B细胞易位基因2过表达增加人乳腺癌细胞T-47D的放射敏感性的研究

Effects of BTG2 Overexpression on the Radiosensitivity of Human Breast Cancer T-47D Cells

目的研究B细胞易位基因2(BTG2)对人乳腺癌细胞T-47D放射敏感性的影响。方法应用脂质体转染的方法提高BTG2基因在人乳腺癌细胞T-47D的表达水平,利用克隆形成实验研究转染后细胞的放射敏感性改变,采用流式细胞术对细胞周期变化进行分析,应用Western blot方法研究相关蛋白的变化。结果克隆形成细胞生存实验结果表明,BTG2稳定高表达的T-47D/BTG2细胞在不同剂量X射线照射后,其存活分数明显低于X射线照射后的未转染的T-47D/Parental(母)细胞以及"空质粒"转染的对照T-47D/Neo细胞的存活分数。细胞平均致死剂量(D_0)值在T-47D/Parental细胞、T-47D/Neo细胞和T-47D/BTG2细胞分别为1.84,1.86和1.57。流式细胞实验结果显示,与T-47D/母细胞和T-47D/Neo细胞相比,T-47D/BTG2细胞在受照射后出现明显的细胞凋亡sub-G1峰。另外,BTG2高表达上调了Bad和Bax促凋亡蛋白的表达水平和下调了协作性的致癌基因Cyclin D1的表达水平。结论增加BTG2基因的表达水平可以明显提高凋亡相关蛋白水平,增加放射治疗所诱导的细胞凋亡,从而提高人类乳腺癌细胞T-47D对电离辐射放射治疗的敏感性。BTG2可能是调节人类乳腺癌细胞放射治疗的有效靶点之一。

Objective To investigate the effects of BTG2 overexpression on the radiosensitivity of human breast cancer T-47D cells.Methods T-47D cells with overexpression of BTG2 were developed via stable transfection of full-length human BTG2 cDNA which was inserted into a mammalian expression plasmid pcDNA3.Cell survival was determined by clonogenic survival assay.Cell cycle distribution and apoptosis were analyzed by flow cytometry.Protein expression was assayed by Western blot assay.Results With different doses（0-6 Gy） of X-ray irradiation,the survival fraction in T-47D/BTG2 cells with overxpression of the BTG2 gene was significantly lower than that of un-transfected T-47D/Parental cells and T-47D/Neo cells which were transfected with an＂empty＂pcDNA3 control vector.The data with Flow cytometry indicate dramatic increase of the apoptotic sub-G1 cell population in T-47D/BTG2 cells compared with T-47D/Parental and T-47D/Neo cells following exposure to irradiation in a dose of 6 Gy. BTG2 further enhanced radiation-mediated increase of Bad and Bax apoptotic protein expression as well as decrease of Cyclin D1 protein expression.Conclusion The present study,for the first time,demonstrate that overexpression of BTG2 increases the sensitivity of human breast cancer T-47D cells to ionizing radiation, accompanied with a promotion of radiation-caused apoptosis induction.These findings suggest that BTG2 may be a new and important target in radiotherapy of human breast cancer.