疱疹病毒Bcl2同源分子壓制核苷酸剪切修復能力以及干擾錯誤配對辨識程序

Abstract

許多疱疹病毒和癌症的發生有密切關聯性，例如EB 病毒與淋巴瘤、鼻咽癌、胃癌等多種癌症有關，另卡波西氏肉瘤疱疹病毒(KSHV)會造成卡波西氏肉瘤的發生。 癌症發生之時往往會伴隨著大量的基因體不安定性。本實驗室先前發現某些EB病毒蛋白質表現會造成基因體不安定的情形，而在KSHV也已經被證實其潛伏期蛋白表現也會造成基因體的不穩定性。依據以上結果而讓我們對於這兩個人類致癌性疱疹病毒在溶解期所表現的其他蛋白質是否會造成基因體不安定性有著高度的興趣，這是一個過去從未被探討過的領域。 我們實驗室經初步的實驗觀察EB病毒所轉譯出的Bcl2同源分子BHRF1有可能會促進基因體的不安定性，因而選擇在疱疹病毒中EB病毒及KSHV在溶解期所轉譯出的Bcl-2同源分子(EBV的BHRF1,BALF1以及KSHV的KsBcl2)進行基因體不安定性的研究，透過和宿主細胞的交互作用來了解其造成基因體不安定性的可能原因。在本實驗中，我們先行構築了可以表現Bcl2及其同源分子的表現質體，將其轉染至H1299細胞，並證實此組蛋白質表現質體可以在H1299細胞中大量表現。Bcl2及其同源分子可以增加細胞對於DNA損傷藥物的耐受力。進ㄧ步利用宿主細胞再活化實驗，針對核苷酸修復系統也發現H1299細胞表現Bcl2及其同源分子時會降低細胞對於紫外線造成的DNA缺損的修復力。也針對錯誤配對修復系統進行研究。利用ELISA的方法在僅轉染Bcl2及其同源分子表現質體以及在轉染之後暴露在DNA損傷的藥物的情形下，發現會降低錯誤配對辨識的能力。並且利用西方墨點法監測H1299細胞在轉染Bcl2以及病毒同源分子之後暴露在DNA損傷藥物的環境下，發現Bcl2，BHRF1表現會使得參與錯誤配對系統的hMsh2以及hMsh6的蛋白質表現有下降的情形推測因此而促使其錯誤配對修復辨識能力下降：基於以上結果我們認為EB病毒的病毒蛋白BHRF1以及卡波西氏肉瘤疱疹病毒的KsBcl2均會抑制DNA修復機制，進而導致基因體的穩定性遭受破壞，可能進而促進細胞的癌化。

Several herpesviruses are associated with cancer formation. Two human oncogenic herpesviruses, namely Epstein-Barr virus (EBV) and Kaposi’s Sarcoma Associated Herpesvirus (KSHV), have been linked to a number of malignancies including lymphomas, nasopharyngeal carcinoma, gastric carcinoma, and Kaposi’s sarcoma. The hallmark of tumor cells is genomic instability. Our previous finding provides an insight about the role of EBV viral proteins in DNA repair suppression to promote genomic instability. Studies carried out in other laboratories also demonstrated that infection of KSHV increased genomic instability. Based on these findings, we are interested in identifying EBV and KSHV encoded viral proteins that can lead to genomic instability. EBV and KSHV carry Bcl2 homologues and whether these homologues supress DNA repair activity has not been elucidated yet. Preliminary data in our laboratory indicate BHRF1 may promote genomic instability. By this finding, we have been interested in illustrating how viral Bcl2 homologues cause genomic instability. We chose BHRF1, BALF1 (Bcl2 homologues of EBV), and KsBcl2 (Bcl2 homologue of KSHV) to further study their activity in promotion of genomic instability. First, we constructed Bcl2 and viral Bcl2 homologues expression plasmids and checked their expressions in H1299 cell. Bcl2 and Viral Bcl2 homologues expressed in H1299 cell correctly by recombinant plasmids constructed. Bcl2 and viral Bcl2 expressions in H1299 prevent cell death by MNNG treatment. We monitored nucleotide excision repair efficiency by host cell reactivation assay. Results showed that viral Bcl2 homologues over-expressed in H1299 cell attenuated the capacity of UV-induced DNA repair. In the other repair process, we focused on mismatch repair (MMR). Using ELISA assay to investigate MutSα activity. We revealed that Bcl2 and viral Bcl2 homologues (BHRF1 and BALF1 in EBV, KsBcL2 in KSHV) potently suppressed MMR in association with decreased MutS alpha activity through the down-regulation of Msh2 and Msh6 expression in protein level. Based on these results, viral Bcl2 homologues may potentially contribute to the induction of genetic instability and subsequent carcinogenesis.