BRAFV600E Inhibitor Radiosensitizes Thyroid Cancer-Response.
BRAFV600E Inhibitor Radiosensitizes Thyroid Cancer-Response.
复制标题
BRAFV600E 抑制剂可提高甲状腺癌的放射敏感性。
DOI：
10.1158/1078-0432.ccr-19-2705
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发表时间：
2019
期刊：
影响因子：
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通讯作者：
Williams,TerenceM
中科院分区：
文献类型：
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作者：
Williams,TerenceM
我们感谢EL Göttgens等人对我们的出版物提供了有见地的评论。我们同意维罗非尼通常会诱导BRAF突变肿瘤细胞的G1期阻滞，但这通常发生在12-24小时或更长的时间内。在图3A中，我们观察到辐射后仅1小时内γ H2 AX的水平较高，其中用维罗非尼预处理3小时（总共= 4小时的维罗非尼），这表明维罗非尼对改变双链断裂的修复有直接作用。此外，与G1晚期相比，G1停滞的细胞在G1的早期部分可能具有辐射抗性，而G1晚期则更具辐射敏感性（因为它们接近早期S期），因此尚不清楚细胞周期状态的改变是否在放射增敏中发挥直接作用。然而，我们同意细胞周期分布向G1阻滞状态的变化可能优先导致NHEJ DNA修复途径的参与，这可以解释我们的核灶修复结果。尽管如此，我们发现突变体BRAF-V600 E的表达在不存在维罗非尼的情况下导致我们的GFP报告系统中NHEJ修复活性增加，但不导致HR修复（图4C）。在测试BRAF-V600 E和维罗非尼对GFP报告系统中NHEJ或HR活性的影响的实验中，我们不是通过辐射诱导DNA损伤，而是通过内切核酸酶I-SceI诱导，其在我们的报告DNA中的识别序列处诱导双链断裂。我们在用腺病毒I-SceI处理的同时用载体或维罗非尼处理的原因是为了在24小时的过程中允许感染和I-SceI酶表达的时间。实际上，这是在I-SceI诱导DNA损伤之前用vemurafenib预处理的一段时间。如图4C的右图所示，我们在空载体（对照）转染的细胞中没有观察到用维罗非尼处理42小时（18+ 24小时）的细胞的NHEJ修复活性的增加，这与G1分数增加解释DNA修复差异的作用相反。然而，在表达BRAF-V600 E的细胞中，在不存在维罗非尼的情况下存在较高水平的基线NHEJ修复活性，当用维罗非尼处理细胞42小时时，其显著降低。
We thank EL Göttgens et al. on providing insightful comments on our publication. We agree that vemurafenib will commonly induce G1 arrest in BRAF mutant tumor cells, but that this typically happens over the course of 12–24 hrs or longer. We were observing higher levels of γH2AX within just one hour after radiation with 3 hours of pre-treatment with vemurafenib (total= 4 hours of vemurafenib) in Figure 3A, arguing for a direct effect of vemurafenib on altering repair of double-stranded breaks. In addition, G1 arrested cells can be radioresistant in the earlier portions of G1, compared to late G1 when they are more radiosensitive (as they approach early S phase), so it’s not clear that altered cell cycle state is playing a direct role in radiosensitization. However, we do agree that changes in cell cycle distribution towards a G1 arrested state could preferentially result in engagement of NHEJ DNA repair pathways which can account for our nuclear foci repair results. Despite this, we find that expression of mutant BRAF-V600E results in increased NHEJ repair activity in our GFP reporter system in the absence of vemurafenib, but not HR repair (Fig. 4C). In this experiment testing the effect of BRAF-V600E and vemurafenib on NHEJ or HR activity in our GFP reporter system, we are not inducing DNA damage by radiation, but rather by the endonuclease I-SceI, which induces double-strand breaks in our reporter DNA at the recognition sequence. The reason we treated with vehicle or vemurafenib at the same time as treatment with adenoviral I-SceI was to allow for time of infection and expression of I-SceI enzyme over the course of 24 hours. In effect, this served as a period of pre-treatment with vemurafenib prior to DNA damage induction by I-SceI. As shown in the right panel of Fig. 4C, we did not observe an increase in NHEJ repair activity of cells treated with 42 hrs (18+ 24 hrs) of vemurafenib in the empty vector (control) transfected cells, arguing against a role for an increase in G1 fraction accounting for the differences in DNA repair. However, in cells expressing BRAF-V600E, there was a higher level of baseline NHEJ repair activity in the absence of vemurafenib, which was significantly reduced when cells were treated for 42 hrs with vemurafenib.