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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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Bi-transgenic hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal mice do not harbor detectable gliomas after induction of d/n-ATF5. (a, b) PDGF-B-HA/p53-shRNA-induced glioma in a bi-transgenic mouse expresses ATF5, but not the d/n-ATF5/β-gal transgene. The mouse was continuously maintained on doxycycline from conception to suppress d/n-ATF5 induction and was killed 135 days post-stereotactic injection with PDGF-B-HA/p53-shRNA retrovirus. Brain sections were stained with rabbit anti-ATF5 antibody (red) and chicken anti-β-gal antibody (green). Note strong immunostaining for ATF5 and lack of β-gal immunostaining in tumor (a) and only weak scattered, low-level ATF5 immunoreactivity in the contralateral hemisphere (b). H&E staining for (a) is from the same brain shown in Figure 3c′, and H&E staining for (b) is from the same brain shown in Figure 3d′. Scale bar=6 μm. (c, d) Immunostaining for the HA tag (red) and GFAP (green) detects a glioma, as shown by H&E staining, in a mouse in which expression of d/n-ATF5 was suppressed (c), but not in a mouse in which d/n-ATF5 was induced (d). The right-hand panels show H&E staining to confirm the presence or absence of a glioma. Boxes within the H&E photos show where the anti-HA and anti-GFAP immunofluorescence images were taken. Scale bar=40 μm for H&E photos and 12 μm for immunofluorescence images in (c) and (d). The bi-transgenic mouse in (c) was continuously maintained on doxycycline from conception to suppress d/n-ATF5 induction and was killed 135 days post-stereotactic injection with PDGF-B-HA/p53-shRNA retrovirus. Note HA and GFAP immunostaining in tumor remote from site of injection (c). The bi-transgenic mouse in (d) was maintained on doxycycline from conception to 90 days post-injection with PDGF-B-HA/p53-shRNA retrovirus. Doxycycline was then withdrawn from the diet for 45 days to induce d/n-ATF5 and the animal was killed. Examination of serial brain sections revealed scattered HA-stained cells as shown, only at the site of viral injection. GFAP+ cells at upper right reflect astrocytic activation in response to the injection wound. Paraffin-embedded sections. (e, f) Evidence for eradication of a pre-existing tumor by induction of d/n-ATF5. The bi-transgenic mouse was maintained on doxycycline from conception to 92 days post-injection with PDGF-B-HA/p53-shRNA retrovirus. Doxycycline was then withdrawn from the diet for 50 days to induce d/n-ATF5. (e) Normal cells in contralateral hemisphere, but (f) lesions in the neutrophil remote from the viral injection site in the ipsilateral hemisphere. Scale bar=20 μm. (g) Co-expression of cleaved caspase-3 (CC-3) and d/n-ATF5 in a tumor remnant in the ‘eradication/regression' paradigm. A bi-transgenic mouse was maintained on doxycycline from conception to 90 days after injection with PDGF-B-HA/p53-shRNA retrovirus. Doxycycline was then withdrawn from the diet for 50 days to induce d/n-ATF5 and the animal was killed. Brain sections that contained a remnant of a glioma were co-stained for Flag-tagged d/n-ATF5 (green) and cleaved-caspase-3 (red). Scale bar=20 μm. Diagrams show the brain areas for stereotactic injection site (arrow) with corresponding panel letter (diagrams adapted from http://www.hms.harvard.edu/research/brain/atlas).
