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Retinoblastoma loss modulates DNA damage response favoring tumor progression.

Seoane M, Iglesias P, Gonzalez T, Dominguez F, Fraga M, Aliste C, Forteza J, Costoya JA - PLoS ONE (2008)

Bottom Line: It has been described in premalignant lesions that OIS requires DNA damage response (DDR) activation, safeguard of the integrity of the genome.In human gliomas most of the genetic alterations that have been previously identified result in abnormal activation of cell growth signaling pathways and deregulation of cell cycle, features recapitulated in our model by oncogenic Ras expression and retinoblastoma (Rb) inactivation respectively.Moreover, Rb loss inactivates the stress kinase DDR-associated p38MAPK by specific Wip1-dependent dephosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology Lab, Departamento de Fisioloxia, Facultade de Medicina, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.

ABSTRACT
Senescence is one of the main barriers against tumor progression. Oncogenic signals in primary cells result in oncogene-induced senescence (OIS), crucial for protection against cancer development. It has been described in premalignant lesions that OIS requires DNA damage response (DDR) activation, safeguard of the integrity of the genome. Here we demonstrate how the cellular mechanisms involved in oncogenic transformation in a model of glioma uncouple OIS and DDR. We use this tumor type as a paradigm of oncogenic transformation. In human gliomas most of the genetic alterations that have been previously identified result in abnormal activation of cell growth signaling pathways and deregulation of cell cycle, features recapitulated in our model by oncogenic Ras expression and retinoblastoma (Rb) inactivation respectively. In this scenario, the absence of pRb confers a proliferative advantage and activates DDR to a greater extent in a DNA lesion-independent fashion than cells that express only HRas(V12). Moreover, Rb loss inactivates the stress kinase DDR-associated p38MAPK by specific Wip1-dependent dephosphorylation. Thus, Rb loss acts as a switch mediating the transition between premalignant lesions and cancer through DDR modulation. These findings may have important implications for the understanding the biology of gliomas and anticipate a new target, Wip1 phosphatase, for novel therapeutic strategies.

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Mouse astrocytes are defective for HRasv12-induced cell cycle arrest.a, Growth curve analysis of early-passage cRbloxP/loxP conditional astrocytes co-infected with PIG/pBABE (vector), PIG/pBABE-HRasV12, PIG-Cre/pBABE and PIG-Cre/pBABE-HRasV12 retroviral vectors. After infection, cells were plated in triplicate and the cells were fixed on the indicated days for subsequent staining with crystal violet. Each time point represents the mean±s.d. of total cumulative cell number from at least three independent experiments. b, Measurement of the proliferation of astrocytes by BrdU incorporation assay (panel c). c, Cells were labeled with BrdU for 5 h on day 5 after puromycin selection. d, Senescence assays in cRbloxP/loxP and cRb−/−. The y-axis represents the percentage of SA-β-galactosidase-positive cells (mean and s.d.) from at least three independent experiments. e, A representative result of three independent experiments is shown. Photographs are at the same magnification.
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pone-0003632-g001: Mouse astrocytes are defective for HRasv12-induced cell cycle arrest.a, Growth curve analysis of early-passage cRbloxP/loxP conditional astrocytes co-infected with PIG/pBABE (vector), PIG/pBABE-HRasV12, PIG-Cre/pBABE and PIG-Cre/pBABE-HRasV12 retroviral vectors. After infection, cells were plated in triplicate and the cells were fixed on the indicated days for subsequent staining with crystal violet. Each time point represents the mean±s.d. of total cumulative cell number from at least three independent experiments. b, Measurement of the proliferation of astrocytes by BrdU incorporation assay (panel c). c, Cells were labeled with BrdU for 5 h on day 5 after puromycin selection. d, Senescence assays in cRbloxP/loxP and cRb−/−. The y-axis represents the percentage of SA-β-galactosidase-positive cells (mean and s.d.) from at least three independent experiments. e, A representative result of three independent experiments is shown. Photographs are at the same magnification.

Mentions: Unexpectedly, we observed that the astrocytes infected with PIG-Cre/pBABE-HRasV12 retroviral vector (hereafter cRb−/−/RasV12) showed after one week an increased proliferation rate when compared with the remaining groups. Astrocytes infected with PIG-Cre/pBABE (hereafter cRb−/−) and PIG/pBABE (hereafter cRbloxP/loxP) were used as control groups. To our surprise, astrocytes infected with PIG/pBABE-HRasV12 (hereafter cRbloxP/loxP/RasV12) also displayed a dramatic increment when compared with the control group, although at a less extent than cRbloxP/loxP/RasV12 astrocytes. Proliferative differences between astrocytic populations can be easily spotted in Figure 1A. To further support these data, we carried out a BrdU incorporation assay on all of the experimental groups, as shown in Figures 1B and 1C, that confirmed our initial observation.


