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DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

Dolman ME, van der Ploeg I, Koster J, Bate-Eya LT, Versteeg R, Caron HN, Molenaar JJ - PLoS ONE (2015)

Bottom Line: Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on.Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone.Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.

ABSTRACT
Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

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PRKDC knockdown in NGP cells confirms DNA-PKcs-dependent radiosensitizing activity of NU7026.(A) PRKDC mRNA expression in NGP cells at 144 h after transduction with nonspecific control shRNA (SHC002) or PRKDC shRNA. PRKDC mRNA levels were determined by qPCR and ΔCt values were normalized to the household gene β-actin. Normalized ΔCt values of untreated non-transduced NGP cells (no virus) were set at zero, giving ΔΔCt. Data represent the mean (n = 4) +/- SD. (B) FACS analysis of the effects of treatment with 0.63 Gy IR on the sub-G1 fraction of normal NGP cells and NGP cells transduced with SHC002 or PRKDC shRNA. Results are obtained at 96 h after IR-exposure of the cells. (C) Sub-G1 fraction of NGP cells transduced with SHC002 or PRKDC shRNA after non-treatment and after treatment with 0.63 Gy IR, at 48, 72 and 96 h after irradiation. (D) Western Blot analysis of protein levels of DNA-PKcs and cleaved PARP in non-irradiated and irradiated normal NGP cells and NGP cells transduced with SHC002 or PRKDC shRNA. Results were obtained at 120 h after irradiation, which was equal to 168 h after transduction. β-actin protein levels were used as loading control.
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pone.0145744.g006: PRKDC knockdown in NGP cells confirms DNA-PKcs-dependent radiosensitizing activity of NU7026.(A) PRKDC mRNA expression in NGP cells at 144 h after transduction with nonspecific control shRNA (SHC002) or PRKDC shRNA. PRKDC mRNA levels were determined by qPCR and ΔCt values were normalized to the household gene β-actin. Normalized ΔCt values of untreated non-transduced NGP cells (no virus) were set at zero, giving ΔΔCt. Data represent the mean (n = 4) +/- SD. (B) FACS analysis of the effects of treatment with 0.63 Gy IR on the sub-G1 fraction of normal NGP cells and NGP cells transduced with SHC002 or PRKDC shRNA. Results are obtained at 96 h after IR-exposure of the cells. (C) Sub-G1 fraction of NGP cells transduced with SHC002 or PRKDC shRNA after non-treatment and after treatment with 0.63 Gy IR, at 48, 72 and 96 h after irradiation. (D) Western Blot analysis of protein levels of DNA-PKcs and cleaved PARP in non-irradiated and irradiated normal NGP cells and NGP cells transduced with SHC002 or PRKDC shRNA. Results were obtained at 120 h after irradiation, which was equal to 168 h after transduction. β-actin protein levels were used as loading control.

Mentions: Small-molecule inhibitors, also when they are called specific, often possess off–target effects. To further investigate if the radiosensitizing effects observed for NU7026 are caused by DNA-PKcs inhibition, validation studies were performed using NGP cells transduced with a PRKDC shRNA or a nonspecific control shRNA (SHC002). PRKDC gene silencing was observed at 144 h after transduction of the NGP cells with PRKDC shRNA, while SHC002 did not affect the gene expression level of PRKDC (Fig 6A). Effects on gene level were confirmed on protein level by Western Blot analysis of DNA-PKcs (Fig 6B).


DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

Dolman ME, van der Ploeg I, Koster J, Bate-Eya LT, Versteeg R, Caron HN, Molenaar JJ - PLoS ONE (2015)

PRKDC knockdown in NGP cells confirms DNA-PKcs-dependent radiosensitizing activity of NU7026.(A) PRKDC mRNA expression in NGP cells at 144 h after transduction with nonspecific control shRNA (SHC002) or PRKDC shRNA. PRKDC mRNA levels were determined by qPCR and ΔCt values were normalized to the household gene β-actin. Normalized ΔCt values of untreated non-transduced NGP cells (no virus) were set at zero, giving ΔΔCt. Data represent the mean (n = 4) +/- SD. (B) FACS analysis of the effects of treatment with 0.63 Gy IR on the sub-G1 fraction of normal NGP cells and NGP cells transduced with SHC002 or PRKDC shRNA. Results are obtained at 96 h after IR-exposure of the cells. (C) Sub-G1 fraction of NGP cells transduced with SHC002 or PRKDC shRNA after non-treatment and after treatment with 0.63 Gy IR, at 48, 72 and 96 h after irradiation. (D) Western Blot analysis of protein levels of DNA-PKcs and cleaved PARP in non-irradiated and irradiated normal NGP cells and NGP cells transduced with SHC002 or PRKDC shRNA. Results were obtained at 120 h after irradiation, which was equal to 168 h after transduction. β-actin protein levels were used as loading control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4696738&req=5

pone.0145744.g006: PRKDC knockdown in NGP cells confirms DNA-PKcs-dependent radiosensitizing activity of NU7026.(A) PRKDC mRNA expression in NGP cells at 144 h after transduction with nonspecific control shRNA (SHC002) or PRKDC shRNA. PRKDC mRNA levels were determined by qPCR and ΔCt values were normalized to the household gene β-actin. Normalized ΔCt values of untreated non-transduced NGP cells (no virus) were set at zero, giving ΔΔCt. Data represent the mean (n = 4) +/- SD. (B) FACS analysis of the effects of treatment with 0.63 Gy IR on the sub-G1 fraction of normal NGP cells and NGP cells transduced with SHC002 or PRKDC shRNA. Results are obtained at 96 h after IR-exposure of the cells. (C) Sub-G1 fraction of NGP cells transduced with SHC002 or PRKDC shRNA after non-treatment and after treatment with 0.63 Gy IR, at 48, 72 and 96 h after irradiation. (D) Western Blot analysis of protein levels of DNA-PKcs and cleaved PARP in non-irradiated and irradiated normal NGP cells and NGP cells transduced with SHC002 or PRKDC shRNA. Results were obtained at 120 h after irradiation, which was equal to 168 h after transduction. β-actin protein levels were used as loading control.
Mentions: Small-molecule inhibitors, also when they are called specific, often possess off–target effects. To further investigate if the radiosensitizing effects observed for NU7026 are caused by DNA-PKcs inhibition, validation studies were performed using NGP cells transduced with a PRKDC shRNA or a nonspecific control shRNA (SHC002). PRKDC gene silencing was observed at 144 h after transduction of the NGP cells with PRKDC shRNA, while SHC002 did not affect the gene expression level of PRKDC (Fig 6A). Effects on gene level were confirmed on protein level by Western Blot analysis of DNA-PKcs (Fig 6B).

Bottom Line: Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on.Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone.Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.

ABSTRACT
Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

Show MeSH
Related in: MedlinePlus