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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

Retrovirus encoding PDGF-B-HA and p53-short hairpin RNA (shRNA) induces gliomas within mouse brains, and knocks down p53. (a) Scheme of self-inactivating retroviral construct used to induce gliomas. The 5′-long terminal repeat is intact, but the 3′-deleted long terminal repeat possesses a deletion to prevent self-replication. The U6 pol III promoter drives transcription of p53-shRNA, whereas the cytomegalovirus promoter drives transcription of the bicistronic PDGF-B-HA-IRES-DsRed-Express reporter. (b) Frozen section. H&E staining reveals a glioma induced in a doxycycline-treated hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal bi-transgenic mouse 100 days after stereotactic injection of retrovirus expressing PDGF-B-HA and p53-shRNA. Area of glioma is enclosed within black line. (c, c′–f, f′) Tumor cells induced by PDGF-B-HA/p53-shRNA express the HA tag and have dark hyperchromatic nuclei. The bi-transgenic mouse was continuously maintained with doxycycline from conception and was killed 135 days after injection with PDGF-B-HA/p53-shRNA retrovirus. Paraffin-embedded brain sections of PDGF-B-HA/p53-shRNA were immunostained with rabbit anti-HA antibody (red) and 4′,6-diamidino-2-phenylindole (blue) (c–f) or H&E (c′–f′). c, c′, e and e′ show PDGF-B-HA/p53-shRNA-induced tumors, whereas d, d′, f and f′ are from the corresponding tumor-free contralateral hemisphere. Scale bar=12 μm. (g–i) Tumors induced by PDGF-B-HA/p53-shRNA express the HA tag, but not p53. Photos show immunostained (for HA and p53) paraffin-embedded brain sections from a bi-transgenic mouse continuously exposed to doxycycline from conception and killed 135 days after retroviral injection. The HA+, p53− sections in (g) and (h) include tumor tissue, whereas the p53+ (green) section in (i) was outside the tumor margins. Scale bar=5 μm. (j, k) Gliomas induced by PDGF-B-HA/p53-shRNA have a proliferative index of 100% calculated from Ki67 immunostaining. A monotransgenic hGFAP-tTA mouse was continuously fed with doxycycline from conception and killed 31 days after stereotactic injection with PDGF-B-HA/p53-shRNA retrovirus. Paraffin-embedded brain sections were immunostained for Ki67 antiserum with visualization by immunoperoxidase (brown). (j) Ki67+ cells in a glioma induced in the injected hemisphere, whereas (k) shows the absence of Ki67 staining in normal tissue in the contralateral hemisphere. Scale bar=10 μm. Diagrams of brain sections in (c)–(k) show sites of retroviral injection (arrows) and corresponding areas where photographs were taken (vertical lines and letters). Diagrams are adapted from http://www.hms.harvard.edu/research/brain/atlas.
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fig3: Retrovirus encoding PDGF-B-HA and p53-short hairpin RNA (shRNA) induces gliomas within mouse brains, and knocks down p53. (a) Scheme of self-inactivating retroviral construct used to induce gliomas. The 5′-long terminal repeat is intact, but the 3′-deleted long terminal repeat possesses a deletion to prevent self-replication. The U6 pol III promoter drives transcription of p53-shRNA, whereas the cytomegalovirus promoter drives transcription of the bicistronic PDGF-B-HA-IRES-DsRed-Express reporter. (b) Frozen section. H&E staining reveals a glioma induced in a doxycycline-treated hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal bi-transgenic mouse 100 days after stereotactic injection of retrovirus expressing PDGF-B-HA and p53-shRNA. Area of glioma is enclosed within black line. (c, c′–f, f′) Tumor cells induced by PDGF-B-HA/p53-shRNA express the HA tag and have dark hyperchromatic nuclei. The bi-transgenic mouse was continuously maintained with doxycycline from conception and was killed 135 days after injection with PDGF-B-HA/p53-shRNA retrovirus. Paraffin-embedded brain sections of PDGF-B-HA/p53-shRNA were immunostained with rabbit anti-HA antibody (red) and 4′,6-diamidino-2-phenylindole (blue) (c–f) or H&E (c′–f′). c, c′, e and e′ show PDGF-B-HA/p53-shRNA-induced tumors, whereas d, d′, f and f′ are from the corresponding tumor-free contralateral hemisphere. Scale bar=12 μm. (g–i) Tumors induced by PDGF-B-HA/p53-shRNA express the HA tag, but not p53. Photos show immunostained (for HA and p53) paraffin-embedded brain sections from a bi-transgenic mouse continuously exposed to doxycycline from conception and killed 135 days after retroviral injection. The HA+, p53− sections in (g) and (h) include tumor tissue, whereas the p53+ (green) section in (i) was outside the tumor margins. Scale bar=5 μm. (j, k) Gliomas induced by PDGF-B-HA/p53-shRNA have a proliferative index of 100% calculated from Ki67 immunostaining. A monotransgenic hGFAP-tTA mouse was continuously fed with doxycycline from conception and killed 31 days after stereotactic injection with PDGF-B-HA/p53-shRNA retrovirus. Paraffin-embedded brain sections were immunostained for Ki67 antiserum with visualization by immunoperoxidase (brown). (j) Ki67+ cells in a glioma induced in the injected hemisphere, whereas (k) shows the absence of Ki67 staining in normal tissue in the contralateral hemisphere. Scale bar=10 μm. Diagrams of brain sections in (c)–(k) show sites of retroviral injection (arrows) and corresponding areas where photographs were taken (vertical lines and letters). Diagrams are adapted from http://www.hms.harvard.edu/research/brain/atlas.

