes defects in hematopoietic differentiation and leads to myeloproliferation, suggesting an essential role of ASXL2 in normal and malignant hematopoiesis. *LH and NH contributed equally Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

Context Dependent Role of Polycomb Repressive Complex 2 in Acute Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 610-610
Author(s):  
Etienne Danis ◽  
Taylor Yamauchi ◽  
Kristen Echanique ◽  
Jessica Haladyna ◽  
Huafeng Xie ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Acute Leukemia ◽  
Murine Models ◽  
Polycomb Repressive Complex ◽  
Cell Precursor ◽  
Polycomb Repressive Complex 2

Abstract Polycomb Repressive Complex 2 (PRC2) is a multi-protein complex with important roles in development and cancer. Both hyper- and hypoactivity of PRC2 are associated with blood-related malignancies. Activating mutations of the PRC2 methyltransferase EZH2 have been found in human B-lineage lymphomas. Inactivating mutations of PRC2 components EZH2, EED and SUZ12 have been described in early T-cell Precursor ALL (ETP-ALL) and inactivating PRC2-alterations are found in Myelodysplastic and Myeloproliferative Syndromes. The mechanisms underlying this paradox are incompletely understood. We here investigate the context dependent role of PRC2 in murine models. We initially studied PRC2 in normal hematopoiesis: Chip-seq analysis of the PRC2-mediated H3K27me3 chromatin mark demonstrates that many genes highly expressed in immature hematopoiesis gain H3K27me3 in the developmental transition from more immature Lin-Sca1+Kit+ (LSK) cells to lineage committed Granulocyte Macrophage Progenitors (GMPs). Transcription of these genes is enriched in EZH2ko GMP compared to EZH2ffGMP by Gene Set Enrichment Analysis (GSEA). These data suggest that PRC2 is important for the silencing of immature gene expression programs in the developmental transition from LSK to GMP. We next analyzed the role of PRC2 in two murine models of acute leukemia: MLL-AF9 driven leukemia, and a model of early T-cell precursor T-ALL (ETP-ALL). In MLL-AF9 leukemia, we previously found that inactivation of Eed completely abrogate leukemogenesis in vitro and in vivo. We now report that genetic inactivation of the tumor suppressor Cdkn2a (a canonical PRC2 target) partially rescued MLL-AF9 mediated leukemia in vitro and in vivo. However, Cdkn2akoEEDko MLL-AF9 leukemia remained compromised. In vitro growth was reduced to approximately 10% of Eedff controls. While control MLL-AF9 leukemia developed in vivo in 100% of the recipients, Cdkn2akoEEDkoMLL-AF9 leukemia developed with significantly prolonged latency and incomplete penetrance (25%). RNAseq analysis revealed that high level expression of genes with established roles in MLL-AF9 leukemia such as HoxA9, Cdk6 and Jmjd1c unexpectedly depends on Eed. These data are in keeping with the absence of alterations in PRC2-components in human MLL-rearranged leukemia. In contrast, PRC2 core components (EZH2/EED/SUZ12) are deleted or mutated in > 40% of ETP-ALL. ETP-ALL also often has direct or indirect activation of the RAS-pathway, and carries frequent deletions of the CDKN2A locus. To model the effects of EED and EZH2-inactivation in ETP-ALL, we established Cdkn2akoEedff vs Cdkn2akoEedko, and Cdkn2akoEzh2ff vs Cdkn2akoEZH2koleukemias by transduction with NRASQ61K followed by expansion on OP9DL1 stroma cells to activate T-lineage differentiation via Notch-signaling. Cdkn2ako NRASQ61K leukemia showed an immunophenotype similar to human ETP-ALL (positive for c-Kit, CD5 and myeloid markers and mostly negative for CD4/8). Inactivation of Eed or Ezh2 in this model led to a shortening of latency (p=0.03 for Eed, p=0.0001 for Ezh2). RNAseq revealed enrichment of genes associated with murine DN1 thymocytes and with human ETP-ALL in Eedko vs Eedff Cdkn2ako NRASQ61K leukemia. These genesets showed even more pronounced enrichment in Ezh2kocompared to Ezh2ff Cdkn2ako NRASQ61K leukemia. Genes highly expressed in early hematopoiesis were enriched in Eedko and Ezh2ko cells in both, the MLL-AF9 and NRASQ61K leukemia models. However, there was an opposing effect on HoxA9 gene expression, with PRC2 inactivation leading to decreased HoxA9 expression in MLL-AF9, and increased HoxA9 expression in Cdkn2ako NRASQ61K leukemia. Decreased HoxA9 has been shown to impair MLL-AF9 leukemia growth. To test the functional significance of elevated HoxA9-levels in the Eedko and EZH2koNRASQ61K leukemias, we co-expressed HoxA9 and NRASQ61K in the presence of intact Eed and Ezh2 loci. Preliminary data suggest that HoxA9 accelerates leukemia development in this setting. Alterations in chromatin modifiers, including PRC2, are frequent in leukemia and lymphoma. Our data demonstrate that manipulation of PRC2 can have opposite effects on leukemia phenotype and expression of key PRC2-repressed genes such as HoxA9 in the context of different tumors. We are currently characterizing the mechanisms leading to divergent outcomes of PRC2 manipulation in MLL-AF9 leukemia compared to NRASQ61K ETP-like leukemia. Disclosures Armstrong: Epizyme : Consultancy.

