Down regulation of BRCA2 causes radio-sensitization of human tumor cells in vitro and in vivo.
In order to study the role of BRCA2 protein in homologous recombination repair and radio-sensitization, we utilized RNA interference strategy in vitro and in vivo with human tumor cells. HeLa cells transfected with small-interfering BRCA2 NA (BRCA2 siRNA) (Qiagen) as well as negative-control siRNA for 48 h were irradiated, and several critical end points were examined. The radiation cell survival level was significantly reduced in HeLa cells with BRCA2 siRNA when compared with mock- or negative-control siRNA transfected cells. DNA double strand break repair as measured by constant field gel-electrophoresis showed a clear inhibition in cells with BRCA2 siRNA, while little inhibition was observed in cells with negative control siRNA. Our immuno-staining experiments revealed a significant delay in Rad51 foci formation in cells with BRCA2 siRNA when compared with the control populations. However, none of the non-homologous end joining proteins nor the phosphorylation of DNA-dependent protein kinase catalytic subunit was affected in cells transfected with BRCA2 siRNA. In addition, the combined treatment with radiation and BRCA2 siRNA in xenograft model with HeLa cells showed an efficient inhibition of in vivo tumor growth. Our results demonstrate down-regulation of BRCA2 leads to radio-sensitization mainly through the inhibition of homologous recombination repair type double-strand break repair; a possibility of using BRCA2 siRNA as an effective radiosensitizer in tumor radiotherapy may arise.
Yu
Dong
D
Heavy-Ion Radiobiology Research Group, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi, 263-8555, Japan.
Sekine
Emiko
E
Fujimori
Akira
A
Ochiya
Takahiro
T
Okayasu
Ryuichi
R
D000818:Animals; D024682:BRCA2 Protein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology*; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics; D015536:Down-Regulation; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D051135:Rad51 Recombinase / Q000032:analysis / Q000378:metabolism; D011836:Radiation Tolerance / Q000187:drug effects* / Q000235:genetics*; D023041:Xenograft Model Antitumor Assays
D024682:BRCA2 Protein; D034741:RNA, Small Interfering; C495153:RAD51 protein, human; D051135:Rad51 Recombinase