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Radiosensitization and growth inhibition of cancer cells mediated by an scFv antibody gene against DNA-PKcs in vitro and in vivo.

Du L, Zhou LJ, Pan XJ, Wang YX, Xu QZ, Yang ZH, Wang Y, Liu XD, Zhu MX, Zhou PK - Radiat Oncol (2010)

Bottom Line: Transfection of HeLa cells with the anti-DPK3-scFv gene resulted in an increased sensitivity to IR, decreased repair capability of DNA double-strand breaks (DSB) detected by comet assay and immunofluorescence detection of gammaH2AX foci.Moreover, the kinase activity of DNA-PKcs was inhibited by anti-DPK3-scFv, which was displayed by the decreased phosphorylation levels of its target Akt/S473 and the autophosphorylation of DNA-PKcs on S2056 induced by radiation.Measurement of the growth and apoptosis rates showed that anti-DPK3-scFv enhanced the sensitivity of tumours transplanted in Balb/c athymic mice to radiation therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, 27 Taiping Road, Haidian District, Beijing 100850, China.

ABSTRACT

Background: Overexpression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is commonly occurred in cancers and causes radioresistance and poor prognosis. In present study, the single-chain variable antibody fragments (scFv) targeting DNA-PKcs was developed for the application of radiosensitization in vitro and in vivo. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody.

Methods: DNA-PKcs epitopes were predicted and cloned. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody. DNA damage repair was analyzed by comet assay and immunofluorescence detection of gammaH2AX foci. The radiosensitization in vivo was determined on Balb/c athymic mice transplanted tumours of HeLa cells.

Results: Four epitopes of DNA-PKcs have been predicted and expressed as the antigens, and a specific human anti-DNA-PKcs scFv antibody gene, anti-DPK3-scFv, was obtained by screening the phage antibody library using the DNA-PKcs peptide DPK3. The specificity of anti-DPK3-scFv was verified, in vitro. Transfection of HeLa cells with the anti-DPK3-scFv gene resulted in an increased sensitivity to IR, decreased repair capability of DNA double-strand breaks (DSB) detected by comet assay and immunofluorescence detection of gammaH2AX foci. Moreover, the kinase activity of DNA-PKcs was inhibited by anti-DPK3-scFv, which was displayed by the decreased phosphorylation levels of its target Akt/S473 and the autophosphorylation of DNA-PKcs on S2056 induced by radiation. Measurement of the growth and apoptosis rates showed that anti-DPK3-scFv enhanced the sensitivity of tumours transplanted in Balb/c athymic mice to radiation therapy.

Conclusion: The antiproliferation and radiosensitizing effects of anti-DPK3-scFv via targeting DNA-PKcs make it very appealing for the development as a novel biological radiosensitizer for cancer therapeutic potential.

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Effect of anti-DPK3-scFv on cellular survival. (A) Immunohybridization analysis of anti-DPK3-scFv expression in HeLa cell clones (C1 - C5) stably transfected with His-anti-DPK3-scFv-2 using an anti-His antibody. (B) Growth rates for HeLa, HeLa-pcDNA and DPK3-scFv-2-transfected HeLa cells (HeLa-DPK3-scFv) under normal growing condition. (C) Clonogenic assays of cells survivals for HeLa, HeLa-pcDNA and the clones (scFv-C3, C4, C5) of DPK3-scFv-2-transfected HeLa cells after 4 Gy γ-ray irradiation. * P < 0.05, #P < 0.01 as compared with control HeLa-pcDNA cells. (D) Cell survival curves of HeLa, HeLa-pcDNA and HeLa-DPK3-scFv cells (clone 2) post-irradiation. (E) Caspase-3 activity assays of HeLa, HeLa-pcDNA and HeLa-DPK3-scFv cells post-4 Gy irradiation. * P < 0.05 as compared with control HeLa or HeLa-pcDNA cells.
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Figure 3: Effect of anti-DPK3-scFv on cellular survival. (A) Immunohybridization analysis of anti-DPK3-scFv expression in HeLa cell clones (C1 - C5) stably transfected with His-anti-DPK3-scFv-2 using an anti-His antibody. (B) Growth rates for HeLa, HeLa-pcDNA and DPK3-scFv-2-transfected HeLa cells (HeLa-DPK3-scFv) under normal growing condition. (C) Clonogenic assays of cells survivals for HeLa, HeLa-pcDNA and the clones (scFv-C3, C4, C5) of DPK3-scFv-2-transfected HeLa cells after 4 Gy γ-ray irradiation. * P < 0.05, #P < 0.01 as compared with control HeLa-pcDNA cells. (D) Cell survival curves of HeLa, HeLa-pcDNA and HeLa-DPK3-scFv cells (clone 2) post-irradiation. (E) Caspase-3 activity assays of HeLa, HeLa-pcDNA and HeLa-DPK3-scFv cells post-4 Gy irradiation. * P < 0.05 as compared with control HeLa or HeLa-pcDNA cells.

