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Regulated ATF5 loss-of-function in adult mice blocks formation and causes regression/eradication of gliomas.

Arias A, Lamé MW, Santarelli L, Hen R, Greene LA, Angelastro JM - Oncogene (2011)

Bottom Line: However, it was unknown whether interference with ATF5 function can prevent or promote regression/eradication of malignant gliomas in vivo.In this model, d/n-ATF5 expression is controlled by doxycycline and the promoter for GFAP, a marker for stem/progenitor cells as well as gliomas.Induction of d/n-ATF5 before delivery of PDGF-B/p53-shRNA virus greatly reduced the proportion of mice that formed tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of California, Davis School of Veterinary Medicine, Davis, CA 95616, USA.

ABSTRACT
Glioblastomas are among the most incurable cancers. Our past findings indicated that glioblastoma cells, but not neurons or glia, require the transcription factor ATF5 (activating transcription factor 5) for survival. However, it was unknown whether interference with ATF5 function can prevent or promote regression/eradication of malignant gliomas in vivo. To address this issue, we created a mouse model by crossing a human glial fibrillary acidic protein (GFAP) promoter-tetracycline transactivator mouse line with tetracycline operon-dominant negative-ATF5 (d/n-ATF5) mice to establish bi-transgenic mice. In this model, d/n-ATF5 expression is controlled by doxycycline and the promoter for GFAP, a marker for stem/progenitor cells as well as gliomas. Endogenous gliomas were produced with high efficiency by retroviral delivery of platelet-derived growth factor (PDGF)-B and p53-short hairpin RNA (shRNA) in adult bi-transgenic mice in which expression of d/n-ATF5 was spatially and temporally regulated. Induction of d/n-ATF5 before delivery of PDGF-B/p53-shRNA virus greatly reduced the proportion of mice that formed tumors. Moreover, d/n-ATF5 induction after tumor formation led to regression/eradication of detectable gliomas without evident damage to normal brain cells in all 24 mice assessed.

