School of Radiation Medicine and Public Health, Medical College of Soochow University, Suzhou, Jiangsu, P. R. China.
Int J Radiat Biol. 2011 Mar;87(3):263-73. doi: 10.3109/09553002.2011.530335. Epub 2010 Nov 10.
To investigate the effect of ubiquitin-like with plant homeodomain (PHD) and ring finger domains 1 (UHRF1) overexpression on radiosensitivity to X-rays in human breast cancer MDA-MB-231 cells.
Cell survival was determined by colony formation assay; cell cycle distribution was measured by flow cytometry; apoptosis was evaluated by DNA fragmentation assay and Annexin V apoptosis detection kit; protein expression was analysed by Western blot assay; chromosome aberrations (centric rings and dicentrics) were assayed by conventional chromosome analysis.
A significant decrease of radiosensitivity to X-rays was observed in MDA-MB-231 cells transfected with a full-length of human UHRF1 cDNA (MDA-MB-231/UHRF1) compared to the control cells (MDA-MB-231/parental and MDA-MB-231/pcDNA3 [mammalian expression vector]), and the similar results were observed in MDA-MB-468 cells. In contrast, a decreased expression of UHRF1 by a specific UHRF1-small interfering RNA (siRNA) significantly enhanced cell radiosensitivity. The UHRF1-mediated radioresistance was correlated with a G2(Ra)/M arrest, a decreased induction of apoptosis, a down-regulation of the pro-apoptotic protein anti-B cell lymphoma/leukemia 2 (bcl-2) associated X protein (Bax) and a up-regulation of the DNA damage repair proteins Lupus Ku autoantigen protein p70 (Ku-70) and Lupus Ku autoantigen protein p80 (Ku-80). Furthermore, chromosomal aberrations (centric rings and dicentrics) by X-rays were less in MDA-MB-231/UHRF1 than in MDA-MB-231/parental and MDA-MB-231/pcDNA3 control cells.
These results suggested that UHRF1 may be a new target in the radiotherapy of breast cancer via affecting apoptosis and DNA damage repair.
研究泛素样含植物同源结构域（PHD）和指环结构域 1（UHRF1）过表达对人乳腺癌 MDA-MB-231 细胞 X 射线放射敏感性的影响。
细胞存活率通过集落形成实验测定；细胞周期分布通过流式细胞术检测；细胞凋亡通过 DNA 片段化检测和 Annexin V 凋亡检测试剂盒评估；蛋白表达通过 Western blot 分析；染色体畸变（着丝粒环和双着丝粒）通过常规染色体分析检测。
与对照细胞（MDA-MB-231/亲本和 MDA-MB-231/pcDNA3 [哺乳动物表达载体]）相比，转染全长人 UHRF1 cDNA 的 MDA-MB-231 细胞（MDA-MB-231/UHRF1）对 X 射线的放射敏感性显著降低，MDA-MB-468 细胞也观察到类似结果。相反，特异性 UHRF1-siRNA 下调 UHRF1 表达显著增强细胞放射敏感性。UHRF1 介导的放射抗性与 G2（Ra）/M 期阻滞、凋亡诱导减少、促凋亡蛋白抗 B 细胞淋巴瘤/白血病 2（bcl-2）相关 X 蛋白（Bax）下调和 DNA 损伤修复蛋白狼疮 Ku 自身抗原蛋白 p70（Ku-70）和狼疮 Ku 自身抗原蛋白 p80（Ku-80）上调相关。此外，X 射线引起的 MDA-MB-231/UHRF1 染色体畸变（着丝粒环和双着丝粒）少于 MDA-MB-231/亲本和 MDA-MB-231/pcDNA3 对照细胞。
这些结果表明，UHRF1 可能通过影响细胞凋亡和 DNA 损伤修复成为乳腺癌放射治疗的新靶点。
