Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle

Allera-Moreau, Camille; Delluc-Clavieres, A.; Castano, Caroline; Berghe, L. Van Den; Golzio, Muriel; Moreau, Marc; Teissié, Justin; Arnal, Jean‐François; Prats, Anne-Catherine

doi:10.1186/1472-6750-7-74

Cited by 18 publications

(17 citation statements)

References 34 publications

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“…These data are consistent with the known requirement for SAPK2/p38 activity for c-MYC IRES activity, and more recently SAPK2/p38 activity has been implicated in the regulation of XIAP IRES activity (15, 38). While long term stable expression of IRES containing dicistronic reporters has been achieved in mice (39), our efforts thus far in generating stable long term expression of cyclin D1 and c-MYC IRES baring dicistronic reporters in cell lines have been unsuccessful, hampering the ability to directly assess IRES activities in xenografts. Inducible expression of these reporters may permit analyses of IRES activities in tumors.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of SAPK2/p38 Enhances Sensitivity to mTORC1 Inhibition by Blocking IRES-Mediated Translation Initiation in Glioblastoma

Cloninger

Bernath

Bashir

et al. 2011

Molecular Cancer Therapeutics

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A variety of mechanisms confer hypersensitivity of tumor cells to the macrolide rapamycin, the prototypic mTORC1 inhibitor. Several studies have demonstrated that the status of the AKT kinase plays a critical role in determining hypersensitivity. Cancer cells in which AKT activity is elevated are exquisitely sensitive to mTORC1 inhibitors while cells in which the kinase is quiescent are relatively resistant. Our previous work has demonstrated that a transcript specific protein synthesis salvage pathway is operative in cells with quiescent AKT levels, maintaining the translation of crucial mRNAs involved in cell cycle progression in the face of global eIF-4E-mediated translation inhibition. The activation of this salvage pathway is dependent on SAPK2/p38-mediated activation of IRES-dependent initiation of the cyclin D1 and c-MYC mRNAs resulting in the maintenance of their protein expression levels. Here we demonstrate that both genetic and pharmacological inhibition of SAPK2/p38 in glioblastoma multiforme (GBM) cells significantly reduces rapamycin induced IRES-mediated translation initiation of cyclin D1 and c-MYC resulting in increased G1 arrest in vitro and inhibition of tumor growth in xenografts. Moreover, we observed that the AKT-dependent signaling alterations seen in vitro are also displayed in engrafted tumors cells and were able to demonstrate that combined inhibitor treatments markedly reduced the mRNA translational state of cyclin D1 and c-MYC transcripts in tumors isolated from mice. These data support the combined use of SAPK2/p38 and mTORC1 inhibitors to achieve a synergistic anti-tumor therapeutic response, particularly in rapamycin resistant quiescent AKT-containing cells.

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Section: Discussionmentioning

confidence: 99%

Inhibition of SAPK2/p38 Enhances Sensitivity to mTORC1 Inhibition by Blocking IRES-Mediated Translation Initiation in Glioblastoma

Cloninger

Bernath

Bashir

et al. 2011

Molecular Cancer Therapeutics

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“…Although a gene transcribed upstream of IRES can be translated at a much higher level than the downstream gene, 22 there was evidence that the ratio of translation between each gene remained pretty much stable. 23,24 The bicistronic FM-1 lentivector in our study enables the independent expression of the BLI gene in conjunction with the fluorescent probe Venus, a variant of GFP.…”

Section: Discussionmentioning

confidence: 99%

Comparison of red-shifted firefly luciferase Ppy RE9 and conventional Luc2 as bioluminescence imaging reporter genes for <italic>in vivo</italic> imaging of stem cells

Liang

2012

J. Biomed. Opt

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Abstract. One critical issue for noninvasive imaging of transplanted bioluminescent cells is the large amount of light absorption in tissue when emission wavelengths below 600 nm are used. Luciferase with a red-shifted spectrum can potentially bypass this limitation. We assessed and compared a mutant of firefly luciferase (Ppy RE9, PRE9) against the yellow luciferase luc2 gene for use in cell transplantation studies. C17.2 neural stem cells expressing PRE9-Venus and luc2-Venus were sorted by flow cytometry and assessed for bioluminescence in vitro in culture and in vivo after transplantation into the brain of immunodeficient Rag2-/-mice. We found that the luminescence from PRE9 was stable, with a peak emission at 620 nm, shifted to the red compared to that of luc2. The emission peak for PRE9 was pH-independent, in contrast to luc2, and much less affected by tissue absorbance compared to that of luc2. However, the total emitted light radiance from PRE9 was substantially lower than that of luc2, both in vitro and in vivo. We conclude that PRE9 has favorable properties as compared to luc2 in terms of pH independence, redshifted spectrum, tissue light penetration, and signal quantification, justifying further optimization of protein expression and enzymatic activity.

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“…To address this issue, several groups have sought to identify alternative cellular and viral IRES elements that initiate protein expression more efficiently than EMCV IRES. 15,[19][20][21][22][23][24][25][26] Cellular IRES elements such as eukaryotic initiation factor 4G, 15 immunoglobulin heavy chain binding protein, 15 c-myc proto-oncogene, 15 vascular endothelial growth factor 15 and fibroblast growth factor-1 27 IRES have been reported to show higher efficiency than EMCV IRES. In this study, we searched to identify an IRES(s) with higher translation efficiency than the EMCV IRES both in vitro and in vivo.…”

Section: Introductionmentioning

confidence: 99%

In vitro and in vivo comparison of viral and cellular internal ribosome entry sites for bicistronic vector expression

et al. 2011

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Bicistronic vectors are essential to achieve efficient expression of multiple genes in gene therapy protocols and biomedical applications. Internal ribosome entry site (IRES) elements have been utilized to initiate expression of an additional protein from a bicistronic vector. The IRES element commonly used in current bicistronic vectors originates from the encephalomyocarditis virus (EMCV). As IRES-mediated translation is dependent on availability of IRES trans-acting factors, which vary between cell types and species, adequate gene expression from the EMCV IRES element is not always achieved. To identify a novel IRES element that mediates gene expression consistently with a higher efficiency than the EMCV IRES, we tested 13 bicistronic reporter constructs containing different viral and cellular IRES elements. The in vitro screening in human and mouse fibroblast and hepatocarcinoma cells revealed that the vascular endothelial growth factor and type 1 collagen-inducible protein (VCIP) IRES was the only IRES element that directed translation more efficiently than the EMCV IRES in all cell lines. Furthermore, the VCIP IRES initiated greater reporter expression levels than the EMCV IRES in transfected mouse livers. These results suggest that VCIP-IRES containing vectors improve gene expression compared with those harboring an EMCV-IRES. This could increase the potential benefits of bicistronic vectors for experimental and therapeutic purposes.

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Long term expression of bicistronic vector driven by the FGF-1 IRES in mouse muscle

Cited by 18 publications

References 34 publications

Inhibition of SAPK2/p38 Enhances Sensitivity to mTORC1 Inhibition by Blocking IRES-Mediated Translation Initiation in Glioblastoma

Inhibition of SAPK2/p38 Enhances Sensitivity to mTORC1 Inhibition by Blocking IRES-Mediated Translation Initiation in Glioblastoma

Comparison of red-shifted firefly luciferase Ppy RE9 and conventional Luc2 as bioluminescence imaging reporter genes for <italic>in vivo</italic> imaging of stem cells

In vitro and in vivo comparison of viral and cellular internal ribosome entry sites for bicistronic vector expression

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