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Related in: MedlinePlus

Regulated expression of d/n-ATF5 in bi-transgenic hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal mice. (a) Scheme for generation and utilization of bi-transgenic hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal mice. The hGFAP promoter drives the tTA, and the tTA binds to TETO in the absence of doxycycline (Dox) to induce bi-directional co-expression of the Flag-d/n-ATF5 and β-gal (LacZ) transgenes. (b) Regulated expression of Flag-d/n-ATF5 (left-hand panels) and β-gal (right-hand panels) in the cortex, SVZ and CC of bi-transgenic mice. One bi-transgenic mouse was maintained on Dox at conception and killed 63 days after birth (+Dox), whereas the other was maintained on Dox from conception until 35 days after birth, and then withdrawn from the drug and killed 28 days later (−Dox). Colorimetric staining of Flag-tagged d/n-ATF5 in the cortex (sagittal section) was achieved with anti-FLAG M2 antibody and Vulcan Fast Red. Scale bar=10 μm. X-gal substrate was used for β-gal colorimetric detection. V=lateral ventricle; CC=corpus callosum; Cx=cortex; SVZ=subventricular zone. Scale bar=20 μm. (c) Coordinately regulated expression of Flag-d/n-ATF5 and β-gal within the SVZ and near the CA1 region of the hippocampus (orthogonal view) in a 79-day-old bi-transgenic mouse raised without Dox since conception. Immunofluorescence staining was carried out with anti-Flag (for detection of Flag-d/n-ATF5; green) and β-gal (red). Arrows in the merged panel indicate cell colocalization. The confocal (2 μm) orthogonal section image shows the x–y and y–z location of both β-gal and Flag-d/n-ATF5 within the same cell. (d) Regulated expression of β-gal in GFAP+ cells within the cortex of a bi-transgenic mouse. The animal was maintained on Dox from conception until 204 days after birth and the drug was then withdrawn until being killed 50 days later. Panels show immunostaining with anti-GFAP (red) and anti-β-gal (green) and arrows show the locations of cells in which both are co-expressed. (e) Regulated expression of β-gal in GFAP+ cells within the SVZ of a bi-transgenic mouse. The mouse was maintained on doxycycline from conception until 167 days after birth and the drug was then withdrawn until being killed 42 days later. Panels show immunostaining with anti-GFAP (green) and anti-β-gal (red) and arrows show the locations of cells in which both are co-expressed. Diagrams show the brain areas where photographs were taken (adapted from BrainMaps.org and http://www.hms.harvard.edu/research/brain/atlas). Scale bar=20 μm (c–e).
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fig2: Regulated expression of d/n-ATF5 in bi-transgenic hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal mice. (a) Scheme for generation and utilization of bi-transgenic hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal mice. The hGFAP promoter drives the tTA, and the tTA binds to TETO in the absence of doxycycline (Dox) to induce bi-directional co-expression of the Flag-d/n-ATF5 and β-gal (LacZ) transgenes. (b) Regulated expression of Flag-d/n-ATF5 (left-hand panels) and β-gal (right-hand panels) in the cortex, SVZ and CC of bi-transgenic mice. One bi-transgenic mouse was maintained on Dox at conception and killed 63 days after birth (+Dox), whereas the other was maintained on Dox from conception until 35 days after birth, and then withdrawn from the drug and killed 28 days later (−Dox). Colorimetric staining of Flag-tagged d/n-ATF5 in the cortex (sagittal section) was achieved with anti-FLAG M2 antibody and Vulcan Fast Red. Scale bar=10 μm. X-gal substrate was used for β-gal colorimetric detection. V=lateral ventricle; CC=corpus callosum; Cx=cortex; SVZ=subventricular zone. Scale bar=20 μm. (c) Coordinately regulated expression of Flag-d/n-ATF5 and β-gal within the SVZ and near the CA1 region of the hippocampus (orthogonal view) in a 79-day-old bi-transgenic mouse raised without Dox since conception. Immunofluorescence staining was carried out with anti-Flag (for detection of Flag-d/n-ATF5; green) and β-gal (red). Arrows in the merged panel indicate cell colocalization. The confocal (2 μm) orthogonal section image shows the x–y and y–z location of both β-gal and Flag-d/n-ATF5 within the same cell. (d) Regulated expression of β-gal in GFAP+ cells within the cortex of a bi-transgenic mouse. The animal was maintained on Dox from conception until 204 days after birth and the drug was then withdrawn until being killed 50 days later. Panels show immunostaining with anti-GFAP (red) and anti-β-gal (green) and arrows show the locations of cells in which both are co-expressed. (e) Regulated expression of β-gal in GFAP+ cells within the SVZ of a bi-transgenic mouse. The mouse was maintained on doxycycline from conception until 167 days after birth and the drug was then withdrawn until being killed 42 days later. Panels show immunostaining with anti-GFAP (green) and anti-β-gal (red) and arrows show the locations of cells in which both are co-expressed. Diagrams show the brain areas where photographs were taken (adapted from BrainMaps.org and http://www.hms.harvard.edu/research/brain/atlas). Scale bar=20 μm (c–e).

Mentions: To study ATF5 function, we have created a specific interfering d/n form of the protein that lacks the N-terminal acidic activation and DNA-binding domains and that contains an enhanced bZip domain (Vinson et al., 1993; Krylov et al., 1995; Moitra et al., 1998; Moll et al., 2000; Angelastro et al., 2006). Flag-tagged d/n-ATF5 blocks ATF5 function and consequently accelerates differentiation of neural progenitors and induces apoptosis of glioblastoma and other neoplastic cells both in vitro and in vivo (Angelastro et al., 2003, 2005, 2006; Mason et al., 2005; Monaco et al., 2007). For the current studies, we generated a transgenic mouse line in which Flag-tagged-d/n-ATF5 and β-galactosidase (β-gal) (product of LacZ) are driven as separate gene products under the control of the pBi3-tetracycline operon (TETO) operator (Baron et al., 1995). This line was crossed with an hGFAP-tetracycline transactivator (tTA) transgenic mouse line (Pascual et al., 2005; Sweger et al., 2007) in which the hGFAP promoter drives the tTA protein (Gossen and Bujard, 1992; Gossen et al., 1994). This line has been successfully employed to spatially and temporally regulate transgenes such as d/n SNARE, GPCR Ro1 RASSL, Gq-coupled receptor and ErbB2 in neural progenitors and astrocytes (Pascual et al., 2005; Fiacco et al., 2007; Ghashghaei et al., 2007; Sweger et al., 2007). In this ‘Tet-off' system, in the absence of doxycycline, the bi-transgenic mice should express d/n-ATF5 and β-gal in cells such as neuroprogenitors and gliomas in which the hGFAP promoter is active; conversely in the presence of dietary doxycycline, d/n-ATF5 expression should be suppressed (Figure 2a).


