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Study of DNA repair mechanisms that affect chemosensitivity of breast cancer

影响乳腺癌化疗敏感性的DNA修复机制研究

基本信息

批准号：
18591446
负责人：
OHTA Tomohiko
金额：
$ 2.55万
依托单位：
St.Marianna University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591446/
关键词：
BRCA1 BARD1 familial breast cancer ubiquitin ligase Nucleophosmin RING finger RPB8 Nucleophosmin

项目摘要

I have investigated the role of substrate ubiquitination by the breast and ovarian tumor suppressor BRCA1 on DNA damage response to elucidate the DNA damage repair mechanisms underlying chemosensitivity of breast cancer I previously identified several substrates including nucleophosmin (NPM1/B23) and RPB8, a common subunit of RNA polymerases. I have mainly investigated RPB8 in 2006 and found that 1) endogenous RPB8 interacts with BARD1, 2) RPB8 polyubiquitination by purified BRCAl-BARD1 in vitro, 3) Lys6-linked ubiquitin polymer formation in the RPB8 ubiquitination, 4) inhibition of in vivo RPB8 ubiquitination after UV irradiation by siRNA knockdown of BRCA1, 5) destabilization of endogenous RPB8 protein in vivo by siRNA knockdown of BRCA1, 6) HeLa cell lines stably expressing a ubiquitin-resistant form of RPB8 exhibited UV hypersensitivity accompanied by up-regulated caspase activity and p53 phosphorylation at Ser15. In 2006 I have mainly investigated NPM1. I established a cell lines stably expressing NPM1 mutant that is incapable of being ubiquitinated by BRCA1 and investigated its biological significance. However I finally concluded that the cells did not express the phenotype caused by the deficiency of the ubiquitination. I then reconstituted an in vitro NPM1 ubiquitination system using only purified recombinant proteins to investigate the effect of NPM1 ubiquitination on histone chaperone activity of NPM1. I further created recombinant proteins including NPM1, BRCA1-BARD1 heterodimer, Ube2w, Ubc13, and Mms2 and established Lys63-linked NPM1 polyubiquitination system to study DNA damage response.

我研究了乳腺癌和卵巢肿瘤抑制因子 BRCA1 的底物泛素化对 DNA 损伤反应的作用，以阐明乳腺癌化疗敏感性背后的 DNA 损伤修复机制。我之前鉴定了几种底物，包括核磷蛋白 (NPM1/B23) 和 RPB8（RNA 聚合酶的常见亚基）。我在2006年主要研究了RPB8，发现1）内源性RPB8与BARD1相互作用，2）体外纯化的BRCA1-BARD1对RPB8进行多泛素化，3）RPB8泛素化中Lys6连接的泛素聚合物形成，4）通过siRNA敲低UV照射后抑制体内RPB8泛素化 BRCA1, 5) 通过 siRNA 敲低 BRCA1, 6) 稳定表达泛素抗性形式的 RPB8 的 HeLa 细胞系表现出紫外线超敏性，并伴有 caspase 活性上调和 Ser15 位点的 p53 磷酸化。 2006年我主要研究了NPM1。我建立了稳定表达 NPM1 突变体的细胞系，该突变体无法被 BRCA1 泛素化，并研究了其生物学意义。但我最终得出的结论是，细胞没有表达由于泛素化缺陷而导致的表型。然后，我仅使用纯化的重组蛋白重建了体外 NPM1 泛素化系统，以研究 NPM1 泛素化对 NPM1 组蛋白伴侣活性的影响。我进一步创建了包括NPM1、BRCA1-BARD1异二聚体、Ube2w、Ubc13和Mms2在内的重组蛋白，并建立了Lys63连接的NPM1多聚泛素化系统来研究DNA损伤反应。