Volume 130, Issue 3, Pages (September 2013)

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Volume 130, Issue 3, Pages 601-608 (September 2013) Radiation-inducible protein RbAp48 contributes to radiosensitivity of cervical cancer cells Lin Zheng, Wei Tang, Fanhua Wei, Hong Wang, Juan Liu, Yi Lu, Yufeng Cheng, Xiaohui Bai, Xiuping Yu, Weiming Zhao Gynecologic Oncology Volume 130, Issue 3, Pages 601-608 (September 2013) DOI: 10.1016/j.ygyno.2013.06.002 Copyright © 2013 Terms and Conditions

Fig. 1 Irradiation increases the expression of RbAp48 in cervical cancer cell lines. (A) Real-time RT-PCR assay of RbAp48 mRNA levels in SiHa, Caski and HeLa cells treated with doses of radiation. Expression of RbAp48 in each sample was normalized to that of β-actin. The normalized values were then calibrated against untreated cell values arbitrarily set to 1. Data are mean±SD from 3 independent experiments. **, P<0.01; ***, P<0.001 versus the no treated cells. (B) Western blot assay of RbAp48 protein levels in SiHa, Caski and HeLa cells treated with radiation. β-Tubulin was a loading control. (C) The RbAp48/tubulin ratio for each was analyzed by densitometry and shown as bar graphs. Gynecologic Oncology 2013 130, 601-608DOI: (10.1016/j.ygyno.2013.06.002) Copyright © 2013 Terms and Conditions

Fig. 2 Knockdown of RbAp48 reduces radiosensitivity of cervical cancer cells. (A) Western blot assay of RbAp48 protein level in cervical cancer cells transfected with RbAp48 siRNA (si-RbAp48-1, -2) or control siRNA (si-control). β-Tubulin was a loading control. Assay for cell proliferation (B), cell counting (C), survival (D) and soft agar colony formation (E) of RbAp48-knockdown (si-RbAp48) and control cervical cancer cells (si-control) treated with doses of radiation. Data are mean±SD from 3 independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001 versus the si-control group. Gynecologic Oncology 2013 130, 601-608DOI: (10.1016/j.ygyno.2013.06.002) Copyright © 2013 Terms and Conditions

Fig. 3 Overexpression of RbAp48 enhances radiosensitivity of cervical cancer cells. (A) Western blot assay of RbAp48 protein level in RbAp48-overexpressing cervical cancer cell lines (pcDNA3.1-RbAp48) and control cell lines (pcDNA3.1). β-Tubulin was a loading control. Assay for cell proliferation (B), cell counting (C), survival (D) and soft agar colony formation (E) of RbAp48-overexpressing cell lines (pcDNA3.1-RbAp48) and control cell lines (pcDNA3.1) treated with doses of radiation. Data are mean±SD from 3 independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001 versus the pcDNA3.1 group. Gynecologic Oncology 2013 130, 601-608DOI: (10.1016/j.ygyno.2013.06.002) Copyright © 2013 Terms and Conditions

Fig. 4 RbAp48 modulates radiation-induced G2/M cell-cycle arrest and apoptosis in cervical cancer cells. Plasmid overexpression of RbAp48 in cervical cancer cell lines enhanced (A) and siRNA knockout inhibited (B) radiation-induced G2/M phase arrest. Plasmid overexpression of RbAp48 in cervical cancer cell lines enhanced (C) and siRNA knockout inhibited (D) radiation-induced apoptosis. Data are mean±SD from 3 independent experiments. **, P<0.01; ***, P<0.001 versus radiation treated pcDNA3.1 or si-control group, respectively. Gynecologic Oncology 2013 130, 601-608DOI: (10.1016/j.ygyno.2013.06.002) Copyright © 2013 Terms and Conditions

Fig. 5 Altered expression of RbAp48 regulates mRNA levels of p53, Rb and caspase-8 with radiation of cervical cancer cells. Real-time RT-PCR assay of mRNA levels of p53 (A), Rb (B) and caspase-8 (C) in SiHa, Caski and HeLa cells with various treatments. Expression of p53, Rb and caspase-8 in each sample was normalized to that of β-actin. The normalized values were then calibrated against the pcDNA3.1 stable transfected (pcDNA3.1) or control siRNA transfected (si-control) values that were arbitrarily set to 1. Data are mean±SD from 3 independent experiments. **P<0.01; ***P<0.001 versus radiation treated pcDNA3.1 or si-control group, respectively. Gynecologic Oncology 2013 130, 601-608DOI: (10.1016/j.ygyno.2013.06.002) Copyright © 2013 Terms and Conditions

Fig. 6 Ad5-RbAp48 infection enhanced radiosensitivity of cervical cancer cells in vivo. (A) Tumor growth curves for mice with SiHa or HeLa implantation with and without adenovirus infection and irradiation. Data are mean tumor volume±SEM. *, P<0.05; **, P<0.01; ***, P<0.001 versus group with Ad5-EGFP infection plus radiation. (B, C) Photographs and weights of tumors from groups of mice. Horizontal bars in C indicate mean tumor weight±SEM. ***P<0.001 versus group with Ad5-EGFP infection plus radiation. Gynecologic Oncology 2013 130, 601-608DOI: (10.1016/j.ygyno.2013.06.002) Copyright © 2013 Terms and Conditions

Supplemental Fig. 1 Ad5-RbAp48 infection enhances radiosensitivity of cervical cancer cells. (A) Western blot assay of RbAp48 protein level in adenovirus-infected cervical cancer cells. β-Tubulin was an internal control. (B, C) Assay for cell proliferation and survival of Ad5-RbAp48 infected (Ad5-RbAp48) cell lines and control cell lines (Ad5-EGFP) treated with doses of radiation. Data are mean±SD from 3 independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001 versus the Ad5-EGFP group. Gynecologic Oncology 2013 130, 601-608DOI: (10.1016/j.ygyno.2013.06.002) Copyright © 2013 Terms and Conditions

Supplemental Fig. 2 Comparison of sensitization of Ad5-RbAp48 infection and cisplatin for radiation therapy. Photographs (A) and weights (B) of tumors from groups of mice. Horizontal bars in (B) indicate mean tumor weight±SEM. **, P<0.01 versus group with Ad5-RbAp48 infection plus radiation; #, P<0.05 versus group with cisplatin treatment plus radiation. Gynecologic Oncology 2013 130, 601-608DOI: (10.1016/j.ygyno.2013.06.002) Copyright © 2013 Terms and Conditions