The effect of p21 antisense oligodeoxynucleotides on the radiosensitivity of nasopharyngeal carcinoma cells with normal p53 function
The effect of p21 antisense oligodeoxynucleotides on the radiosensitivity of nasopharyngeal carcinoma cells with normal p53 function
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p21反义寡核苷酸对p53功能正常鼻咽癌细胞放射敏感性的影响
DOI：
10.1016/j.cellbi.2005.11.010
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发表时间：
2006-03-01
影响因子：
3.9
通讯作者：
Huang, WL
中科院分区：
文献类型：
--
作者：
Liu, XF;Xia, YF;Huang, WL
目的：p21（WAF1/CIP1）被p53转录激活，是G期到S期进展所必需的。p21在DNA损伤后的DNA修复中起关键作用。因此，p21缺陷的细胞可能导致辐射诱导的细胞凋亡增强和辐射敏感性提高。在p53功能正常的CNE-1-wtp53鼻咽癌细胞系中，我们验证了p21反义寡脱氧核苷酸（p21 AS ODNs）可以降低p21的表达水平并增加放射敏感性的假设。方法与材料：合成p21反义寡脱氧核苷酸（p21 AS ODNs）和随机对照寡脱氧核苷酸（p21 RD ODNs）。p21 AS ODNs序列：5'- tgtcatgctggtctgcccc -3'；p21 RD ODNs序列：5‘-CCGGTGAACGAGCGAGCACA-3’。将p21 AS ODNs和p21 RD ODNs转染CNE-1-wtp53鼻咽癌细胞系。Western blotting分析P21蛋白表达水平。流式细胞术观察细胞周期进展和凋亡细胞。克隆生存试验测定存活比例。计算单发多目标模型的参数D-o、Dq、N和线性二次模型的参数alpha、beta、alpha/beta、SF2。采用BALB/c裸鼠在体内研究p21 AS ODNs对鼻咽异种移植物放射敏感性的影响。结果：荧光显微镜主要在血浆中检测到p21 AS ODNs。辐照后，转染P21 AS ODNs的细胞比转染P21 RD ODNs的细胞P21蛋白水平显著降低，凋亡细胞数量显著增加。辐射后24 h转染p21 AS ODNs的细胞G(1)阻滞率下降，48 h后G(2)阻滞率下降。与对照细胞相比，p21 AS ODNs转染细胞的D-o、Dq SF值降低，a值升高。仅10 Gy X射线照射对肿瘤的抑制率为39.1%，照射前注射p21 AS ODNs对肿瘤的抑制率为51.4%。但两组间差异无统计学意义（P < 0.05）。结论：p21反义寡脱氧核苷酸在体外可抑制CNE-1-wtp53鼻咽癌细胞系p21蛋白表达、G缺失、阻滞、细胞凋亡增加，在体内可抑制肿瘤生长。反义寡脱氧核苷酸可能成为增强具有正常p53功能的鼻咽癌细胞放射敏感性的有希望的策略。(C) 2005国际细胞生物学联合会。Elsevier Ltd.出版。版权所有。
Objective: p21(WAF1/CIP1) is transcriptionally activated by p53 and is required for G, to S phase progression. p21 plays a critical role in DNA repair after DNA damage. Thus, cells with defective p21 may result in an enhancement of radiation induced apoptosis and improved radiosensitivity. We tested the hypothesis that p21 antisense oligodeoxynucleotides (p21 AS ODNs) can be used to reduce p21 expression level and increase radiosensitivity in CNE-1-wtp53 nasopharyngeal carcinoma cell line with normal p53 function.Methods and materials: The p21 antisense oligodeoxynucleotides (p21 AS ODNs) and the random control oligodeoxynucleotides (p21 RD ODNs) were synthesized. p21 AS ODNs sequence: 5'-TGTCATGCTGGTCTGCCGCC-3'; p21 RD ODNs sequence: 5'-CCGGTGAACGAGCGAGCACA-3'. p21 AS ODNs and p21 RD ODNs were transfected into CNE-1-wtp53 nasopharyngeal carcinoma cell line. The protein expression levels of P21 were evaluated using Western blotting analysis. Cell cycle progression and apoptotic cells were assessed by flow cytometric analysis. The clonogenic survival assay was performed to determine the survival fraction. The parameters D-o, Dq, and N for the single-hit multi-target model and the parameters alpha, beta, alpha/beta, and SF2 for the linear-quadratic model were calculated. BALB/c nude mice were used to investigate the effect of p21 AS ODNs on the radiosensitivity of nasopharyngeal xenografts in vivo. Results: p21 AS ODNs were detected mainly in plasma with fluorescence microscopy investigation. P21 protein level dramatically decreased and the amount of apoptotic cells increased in p21 AS ODNs transfected cells than in p21 RD ODNs transfected cells after irradiation. The percentage of G(1) arrest decreased in p21 AS ODNs transfected cells 24 h after radiation, then G(2) arrest decreased 48 h after radiation. The values of D-o, Dq SF, decreased and a value increased in p21 AS ODNs transfected cells than in control cells. The inhibition rate in tumor xenografts exposed to X ray of 10 Gy alone was 39.1%, while it was 51.4% in xenografts injected with p21 AS ODNs before exposure to radiation. Unfortunately, there was no significant difference between these two groups (P < 0.05).Conclusion: p21 Antisense oligodeoxynucleotides led to inhibition of P21 protein expression, loss of G, arrest, increase of apoptosis in CNE-1-wtp53 nasopharyngeal carcinoma cell line in vitro and inhibited tumor growth in vivo. Antisense oligodeoxynucleotides may become a promising strategy to enhance radiosensitivity in nasopharyngeal carcinoma cells with normal p53 function. (C) 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.