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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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HRasV12-dependent production of reactive oxygen species (ROS) and associated chromosomal instability.a, cells were assayed for dihydroethidium (DHE) fluorescence, indicative of ROS production and visualized by fluorescent microscopy. Under identical imaging conditions, DHE oxidation was significantly increased in cRbloxP/loxP/RasV12 and cRb−/−/RasV12 astrocytes. b, cRb−/−/RasV12 and cRbloxP/loxP/RasV12 cells are significantly more aneuploid than cRbloxP/loxP and cRb−/− cells. c, Absolute chromosome numbers of cRbloxP/loxP, cRbloxP/loxP/RasV12, cRb−/− and cRb−/−/RasV12 cells, showing the presence of diploid and tetraploid populations. Representative data of at least three independent experiments. d, Band G (Wright S) and telomere fluorescence in situ hybridization (T-Fish) was performed using a Cy3-labeled peptide nucleic acid (PNA) telomeric probe. Representative metaphase spreads from cRbloxP/loxP and cRb−/−, partial representative metaphase spreads from cRbloxP/loxP/RasV12 and cRb−/−/RasV12 stained with Wright and hybridized with Cy3 telomeric probe are shown. The regions of metaphase spreads from cRbloxP/loxP/RasV12 and cRb−/−/RasV12 are magnified in the lower panel to show centromere fragments (CF), acentric fragment (AF), chromatide type break (CTB), telomeric fusion (TF), robertsonian translocation (Rob) and other type of translocation (T). X indicates quadrivalent (Qua) found in cRbloxP/loxP/RasV12.
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pone-0003632-g003: HRasV12-dependent production of reactive oxygen species (ROS) and associated chromosomal instability.a, cells were assayed for dihydroethidium (DHE) fluorescence, indicative of ROS production and visualized by fluorescent microscopy. Under identical imaging conditions, DHE oxidation was significantly increased in cRbloxP/loxP/RasV12 and cRb−/−/RasV12 astrocytes. b, cRb−/−/RasV12 and cRbloxP/loxP/RasV12 cells are significantly more aneuploid than cRbloxP/loxP and cRb−/− cells. c, Absolute chromosome numbers of cRbloxP/loxP, cRbloxP/loxP/RasV12, cRb−/− and cRb−/−/RasV12 cells, showing the presence of diploid and tetraploid populations. Representative data of at least three independent experiments. d, Band G (Wright S) and telomere fluorescence in situ hybridization (T-Fish) was performed using a Cy3-labeled peptide nucleic acid (PNA) telomeric probe. Representative metaphase spreads from cRbloxP/loxP and cRb−/−, partial representative metaphase spreads from cRbloxP/loxP/RasV12 and cRb−/−/RasV12 stained with Wright and hybridized with Cy3 telomeric probe are shown. The regions of metaphase spreads from cRbloxP/loxP/RasV12 and cRb−/−/RasV12 are magnified in the lower panel to show centromere fragments (CF), acentric fragment (AF), chromatide type break (CTB), telomeric fusion (TF), robertsonian translocation (Rob) and other type of translocation (T). X indicates quadrivalent (Qua) found in cRbloxP/loxP/RasV12.

Mentions: As we previously described, although astrocytes harboring HRasV12 did not enter senescence we wanted to confirm whether DNA damage provoked by DNA replication stress, and usually associated with oncogene-induced senescence, was present. On the other hand, HRasV12 is also known to cause an increase in reactive oxygen species (ROS) production [36] as a result of replication stress. ROS has been traditionally considered as a toxic by-product of cellular metabolism, but it has been appreciated that they are actively involved in oncogenic signaling in cellular transformation and cancer. Increased intracellular levels of ROS have also been reported to mediate some biological effects of oncogenic HRasV12, such as the onset of premature senescence in primary cells, the generation of genomic instability [37], and malignant transformation [38]. Furthermore, high levels of ROS have been detected in several human cancer cell lines as well as in human tumors from different tissues. Taken together these reports, we wanted to analyze in astrocytes the relationship between oxidative stress and glioma. We observed similar oncogene-induced ROS accumulation in cRbloxP/loxP/RasV12 and cRb−/−/RasV12 astrocytes, but not in those that had lost only Rb (Figure 3A). Likewise, we found signs of chromosomal instability (Figure 3B–D) in the groups that expressed HRasV12 and displayed ROS accumulation. One of the most remarkable features of cRbloxP/loxP/RasV12 and cRb−/−/RasV12 astrocytes is the abundance of centromeric instability manifested as centromere fragments (CF) (Figure 3D and Figure S1). We also found Robertsonian centromere fusion (Rob) in cRbloxP/loxP/RasV12 and cRb−/−/RasV12 astrocytes but not in cRb−/− and wildtype astrocytes (Figure S1). In addition, there were numerical chromosome abnormalities in the experimental groups that expressed oncogenic Ras. These astrocytes presented >80 chromosomes per cell, doubling the chromosome number found in control cells (Figure 3B and 3C). Hence, these numerical and structural chromosome abnormalities show that activated Ras is sufficient to induce chromosomal instability in the absence of other signals, suggesting that Ras-induced chromosomal instability arises as consequence of ROS accumulation and DNA replication stress. In addition, there is not a significant difference between cRbloxP/loxP/RasV12 and cRb−/−/RasV12 astrocytes implying that the absence of pRb has not effect on DNA lesions, either quantitative or qualitative.