Mentions: For our study, we constructed one control retrovirus that encoded short-hairpin-p53 RNA interference (p53-shRNA) and the DsRed fluorescent reporter, as well as a tumorigenic retrovirus also encoding PDGF-B-HA (hemagglutinin tagged), besides p53-shRNA and DsRed. The human U6 pol III drives the expression of p53-shRNA, whereas the cytomegalovirus promoter drives DsRed for the control retrovirus and PDGF-B-HA and DsRed for the tumorigenic retrovirus (Figure 3a). The retroviruses were stereotactically injected into the SVZ and corpus callosum of the left cerebral hemispheres of the mice at about 10 weeks of age. Injection of the left hemisphere permitted comparison with the unaffected right hemisphere. Brains were harvested at 40–180 days after viral injection, fixed, serially sectioned and stained and examined for the presence of tumors. In some cases, brains were paraffin embedded before sectioning to enhance the detection of tumor tissue.


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)

Retrovirus encoding PDGF-B-HA and p53-short hairpin RNA (shRNA) induces gliomas within mouse brains, and knocks down p53. (a) Scheme of self-inactivating retroviral construct used to induce gliomas. The 5′-long terminal repeat is intact, but the 3′-deleted long terminal repeat possesses a deletion to prevent self-replication. The U6 pol III promoter drives transcription of p53-shRNA, whereas the cytomegalovirus promoter drives transcription of the bicistronic PDGF-B-HA-IRES-DsRed-Express reporter. (b) Frozen section. H&E staining reveals a glioma induced in a doxycycline-treated hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal bi-transgenic mouse 100 days after stereotactic injection of retrovirus expressing PDGF-B-HA and p53-shRNA. Area of glioma is enclosed within black line. (c, c′–f, f′) Tumor cells induced by PDGF-B-HA/p53-shRNA express the HA tag and have dark hyperchromatic nuclei. The bi-transgenic mouse was continuously maintained with doxycycline from conception and was killed 135 days after injection with PDGF-B-HA/p53-shRNA retrovirus. Paraffin-embedded brain sections of PDGF-B-HA/p53-shRNA were immunostained with rabbit anti-HA antibody (red) and 4′,6-diamidino-2-phenylindole (blue) (c–f) or H&E (c′–f′). c, c′, e and e′ show PDGF-B-HA/p53-shRNA-induced tumors, whereas d, d′, f and f′ are from the corresponding tumor-free contralateral hemisphere. Scale bar=12 μm. (g–i) Tumors induced by PDGF-B-HA/p53-shRNA express the HA tag, but not p53. Photos show immunostained (for HA and p53) paraffin-embedded brain sections from a bi-transgenic mouse continuously exposed to doxycycline from conception and killed 135 days after retroviral injection. The HA+, p53− sections in (g) and (h) include tumor tissue, whereas the p53+ (green) section in (i) was outside the tumor margins. Scale bar=5 μm. (j, k) Gliomas induced by PDGF-B-HA/p53-shRNA have a proliferative index of 100% calculated from Ki67 immunostaining. A monotransgenic hGFAP-tTA mouse was continuously fed with doxycycline from conception and killed 31 days after stereotactic injection with PDGF-B-HA/p53-shRNA retrovirus. Paraffin-embedded brain sections were immunostained for Ki67 antiserum with visualization by immunoperoxidase (brown). (j) Ki67+ cells in a glioma induced in the injected hemisphere, whereas (k) shows the absence of Ki67 staining in normal tissue in the contralateral hemisphere. Scale bar=10 μm. Diagrams of brain sections in (c)–(k) show sites of retroviral injection (arrows) and corresponding areas where photographs were taken (vertical lines and letters). Diagrams are adapted from http://www.hms.harvard.edu/research/brain/atlas.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3277917&req=5