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fig4: Bi-transgenic hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal mice do not harbor detectable gliomas after induction of d/n-ATF5. (a, b) PDGF-B-HA/p53-shRNA-induced glioma in a bi-transgenic mouse expresses ATF5, but not the d/n-ATF5/β-gal transgene. The mouse was continuously maintained on doxycycline from conception to suppress d/n-ATF5 induction and was killed 135 days post-stereotactic injection with PDGF-B-HA/p53-shRNA retrovirus. Brain sections were stained with rabbit anti-ATF5 antibody (red) and chicken anti-β-gal antibody (green). Note strong immunostaining for ATF5 and lack of β-gal immunostaining in tumor (a) and only weak scattered, low-level ATF5 immunoreactivity in the contralateral hemisphere (b). H&E staining for (a) is from the same brain shown in Figure 3c′, and H&E staining for (b) is from the same brain shown in Figure 3d′. Scale bar=6 μm. (c, d) Immunostaining for the HA tag (red) and GFAP (green) detects a glioma, as shown by H&E staining, in a mouse in which expression of d/n-ATF5 was suppressed (c), but not in a mouse in which d/n-ATF5 was induced (d). The right-hand panels show H&E staining to confirm the presence or absence of a glioma. Boxes within the H&E photos show where the anti-HA and anti-GFAP immunofluorescence images were taken. Scale bar=40 μm for H&E photos and 12 μm for immunofluorescence images in (c) and (d). The bi-transgenic mouse in (c) was continuously maintained on doxycycline from conception to suppress d/n-ATF5 induction and was killed 135 days post-stereotactic injection with PDGF-B-HA/p53-shRNA retrovirus. Note HA and GFAP immunostaining in tumor remote from site of injection (c). The bi-transgenic mouse in (d) was maintained on doxycycline from conception to 90 days post-injection with PDGF-B-HA/p53-shRNA retrovirus. Doxycycline was then withdrawn from the diet for 45 days to induce d/n-ATF5 and the animal was killed. Examination of serial brain sections revealed scattered HA-stained cells as shown, only at the site of viral injection. GFAP+ cells at upper right reflect astrocytic activation in response to the injection wound. Paraffin-embedded sections. (e, f) Evidence for eradication of a pre-existing tumor by induction of d/n-ATF5. The bi-transgenic mouse was maintained on doxycycline from conception to 92 days post-injection with PDGF-B-HA/p53-shRNA retrovirus. Doxycycline was then withdrawn from the diet for 50 days to induce d/n-ATF5. (e) Normal cells in contralateral hemisphere, but (f) lesions in the neutrophil remote from the viral injection site in the ipsilateral hemisphere. Scale bar=20 μm. (g) Co-expression of cleaved caspase-3 (CC-3) and d/n-ATF5 in a tumor remnant in the ‘eradication/regression' paradigm. A bi-transgenic mouse was maintained on doxycycline from conception to 90 days after injection with PDGF-B-HA/p53-shRNA retrovirus. Doxycycline was then withdrawn from the diet for 50 days to induce d/n-ATF5 and the animal was killed. Brain sections that contained a remnant of a glioma were co-stained for Flag-tagged d/n-ATF5 (green) and cleaved-caspase-3 (red). Scale bar=20 μm. Diagrams show the brain areas for stereotactic injection site (arrow) with corresponding panel letter (diagrams adapted from http://www.hms.harvard.edu/research/brain/atlas).

Mentions: The glioma cells were typically diffuse with hyperchromatic irregular nuclei and a sharp reduction in neuronal density (hematoxylin and eosin (H&E); Figures 3b, c′ and e′); about 10% exhibited pseudopalisading cells around necrotic foci. The tumors were also highly invasive as evidenced by one example in which tumor cells migrated away from the injection site in the septo-diencephalic region to the caudal mesencephalon region (Figure 3e; see also Figures 3c and 4a, c and Supplementary Figure S4 for additional examples). The appearance of tumor cells far from the site of viral injection was not due to diffusion of the virus. Injection of control p53-shRNA/DsRed retroviruses generated infected cells only at the injection site and these were not invasive (Supplementary Figure S5; see also Assanah et al. (2006)). The high proliferative activity (100% index) of the gliomas was indicated by the presence of marked Ki67 staining, which contrasted with the paucity of such staining in the contralateral hemisphere (0% index) (Figures 3j and k). As evidence that the tumor cells were derived from cells that took up the virus, they expressed the HA tag (Figures 3c, e and g) and were negative for p53 immunostaining (Figure 3h). This contrasted with normal cells on the contralateral side or that were remote from the tumor cells, which were HA− and p53+ (Figures 3d, f and i; distal to the tumor). These results were corroborated by staining for DsRed (data not shown). In contrast to normal contralateral brain tissue that has only a small proportion of cells immunoreactive for PDGF-A receptors (Supplementary Figure S4A), the gliomas were strongly positive for PDGF-A receptor expression (Supplementary Figure S4B, C). The glioma cells were also positive for the expression of GFAP (Figure 4c) and, significantly, for ATF5 (Figures 4a and b). Immunofluorescent microscopy for β-gal expression in the tumors (as well as in ipsilateral normal brain tissue) in the doxycycline-treated bi-transgenics showed no immunoreactivity, confirming that the d/n-ATF5/LacZ transgene was not activated in presence of the drug (Figures 4a and b). Finally, another indication of tumor formation in response to the p53-shRNA/PDGF-B-HA/DsRed retrovirus was moribund behavior. Within the 180-day course of the study, six of the 16 virally injected bi-transgenic animals treated with doxycycline since conception to suppress d/n-ATF5 were judged by veterinary staff to have become moribund and were immediately killed (Figure 5b). All of these were found to harbor gliomas. Taken together, these data provide evidence that the p53-shRNA/PDGF-B-HA/DsRed retrovirus effectively and rapidly induces gliomas in bi-transgenic mice and that this model provides an effective system in which to assess the effects of activating d/n-ATF5 on tumor formation and maintenance.


