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


CONCLUSIONS
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.
Du
Li
L
Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, 27 Taiping Road, Haidian District, Beijing 100850, China.
Zhou
Li-Jun
LJ
Pan
Xiu-Jie
XJ
Wang
Yu-Xiao
YX
Xu
Qin-Zhi
QZ
Yang
Zhi-Hua
ZH
Wang
Yu
Y
Liu
Xiao-Dan
XD
Zhu
Mao-Xiang
MX
Zhou
Ping-Kun
PK
D000818:Animals; D000918:Antibody Specificity; D049109:Cell Proliferation; D020552:Comet Assay; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000502:physiology; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors*; D015316:Genetic Therapy / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007167:Immunotherapy / Q000379:methods; D020287:In Situ Nick-End Labeling; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000235:genetics* / Q000628:therapy*; D019151:Peptide Library; D011836:Radiation Tolerance / Q000235:genetics*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000627:therapeutic use*; D014162:Transfection; D023041:Xenograft Model Antitumor Assays
D019151:Peptide Library; D057127:Single-Chain Antibodies; D051747:DNA-Activated Protein Kinase