外源性p53对不同LET辐射诱导人黑色素瘤细胞系A375死亡途径的影响

目的 观察外源性P53蛋白对不同传能线密度(LET)射线辐照诱导肿瘤细胞凋亡和坏死的影响,并探讨其可能的机制。方法 人黑色素瘤细胞系A375(wild-type p53)经携带人野生型p53基因的腺病毒载体(AdCMV-p53)感染后分别给予X射线和碳离子束照射,采用克隆形成法测定细胞辐射敏感性,Hoechst 33258和吖啶橙-溴化乙锭双染荧光显微镜下观察细胞凋亡和坏死。结果1高LET辐照时,A375细胞和转导人野生型p53基因的A375细胞(A375/p53)的辐射敏感性没有明显差异;2虽然辐射诱导细胞凋亡比例的增加依赖于LET升高,但是无论高LET或低LET,外源性P53蛋白均可有效诱导细胞凋亡。3高LET辐照时,A375细胞的坏死细胞明显高于A375/p53细胞。结论 尽管高LET辐射对A375和A375/p53细胞的存活无明显影响,但是对细胞凋亡的诱导却部分依赖于P53蛋白的功能,P53蛋白可能在调节细胞死亡类型中发挥重要作用。这对临床应用高LET辐射联合p53基因治疗恶性黑色素瘤有一定参考意义。

Cell death pathways of melanoma cells induced by irradiation at different LET combined with p53 gene therapy

Objective To investigate the effect of exogenous wild type P53 on the radiation induced cells apoptosis and necrosis at different levels of linear energy transfer(LET),and evaluate its mechanisms.Methods The human melanoma cell line A375,with wild-type p53 gene was used,as well as the transfectant A375 cells (A375/p53) with adenoviral vector containing the wild-type p53 gene.These cells were exposed to X-rays and accelerated carbon-ion(C-)beams.Cellular radiosensitivity was determined by using clonogenic assay.Apoptotic and necrotic cell death rate was determined morphologically by dual-staining(acridine orange,ethidium bromide) for fluorescence microscope.Results ①There was no significant difference in survival fraction between A375 cells and A375/p53 cells irradiated by C-beams with greater than 32 KeV/μm.②Apoptosis rates in the two kinds of cells increased in a LET-dependent manner, exogenous wild-type P53 induced cell apoptosis more efficiently in A375/p53 than that in A375 cells with X-rays or high-LET irradiation.③The necrosis rates in A375 ceils exposed to high-LET irradiation increased significantly(P<0.05)in comparison with A375/p53 cells.Conclusions Apoptosis induction is p53 dependent partly by high-LET irradiation,and exogenous wildtype P53 plays an important role in modulating cell death type,although there was no significant difference in cellular radiosensitivities.The observation in the study indicates the potential application of high-LET radiation combined with p53 in the management of human melanoma.