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)

Bi-transgenic hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal mice do not harbor detectable gliomas after induction of d/n-ATF5. (a, b) PDGF-B-HA/p53-shRNA-induced glioma in a bi-transgenic mouse expresses ATF5, but not the d/n-ATF5/β-gal transgene. The mouse was continuously maintained on doxycycline from conception to suppress d/n-ATF5 induction and was killed 135 days post-stereotactic injection with PDGF-B-HA/p53-shRNA retrovirus. Brain sections were stained with rabbit anti-ATF5 antibody (red) and chicken anti-β-gal antibody (green). Note strong immunostaining for ATF5 and lack of β-gal immunostaining in tumor (a) and only weak scattered, low-level ATF5 immunoreactivity in the contralateral hemisphere (b). H&E staining for (a) is from the same brain shown in Figure 3c′, and H&E staining for (b) is from the same brain shown in Figure 3d′. Scale bar=6 μm. (c, d) Immunostaining for the HA tag (red) and GFAP (green) detects a glioma, as shown by H&E staining, in a mouse in which expression of d/n-ATF5 was suppressed (c), but not in a mouse in which d/n-ATF5 was induced (d). The right-hand panels show H&E staining to confirm the presence or absence of a glioma. Boxes within the H&E photos show where the anti-HA and anti-GFAP immunofluorescence images were taken. Scale bar=40 μm for H&E photos and 12 μm for immunofluorescence images in (c) and (d). The bi-transgenic mouse in (c) was continuously maintained on doxycycline from conception to suppress d/n-ATF5 induction and was killed 135 days post-stereotactic injection with PDGF-B-HA/p53-shRNA retrovirus. Note HA and GFAP immunostaining in tumor remote from site of injection (c). The bi-transgenic mouse in (d) was maintained on doxycycline from conception to 90 days post-injection with PDGF-B-HA/p53-shRNA retrovirus. Doxycycline was then withdrawn from the diet for 45 days to induce d/n-ATF5 and the animal was killed. Examination of serial brain sections revealed scattered HA-stained cells as shown, only at the site of viral injection. GFAP+ cells at upper right reflect astrocytic activation in response to the injection wound. Paraffin-embedded sections. (e, f) Evidence for eradication of a pre-existing tumor by induction of d/n-ATF5. The bi-transgenic mouse was maintained on doxycycline from conception to 92 days post-injection with PDGF-B-HA/p53-shRNA retrovirus. Doxycycline was then withdrawn from the diet for 50 days to induce d/n-ATF5. (e) Normal cells in contralateral hemisphere, but (f) lesions in the neutrophil remote from the viral injection site in the ipsilateral hemisphere. Scale bar=20 μm. (g) Co-expression of cleaved caspase-3 (CC-3) and d/n-ATF5 in a tumor remnant in the ‘eradication/regression' paradigm. A bi-transgenic mouse was maintained on doxycycline from conception to 90 days after injection with PDGF-B-HA/p53-shRNA retrovirus. Doxycycline was then withdrawn from the diet for 50 days to induce d/n-ATF5 and the animal was killed. Brain sections that contained a remnant of a glioma were co-stained for Flag-tagged d/n-ATF5 (green) and cleaved-caspase-3 (red). Scale bar=20 μm. Diagrams show the brain areas for stereotactic injection site (arrow) with corresponding panel letter (diagrams adapted from http://www.hms.harvard.edu/research/brain/atlas).
