| 1. |
- Chen, Dongfeng, et al.
(author)
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Glioma Cell Proliferation Controlled by ERK Activity-Dependent Surface Expression of PDGFRA.
- 2014
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:1
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Journal article (peer-reviewed)abstract
- Increased PDGFRA signaling is an essential pathogenic factor in many subtypes of gliomas. In this context the cell surface expression of PDGFRA is an important determinant of ligand sensing in the glioma microenvironment. However, the regulation of spatial distribution of PDGFRA in glioma cells remains poorly characterized. Here, we report that cell surface PDGFRA expression in gliomas is negatively regulated by an ERK-dependent mechanism, resulting in reduced proliferation of glioma cells. Glioma tumor tissues and their corresponding cell lines were isolated from 14 patients and analyzed by single-cell imaging and flow cytometry. In both cell lines and their corresponding tumor samples, glioma cell proliferation correlated with the extent of surface expression of PDGFRA. High levels of surface PDGFRA also correlated to high tubulin expression in glioma tumor tissue in vivo. In glioma cell lines, surface PDGFRA declined following treatment with inhibitors of tubulin, actin and dynamin. Screening of a panel of small molecule compounds identified the MEK inhibitor U0126 as a potent inhibitor of surface PDGFRA expression. Importantly, U0126 inhibited surface expression in a reversible, dose- and time-dependent manner, without affecting general PDGFRA expression. Treatment with U0126 resulted in reduced co-localization between PDGFRA and intracellular trafficking molecules e.g. clathrin, RAB11 and early endosomal antigen-1, in parallel with enhanced co-localization between PDGFRA and the Golgi cisternae maker, Giantin, suggesting a deviation of PDGFRA from the endosomal trafficking and recycling compartment, to the Golgi network. Furthermore, U0126 treatment in glioma cells induced an initial inhibition of ERK1/2 phosphorylation, followed by up-regulated ERK1/2 phosphorylation concomitant with diminished surface expression of PDGFRA. Finally, down-regulation of surface PDGFRA expression by U0126 is concordant with reduced glioma cell proliferation. These findings suggest that manipulation of spatial expression of PDGFRA can potentially be used to combat gliomas.
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| 2. |
- Na, Manli, et al.
(author)
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Adenovirus assembly is impaired by BMI1-related histone deacetylase activity.
- 2014
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In: Virology. - : Elsevier BV. - 1096-0341 .- 0042-6822. ; 456:Apr 17, s. 227-237
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Journal article (peer-reviewed)abstract
- Polycomb ring finger oncogene BMI1 (B cell-specific Moloney murine leukemia virus integration site 1) plays a critical role in development of several types of cancers. Here, we report an inverse relationship between levels of BMI1 expression and adenovirus (Ad) progeny production. Enforced BMI1 expression in A549 cells impaired Ad progeny production. In contrast, knocking-down of endogenous BMI1 expression enhanced progeny production of a conditionally replicating Ad and wild-type Ad5 and Ad11p. Ad vectors overexpressing BMI1 were not impaired in the replication of progeny genomes and in the expression of E1A and Ad structural proteins. However, 293 cells infected by Ad vector overexpressing BMI1 contained a large proportion of morphologically irregular Ad particles. This effect was reversed in 293 cells pre-treated with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) in parallel with the production of infectious Ad particles. Our findings suggest an inhibitory role of BMI1 in Ad morphogenesis that can be implied in Ad tropism and Ad-mediated cancer therapy.
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| 3. |
- Na, Manli, et al.
(author)
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Design of Ad5F35 vectors for coordinated dual gene expression in candidate human hematopoietic stem cells.
