Downregulation of Hus1 by antisense oligonucleotides enhances the sensitivity of human lung carcinoma cells to cisplatin
Downregulation of Hus1 by antisense oligonucleotides enhances the sensitivity of human lung carcinoma cells to cisplatin
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DOI：
10.1002/cncr.10383
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发表时间：
2002-03-15
期刊：
影响因子：
6.2
通讯作者：
Cohen, D
中科院分区：
文献类型：
--
作者：
Kinzel, B;Hall, J;Cohen, D
背景资料。在裂殖酵母中，Hus1是辐射敏感(Rad)机制的一个组成部分，已被发现在DNA修复和细胞周期G(2)/M检查点控制通路中发挥作用。HUS1已被证明存在于一个离散的复合体中，至少有两个Rad家族成员，Rad1和Rad9。此外，Hus1对于检查点激活是必不可少的，因为Hus1突变体无法阻止细胞周期对DNA损伤或未复制的DNA做出反应。方法以脂质体为载体，将2‘-O-甲氧乙基修饰的Flus1反义寡核苷酸导入人H1299非小细胞肺癌细胞，观察其在细胞周期调控中的作用及相关性。作者从转基因细胞中制备RNA，并用实时定量聚合酶链式反应分析Hus1的表达水平。结果：将HUS1反义寡核苷酸导入p53基因缺失的H1299细胞，可显著下调HUS1基因的表达，最高可下调80%；RNA分析显示，当HUS1反义寡核苷酸的浓度为200 nM时，HUS1的活性最高，IC50为90 nM。含有3个错配的反义寡核苷酸的设计和转染对HUS1的mRNA水平没有或仅有轻微的影响，显示了HUS1mRNA下调的特异性。未转染的H1299细胞对顺铂的IC50为20微米，经HUS1反义寡核苷酸处理后，IC50仅为7微米。结论：H1299细胞的增殖和活力实验表明，特定的反义寡核苷酸下调HUS1使人细胞对DNA损伤剂顺铂的处理敏感。(C)2002年美国癌症协会
BACKGROUND. In Schizosaccharomyces pombe, Hus1 is a component of the radiation sensitive (Rad) machinery that has been identified as playing a role in DNA repair and cell cycle G(2)/M checkpoint control pathways. Hus1 has been shown to exist in a discrete complex with at least two Rad family members, Rad1 and Rad9. Furthermore, Hus1 is essential for checkpoint activation, since Hus1 mutants fail to arrest the cell cycle in response to DNA damage or unreplicated DNA. To establish the role and relevance of human Hus1 in cell cycle regulation, the authors applied antisense technology to selectively downregulate the expression of Hus1 mRNA.METHODS. Transfection of 2'-O-methoxyethyl-modified Flus1 antisense oligoribonucleotides into human H1299 nonsmall lung carcinoma cells was performed using Lipofectin as the carrier. The authors prepared RNA from transfected cells, and levels of Hus1 expression were analyzed by real time polymerase chain reaction. The growth and viability of cells treated with Hus1 antisense oligonucleotides in the presence or absence of cisplatin were analyzed and compared to controls.RESULTS. Transfection of selected Hus1 antisense oligonucleotides into p53 deficient H1299 cells resulted in significant downregulation of Hus1 mRNA, up to 80%; RNA analyses reveal a maximal Hus1 antisense activity at a concentration of 200 nM with an IC50 determined to be 90 nM. The design and transfection of oligonucleotides containing three mismatches to their corresponding antisense counterparts had no or only minor effects on Hus1 mRNA levels, showing the specificity of Hus1 mRNA downregulation. The cisplatin IC50 in untransfected H1299 cells was found to be 20 muM and could be reduced significantly to only 7 muM after transfection of a Hus1 antisense oligonucleotide.CONCLUSIONS. Experiments addressing the proliferation and viability of transfected H1299 cells suggest that downregulation of Hus1 by specific antisense oligonucleotides sensitizes human cells to treatment with the DNA damaging agent cisplatin. (C) 2002 American Cancer Society