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Knockdown of EphB1 receptor decreases medulloblastoma cell growth and migration and increases cellular radiosensitization.

Bhatia S, Baig NA, Timofeeva O, Pasquale EB, Hirsch K, MacDonald TJ, Dritschilo A, Lee YC, Henkemeyer M, Rood B, Jung M, Wang XJ, Kool M, Rodriguez O, Albanese C, Karam SD - Oncotarget (2015)

Bottom Line: EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle.In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased β1-integrin expression and levels of phosphorylated Src.Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA.

ABSTRACT
The expression of members of the Eph family of receptor tyrosine kinases and their ephrin ligands is frequently dysregulated in medulloblastomas. We assessed the expression and functional role of EphB1 in medulloblastoma cell lines and engineered mouse models. mRNA and protein expression profiling showed expression of EphB1 receptor in the human medulloblastoma cell lines DAOY and UW228. EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle. It also modulated the expression of proliferation, and cell survival markers. In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased β1-integrin expression and levels of phosphorylated Src. Furthermore, EphB1 knockdown enhanced cellular radiosensitization of medulloblastoma cells in culture and in a genetically engineered mouse medulloblastoma model. Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors. Taken together, our findings establish that EphB1 plays a key role in medulloblastoma cell growth, viability, migration, and radiation sensitivity, making EphB1 a promising therapeutic target.

No MeSH data available.


Related in: MedlinePlus

Knockdown of EphB1 receptor sensitizes DAOY cells to ionizing radiationDAOY cells transiently transfected with either non-specific siRNA (Ns-siRNA) or EphB1-targeting siRNA were irradiated at 24 h post-transfection with 10 Gy. At 96 h post-transfection, MTT reagent was added to samples and optical density measured after 24 h incubation. Data shown are normalized values and represent average ± standard error with **p < 0.001, *p < 0.05.
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Figure 6: Knockdown of EphB1 receptor sensitizes DAOY cells to ionizing radiationDAOY cells transiently transfected with either non-specific siRNA (Ns-siRNA) or EphB1-targeting siRNA were irradiated at 24 h post-transfection with 10 Gy. At 96 h post-transfection, MTT reagent was added to samples and optical density measured after 24 h incubation. Data shown are normalized values and represent average ± standard error with **p < 0.001, *p < 0.05.

Mentions: To investigate whether EphB1 also plays a role in radiation sensitivity, we compared the responses of the EphB1-knockdown vs. control siRNA in DAOY cells. Using a single dose of 10 Gy of ionizing radiation, cell growth was determined at 96 h post transfection using an MTT assay. The percentage of cell growth was reduced by approximately 26% in the EphB1 knockdown cells vs. the non-specific siRNA (Ns-siRNA)-treated DAOY cells (Figure 6). Furthermore, cell growth was reduced by approximately 32% in the irradiated EphB1 knockdown cells (Figure 6). In addition, analysis of cell cycle profiles by flow cytometry in DAOY cells indicated that knockdown of EphB1 expression enhanced the percentage of cells in G1 phase in both non-irradiated and irradiated (10 Gy) samples at both 48 h and 72 h post-irradiation, with a more dramatic effect observed at 72 h (Figure 7).


Knockdown of EphB1 receptor decreases medulloblastoma cell growth and migration and increases cellular radiosensitization.

Bhatia S, Baig NA, Timofeeva O, Pasquale EB, Hirsch K, MacDonald TJ, Dritschilo A, Lee YC, Henkemeyer M, Rood B, Jung M, Wang XJ, Kool M, Rodriguez O, Albanese C, Karam SD - Oncotarget (2015)

Knockdown of EphB1 receptor sensitizes DAOY cells to ionizing radiationDAOY cells transiently transfected with either non-specific siRNA (Ns-siRNA) or EphB1-targeting siRNA were irradiated at 24 h post-transfection with 10 Gy. At 96 h post-transfection, MTT reagent was added to samples and optical density measured after 24 h incubation. Data shown are normalized values and represent average ± standard error with **p < 0.001, *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496193&req=5

Figure 6: Knockdown of EphB1 receptor sensitizes DAOY cells to ionizing radiationDAOY cells transiently transfected with either non-specific siRNA (Ns-siRNA) or EphB1-targeting siRNA were irradiated at 24 h post-transfection with 10 Gy. At 96 h post-transfection, MTT reagent was added to samples and optical density measured after 24 h incubation. Data shown are normalized values and represent average ± standard error with **p < 0.001, *p < 0.05.
Mentions: To investigate whether EphB1 also plays a role in radiation sensitivity, we compared the responses of the EphB1-knockdown vs. control siRNA in DAOY cells. Using a single dose of 10 Gy of ionizing radiation, cell growth was determined at 96 h post transfection using an MTT assay. The percentage of cell growth was reduced by approximately 26% in the EphB1 knockdown cells vs. the non-specific siRNA (Ns-siRNA)-treated DAOY cells (Figure 6). Furthermore, cell growth was reduced by approximately 32% in the irradiated EphB1 knockdown cells (Figure 6). In addition, analysis of cell cycle profiles by flow cytometry in DAOY cells indicated that knockdown of EphB1 expression enhanced the percentage of cells in G1 phase in both non-irradiated and irradiated (10 Gy) samples at both 48 h and 72 h post-irradiation, with a more dramatic effect observed at 72 h (Figure 7).

Bottom Line: EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle.In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased β1-integrin expression and levels of phosphorylated Src.Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA.

ABSTRACT
The expression of members of the Eph family of receptor tyrosine kinases and their ephrin ligands is frequently dysregulated in medulloblastomas. We assessed the expression and functional role of EphB1 in medulloblastoma cell lines and engineered mouse models. mRNA and protein expression profiling showed expression of EphB1 receptor in the human medulloblastoma cell lines DAOY and UW228. EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle. It also modulated the expression of proliferation, and cell survival markers. In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased β1-integrin expression and levels of phosphorylated Src. Furthermore, EphB1 knockdown enhanced cellular radiosensitization of medulloblastoma cells in culture and in a genetically engineered mouse medulloblastoma model. Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors. Taken together, our findings establish that EphB1 plays a key role in medulloblastoma cell growth, viability, migration, and radiation sensitivity, making EphB1 a promising therapeutic target.

No MeSH data available.


Related in: MedlinePlus