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fig4: Bi-transgenic hGFAP-tTA × pBi-TETO-3-Flag-d/n-ATF5/β-gal mice do not harbor detectable gliomas after induction of d/n-ATF5. (a, b) PDGF-B-HA/p53-shRNA-induced glioma in a bi-transgenic mouse expresses ATF5, but not the d/n-ATF5/β-gal transgene. The mouse was continuously maintained on doxycycline from conception to suppress d/n-ATF5 induction and was killed 135 days post-stereotactic injection with PDGF-B-HA/p53-shRNA retrovirus. Brain sections were stained with rabbit anti-ATF5 antibody (red) and chicken anti-β-gal antibody (green). Note strong immunostaining for ATF5 and lack of β-gal immunostaining in tumor (a) and only weak scattered, low-level ATF5 immunoreactivity in the contralateral hemisphere (b). H&E staining for (a) is from the same brain shown in Figure 3c′, and H&E staining for (b) is from the same brain shown in Figure 3d′. Scale bar=6 μm. (c, d) Immunostaining for the HA tag (red) and GFAP (green) detects a glioma, as shown by H&E staining, in a mouse in which expression of d/n-ATF5 was suppressed (c), but not in a mouse in which d/n-ATF5 was induced (d). The right-hand panels show H&E staining to confirm the presence or absence of a glioma. Boxes within the H&E photos show where the anti-HA and anti-GFAP immunofluorescence images were taken. Scale bar=40 μm for H&E photos and 12 μm for immunofluorescence images in (c) and (d). The bi-transgenic mouse in (c) was continuously maintained on doxycycline from conception to suppress d/n-ATF5 induction and was killed 135 days post-stereotactic injection with PDGF-B-HA/p53-shRNA retrovirus. Note HA and GFAP immunostaining in tumor remote from site of injection (c). The bi-transgenic mouse in (d) was maintained on doxycycline from conception to 90 days post-injection with PDGF-B-HA/p53-shRNA retrovirus. Doxycycline was then withdrawn from the diet for 45 days to induce d/n-ATF5 and the animal was killed. Examination of serial brain sections revealed scattered HA-stained cells as shown, only at the site of viral injection. GFAP+ cells at upper right reflect astrocytic activation in response to the injection wound. Paraffin-embedded sections. (e, f) Evidence for eradication of a pre-existing tumor by induction of d/n-ATF5. The bi-transgenic mouse was maintained on doxycycline from conception to 92 days post-injection with PDGF-B-HA/p53-shRNA retrovirus. Doxycycline was then withdrawn from the diet for 50 days to induce d/n-ATF5. (e) Normal cells in contralateral hemisphere, but (f) lesions in the neutrophil remote from the viral injection site in the ipsilateral hemisphere. Scale bar=20 μm. (g) Co-expression of cleaved caspase-3 (CC-3) and d/n-ATF5 in a tumor remnant in the ‘eradication/regression' paradigm. A bi-transgenic mouse was maintained on doxycycline from conception to 90 days after injection with PDGF-B-HA/p53-shRNA retrovirus. Doxycycline was then withdrawn from the diet for 50 days to induce d/n-ATF5 and the animal was killed. Brain sections that contained a remnant of a glioma were co-stained for Flag-tagged d/n-ATF5 (green) and cleaved-caspase-3 (red). Scale bar=20 μm. Diagrams show the brain areas for stereotactic injection site (arrow) with corresponding panel letter (diagrams adapted from http://www.hms.harvard.edu/research/brain/atlas).
Mentions: The glioma cells were typically diffuse with hyperchromatic irregular nuclei and a sharp reduction in neuronal density (hematoxylin and eosin (H&E); Figures 3b, c′ and e′); about 10% exhibited pseudopalisading cells around necrotic foci. The tumors were also highly invasive as evidenced by one example in which tumor cells migrated away from the injection site in the septo-diencephalic region to the caudal mesencephalon region (Figure 3e; see also Figures 3c and 4a, c and Supplementary Figure S4 for additional examples). The appearance of tumor cells far from the site of viral injection was not due to diffusion of the virus. Injection of control p53-shRNA/DsRed retroviruses generated infected cells only at the injection site and these were not invasive (Supplementary Figure S5; see also Assanah et al. (2006)). The high proliferative activity (100% index) of the gliomas was indicated by the presence of marked Ki67 staining, which contrasted with the paucity of such staining in the contralateral hemisphere (0% index) (Figures 3j and k). As evidence that the tumor cells were derived from cells that took up the virus, they expressed the HA tag (Figures 3c, e and g) and were negative for p53 immunostaining (Figure 3h). This contrasted with normal cells on the contralateral side or that were remote from the tumor cells, which were HA− and p53+ (Figures 3d, f and i; distal to the tumor). These results were corroborated by staining for DsRed (data not shown). In contrast to normal contralateral brain tissue that has only a small proportion of cells immunoreactive for PDGF-A receptors (Supplementary Figure S4A), the gliomas were strongly positive for PDGF-A receptor expression (Supplementary Figure S4B, C). The glioma cells were also positive for the expression of GFAP (Figure 4c) and, significantly, for ATF5 (Figures 4a and b). Immunofluorescent microscopy for β-gal expression in the tumors (as well as in ipsilateral normal brain tissue) in the doxycycline-treated bi-transgenics showed no immunoreactivity, confirming that the d/n-ATF5/LacZ transgene was not activated in presence of the drug (Figures 4a and b). Finally, another indication of tumor formation in response to the p53-shRNA/PDGF-B-HA/DsRed retrovirus was moribund behavior. Within the 180-day course of the study, six of the 16 virally injected bi-transgenic animals treated with doxycycline since conception to suppress d/n-ATF5 were judged by veterinary staff to have become moribund and were immediately killed (Figure 5b). All of these were found to harbor gliomas. Taken together, these data provide evidence that the p53-shRNA/PDGF-B-HA/DsRed retrovirus effectively and rapidly induces gliomas in bi-transgenic mice and that this model provides an effective system in which to assess the effects of activating d/n-ATF5 on tumor formation and maintenance.

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