Retinoblastoma loss modulates DNA damage response favoring tumor progression.

Seoane M, Iglesias P, Gonzalez T, Dominguez F, Fraga M, Aliste C, Forteza J, Costoya JA - PLoS ONE (2008)

Mouse astrocytes are defective for HRasv12-induced cell cycle arrest.a, Growth curve analysis of early-passage cRbloxP/loxP conditional astrocytes co-infected with PIG/pBABE (vector), PIG/pBABE-HRasV12, PIG-Cre/pBABE and PIG-Cre/pBABE-HRasV12 retroviral vectors. After infection, cells were plated in triplicate and the cells were fixed on the indicated days for subsequent staining with crystal violet. Each time point represents the mean±s.d. of total cumulative cell number from at least three independent experiments. b, Measurement of the proliferation of astrocytes by BrdU incorporation assay (panel c). c, Cells were labeled with BrdU for 5 h on day 5 after puromycin selection. d, Senescence assays in cRbloxP/loxP and cRb−/−. The y-axis represents the percentage of SA-β-galactosidase-positive cells (mean and s.d.) from at least three independent experiments. e, A representative result of three independent experiments is shown. Photographs are at the same magnification.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2573954&req=5

pone-0003632-g001: Mouse astrocytes are defective for HRasv12-induced cell cycle arrest.a, Growth curve analysis of early-passage cRbloxP/loxP conditional astrocytes co-infected with PIG/pBABE (vector), PIG/pBABE-HRasV12, PIG-Cre/pBABE and PIG-Cre/pBABE-HRasV12 retroviral vectors. After infection, cells were plated in triplicate and the cells were fixed on the indicated days for subsequent staining with crystal violet. Each time point represents the mean±s.d. of total cumulative cell number from at least three independent experiments. b, Measurement of the proliferation of astrocytes by BrdU incorporation assay (panel c). c, Cells were labeled with BrdU for 5 h on day 5 after puromycin selection. d, Senescence assays in cRbloxP/loxP and cRb−/−. The y-axis represents the percentage of SA-β-galactosidase-positive cells (mean and s.d.) from at least three independent experiments. e, A representative result of three independent experiments is shown. Photographs are at the same magnification.
Mentions: Unexpectedly, we observed that the astrocytes infected with PIG-Cre/pBABE-HRasV12 retroviral vector (hereafter cRb−/−/RasV12) showed after one week an increased proliferation rate when compared with the remaining groups. Astrocytes infected with PIG-Cre/pBABE (hereafter cRb−/−) and PIG/pBABE (hereafter cRbloxP/loxP) were used as control groups. To our surprise, astrocytes infected with PIG/pBABE-HRasV12 (hereafter cRbloxP/loxP/RasV12) also displayed a dramatic increment when compared with the control group, although at a less extent than cRbloxP/loxP/RasV12 astrocytes. Proliferative differences between astrocytic populations can be easily spotted in Figure 1A. To further support these data, we carried out a BrdU incorporation assay on all of the experimental groups, as shown in Figures 1B and 1C, that confirmed our initial observation.

Bottom Line: It has been described in premalignant lesions that OIS requires DNA damage response (DDR) activation, safeguard of the integrity of the genome.In human gliomas most of the genetic alterations that have been previously identified result in abnormal activation of cell growth signaling pathways and deregulation of cell cycle, features recapitulated in our model by oncogenic Ras expression and retinoblastoma (Rb) inactivation respectively.Moreover, Rb loss inactivates the stress kinase DDR-associated p38MAPK by specific Wip1-dependent dephosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology Lab, Departamento de Fisioloxia, Facultade de Medicina, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.

ABSTRACT
Senescence is one of the main barriers against tumor progression. Oncogenic signals in primary cells result in oncogene-induced senescence (OIS), crucial for protection against cancer development. It has been described in premalignant lesions that OIS requires DNA damage response (DDR) activation, safeguard of the integrity of the genome. Here we demonstrate how the cellular mechanisms involved in oncogenic transformation in a model of glioma uncouple OIS and DDR. We use this tumor type as a paradigm of oncogenic transformation. In human gliomas most of the genetic alterations that have been previously identified result in abnormal activation of cell growth signaling pathways and deregulation of cell cycle, features recapitulated in our model by oncogenic Ras expression and retinoblastoma (Rb) inactivation respectively. In this scenario, the absence of pRb confers a proliferative advantage and activates DDR to a greater extent in a DNA lesion-independent fashion than cells that express only HRas(V12). Moreover, Rb loss inactivates the stress kinase DDR-associated p38MAPK by specific Wip1-dependent dephosphorylation. Thus, Rb loss acts as a switch mediating the transition between premalignant lesions and cancer through DDR modulation. These findings may have important implications for the understanding the biology of gliomas and anticipate a new target, Wip1 phosphatase, for novel therapeutic strategies.

Show MeSH
Related in: MedlinePlus