Stable suppression of the R2 subunit of ribonucleotide reductase by R2-targeted short interference RNA sensitizes p53(-/-) HCT-116 colon cancer cells to DNA-damaging agents and ribonucleotide reductase inhibitors
Stable suppression of the R2 subunit of ribonucleotide reductase by R2-targeted short interference RNA sensitizes p53(-/-) HCT-116 colon cancer cells to DNA-damaging agents and ribonucleotide reductase inhibitors
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DOI：
10.1074/jbc.m402056200
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发表时间：
2004-06-25
影响因子：
4.8
通讯作者：
Sartorelli, AC
中科院分区：
文献类型：
--
作者：
Lin, ZP;Belcourt, MF;Sartorelli, AC
核糖核苷酸还原酶催化脱氧核糖核苷二磷酸的产生，脱氧核糖核苷三磷酸的前体用于DNA合成。哺乳动物核糖核苷酸还原酶（RNR）是由两个不同的同源二聚体R1和R2或p53 R2组成的四聚体，它们被认为分别参与DNA复制和修复。我们已经证明，阿霉素和顺铂的DNA损伤导致p53（-/-）HCT-116人结肠癌细胞中R2蛋白的稳定升高，但诱导p53（+/+）细胞中蛋白的降解。为了评估R2参与DNA损伤的反应，用转录针对R2 mRNA的短发夹/短干扰RNA的表达载体稳定转染p53（-/-）HCT-116细胞。稳定转染的克隆表现出R2蛋白的显著减少，而细胞生长速率没有变化。此外，短干扰RNA介导的R2蛋白的减少导致对DNA损伤剂顺铂以及RNR抑制剂Trifecta（R）和羟基脲的敏感性显著增加。p53 R2的异位表达部分逆转了顺铂的细胞毒性，但RNR抑制剂对R2敲低细胞的细胞毒性没有逆转。对顺铂和RNR抑制剂的敏感性增加与由R2靶向短干扰RNA的稳定表达引起的dATP和dGTP水平的抑制相关。这些结果表明，DNA损伤导致R2蛋白和dNTPs水平升高，从而提高了p53（-/-）HCT-116细胞的存活率。这些发现提供了证据表明，R2- RNR可用于提供dNTPs，用于修复p53依赖性p53 R2诱导受损的细胞中的DNA损伤。
Ribonucleotide reductase catalyzes the production of deoxyribonucleoside diphosphates, the precursors of deoxyribonucleoside triphosphates for DNA synthesis. Mammalian ribonucleotide reductase (RNR) is a tetramer consisting of two non-identical homodimers, R1 and either R2 or p53R2, which are considered to be involved in DNA replication and repair, respectively. We have demonstrated that DNA damage by doxorubicin and cisplatin caused a steady elevation of the R2 protein in p53(-/-) HCT-116 human colon carcinoma cells but induced degradation of the protein in p53(+/+) cells. To evaluate the involvement of R2 in response to DNA damage, p53(-/-) HCT-116 cells were stably transfected with an expression vector transcribing short hairpin/short interference RNA directed against R2 mRNA. Stably transfected clones exhibited a pronounced reduction of the R2 protein with no change in the cellular growth rate. Furthermore, short interference RNA-mediated reduction of the R2 protein caused a marked increase in sensitivity to the DNA-damaging agent cisplatin as well as to the RNR inhibitors Triapine(R) and hydroxyurea. Ectopic expression of p53R2 partially reversed the cytotoxicity of cisplatin but not that of RNR inhibitors to R2 knockdown cells. The increase in sensitivity to cisplatin and RNR inhibitors was correlated with the suppression of dATP and dGTP levels caused by stable expression of R2-targeted short interference RNA. These results indicated that DNA damage resulted in elevated levels of the R2 protein and dNTPs and, consequently, enhanced the survival of p53(-/-) HCT-116 cells. The findings provide evidence that R2- RNR can be employed to supply dNTPs for the repair of DNA damage in cells with an impaired p53-dependent induction of p53R2.