CHEK2 Y390C突变对DNA双链断裂损伤中不同p53状态乳腺癌细胞增殖和凋亡的影响

CHEK2 has been found to be a breast cancer susceptibility gene in several populations. The gene encodes for a cell-cycle checkpoint kinase involved in the cellular response to DNA damage through regulation of the cell cycle and apoptosis. In our previous study, the novel missense mutation Y390C was detected and found to be significantly associated with a higher risk of breast cancer, especially early-onset breast cancer, in Chinese patients. P53 is one of the most important substrates of CHEK2, and possible pathogenic effects of CHEK2 Y390C on p53 remain to be corroborated. We hypothesize that the decreased kinase activity of CHEK2 Y390C may result in an impaired DNA damage response and altered cell proliferation and apoptosis. Based on this hypothesis, we plan to study the effect of the CHEK2 Y390C mutation on p53 function, cell proliferation, and apoptosis in breast cancer cells with variations in p53 status. We will investigate response to chemotherapy-induced DNA double-strand breaks by means of immunoblotting, immuno-fluorescence staining, nuclear morphology and flow cytometry. Through this investigation we hope to explain possible mechanisms for effects of the CHEK2 Y390C mutation on apoptosis and cell cycle profiles in breast cancer cells, and whether CHEK2- and P53-deficiency have synergic effects on breast carcinogenesis. We hope to provide a scientific basis for the evidence that Chinese individuals with CHEK2 variants are more susceptible to breast cancer than the general population.

细胞周期检验点激酶2（CHEK2）是乳腺癌遗传易感基因，其编码蛋白通过调节细胞周期和凋亡参与DNA损伤的修复。我们前期研究发现新突变CHEK2 Y390C，并证实其与国人早发性乳腺癌明显相关。p53是CHEK2重要底物，Y390C突变的致病作用以及对p53功能的影响不明。我们推断CHEK2 Y390C使DNA损伤修复能力下降而导致细胞增殖和凋亡活性改变；基于此假设，我们选用不同p53状态的乳腺癌细胞作为研究对象，采用化疗药物诱导DNA双链断裂损伤，利用免疫印迹，免疫荧光染色、核形态学观察以及流式细胞术等技术，研究CHEK2 Y390C突变对p53功能以及细胞增殖和凋亡的影响。通过上述研究，力图阐明新突变CHEK2 Y390C对乳腺癌细胞的恶性表型的影响，CHEK2联合p53功能缺陷对乳腺癌发生是否具有协同作用，为CHEK2在国人乳腺癌遗传易感性的作用提供了基础。

一些遗传易感基因，如BRCA1/2和CHEK2突变可以导致家族性乳腺癌发病年龄提早。和高加索女性相比，我国乳腺癌的有发病年龄早而且小于40岁发病比例高的特点。但是，我国女性中检测到BRCA1/2和CHEK2的突变率很低，甚至缺如。我们推断CHEK2突变可能为继BRCA1以外与中国的遗传性乳腺癌的发病密切相关。我们在研究中发现高危的乳腺癌人群中发现了新的错义突变CHEK2 Y390C (1169A>G)，该突变在高危乳腺癌与健康女性人群中的分布存在显著性差异（8.0% v.s 0.8%; P =0.0002）。其中Y390C突变的携带者中有4位伴随乳腺癌或者卵巢癌家族史,不伴有家族史的Y390C携带者的发病年龄提早，甚至小于35岁。CHEK2激酶390位的酪氨酸是高度保守的，突变成半胱氨酸后，降低了CHEK2对DNA损伤药物的修复功能。在DNA损伤性化疗药物作用下，Y390C突变后对细胞灭活CDC25A或者激活P53出现异常，同时下调了P53下游基因P21和Puma的表达。因此该突变可能通过细胞周期以及凋亡来影响CHEK2发挥正常的DNA损伤调节作用。以上研究发现新的突变位点与乳腺癌的发病密切相关，可能成为国人特有的遗传易感性多态位点。