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)

HRasV12-dependent production of reactive oxygen species (ROS) and associated chromosomal instability.a, cells were assayed for dihydroethidium (DHE) fluorescence, indicative of ROS production and visualized by fluorescent microscopy. Under identical imaging conditions, DHE oxidation was significantly increased in cRbloxP/loxP/RasV12 and cRb−/−/RasV12 astrocytes. b, cRb−/−/RasV12 and cRbloxP/loxP/RasV12 cells are significantly more aneuploid than cRbloxP/loxP and cRb−/− cells. c, Absolute chromosome numbers of cRbloxP/loxP, cRbloxP/loxP/RasV12, cRb−/− and cRb−/−/RasV12 cells, showing the presence of diploid and tetraploid populations. Representative data of at least three independent experiments. d, Band G (Wright S) and telomere fluorescence in situ hybridization (T-Fish) was performed using a Cy3-labeled peptide nucleic acid (PNA) telomeric probe. Representative metaphase spreads from cRbloxP/loxP and cRb−/−, partial representative metaphase spreads from cRbloxP/loxP/RasV12 and cRb−/−/RasV12 stained with Wright and hybridized with Cy3 telomeric probe are shown. The regions of metaphase spreads from cRbloxP/loxP/RasV12 and cRb−/−/RasV12 are magnified in the lower panel to show centromere fragments (CF), acentric fragment (AF), chromatide type break (CTB), telomeric fusion (TF), robertsonian translocation (Rob) and other type of translocation (T). X indicates quadrivalent (Qua) found in cRbloxP/loxP/RasV12.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003632-g003: HRasV12-dependent production of reactive oxygen species (ROS) and associated chromosomal instability.a, cells were assayed for dihydroethidium (DHE) fluorescence, indicative of ROS production and visualized by fluorescent microscopy. Under identical imaging conditions, DHE oxidation was significantly increased in cRbloxP/loxP/RasV12 and cRb−/−/RasV12 astrocytes. b, cRb−/−/RasV12 and cRbloxP/loxP/RasV12 cells are significantly more aneuploid than cRbloxP/loxP and cRb−/− cells. c, Absolute chromosome numbers of cRbloxP/loxP, cRbloxP/loxP/RasV12, cRb−/− and cRb−/−/RasV12 cells, showing the presence of diploid and tetraploid populations. Representative data of at least three independent experiments. d, Band G (Wright S) and telomere fluorescence in situ hybridization (T-Fish) was performed using a Cy3-labeled peptide nucleic acid (PNA) telomeric probe. Representative metaphase spreads from cRbloxP/loxP and cRb−/−, partial representative metaphase spreads from cRbloxP/loxP/RasV12 and cRb−/−/RasV12 stained with Wright and hybridized with Cy3 telomeric probe are shown. The regions of metaphase spreads from cRbloxP/loxP/RasV12 and cRb−/−/RasV12 are magnified in the lower panel to show centromere fragments (CF), acentric fragment (AF), chromatide type break (CTB), telomeric fusion (TF), robertsonian translocation (Rob) and other type of translocation (T). X indicates quadrivalent (Qua) found in cRbloxP/loxP/RasV12.
Mentions: As we previously described, although astrocytes harboring HRasV12 did not enter senescence we wanted to confirm whether DNA damage provoked by DNA replication stress, and usually associated with oncogene-induced senescence, was present. On the other hand, HRasV12 is also known to cause an increase in reactive oxygen species (ROS) production [36] as a result of replication stress. ROS has been traditionally considered as a toxic by-product of cellular metabolism, but it has been appreciated that they are actively involved in oncogenic signaling in cellular transformation and cancer. Increased intracellular levels of ROS have also been reported to mediate some biological effects of oncogenic HRasV12, such as the onset of premature senescence in primary cells, the generation of genomic instability [37], and malignant transformation [38]. Furthermore, high levels of ROS have been detected in several human cancer cell lines as well as in human tumors from different tissues. Taken together these reports, we wanted to analyze in astrocytes the relationship between oxidative stress and glioma. We observed similar oncogene-induced ROS accumulation in cRbloxP/loxP/RasV12 and cRb−/−/RasV12 astrocytes, but not in those that had lost only Rb (Figure 3A). Likewise, we found signs of chromosomal instability (Figure 3B–D) in the groups that expressed HRasV12 and displayed ROS accumulation. One of the most remarkable features of cRbloxP/loxP/RasV12 and cRb−/−/RasV12 astrocytes is the abundance of centromeric instability manifested as centromere fragments (CF) (Figure 3D and Figure S1). We also found Robertsonian centromere fusion (Rob) in cRbloxP/loxP/RasV12 and cRb−/−/RasV12 astrocytes but not in cRb−/− and wildtype astrocytes (Figure S1). In addition, there were numerical chromosome abnormalities in the experimental groups that expressed oncogenic Ras. These astrocytes presented >80 chromosomes per cell, doubling the chromosome number found in control cells (Figure 3B and 3C). Hence, these numerical and structural chromosome abnormalities show that activated Ras is sufficient to induce chromosomal instability in the absence of other signals, suggesting that Ras-induced chromosomal instability arises as consequence of ROS accumulation and DNA replication stress. In addition, there is not a significant difference between cRbloxP/loxP/RasV12 and cRb−/−/RasV12 astrocytes implying that the absence of pRb has not effect on DNA lesions, either quantitative or qualitative.

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