Abstract
摘要:細胞凋亡為自然生物界裡一種去除已被嚴重傷害之細胞的死亡機制,其在演化上具有高度保留性,且對於正常胚胎發育及維持組織恆定性非常重要。此機制的失調會導致許多疾病,包括組織形態及發生的異常、癌症、自體免疫疾病及神經退化疾病。是以,深入徹底瞭解細胞凋亡的調控機制可望帶來一些疾病治療的突破方法及契機。目前已發現許多調控細胞凋亡機制之分子,其中,BCL-2 家族分子在調控以粒腺體為主之細胞凋亡過程中扮演了極重要的角色。我們近期發現了一個新的BCL-2家族分子,將之命名為Blm-s(Bcl-2likemolecule,shorttranscriptisoform)。我們的研究結果顯示BLM-s在經由DNA 雙股斷裂的不可逆刺激下其RNA 的表現會經由ATM/p53 及JNK/AP1 訊息傳導路線被誘發,且BLM-s 蛋白質通常會跑到粒腺體或內質網,並經由粒腺體釋放cytochromec 的路徑來促進細胞凋亡。基於腫瘤細胞常常利用BCL-2 家族分子發展出各種對抗細胞凋亡的機制,我們初步以免疫組織染色方法篩選BLM-s 在人類腫瘤組織列陣片的表現情形。出乎我們的意料,我們發現致死分子BLM-s 特殊的表現在唾液組織的惡性腺性瘤; 而且,BLM-s 不在其正常該有的粒腺體或內質網,反而進入腫瘤細胞核中。另外,我們亦發現人類胚胎發育時期的腦神經細胞會因某種未知的細胞生理壓力驅使細胞核表現BLM-s。據此,我們推測BLM-s 進入細胞核與細胞生理或生存壓力具有某種程度的因果關係。此外,BLM-s 蛋白質結構分析顯示其並不擁有入核的訊息,是以我們猜想BLM-s 可能需要藉由與其他分子交互作用來導致其進入細胞核。為了尋找BLM-s 的作用分子,我們運用酵母菌二維雜交系統找出數十個BLM-s 作用分子,其中,具有修復DNA 雙股斷裂的Ku70 引起我們的特別注意。初步實驗結果顯示同時表現BLM-s 和Ku70 在細胞中可導致BLM-s 與Ku70 共同入核,並保護細胞免於因過度表現致死分子BLM-s 所引發的細胞凋亡。據此,我們提出我們的假設∶Ku70 藉由與BLM-s 形成蛋白複合體導致BLM-s 進入細胞核來拮抗其細胞凋亡之機制作用。為了驗證我們的假設,我們特別針對幾個相關問題提出數個實驗方法來解決回答。我們欲找出Ku70 如何調控BLM-s 入核的分子與細胞生物機制(Specificaim-1); 同時入核的BLM-s/Ku70 蛋白複合體是否影響BLM-s 與Ku70 各自調控的生物功能? (Specificaim-2) ;細胞是遭受什麼樣的細胞生理或生存壓力及其訊息傳導路徑導致BLM-s 進入細胞核?(Specificaim-3)。另外,為了轉譯醫學的應用,我們提出建立以BLM-s 免疫組織核染色表現來輔助診斷唾液組織的惡性腺性瘤(Specificaim-4)。
Abstract: Apoptosis is a natural program of removing unwanted or damaged cells and is thus crucial fornormal embryo development and maintenance of tissue homeostasis. Dysregulation of apoptosisresults in multiple diseases including morphogenetic abnormalities, cancer, autoimmune diseaseand neurodegenerative disorders. Thus, understanding the molecular regulation of apoptosis hasgreat therapeutic implications. Many components of the death machinery have been identified,among which the BCL-2 family members play a pivotal role as a mitochondria gate-keeper forensuing cell death. We have identified and published a novel apoptotic gene, Blm-s (Bcl-2 likemolecule, short transcript isoform), which transcript is upregulated by DSBs transduced byATM/p53 and JNK/AP1 signaling pathway. BLM-s normally locates at mitochondria and ER, andmediates apoptosis via mitochondrial cytochrome c release. Recently, by tumor tissue arrayscreening, we unexpectedly uncovered a specific association of nuclear relocation of BLM-sprotein and a salivary grand tumor. Preliminary studies revealed that human fetal cortical neuronsalso express nuclear BLM-s and are non-apoptotic under stress condition. These finding indicatesa significant correlation of nuclear BLM-s with several pathological conditions such as stress andtumor. However, due to lack of the nuclear localization signal in BLM-s, BLM-s is hypothesized tointeract with other molecules for shuttling from the mitochondria/ER into the nucleus. To search forBLM-s interacting molecules, we have performed yeast-two-hybrid screening and identified Ku70,an evolutionary conserved gene involved mainly in the NHEJ pathway for DSBs repair, formingprotein complex with BLM-s. Cells co-expressing BLM-s and Ku70 reveals nuclear co-localizationof both molecules and BLM-s-mediated apoptosis is suppressed by Ku70 overexpression.Accordingly, we hypothesize that Ku70 effects the inactivation of BLM-s-mediated apoptosis vianuclear localization of BLM-s. We aim to investigate the molecular and cellular basis for BLM-snuclear relocation. Specifically, we ask how Ku70 regulates BLM-s shuttling into the nucleus(Specific aim-1) and whether nuclear BLM-s/Ku70 protein complex affects the biological processesmodulated by each respective molecule (Specific aim-2). We will identify the specific cell stresscondition and its transducing signaling pathway to effect nuclear localization of BLM-s (Specificaim-3). For the translational research, we will establish BLM-s immunohistochemistry as anadjuvant diagnostic tool for salivary adenoid cystic carcinoma (Specific aim-4).
Keyword(s)
BLM-s
Ku70
細胞凋亡拮抗
細胞核之進出
細胞生理/生存壓力
BLM-s
Ku70
inactivation of apoptosis
nuclear localization
cell stress