scholarly journals The Use of Ultrasound and Micelles in Cancer Treatment

2008 ◽  
Vol 8 (5) ◽  
pp. 2205-2215 ◽  
Author(s):  
Ghaleb A. Husseini ◽  
William G. Pitt
Keyword(s):  
Drug Delivery ◽  
Polymeric Micelles ◽  
Tumor Model ◽  
Therapeutic Window ◽  
Rat Tumor Model ◽  
Drug Potency ◽  
Potential Benefits

The high toxicity of potent chemotherapeutic drugs like Doxorubicin (Dox) limits the therapeutic window in which they can be applied. This window can be expanded by controlling the drug delivery in both space and time such that non-targeted tissues are not adversely affected. Recent research has shown that ultrasound (US) can be used to control the release of Dox and other hydrophobic drugs from polymeric micelles in both time and space. It has also been shown using an in vivo rat tumor model that Dox activity can be enhanced by ultrasound in one region, while in an adjacent region there is little or no effect of the drug. In this article, we review the in vivo and in vitro research being conducted in the area of using ultrasound to enhance and target micellar drug delivery to cancerous tissues. Additionally, we summarize our previously published mathematical models that attempt to represent the release and re-encapsulation phenomena of Dox from Pluronic® P105 micelles upon the application of ultrasound. The potential benefits of such controlled chemotherapy compels a thorough investigation of the role of ultrasound (US) and the mechanisms by which US accomplishes drug release and/or enhances drug potency. Therefore we will summarize our findings related to the mechanism involved in acoustically activated micellar drug delivery to tumors.

scholarly journals Gle1 Regulates RNA Binding of the DEAD-Box Helicase Ded1 in Its Complex Role in Translation Initiation

2017 ◽  
Vol 37 (21) ◽  
Author(s):  
Peyman P. Aryanpur ◽  
Chelsea A. Regan ◽  
John M. Collins ◽  
Telsa M. Mittelmeier ◽  
David M. Renner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Binding ◽  
Mrna Export ◽  
Binding Assays ◽  
Cellular Mechanisms ◽  
Ribonucleoprotein Complexes ◽  
Dead Box

ABSTRACT DEAD-box proteins (DBPs) are required in gene expression to facilitate changes to ribonucleoprotein complexes, but the cellular mechanisms and regulation of DBPs are not fully defined. Gle1 is a multifunctional regulator of DBPs with roles in mRNA export and translation. In translation, Gle1 modulates Ded1, a DBP required for initiation. However, DED1 overexpression causes defects, suggesting that Ded1 can promote or repress translation in different contexts. Here we show that GLE1 expression suppresses the repressive effects of DED1 in vivo and Gle1 counteracts Ded1 in translation assays in vitro. Furthermore, both Ded1 and Gle1 affect the assembly of preinitiation complexes. Through mutation analysis and binding assays, we show that Gle1 inhibits Ded1 by reducing its affinity for RNA. Our results are consistent with a model wherein active Ded1 promotes translation but inactive or excess Ded1 leads to translation repression. Gle1 can inhibit either role of Ded1, positioning it as a gatekeeper to optimize Ded1 activity to the appropriate level for translation. This study suggests a paradigm for finely controlling the activity of DEAD-box proteins to optimize their function in RNA-based processes. It also positions the versatile regulator Gle1 as a potential node for the coordination of different steps of gene expression.

scholarly journals Paraspeckles: Paragons of functional aggregation

2015 ◽  
Vol 210 (4) ◽  
pp. 527-528 ◽  
Author(s):  
Edward Courchaine ◽  
Karla M. Neugebauer
Keyword(s):  
Gene Expression ◽  
Phase Separation ◽  
Low Complexity ◽  
Nuclear Body ◽  
Liquid Droplets ◽  
Cell Biol

Low-complexity proteins undergo phase separation in vitro, forming hydrogels or liquid droplets. Whether these form in vivo, and under what conditions, is still unclear. In this issue, Hennig et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201504117) show that formation of the paraspeckle, a nuclear body that regulates gene expression, requires low-complexity prion-like domains (PLDs) within paraspeckle proteins. The same proteins were shown to form hydrogels, shedding light on the role of “functional aggregation” in nuclear substructure.

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