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首页> 外文期刊>Cell Division >Silencing CDK4 radiosensitizes breast cancer cells by promoting apoptosis
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Silencing CDK4 radiosensitizes breast cancer cells by promoting apoptosis

机译：沉默CDK4通过促进凋亡使乳腺癌细胞放射增敏

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Background The discovery of molecular markers associated with various breast cancer subtypes has greatly improved the treatment and outcome of breast cancer patients. Unfortunately, breast cancer cells acquire resistance to various therapies. Mounting evidence suggests that resistance is rooted in the deregulation of the G1 phase regulatory machinery. Methods To address whether deregulation of the G1 phase regulatory machinery contributes to radiotherapy resistance, the MCF10A immortalized human mammary epithelial cell line, ER-PR-Her2+ and ER-PR-Her2- breast cancer cell lines were irradiated. Colony formation assays measured radioresistance, while immunocytochemistry, Western blots, and flow cytometry measured the cell cycle, DNA replication, mitosis, apoptosis, and DNA breaks. Results Molecular markers common to all cell lines were overexpressed, including cyclin A1 and cyclin D1, which impinge on CDK2 and CDK4 activities, respectively. We addressed their potential role in radioresistance by generating cell lines stably expressing small hairpin RNAs (shRNA) against CDK2 and CDK4. None of the cell lines knocked down for CDK2 displayed radiosensitization. In contrast, all cell lines knocked down for CDK4 were significantly radiosensitized, and a CDK4/CDK6 inhibitor sensitized MDA-MB-468 to radiation induced apoptosis. Our data showed that silencing CDK4 significantly increases radiation induced cell apoptosis in cell lines without significantly altering cell cycle progression, or DNA repair after irradiation. Our results indicate lower levels of phospho-Bad at ser136 upon CDK4 silencing and ionizing radiation, which has been shown to signal apoptosis. Conclusion Based on our data we conclude that knockdown of CDK4 activity sensitizes breast cancer cells to radiation by activating apoptosis pathways.
机译：背景技术与各种乳腺癌亚型相关的分子标记的发现极大地改善了乳腺癌患者的治疗和预后。不幸的是，乳腺癌细胞获得了对各种疗法的抗性。越来越多的证据表明，抵抗力根源于G1相调控机制的放松。方法为了研究G1期调控机制的失控是否会导致放疗耐药性，对MCF10A永生化的人乳腺上皮细胞系，ER-PR-Her2 +和ER-PR-Her2-乳腺癌细胞系进行了照射。集落形成测定法测量了放射抗性，而免疫细胞化学，蛋白质印迹和流式细胞术则测量了细胞周期，DNA复制，有丝分裂，细胞凋亡和DNA断裂。结果所有细胞系共有的分子标志物均过表达，其中包括cyclin A1和cyclin D1，分别影响CDK2和CDK4的活性。我们通过产生稳定表达针对CDK2和CDK4的小发夹RNA（shRNA）的细胞系来解决它们在放射抗性中的潜在作用。敲除CDK2的所有细胞系均未显示放射增敏作用。相反，对CDK4敲除的所有细胞系均显着放射致敏，而CDK4 / CDK6抑制剂对MDA-MB-468致辐射诱导的细胞凋亡敏感。我们的数据表明，沉默CDK4可以显着增加辐射诱导的细胞系细胞凋亡，而不会显着改变细胞周期进程或辐射后的DNA修复。我们的结果表明，在CDK4沉默和电离辐射后，ser136处的磷酸化Bad含量较低，这已表明可以凋亡。结论基于我们的数据，我们得出结论，敲低CDK4活性可通过激活细胞凋亡途径使乳腺癌细胞对辐射敏感。

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