Regulated ATF5 loss-of-function in adult mice blocks formation and causes regression/eradication of gliomas.

Arias A, Lamé MW, Santarelli L, Hen R, Greene LA, Angelastro JM - Oncogene (2011)

Regulated expression of d/n-ATF5 in bi-transgenic hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal mice. (a) Scheme for generation and utilization of bi-transgenic hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal mice. The hGFAP promoter drives the tTA, and the tTA binds to TETO in the absence of doxycycline (Dox) to induce bi-directional co-expression of the Flag-d/n-ATF5 and β-gal (LacZ) transgenes. (b) Regulated expression of Flag-d/n-ATF5 (left-hand panels) and β-gal (right-hand panels) in the cortex, SVZ and CC of bi-transgenic mice. One bi-transgenic mouse was maintained on Dox at conception and killed 63 days after birth (+Dox), whereas the other was maintained on Dox from conception until 35 days after birth, and then withdrawn from the drug and killed 28 days later (−Dox). Colorimetric staining of Flag-tagged d/n-ATF5 in the cortex (sagittal section) was achieved with anti-FLAG M2 antibody and Vulcan Fast Red. Scale bar=10 μm. X-gal substrate was used for β-gal colorimetric detection. V=lateral ventricle; CC=corpus callosum; Cx=cortex; SVZ=subventricular zone. Scale bar=20 μm. (c) Coordinately regulated expression of Flag-d/n-ATF5 and β-gal within the SVZ and near the CA1 region of the hippocampus (orthogonal view) in a 79-day-old bi-transgenic mouse raised without Dox since conception. Immunofluorescence staining was carried out with anti-Flag (for detection of Flag-d/n-ATF5; green) and β-gal (red). Arrows in the merged panel indicate cell colocalization. The confocal (2 μm) orthogonal section image shows the x–y and y–z location of both β-gal and Flag-d/n-ATF5 within the same cell. (d) Regulated expression of β-gal in GFAP+ cells within the cortex of a bi-transgenic mouse. The animal was maintained on Dox from conception until 204 days after birth and the drug was then withdrawn until being killed 50 days later. Panels show immunostaining with anti-GFAP (red) and anti-β-gal (green) and arrows show the locations of cells in which both are co-expressed. (e) Regulated expression of β-gal in GFAP+ cells within the SVZ of a bi-transgenic mouse. The mouse was maintained on doxycycline from conception until 167 days after birth and the drug was then withdrawn until being killed 42 days later. Panels show immunostaining with anti-GFAP (green) and anti-β-gal (red) and arrows show the locations of cells in which both are co-expressed. Diagrams show the brain areas where photographs were taken (adapted from BrainMaps.org and http://www.hms.harvard.edu/research/brain/atlas). Scale bar=20 μm (c–e).
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fig2: Regulated expression of d/n-ATF5 in bi-transgenic hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal mice. (a) Scheme for generation and utilization of bi-transgenic hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal mice. The hGFAP promoter drives the tTA, and the tTA binds to TETO in the absence of doxycycline (Dox) to induce bi-directional co-expression of the Flag-d/n-ATF5 and β-gal (LacZ) transgenes. (b) Regulated expression of Flag-d/n-ATF5 (left-hand panels) and β-gal (right-hand panels) in the cortex, SVZ and CC of bi-transgenic mice. One bi-transgenic mouse was maintained on Dox at conception and killed 63 days after birth (+Dox), whereas the other was maintained on Dox from conception until 35 days after birth, and then withdrawn from the drug and killed 28 days later (−Dox). Colorimetric staining of Flag-tagged d/n-ATF5 in the cortex (sagittal section) was achieved with anti-FLAG M2 antibody and Vulcan Fast Red. Scale bar=10 μm. X-gal substrate was used for β-gal colorimetric detection. V=lateral ventricle; CC=corpus callosum; Cx=cortex; SVZ=subventricular zone. Scale bar=20 μm. (c) Coordinately regulated expression of Flag-d/n-ATF5 and β-gal within the SVZ and near the CA1 region of the hippocampus (orthogonal view) in a 79-day-old bi-transgenic mouse raised without Dox since conception. Immunofluorescence staining was carried out with anti-Flag (for detection of Flag-d/n-ATF5; green) and β-gal (red). Arrows in the merged panel indicate cell colocalization. The confocal (2 μm) orthogonal section image shows the x–y and y–z location of both β-gal and Flag-d/n-ATF5 within the same cell. (d) Regulated expression of β-gal in GFAP+ cells within the cortex of a bi-transgenic mouse. The animal was maintained on Dox from conception until 204 days after birth and the drug was then withdrawn until being killed 50 days later. Panels show immunostaining with anti-GFAP (red) and anti-β-gal (green) and arrows show the locations of cells in which both are co-expressed. (e) Regulated expression of β-gal in GFAP+ cells within the SVZ of a bi-transgenic mouse. The mouse was maintained on doxycycline from conception until 167 days after birth and the drug was then withdrawn until being killed 42 days later. Panels show immunostaining with anti-GFAP (green) and anti-β-gal (red) and arrows show the locations of cells in which both are co-expressed. Diagrams show the brain areas where photographs were taken (adapted from BrainMaps.org and http://www.hms.harvard.edu/research/brain/atlas). Scale bar=20 μm (c–e).
Mentions: To study ATF5 function, we have created a specific interfering d/n form of the protein that lacks the N-terminal acidic activation and DNA-binding domains and that contains an enhanced bZip domain (Vinson et al., 1993; Krylov et al., 1995; Moitra et al., 1998; Moll et al., 2000; Angelastro et al., 2006). Flag-tagged d/n-ATF5 blocks ATF5 function and consequently accelerates differentiation of neural progenitors and induces apoptosis of glioblastoma and other neoplastic cells both in vitro and in vivo (Angelastro et al., 2003, 2005, 2006; Mason et al., 2005; Monaco et al., 2007). For the current studies, we generated a transgenic mouse line in which Flag-tagged-d/n-ATF5 and β-galactosidase (β-gal) (product of LacZ) are driven as separate gene products under the control of the pBi3-tetracycline operon (TETO) operator (Baron et al., 1995). This line was crossed with an hGFAP-tetracycline transactivator (tTA) transgenic mouse line (Pascual et al., 2005; Sweger et al., 2007) in which the hGFAP promoter drives the tTA protein (Gossen and Bujard, 1992; Gossen et al., 1994). This line has been successfully employed to spatially and temporally regulate transgenes such as d/n SNARE, GPCR Ro1 RASSL, Gq-coupled receptor and ErbB2 in neural progenitors and astrocytes (Pascual et al., 2005; Fiacco et al., 2007; Ghashghaei et al., 2007; Sweger et al., 2007). In this ‘Tet-off' system, in the absence of doxycycline, the bi-transgenic mice should express d/n-ATF5 and β-gal in cells such as neuroprogenitors and gliomas in which the hGFAP promoter is active; conversely in the presence of dietary doxycycline, d/n-ATF5 expression should be suppressed (Figure 2a).

