Nuclear Function of Abl in DNA Damage Response

描述(由申请人提供)：2009年3月，美国国立卫生研究院宣布恢复法资金可用于竞争性修订申请(NOT-OD-09-058)。作为对这一声明的回应，本申请寻求扩大一个项目的范围，该项目旨在了解核Abl酪氨酸激酶在DNA损伤诱导的细胞死亡反应中的作用。DNA损伤诱导剂，即基因毒素，是癌症治疗中最有效的药物之一。因此，对基因毒素诱导的细胞死亡机制的基本了解有望提高癌症治疗的疗效。我们实验室的研究已经证实，核Abl酪氨酸激酶是DNA损伤后细胞死亡反应的激活剂。我们最近发现，Abl酪氨酸激酶的激活可以调节microRNAs的表达。MicroRNAs是长度在18~25个核苷酸之间的非编码RNA。人类基因组包含500到600个miRNA基因，这些基因的表达在胚胎发育、细胞分化和对遗传毒性应激的反应中受到调节。三个miRNA基因是P53肿瘤抑制基因调控的靶点，它们参与了DNA损伤诱导的细胞死亡。然而，人们对DNA损伤调节其他miRNAs表达的机制知之甚少。拟议的研究将通过探索核Abl的miRNA调节功能来填补这一知识空白。利用恢复法案提供的两年资金，我们将调查Abl磷酸化RNA聚合酶II-CTD以招募核DROSHA-复合体以刺激特定miRNAs表达的假设。我们将(1)研究Abl、酪氨酸磷酸化RNA聚合酶II和DROSHA-复合体与已鉴定的miRNA基因及其pri-miRNA转录本的相互作用，以及(2)通过关注Eya1和Eya3转录因子来确定Abl调控的miRNAs在DNA损伤诱导细胞死亡中的作用，因为它们的miRNAs被预测为Abl调控的miRNA的靶标，最近发现它们通过其酪氨酸磷酸酶活性对抗细胞对DNA损伤的死亡反应。这项拟议的研究将调查一条先前未知的途径，该途径将DNA损伤信号传导到miRNA表达和细胞死亡的调节中。由于Abl在散发性人类癌症中不会发生突变，而且Abl可以激活不依赖于p53的细胞死亡，因此拟议的研究结果将有助于阐明如何利用Abl通过DNA损伤诱导的促凋亡miRNAs来杀死肿瘤细胞。 公共卫生研究成果：拟议中的研究将获得有关DNA损伤如何触发细胞死亡的基础知识。特别是，这项研究将调查DNA损伤诱导剂，如电离辐射、顺铂和阿霉素对microRNAs表达的先前未知的影响，microRNAs是调节蛋白质编码mRNAs功能的非编码RNAs。这项研究的结果将促进我们对细胞对DNA损伤的反应的理解，这一认识将有助于减少有害副作用，同时提高癌症治疗的疗效。

DESCRIPTION (provided by applicant): In March of 2009, NIH announced the availability of Recovery Act funds for competitive revision applications (NOT-OD-09-058). In response to that announcement, this application seeks to expand the scope of a project that is directed at understanding the role of nuclear Abl tyrosine kinase in DNA damage-induced cell death response. DNA damage inducers, i.e., genotoxins, are some of the most effective agents in cancer therapy. Therefore, fundamental understanding of genotoxin-induced cell death mechanisms holds the promise of enhancing the efficacy of cancer therapeutics. Studies from our laboratory have identified the nuclear Abl tyrosine kinase as an activator of cell death response to DNA damage. We have recently found that activation of Abl tyrosine kinase can regulate the expression of microRNAs. MicroRNAs are non-coding RNAs that are between 18~25 nucleotides in length. The human genome contains 500 to 600 miRNA genes, the expression of which is regulated during embryonic development, cellular differentiation and in response to genotoxic stress. Three miRNA genes are targets of regulation by the p53 tumor suppressor and they contribute to DNA damage-induced cell death. However, little is known of the mechanisms by which DNA damage regulates the expression of other miRNAs. The proposed study will fill this knowledge gap by pursuing the miRNA regulatory function of nuclear Abl. With the two-year funding from the Recovery Act, we will investigate the hypothesis that Abl phosphorylates the RNA polymerase II-CTD to recruit a nuclear Drosha-complex to stimulate the expression of specific miRNAs. We will (1) investigate the interactions of Abl, tyrosine phosphorylated RNA polymerase II and the Drosha-complex with the identified miRNA genes and their pri-miRNA transcripts and (2) determine the role of Abl-regulated miRNAs in DNA damage-induced cell death by focusing on the Eya1 and Eya3 transcription factors, as their miRNAs are predicted targets of an Abl-regulated miRNA, and they have recently been shown to antagonize the cell death response to DNA damage through their tyrosine phosphatase activity. The proposed research will investigate a previously unknown pathway that transduces DNA damage signals to the regulation of miRNA expression and cell death. Because Abl is not mutated in sporadic human cancers and because Abl can activate p53-independent cell death, results from the proposed research will shed light on how to exploit the pro-apoptotic miRNAs induced by DNA damage through Abl to kill tumor cells. PUBLIC HEALTH RELENVANCE: The proposed research will acquire fundamental knowledge on how DNA damage triggers cell death. In particular, this research will investigate a previously unknown effect of DNA damage inducing agents, e.g., ionizing radiation, cisplatin and doxorubicin, on the expression of microRNAs, which are non-coding RNAs that regulate the functions of protein-coding mRNAs. Results from this study will advance our understanding of cellular response to DNA damage and this knowledge will help to reduce the harmful side effects while improving the efficacy of cancer therapy.