探討重複表現EB病毒BGLF4激酶對於DNA損傷以及修復路徑之影響

Abstract

EB病毒 (Epstein-Barr virus) 屬於gamma皰疹病毒，感染全球超過90%的人口，並與各種淋巴瘤和上皮腫瘤有關，包括鼻咽癌以及胃癌。先前研究指出EB病毒反覆再活化和鼻咽癌的致病機制息息相關。BGLF4是一種絲胺酸/蘇胺酸-脯氨酸依賴性 (serine/threonine-proline dependent) 蛋白激酶，會藉由磷酸化許多細胞或病毒的受質來促進EB病毒的複製。在去氧羥四環素誘導型 (doxycycline-inducible) NPC-TW01細胞中重複表達BGLF4會導致核纖層 (Nuclear lamina) 形變及微核 (Micronucleus) 的形成，並促進細胞通過狹窄空間。除此之外還會使DNA損傷信號γ-H2AX顯著增加，但53BP1修復信號卻沒有出現。在本論文中發現重複表達BGLF4促進了細胞的移動能力，以及促進細胞脫離培養皿表面且還能夠再重新貼附並生長。此外，在重複表達BGLF4三次後，雖然經過96小時的修補期，仍有71%的細胞有γ-H2AX信號，約有一半的DNA損傷細胞中γ-H2AX信號較少並會伴隨著53BP1信號，另一半γ-H2AX信號較多的DNA損傷細胞則只有看到微弱的或甚至沒有53BP1信號，代表53BP1的招募受到抑制。因此近一步在免疫移漬法中檢查了DNA修復蛋白的蛋白表現量，但53BP1、RB及Rad51蛋白並沒有因為核纖層形變而被降解。在BGLF4重複表達後，RB在對於響應DNA損傷的S612位點以及和G1/S期轉化相關的S780位點的磷酸化沒有增強。此外本研究在胃癌細胞 (AGS) 中建立了去氧羥四環素誘導系統，並發現重複表達BGLF4抑制了細胞生長以及導致DNA損傷信號γ-H2AX的累積，並且有一半的DNA損傷細胞沒有看到53BP1的信號。然而DNA修復蛋白的表現量是否受到BGLF4重複表達的影響仍需近一步的探討。這兩種細胞系統將用於研究BGLF4介導的基因組不穩定對化療敏感性的可能影響，以進一步探索EBV在癌症發展或化療藥物治療中的致病機制。

Epstein-Barr virus (EBV) belongs to the gamma herpesvirus family and infects over 90% of the global population. It is associated with various lymphomas and epithelial tumors, including nasopharyngeal carcinoma (NPC) and gastric cancer (GC). Previous studies have indicated a close relationship between recurrent EBV reactivation enhanced genome instability of EBV positive NPC cells. BGLF4 is a serine/threonine-proline dependent protein kinase that promotes EBV replication by phosphorylating numerous cellular or viral substrates. Repetitive BGLF4 expression in doxycycline-inducible NPC-TW01 Tet-on cells results in nuclear lamina deformation and micronucleus formation and also enhances cell migration through confined spaces. In addition, DNA damage signal γ-H2AX increased significantly without 53BP1 foci in these cells. In this study, repetitive BGLF4 expression enhances cell motility and promotes detachment from the culture dish surface, with the cells exhibiting the ability to reattach and regrow. After 3 cycles of repetitive BGLF4 expression and an additional 96-hour recovery, 71% of cells still exhibited γ-H2AX signals. Approximately half of the DNA-damaged cells with less γ-H2AX foci accompanied by 53BP1 signals, while the other half of the DNA-damaged cells showed strong γ-H2AX signals and only weak or absent of 53BP1 detections. The protein expression levels of DNA repair proteins were examined in the nuclear fraction by immunoblotting. No degradation was observed in 53BP1, RB, and Rad51 due to nuclear lamina deformation. Phosphorylation of RB at Ser612 site, which is related to DNA damage, and Ser780 site, which is associated with the G1/S transition, were not enhanced after repetitive BGLF4 expression after a 96-hour recovery. On the other hand, a doxycycline-inducible system was established in gastric adenocarcinoma (AGS) cell. Repetitive BGLF4 expression in AGS Tet-on cells retarded cell growth, resulted in γ-H2AX accumulation, and the absence of 53BP1 signals in half of the DNA-damaged cells. Further investigation is required to determine the impact of repetitive BGLF4 expression on the expression levels of DNA repair proteins in AGS cells. These two cell systems will be used to examine possible effects of BGLF4-mediated genome instability and chemotherapy resistance to further explore EBV pathogenesis.