Functional differences between wild-type and mutant-type BRCA1-associated protein 1 tumor suppressor against malignant mesothelioma cells.
Functional differences between wild-type and mutant-type BRCA1-associated protein 1 tumor suppressor against malignant mesothelioma cells.
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DOI：
10.1111/cas.12698
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发表时间：
2015-08
期刊：
影响因子：
5.7
通讯作者：
Sekido Y
中科院分区：
文献类型：
--
作者：
Hakiri S;Osada H;Ishiguro F;Murakami H;Murakami-Tonami Y;Yokoi K;Sekido Y
恶性间皮瘤（MM）显示BRCA 1相关蛋白1（BAP 1）基因失活。在这项研究中，我们在从日本MM患者建立的19个细胞系中的5个（26%）中发现了BAP1突变，并检查了WT和突变BAP1之间的功能差异。首先，我们研究了BAP 1的亚细胞定位，证明WT主要存在于细胞核中，而突变体BAP 1存在于细胞的细胞质中。将WT BAP 1载体转导入BAP 1 3′端纯合缺失的MM细胞中，导致细胞增殖和锚定非依赖性细胞生长的抑制，而错义或C端截短形式的BAP 1突变体显示出受损的生长抑制作用。接下来，我们研究了BAP 1如何参与辐射（IR）后的MM细胞存活，这会导致DNA损伤。IR后，我们发现WT和突变体BAP 1都被类似地磷酸化，并且磷酸化BAP 1主要定位于细胞核。有趣的是，BRCA 1蛋白在BAP 1缺失的MM细胞中减少，并且突变体以及WT BAP 1的转导增加BRCA 1蛋白，这表明BAP 1可能部分通过稳定BRCA 1来促进DNA修复。此外，使用BAP 1缺失的MM细胞，我们发现WT BAP 1，甚至错义突变体，与对照载体相比，在IR后赋予更高的存活率。我们的研究结果表明，尽管WT BAP1抑制MM细胞增殖并在IR损伤后恢复细胞存活，但一些突变BAP1也可能适度保留这些功能。
Malignant mesothelioma (MM) shows inactivation of the BRCA1-associated protein 1 (BAP1) gene. In this study, we found BAP1 mutations in 5 (26%) of the 19 cell lines that we established from Japanese MM patients, and examined functional differences between the WT and mutant BAP1. First, we studied the subcellular localization of BAP1, demonstrating that the WT primarily resides in the nucleus and that the mutant BAP1 is found in the cytoplasm of the cells. Transduction of the WT BAP1 vector into MM cells with homozygous deletion at the BAP1 3′ side resulted in both inhibition of cell proliferation and anchorage-independent cell growth, whereas BAP1 mutants of a missense or C-terminal truncated form showed impaired growth inhibitory effects. Next, we studied how BAP1 is involved in MM cell survival after irradiation (IR), which causes DNA damage. After IR, we found that both WT and mutant BAP1 were similarly phosphorylated and phospho-BAP1 localized mainly in the nucleus. Interestingly, BRCA1 proteins were decreased in the MM cells with BAP1 deletion, and transduction of the mutants as well as WT BAP1 increased BRCA1 proteins, suggesting that BAP1 may promote DNA repair partly through stabilizing BRCA1. Furthermore, using the MM cells with BAP1 deletion, we found that WT BAP1, and even a missense mutant, conferred a higher survival rate after IR compared to the control vector. Our results suggested that, whereas WT BAP1 suppresses MM cell proliferation and restores cell survival after IR damage, some mutant BAP1 may also moderately retain these functions.