2010-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/655475摘要：鼻咽癌在台灣是十大癌症之一，然而對於鼻咽癌腫瘤發生分子機制的了解仍很少，鼻咽癌已知有基因體不穩定的現象且與EB 病毒(EBV)的感染有密切關連。先前我們曾報導過EB 病毒EBNA2 蛋白質會透過影響有絲分裂檢查點(mitotic spindle checkpoint)，進而引起癌細胞株及非癌細胞株非整倍体(aneuploidy)及多核(multinuclei)的發生，這些資料顯示EBNA2 蛋白質這個新的功能，可能是造成鼻咽癌基因體不穩定的原因之一。另外，除了引起aneuploidy，我們已發現EBNA2 蛋白質會引起微核(micronuclei)及γ-H2AX 核聚集點(nuclear foci)的產生(見初步結果)，因此我們認為EBNA2 蛋白質會抑制DNA 雙股斷裂的修復，我們還發現EBNA2 蛋白質會抑制非相同性斷端結合(nonhomologous end-joining)，後者為哺乳動物細胞修復DNA 雙股斷裂的主要方式。我們也發現EBNA2 蛋白質會阻止DNA 修復相關MDC1 蛋白質集結到DNA 雙股斷裂點，由於MDC1 是可招來其它DNA 修復蛋白質，例如53BP1 及BRCA1，集結到DNA 雙股斷裂點及單股斷裂點的關鍵蛋白質，因此推測EBNA2 蛋白質阻止MDC1 到達DNA雙股斷裂點可能是EBNA2 蛋白質抑制DNA 雙股斷裂修復的機制。此外，這樣的結果也暗示EBNA2 蛋白質會透過抑制核苷酸切除修復(nucleotide excision repair，NER)系統，進而抑制DNA 單股斷裂的修復。因此，本研究計晝的主要目標如下:(1)研究EBNA2蛋白質抑制DNA 雙股斷裂修復的機制及(2)研究EBNA2 蛋白質是否會抑制NER，若是，則再研究其抑制NER 的機制。為了完成第一個目標，我們將研究EBNA2 蛋白質阻止MDC1 集結到DNA 雙股斷裂點的機轉，同時我們也會研究EBNA2 是否也會阻止其它DNA 修復相關蛋白質，例如53BP1 及BRCA1，集結到DNA 雙股斷裂點。為了完成第二個目標，首先我們會用寄主細胞再活化分析(host-cell reactivation assay)來測試EBNA2蛋白質是否會抑制DNA 單股斷裂的修復(即抑制NER)，若是，則再研究EBNA2 蛋白質是否會抑制DNA 修復相關蛋白質，例如MDC1、53BP1 及BRCA1，集結到DNA 單股斷裂點。細胞修復受損DNA 是維持基因體穩定的必要功能，抑制DNA 修復則會導致基因體不穩定，進而引發腫瘤發生。本研究不僅可闡明鼻咽癌基因體不穩定的產生機制，並可對鼻咽癌形成的病理機轉提供進一步的了解。<br> Abstract: Nasopharyngeal carcinoma (NPC) is one of the ten leading cancers in Taiwan. However,the molecular basis of NPC tumorigenesis is still poorly understood. NPC is known to begenomic unstable and is closely associated with Epstein-Barr virus (EBV) infection.Previously, we reported that EBV EBNA2 protein can induce aneuploidy and multinucleiformation by compromising mitotic spindle checkpoint in cancer cell lines and non-cancercell lines. These data reveal a new function of EBNA2 that may contribute to genomicinstability in NPC. In addition to causing aneuploidy, EBNA2 was found by us to be able toinduce micronuclei formation and -H2AX nuclear foci formation (see Preliminary Results),suggesting that EBNA2 can inhibit the repair of DNA double-strand break (DSB). Indeed, wefound that EBNA2 could inhibit nonhomologous end-joining, a major DSB repair pathway inmammalian cells. We also found that EBNA2 could prevent the recruitment of repair-relatedprotein MDC1 to the sites of DSB. Since MDC1 is the key protein that can recruit otherrepair-related proteins, such as 53BP1 and BRCA1, to the sites of DSB and sites of DNAsingle-strand break. This result suggests that EBNA2 prevention of MDC1 to localize at DSBsites may be the mechanism by which EBNA2 inhibits DSB repair. Moreover, this result alsosuggests that EBNA2 can inhibit the repair of DNA single-strand break, which is repaired bynucleotide excision repair (NER) pathway. Therefore, the major goals of this grant proposalare: (1) to study the molecular mechanisms underlying EBNA2 inhibition of DSB repair; and(2) to study whether EBNA2 can inhibit NER, and if yes, to study the mechanisms underlyingEBNA2 inhibition of NER. To accomplish the first goal, we will study the mechanism bywhich EBNA2 prevents the recruitment of MDC1 to DSB sites. We will also study whetherEBNA2 could prevent the recruitment of other repair-related proteins, such as 53BP1 andBRCA1, to the sites of DSB. To accomplish the second goal, we will first test whetherEBNA2 could inhibit the repair of DNA single-strand breaks (i.e., inhibit NER) by usinghost-cell reactivation assay. If yes, we will study whether EBNA2 could prevent repair-relatedproteins, such as MDC1, 53BP1, and BRCA1, to localize at sites of DNA single-strandbreaks.The ability to repair damaged DNA is essential for maintenance of genomic stability.Inhibition of DNA repair results in genomic instability, which is a driving force oftumorigenesis. Our study proposed here not only can elucidate the mechanism underlyinggenomic instability of NPC but also can provide insights about NPC pathogenesis.