Bottom Line: However, it was unknown whether interference with ATF5 function can prevent or promote regression/eradication of malignant gliomas in vivo.In this model, d/n-ATF5 expression is controlled by doxycycline and the promoter for GFAP, a marker for stem/progenitor cells as well as gliomas.Induction of d/n-ATF5 before delivery of PDGF-B/p53-shRNA virus greatly reduced the proportion of mice that formed tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of California, Davis School of Veterinary Medicine, Davis, CA 95616, USA.

ABSTRACT
Glioblastomas are among the most incurable cancers. Our past findings indicated that glioblastoma cells, but not neurons or glia, require the transcription factor ATF5 (activating transcription factor 5) for survival. However, it was unknown whether interference with ATF5 function can prevent or promote regression/eradication of malignant gliomas in vivo. To address this issue, we created a mouse model by crossing a human glial fibrillary acidic protein (GFAP) promoter-tetracycline transactivator mouse line with tetracycline operon-dominant negative-ATF5 (d/n-ATF5) mice to establish bi-transgenic mice. In this model, d/n-ATF5 expression is controlled by doxycycline and the promoter for GFAP, a marker for stem/progenitor cells as well as gliomas. Endogenous gliomas were produced with high efficiency by retroviral delivery of platelet-derived growth factor (PDGF)-B and p53-short hairpin RNA (shRNA) in adult bi-transgenic mice in which expression of d/n-ATF5 was spatially and temporally regulated. Induction of d/n-ATF5 before delivery of PDGF-B/p53-shRNA virus greatly reduced the proportion of mice that formed tumors. Moreover, d/n-ATF5 induction after tumor formation led to regression/eradication of detectable gliomas without evident damage to normal brain cells in all 24 mice assessed.

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Related in: MedlinePlus