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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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Screening and characterization of positive phage clones of anti-DNA-PKcs segments scFv. (A) Conserved functional domains of DNA-PKcs and the location of segments identified with epitopes predicted in DNA-PKcs. DPK1, DPK2 are located at the N-terminus of DNA-PKcs, and DPK3 and DPK4 are located between the leucine zipper (LZ) and T2609 autophosphorylation cluster. The DPK3 segment includes Ser2056 autophosphorylation cluster. (B) PAGE electrophoresis patterns of the Mva I-digested positive anti-DPK3-scFv clones. (C) Coomassie brilliant blue stained gel of the SDS-PAGE analysis of 10 μg purified DPK3 (~30 kD) and DPK4 (~32 kD) segments. (D) Immunohybridization (western blotting) analysis of purified DPK3 (30 kD) and DPK4 (32 kD) segments using anti-DPK3-scFv-2 antibody. (E) Immunohybridization analysis of DNA-PKcs in HeLa cell protein lysate using anti-DPK3-scFv-2 and anti-DNA-PKcs antibodies.
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Figure 1: Screening and characterization of positive phage clones of anti-DNA-PKcs segments scFv. (A) Conserved functional domains of DNA-PKcs and the location of segments identified with epitopes predicted in DNA-PKcs. DPK1, DPK2 are located at the N-terminus of DNA-PKcs, and DPK3 and DPK4 are located between the leucine zipper (LZ) and T2609 autophosphorylation cluster. The DPK3 segment includes Ser2056 autophosphorylation cluster. (B) PAGE electrophoresis patterns of the Mva I-digested positive anti-DPK3-scFv clones. (C) Coomassie brilliant blue stained gel of the SDS-PAGE analysis of 10 μg purified DPK3 (~30 kD) and DPK4 (~32 kD) segments. (D) Immunohybridization (western blotting) analysis of purified DPK3 (30 kD) and DPK4 (32 kD) segments using anti-DPK3-scFv-2 antibody. (E) Immunohybridization analysis of DNA-PKcs in HeLa cell protein lysate using anti-DPK3-scFv-2 and anti-DNA-PKcs antibodies.

Mentions: DNA-PKcs is approximately 470 kD consisting of 4128 amino acids. Its C-terminus contains a serine/threonine protein kinase domain (3719- 4128) [23]. For screening the specific anti-DNA-PKcs scFv from the phage library, 6 epitopes of DNA-PKcs have been predicted as the antigens. DPK5 and DPK6 peptides are located in the focal adhesion targeting (FAT) domain, which is homologous with other members of the PIKK family [24], so they were rejected. The DPK1 and DPK2 peptides are located in the N-terminus of DNA-PKcs, and DPK3 and DPK4 between the leucine zipper and the autophosphorylation cluster (Figure 1A). The cDNA encoding these peptides were cloned, and the peptides were expressed in and purified from the infected E coli cells. Upon inclusion body protein degeneration and refolding, we have finally successfully obtained the soluble DPK3 peptide of ~30 kD with the purity of >95%.


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)

Screening and characterization of positive phage clones of anti-DNA-PKcs segments scFv. (A) Conserved functional domains of DNA-PKcs and the location of segments identified with epitopes predicted in DNA-PKcs. DPK1, DPK2 are located at the N-terminus of DNA-PKcs, and DPK3 and DPK4 are located between the leucine zipper (LZ) and T2609 autophosphorylation cluster. The DPK3 segment includes Ser2056 autophosphorylation cluster. (B) PAGE electrophoresis patterns of the Mva I-digested positive anti-DPK3-scFv clones. (C) Coomassie brilliant blue stained gel of the SDS-PAGE analysis of 10 μg purified DPK3 (~30 kD) and DPK4 (~32 kD) segments. (D) Immunohybridization (western blotting) analysis of purified DPK3 (30 kD) and DPK4 (32 kD) segments using anti-DPK3-scFv-2 antibody. (E) Immunohybridization analysis of DNA-PKcs in HeLa cell protein lysate using anti-DPK3-scFv-2 and anti-DNA-PKcs antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927608&req=5

Figure 1: Screening and characterization of positive phage clones of anti-DNA-PKcs segments scFv. (A) Conserved functional domains of DNA-PKcs and the location of segments identified with epitopes predicted in DNA-PKcs. DPK1, DPK2 are located at the N-terminus of DNA-PKcs, and DPK3 and DPK4 are located between the leucine zipper (LZ) and T2609 autophosphorylation cluster. The DPK3 segment includes Ser2056 autophosphorylation cluster. (B) PAGE electrophoresis patterns of the Mva I-digested positive anti-DPK3-scFv clones. (C) Coomassie brilliant blue stained gel of the SDS-PAGE analysis of 10 μg purified DPK3 (~30 kD) and DPK4 (~32 kD) segments. (D) Immunohybridization (western blotting) analysis of purified DPK3 (30 kD) and DPK4 (32 kD) segments using anti-DPK3-scFv-2 antibody. (E) Immunohybridization analysis of DNA-PKcs in HeLa cell protein lysate using anti-DPK3-scFv-2 and anti-DNA-PKcs antibodies.
Mentions: DNA-PKcs is approximately 470 kD consisting of 4128 amino acids. Its C-terminus contains a serine/threonine protein kinase domain (3719- 4128) [23]. For screening the specific anti-DNA-PKcs scFv from the phage library, 6 epitopes of DNA-PKcs have been predicted as the antigens. DPK5 and DPK6 peptides are located in the focal adhesion targeting (FAT) domain, which is homologous with other members of the PIKK family [24], so they were rejected. The DPK1 and DPK2 peptides are located in the N-terminus of DNA-PKcs, and DPK3 and DPK4 between the leucine zipper and the autophosphorylation cluster (Figure 1A). The cDNA encoding these peptides were cloned, and the peptides were expressed in and purified from the infected E coli cells. Upon inclusion body protein degeneration and refolding, we have finally successfully obtained the soluble DPK3 peptide of ~30 kD with the purity of >95%.

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