EB病毒EBNA2可以抑制PML nuclear bodies轉移至DNA雙股斷裂點

Epstein-Barr virus EBNA2 Prevents PML Nuclear Bodies from Targeting to DNA Double-Strand Breaks

劉秉鑫(Bing-Sin Liu)

指導教授 ： 王萬波

國立臺灣大學/醫學院/微生物學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.10236

摘要

EB病毒是一種廣泛分佈於全世界的人類皰疹病毒，主要感染人類淋巴球以及上皮細胞，並造成B淋巴球的轉形與不死。目前許多癌症被認為與EB病毒有高度的關連性，包括鼻咽癌、巴氏淋巴瘤、T細胞淋巴瘤、胃癌等。然而EB病毒在致癌過程中所扮演的角色仍不清楚，因此研究EB病毒的癌化機制相當重要。
當細胞感染EB病毒後，Epstein-Barr virus nuclear antigen 2 (EBNA2) 是最早被表現出來的病毒轉錄活化子。研究證實，在EB病毒所引起的B細胞轉形中，EBNA2是必須的因子，且亦能在基因轉殖鼠中誘發腫瘤形成。本實驗室先前的研究結果顯示，EBNA2可能會引起基因體不穩定，進而導致細胞癌化的發生。其中我們發現，EBNA2會抑制DNA修復並造成雙股斷裂在細胞中不斷累積，然而EBNA2引發基因體不穩定的機制仍不清楚。因此本文的主要目的是找出EBNA2抑制DNA修復的可能機制。
Promyelocytic leukemia nuclear bodies (PML NBs)，是細胞核內一個十分動態的結構，易受到熱休克作用、重金屬與DNA受損等因素而改變外型或者是潰散瓦解。此外當細胞遭受游離輻射ionizing radiation (IR)照射後，PML NBs會與DNA斷裂處及許多參與DNA修復的蛋白質如ATR、Mre11、NBS1、TopBP1以及BLM重疊在一起。這些結果顯示PML NBs可能參與DNA修復的作用。
本研究中我們首先發現，當細胞遭受IR游離輻射打斷雙股DNA後，EBNA2會造成Mre11/NBS1 IRIF與PML NBs的位置錯開。進一步確認EBNA2具有抑制PML NBs轉移至雙股斷裂標的γH2AX foci的能力，但Mre11仍可轉移至γH2AX foci，且DNA損傷所引發的NBS1磷酸化不會受到影響。此外也發現，細胞照射IR後在表現EBNA2的細胞株中，PML NBs的顆數明顯少於無EBNA2表現的細胞株，因此可知EBNA2可能會去抑制DNA損傷所引發的PML NBs分裂。由於PML NBs與Mre11、NBS1共同聚集在DNA斷裂處，可能對於雙股修復功能是重要的，因此EBNA2造成Mre11/NBS1 IRIF與PML NBs的位置錯開可能會抑制正常的DNA修復。未來仍須更深入的研究來證實此項論點。

關鍵字

EB病毒 ； EBNA2 ； DNA損傷 ； PML NBs ； MRN complex

並列摘要

Epstein-Barr virus (EBV) is a widespread human herpesvirus that mainly infects human B-lymphocyte and epithelial cells and causes the transformation as well as immortalization of B-lymphocyte. Nowadays many human cancers have been shown to be closely associated with EBV infection, including nasopharyngeal carcinoma (NPC), Burkitt’s lymphoma, T-cell lymphoma, gastric cancers and so on. However, the role of EBV in the development of these cancers still remains unclear. Therefore, it is important to investigate the mechanisms underlying EBV-induced tumorigenesis.
EBNA2, a potent transcriptional activator, is the first viral protein that expressed after EBV infection. EBNA2 has been shown to be required for EBV-transformation of B-cells and can induce tumors in transgenic mice. Recently our lab has found that EBNA2 could cause genomic instability. In particular, we have found that that EBNA2 could inhibit DNA repair and cause the accumulation of DNA double-strand breaks (DSBs). However, the mechanism underlying this is not clear. The aim of this study is to investigate the possible mechanism by which EBNA2 inhibits DNA repair.
Promyelocytic leukaemia nuclear bodies (PML NBs) are highly dynamic subnuclear protein structures that are disrupted or changed in morphology in response to cellular stress such as heat shock, heavy metal exposure, and DNA damage. Moreover, with respect to DNA repair, PML NBs appear to associate with several repair factors, such as ATR, Mre11, NBS1, TopBP1 and BLM, at DSB following ionizing radiation (IR). Together, these data suggest that PML NBs play a role in the repair of DSB.
In this study we discovered that EBNA2 could prevent the colocalization of Mre11 and NBS1 with PML NBs in the cells after IR. EBNA2 did not affect the colocalization of Mre11 with γH2AX foci (markers of DSB) and NBS1 phosphorylation was not altered in the IR-treated cells. Rather, EBNA2 was found to primarily cause the dislocation of PML NBs from DSB after IR. In addition, we also found that after IR, the number of PML NBs in the cells with functional EBNA2 was lower than that in the cells with non-functional EBNA2, suggesting that EBNA2 could inhibit the fission of PML NBs after IR treatment. Since the localization of PML NBs, in company with Mre11 and NBS1, to DSB has been suggested to be important for repairing of IR-induced DSB, it is possible that dislocation of PML NBs from Mre11, NBS1, and DSB by EBNA2 may lead to suppression of DSB repair. Further studies are required to confirm our hypothesis.

並列關鍵字

Epstein-Barr virus ； EBNA2 ； DNA damage ； PML NBs ； MRN complex

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