fig3: Retrovirus encoding PDGF-B-HA and p53-short hairpin RNA (shRNA) induces gliomas within mouse brains, and knocks down p53. (a) Scheme of self-inactivating retroviral construct used to induce gliomas. The 5′-long terminal repeat is intact, but the 3′-deleted long terminal repeat possesses a deletion to prevent self-replication. The U6 pol III promoter drives transcription of p53-shRNA, whereas the cytomegalovirus promoter drives transcription of the bicistronic PDGF-B-HA-IRES-DsRed-Express reporter. (b) Frozen section. H&E staining reveals a glioma induced in a doxycycline-treated hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal bi-transgenic mouse 100 days after stereotactic injection of retrovirus expressing PDGF-B-HA and p53-shRNA. Area of glioma is enclosed within black line. (c, c′–f, f′) Tumor cells induced by PDGF-B-HA/p53-shRNA express the HA tag and have dark hyperchromatic nuclei. The bi-transgenic mouse was continuously maintained with doxycycline from conception and was killed 135 days after injection with PDGF-B-HA/p53-shRNA retrovirus. Paraffin-embedded brain sections of PDGF-B-HA/p53-shRNA were immunostained with rabbit anti-HA antibody (red) and 4′,6-diamidino-2-phenylindole (blue) (c–f) or H&E (c′–f′). c, c′, e and e′ show PDGF-B-HA/p53-shRNA-induced tumors, whereas d, d′, f and f′ are from the corresponding tumor-free contralateral hemisphere. Scale bar=12 μm. (g–i) Tumors induced by PDGF-B-HA/p53-shRNA express the HA tag, but not p53. Photos show immunostained (for HA and p53) paraffin-embedded brain sections from a bi-transgenic mouse continuously exposed to doxycycline from conception and killed 135 days after retroviral injection. The HA+, p53− sections in (g) and (h) include tumor tissue, whereas the p53+ (green) section in (i) was outside the tumor margins. Scale bar=5 μm. (j, k) Gliomas induced by PDGF-B-HA/p53-shRNA have a proliferative index of 100% calculated from Ki67 immunostaining. A monotransgenic hGFAP-tTA mouse was continuously fed with doxycycline from conception and killed 31 days after stereotactic injection with PDGF-B-HA/p53-shRNA retrovirus. Paraffin-embedded brain sections were immunostained for Ki67 antiserum with visualization by immunoperoxidase (brown). (j) Ki67+ cells in a glioma induced in the injected hemisphere, whereas (k) shows the absence of Ki67 staining in normal tissue in the contralateral hemisphere. Scale bar=10 μm. Diagrams of brain sections in (c)–(k) show sites of retroviral injection (arrows) and corresponding areas where photographs were taken (vertical lines and letters). Diagrams are adapted from http://www.hms.harvard.edu/research/brain/atlas.
Mentions: For our study, we constructed one control retrovirus that encoded short-hairpin-p53 RNA interference (p53-shRNA) and the DsRed fluorescent reporter, as well as a tumorigenic retrovirus also encoding PDGF-B-HA (hemagglutinin tagged), besides p53-shRNA and DsRed. The human U6 pol III drives the expression of p53-shRNA, whereas the cytomegalovirus promoter drives DsRed for the control retrovirus and PDGF-B-HA and DsRed for the tumorigenic retrovirus (Figure 3a). The retroviruses were stereotactically injected into the SVZ and corpus callosum of the left cerebral hemispheres of the mice at about 10 weeks of age. Injection of the left hemisphere permitted comparison with the unaffected right hemisphere. Brains were harvested at 40–180 days after viral injection, fixed, serially sectioned and stained and examined for the presence of tumors. In some cases, brains were paraffin embedded before sectioning to enhance the detection of tumor tissue.

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.

Show MeSH
Related in: MedlinePlus