Mentions: HeLa-DPK3-scFv cell clones (C1 to C5) were generated from HeLa cells stably transfected with His-tagged anti-DPK3-scFv-2 gene and the expression level of anti-DPK3-scFv in each clone was shown in Figure 3A. The growth rate of the anti-DPK3-scFv-transfected cells (HeLa-DPK3-scFv) is lightly slower as compared with HeLa cells or the cells transfected with the mock vector (HeLa-pcDNA) (Figure 3B), but this growth difference is not statistically significant. There is no different in cell cycle distribution among these three cell lines under normal growing conditions (data not shown). Survival assay of 4 Gy-irradiated cells determined that anti-DPK3-scFv-transfected cells were more sensitive to γ-ray compared to control cells, with the HeLa-DPK3-scFv-c2 (clone 2) being the most sensitive clone (Figure 3C). Clonogenic assay was further performed for the cells after 0 - 8 Gy γ-ray irradiation. Cell survival curves demonstrate an increased radiosensitivity for the HeLa-DPK3-scFv cells (clone 2) (Figure 3D). There was approximately a 20% dose reduction required for the same level of survival.


Radiosensitization and growth inhibition of cancer cells mediated by an scFv antibody gene against DNA-PKcs in vitro and in vivo.

Du L, Zhou LJ, Pan XJ, Wang YX, Xu QZ, Yang ZH, Wang Y, Liu XD, Zhu MX, Zhou PK - Radiat Oncol (2010)

Effect of anti-DPK3-scFv on cellular survival. (A) Immunohybridization analysis of anti-DPK3-scFv expression in HeLa cell clones (C1 - C5) stably transfected with His-anti-DPK3-scFv-2 using an anti-His antibody. (B) Growth rates for HeLa, HeLa-pcDNA and DPK3-scFv-2-transfected HeLa cells (HeLa-DPK3-scFv) under normal growing condition. (C) Clonogenic assays of cells survivals for HeLa, HeLa-pcDNA and the clones (scFv-C3, C4, C5) of DPK3-scFv-2-transfected HeLa cells after 4 Gy γ-ray irradiation. * P < 0.05, #P < 0.01 as compared with control HeLa-pcDNA cells. (D) Cell survival curves of HeLa, HeLa-pcDNA and HeLa-DPK3-scFv cells (clone 2) post-irradiation. (E) Caspase-3 activity assays of HeLa, HeLa-pcDNA and HeLa-DPK3-scFv cells post-4 Gy irradiation. * P < 0.05 as compared with control HeLa or HeLa-pcDNA cells.
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Figure 3: Effect of anti-DPK3-scFv on cellular survival. (A) Immunohybridization analysis of anti-DPK3-scFv expression in HeLa cell clones (C1 - C5) stably transfected with His-anti-DPK3-scFv-2 using an anti-His antibody. (B) Growth rates for HeLa, HeLa-pcDNA and DPK3-scFv-2-transfected HeLa cells (HeLa-DPK3-scFv) under normal growing condition. (C) Clonogenic assays of cells survivals for HeLa, HeLa-pcDNA and the clones (scFv-C3, C4, C5) of DPK3-scFv-2-transfected HeLa cells after 4 Gy γ-ray irradiation. * P < 0.05, #P < 0.01 as compared with control HeLa-pcDNA cells. (D) Cell survival curves of HeLa, HeLa-pcDNA and HeLa-DPK3-scFv cells (clone 2) post-irradiation. (E) Caspase-3 activity assays of HeLa, HeLa-pcDNA and HeLa-DPK3-scFv cells post-4 Gy irradiation. * P < 0.05 as compared with control HeLa or HeLa-pcDNA cells.
Mentions: HeLa-DPK3-scFv cell clones (C1 to C5) were generated from HeLa cells stably transfected with His-tagged anti-DPK3-scFv-2 gene and the expression level of anti-DPK3-scFv in each clone was shown in Figure 3A. The growth rate of the anti-DPK3-scFv-transfected cells (HeLa-DPK3-scFv) is lightly slower as compared with HeLa cells or the cells transfected with the mock vector (HeLa-pcDNA) (Figure 3B), but this growth difference is not statistically significant. There is no different in cell cycle distribution among these three cell lines under normal growing conditions (data not shown). Survival assay of 4 Gy-irradiated cells determined that anti-DPK3-scFv-transfected cells were more sensitive to γ-ray compared to control cells, with the HeLa-DPK3-scFv-c2 (clone 2) being the most sensitive clone (Figure 3C). Clonogenic assay was further performed for the cells after 0 - 8 Gy γ-ray irradiation. Cell survival curves demonstrate an increased radiosensitivity for the HeLa-DPK3-scFv cells (clone 2) (Figure 3D). There was approximately a 20% dose reduction required for the same level of survival.

