| Abstract: | Antizyme是細胞內調控鳥胺酸去羧化（Ornithine decarboxylase; ODC, EC 4.1.1.17）活性與多胺（polyamines）運送有關的物質。其中之一的Antizyme-1，執行各種細胞內分子功能的各種角色，包括發育的調控、細胞週期、細胞生長與死亡、細胞分化與腫瘤增生。但是，它參與細胞計畫性的死亡機制仍然是未明。為了說明人類Antizyme-1在血液細胞死亡機制，我們研究Antizyme過度表現加強計畫性細胞凋亡。Antizyme在人類前骨髓白血球癌母細胞（Human promyelocytic leukemia HL-60 cell）、人類急性白血病T細胞（Jurkat T cell）與老鼠巨噬細胞（RAW 264.7 cell）的存活率呈現濃度與時間相關性的減少。藉由細胞核濃縮、去氧核醣核酸（DNA）片段化、細胞週期G1前期（sub-G1）上升、粒腺體膜電位的損失、粒腺體細胞色素c釋出至細胞質與凋亡蛋白酵素9（caspase 9）與凋亡蛋白酵素3（caspase 3）的活化。經由誘導方式過度表現Antizyme的細胞在進入到計畫性細胞死亡之前，所有cyclin相關性激（cyclin-dependent kinases；Cdks)與cyclins的蛋白質表現量，除了cyclin D以外並沒有顯著性的減少。然而導入cyclin D1至Jurkat T tetracycline (Tet)-On cell系統使JK-Tet-On細胞過度表現cyclinD1，卻仍然不能挽救進入凋亡期的細胞。Antizyme不影響腫瘤抑制基因p53分子與其下游基因p21分子的表現，但是Antizyme卻會干擾Bcl-2家族的表現。誘導大量的Antizyme進入到粒腺體的結果，造成細胞色素c從粒腺體釋放出至細胞質並且伴隨Bcl-xL的減少與Bax增加。根據這些結果，我們證實Antizyme引起的細胞凋亡主要是經由粒腺體與不相關細胞週期的路徑。進而，Antizyme引起的細胞凋亡不僅藉由Bax的堆積、Bcl-2家族的減少、破壞粒腺體膜電位、釋出細胞色素c至細胞質並且使得凋亡體的Casepases連鎖活化。
Antizymes delicately regulate ornithine decarboxylase (ODC) enzyme activity and polyamine transport. One member of the family, antizyme-1, plays the vital roles in molecular and cellular functions, including developmental regulation, cell cycle, proliferation, cell death, differentiation and tumorigenesis. However, how does it participate in the cell apoptotic mechanism is still unidentified. To elucidate the contribution of human antizyme-1 in haematopoietic cell death, we examine whether inducible overexpression of antizyme enhances the apoptotic cell death. Antizyme reduced the viability in a dose- and time-dependent manner of human leukemia HL-60 cells, acute T leukemia Jurkat cells and mouse macrophage RAW 264.7 cells. The apoptosis-inducing activities were determined by nuclear condensation, DNA fragmentation, sub-G1 appearance, loss of mitochondrial membrane potential (Δym), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Following conditional antizyme overexpression, all protein levels of cyclin-dependent kinases (Cdks) and cyclins are not reduced except cyclin D significantly before their entrance into apoptotic cell death. However, introduced cyclin D1 into Jurkat T tetracycline (Tet)-On cell system still couldn’t rescue cells from apoptosis. Antizyme doesn’t influence the expression of tumor suppressor p53 and its downstream p21, but it interferes in the expressions of Bcl-2 family. Inducible antizyme largely enters mitochondria resulting in cytochrome c release from mitochondria to cytosol following Bcl-xL decrease and Bax increase. According to these data, we suggest that antizyme induces apoptosis mainly through mitochondria-mediated and cell cycle-independent pathway. Furthermore, antizyme induces apoptosis not only by Bax accumulation to decline the function of Bcl-2 family, destroy the Δym, release cytochrome c to cytoplasm but also by the activation of apoptosomal caspase cascade. |
