53BP1缺失补偿DNA同源重组修复增强BCCIP阴性乳腺癌细胞放疗抗性

53BP1 Depletion Enhances Radiotherapy Resistance by Compensating DNA Homologous Recombination Repair Capacity in BCCIP Deficient Breast Cancer Cells

三阴性乳腺癌作为预后较差的乳腺癌亚型,高辐射抗性及分子靶点不明确是影响其放疗效果的主要原因。本研究从一条新的途径,即BCCIP/53BP1途径,研究三阴性乳腺癌高辐射抗性的机制。首先,利用高通量染色体精确分析系统检测X射线照射后53BP1缺失对BCCIP阴性小鼠乳腺癌细胞染色体畸变率的影响,并以免疫荧光染色和蛋白质印迹(Western blot)方法检测53BP1缺失对BCCIP阴性乳腺癌细胞DNA双链断裂损伤恢复效率的影响;随后,采用DR-GFP荧光报告系统和姐妹染色体互换试验检测53BP1对BCCIP阴性乳腺癌细胞同源重组修复效率的调节作用;最后,通过克隆形成试验检测X射线照射后53BP1和BCCIP的共同缺失对乳腺癌细胞存活率的调控效果。结果表明:53BP1缺失下调BCCIP阴性小鼠乳腺癌细胞X射线照射后染色体畸变率的发生;53BP1/BCCIP双缺失乳腺癌细胞受照后,DNA双链断裂标志物γH2AX水平和焦点数量均低于BCCIP单缺失细胞;下调53BP1表达时,受照BCCIP阴性乳腺癌细胞的同源重组修复效率得到明显恢复,并且细胞辐射抗性得到明显增强。综上所述,53BP1缺失通过解除对同源重组修复的抑制,增加了BCCIP阴性乳腺癌细胞DNA双链断裂的修复效率,从而提高了细胞的辐射抗性。研究结果为阐明BCCIP/53BP1通过调控同源重组修复途径影响BCCIP阴性乳腺癌细胞辐射敏感性的作用机制,揭示三阴性乳腺癌放疗抗性的分子机制,以及发现新的放疗增敏靶点,提供了理论依据。

Compared to other subtypes of breast cancers,triple negative breast cancers(TNBC)present poorer clinical out-come and prognosis due to their resistance to therapeutic radiation and undefined molecular signature.The current study in-vestigated the mechanism underlying radiation-resistance of TNBC through a newly discovered BCCIP/53BP1 pathway.An automatic high throughput metaphase screening and analyzing system was used to detect the influence of radiation induced chro-mosomal aberration in BCCIP deficient mouse breast cancer cells by the absence of 53BP1.Immunofluorescent staining and Western blot were then performed to detect the effect of 53BP1 depletion on the repair efficiency of DNA double-strand breaks in BCCIP deficient breast cancer cells.The 53BP1 silencing induced alteration of homologous recombination repair efficiency in BCCIP deficient mouse breast cancer cells were further tested by both DR-GFP report system and sister chromatid exchange assay.Finally,colony formation assay was applied to detect the cell survival rate in 53BP1/BCCIP co-depleted breast cancer cells after radiation exposure.The results showed that the enhanced chromosomal aberration rate in radiation exposed BCCIP deficient mouse breast cancer cells was suppressed by 53BP1 depletion.The level and foci number of DNA double-strand break markerγH2AX in 53BP1/BCCIP co-depleted breast cancer cells were both lower than BCCIP solo-depleted cells.The homolo-gous recombination repair efficiency was significantly recovered in 53BP1 additionally depleted BCCIP null breast cancer cells in response to radiation.The radiation resistant capacity was enhanced by 53BP1 co-deletion in BCCIP deficient cells.Collec-tively,these results suggested that the DNA double-strand break repair efficiency was reinforced in BCCIP deficient breast can-cer cells by removing the inhibition to homologous recombination through 53BP1 simultaneous depletion,and therefore,en-hanced cell radiation resistant capacity.These findings will give new insights in the anti-radiation feature of TNBC,and provide potential biomarkers for its radiotherapeutic intervention.