Radiosensitization of human melanoma cells by ribozyme-mediated inhibition of survivin expression
Radiosensitization of human melanoma cells by ribozyme-mediated inhibition of survivin expression
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DOI：
10.1046/j.1523-1747.2003.12082.x
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发表时间：
2003-04-01
影响因子：
6.5
通讯作者：
Zaffaroni, N
中科院分区：
文献类型：
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作者：
Pennati, M;Binda, M;Zaffaroni, N
Survivin是凋亡蛋白抑制剂家族中结构独特的成员，参与控制细胞分裂和抑制细胞凋亡。survivin在大多数人类肿瘤中过度表达，但在正常成人组织中不表达，只有少数例外，这一概念导致了survivin作为一种有希望的新型抗癌治疗靶点的提议。在这种情况下，我们生成了一种针对survivin mRNA中CUA（110）三联体3'末端的锤头核酶。用携带该核酶序列的pRc/CMV载体稳定转染过表达survivin的两株人黑色素瘤细胞系（JR8和M14）。我们选择了两个被证实内源性表达核酶的多克隆细胞群，它们的survivin蛋白水平明显低于JR8和M14亲本细胞（分别为-60%和-50%）。与JR8和M14细胞相比，表达核酶的细胞对γ辐射的敏感性显著（p < 0.01）增加（通过克隆细胞存活率检测）。此外，在JR8细胞系中，核酶表达细胞的辐射诱导凋亡程度（以碘化丙啶染色细胞中凋亡细胞核的百分比和caspase-3催化活性水平计算）明显高于亲本细胞。这些结果首次证明，survivin表达的衰减使人类黑色素瘤细胞更容易受到γ辐射的影响。
Survivin is a structurally unique member of the inhibitors of apoptosis protein family and is involved in the control of cell division and inhibition of apoptosis. The notion that survivin is overexpressed in most human tumors but absent in normal adult tissues with only a few exceptions has led to the proposal of survivin as a promising therapeutic target for novel anticancer therapies. In this context, we generated a hammerhead ribozyme targeting the 3' end of the CUA(110) triplet in the survivin mRNA. Two human melanoma cell lines (JR8 and M14) overexpressing survivin were stably transfected with the pRc/CMV vector carrying the ribozyme sequence. Two polyclonal cell populations proven to endogenously express ribozyme and characterized by a markedly lower survivin protein level (-60% and -50%, respectively) than JR8 and M14 parental cells were selected for the study. Ribozyme-expressing cells showed a significantly (p < 0.01) increased sensitivity to gamma-irradiation (as detected by clonogenic cell survival) compared to JR8 and M14 cells. Moreover, in the JR8 cell line, the extent of radiation-induced apoptosis (in terms of percentage of apoptotic nuclei in cells stained with propidium iodide and level of caspase-3 catalytic activity) was markedly greater in ribozyme-expressing cells than in parental cells. These results demonstrate for the first time that attenuation of survivin expression renders human melanoma cells more susceptible to gamma-irradiation.