- 2010
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In: Experimental Hematology. - : Elsevier BV. - 1873-2399 .- 0301-472X. ; Apr 8, s. 446-452
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Journal article (peer-reviewed)abstract
- OBJECTIVE: Adenoviral vector mediated gene expression is an attractive approach to manipulate or report gene expression in human hematopoietic stem cells (HSC), when transient gene expression is preferred. Previous studies have demonstrated that fiber retargeted Ad5F35 vectors can mediate efficient gene transfer into human HSCs. In this study, we have investigated the potential of bi-directional promoter controlled Ad5F35 vector for coordinated dual gene expression in candidate HSCs. MATERIAL AND METHODS: We have engineered Ad5F35-DeltaLNGFR-BiDp encoding kinase domain deleted low-affinity NGF receptor (DeltaLNGFR) and green fluorescent protein (GFP) expression cassette controlled by a synthetic bi-directional promoter, which is composed of human phosphoglycerate kinase (PGK) promoter and minimal core promoter from human cytomegalovirus (mCMV). The expression pattern of DeltaLNGFR and GFP following Ad5F35-DeltaLNGFR-BiDp gene transfer in various cell types including candidate HSCs was compared to Ad5F35-DeltaLNGFR-IRES vector encoding PGK promoter controlled bicistronic expression cassette for DeltaLNGFR and GFP. RESULTS AND CONCLUSIONS: Using Ad5F35-DeltaLNGFR-BiDp, we demonstrated a coordinated, high-level dual gene expression in leukemic cells and cord blood CD34(+) cells. However, the ability of Ad5F35-DeltaLNGFR-BiDp-GFP for coordinated dual gene expression varied significantly between re-populating progenitor cells. In NOD/SCID mice bone marrow transplantation assay, sorted CD34(+)/DeltaLNGFR(+)/GFP(+) cells following infection with Ad5F35-DeltaLNGFR-BiDp showed predominantly myeloid lineage reconstitution with limited lymphoid lineage differentiation capacity, whereas the CD34(+)/DeltaLNGFR(+)/GFP(-) cells exhibited both myeloid and lymphoid reconstitution. This study indicates that bi-directional promoter controlled Ad5F35 vector such as Ad5F35-DeltaLNGFR-BiDp can be particularly useful for manipulation of myeloid progenitor cells and potentially also in myeloid lineage leukemic cells.
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| 4. |
- Na, Manli
(author)
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HOST EPIGENETIC REGULATOR BMI1 AND VIRAL FIBER PROTEIN FOR EQUILIBRIUM BETWEEN ADENOVIRUS AND HOST
- 2013
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Doctoral thesis (other academic/artistic)abstract
- Human adenoviruses (Ads) are broadly used in cancer gene therapy, vaccine development and gene delivery. The modified Ad5 or Ad2 viruses have been successfully used as oncolytic agents in pre-clinical studies. However they could not be translated into clinic utility due to a number of limitations including inefficient Ad spread among tumor cells. The cancer cell killing capacity of oncolytic Ad is dependent not only on the efficiency of Ad replication, but also on the efficiency of progeny dispersal and propagation of infection within cancer tissue. In this thesis, we believe that both viral and host cell factors regulate Ad propagation and spread process and consequently affect the co-existence interplay between Ad and host cells. Our projects aim to identify such factors from Ad as well as host cell side, and to clarify their role in regulating Ad propagation and spread. To identity viral factors, we investigated the kinetics of cell killing and Ad propagation following Ad infection at low multiplicity. Our results showed that prior to the release of Ad progenies, Ad infected cells secrete free fiber molecules in an excess, which mask Ad receptor molecules on non-infected bystander host cells, thus preventing these cells from efficient Ad infection and thereby promoting Ad and host cell co-existence. This is advantageous to Ad propagation and persistency of infection compared to the killing of all host cells with rapid kinetics. However, this is disadvantageous if Ad is used as oncolytic agents for therapeutic purposes. To identity host factors, we investigated the effect of polycomb gene BMI1 on Ad propagation. BMI1 is broadly overexpressed in various cancers. By retroviral vector mediated enforced BMI1 overexpression or siRNA mediated down-regulation of endogenous BMI1 expression, we demonstrated an inverse correlation between the Ad progeny production and the levels of BMI1 expression. This effect was not related to the cell cycle status and the receptor dependent Ad infectivity in host cells; nor to the replication of Ad genome and the production of Ad structural proteins. Instead, BMI1 overexpression impaired the morphogenesis of Ad particles, which could be reversed by TSA mediated inhibition of HDAC activity. Our findings indicate overexpression of BMI1 as a limiting factor in cancer therapy based on oncolytic Ad. So inhibition of BMI1 expression or BMI1-related HDAC activity may improve the functionality of oncolytic Ads in cancer therapy. To explore new approaches in Ad vector mediated dual gene expression in human hematopoietic stem cells (HSC), we generated an Ad vector, Ad5F35- ΔLNGFR-BiDp-GFP, encoding kinase domain deleted low-affinity NGF receptor (ΔLNGFR) and green fluorescent protein (GFP) expression cassette controlled by a synthetic bi-directional promoter. Our data showed that Ad5F35-ΔLNGFR-BiDp vector is highly active in directing dual gene expression in HSCs and most leukemic cell types tested, but the relative levels of dual gene expression by this vector is strongly cell-type dependent.
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