Bottom Line: Transfection of HeLa cells with the anti-DPK3-scFv gene resulted in an increased sensitivity to IR, decreased repair capability of DNA double-strand breaks (DSB) detected by comet assay and immunofluorescence detection of gammaH2AX foci.Moreover, the kinase activity of DNA-PKcs was inhibited by anti-DPK3-scFv, which was displayed by the decreased phosphorylation levels of its target Akt/S473 and the autophosphorylation of DNA-PKcs on S2056 induced by radiation.Measurement of the growth and apoptosis rates showed that anti-DPK3-scFv enhanced the sensitivity of tumours transplanted in Balb/c athymic mice to radiation therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, 27 Taiping Road, Haidian District, Beijing 100850, China.

ABSTRACT

Background: Overexpression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is commonly occurred in cancers and causes radioresistance and poor prognosis. In present study, the single-chain variable antibody fragments (scFv) targeting DNA-PKcs was developed for the application of radiosensitization in vitro and in vivo. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody.

Methods: DNA-PKcs epitopes were predicted and cloned. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody. DNA damage repair was analyzed by comet assay and immunofluorescence detection of gammaH2AX foci. The radiosensitization in vivo was determined on Balb/c athymic mice transplanted tumours of HeLa cells.

Results: Four epitopes of DNA-PKcs have been predicted and expressed as the antigens, and a specific human anti-DNA-PKcs scFv antibody gene, anti-DPK3-scFv, was obtained by screening the phage antibody library using the DNA-PKcs peptide DPK3. The specificity of anti-DPK3-scFv was verified, in vitro. Transfection of HeLa cells with the anti-DPK3-scFv gene resulted in an increased sensitivity to IR, decreased repair capability of DNA double-strand breaks (DSB) detected by comet assay and immunofluorescence detection of gammaH2AX foci. Moreover, the kinase activity of DNA-PKcs was inhibited by anti-DPK3-scFv, which was displayed by the decreased phosphorylation levels of its target Akt/S473 and the autophosphorylation of DNA-PKcs on S2056 induced by radiation. Measurement of the growth and apoptosis rates showed that anti-DPK3-scFv enhanced the sensitivity of tumours transplanted in Balb/c athymic mice to radiation therapy.

Conclusion: The antiproliferation and radiosensitizing effects of anti-DPK3-scFv via targeting DNA-PKcs make it very appealing for the development as a novel biological radiosensitizer for cancer therapeutic potential.

Show MeSH
Related in: MedlinePlus