| Abstract: | 鳥胺酸去羧化（Ornithine decarboxylase, ODC, EC 4.1.1.17）催化鳥胺酸（L-ornithine）成為CO2及腐胺（putrescine），為製造多胺酸（polyamines）生合成的速率決定步驟之酵素（rate-limiting enzyme）。多胺酸包括腐胺，亞精胺（spermidine）和精胺（spermine），能幫助單股DNA結構之穩定，進而幫助DNA複製，並具有調控細胞發育、分化、轉形、生長和凋亡等功能。若在細胞內抑制鳥胺酸去羧化的活性將導致多胺酸合成失去平衡，而使細胞內活性氧化合物（reactive oxygen species; ROS）增加，進而造成細胞凋亡（apoptosis）的進行，但對於鳥胺酸去羧化對抗細胞凋亡之機制仍未十分瞭解。本研究將鳥胺酸去羧化的基因轉染入人類前骨髓白血球癌母細胞株（human promyelocytic leukemia HL-60 cell）及人類淋巴癌母細胞株（Jurkat T lymphoblast），使鳥胺酸去羧化高度表現（WT-ODC），發現能夠延遲腫瘤壞死因子a（TNF-a）所引起之細胞凋亡反應而加入鳥胺酸去羧化產物putrescine有類似效果。相反地，若加入鳥胺酸去羧化之不可逆抑制劑DL-alpha-difluoromethylornithine（DFMO）或是將鳥胺酸去羧化活化區域突變使之失去活性（DN-ODC），則會降低其抗細胞凋亡之能力；另一方面，在TNF-a引起細胞凋亡的過程中，同時也觀察到細胞內活性氧的增加及粒線體膜電位下降的情形，與粒線體相關之抗細胞凋亡蛋白Bcl-2與Bcl-XL在處理TNF-a後之表現下降，而cytochrome c釋放至細胞質的量增加，但在轉染鳥胺酸去羧化之細胞中Bcl-2與Bcl-XL表現在處理TNF-a後較其他細胞高，cytochrome c釋放至細胞質的量較少，而下游之劊蛋白群活化隨之受到抑制。此外，在轉染bcl-2基因後同樣觀察到具有對抗細胞凋亡及維持粒線體膜電位之功能，但在清除ROS堆積之能力上和鳥胺酸去羧化比較，其時間點有所不同。
Ornithine decarboxylase (EC 4. 1. 1. 17; ODC), a key enzyme for the synthesis of polyamines, decarboxylates L-ornithine to form putrescine, plays an important role in diverse biological functions including cell development, differentiation, transformation, growth and apoptosis. Repressed ODC could cause cell arrest and induce apoptosis, but the mechanism of anti-apoptosis remains poorly understood. In the present report we first investigated the anti-apoptotic capability of ODC on TNF-a induced apoptosis. TNF-a was able to induce apoptosis on HL-60 and Jurkat cells, and this signal was mediated generation of reactive oxygen species (ROS), disruption of mitochondrial membrane potential (MMP) and activation of caspase cascade including caspase 8, 9 and 3. Use ROS scavengers like N-acetylcysteine (NAC), glutathione (GSH), catalase or ODC’s product putrescine could efficiently attenuated TNF-a-induced apoptosis. To explore the physiological function of ODC in anti-apoptotic process, we generated a cell line embracing an overexpression ODC gene (WT-ODC). Overexpression ODC cells could prevent TNF-a-induced apoptosis but dominant-negative ODC (DN-ODC) not. On the other hand, the suicide inhibitor of ODC, alpha-difluoro-methylornithine (DFMO) was significant decrease ODC’s anti-apoptosis function. The anti-apoptotic mechanism was through reduced the generation of ROS, maintained the potential of mitochondrial membrane and repressed caspase cascade activation. From our data presented, we proposed that ODC was required to against cell death when exposed on TNF-a induced apoptosis, and it also repressed the activation of TNF-a death signal and maintained the mitochondrial membrane potential. At the same time, the famous anti-apoptosis protein Bcl-2 overexpression cells had similar protective function from TNF-a-induced apoptosis. Compared with ODC-overexpression cells, the mechanism of decrease intracellular ROS generation is different. Further, our findings suggested that Bcl-2 and Bcl-XL was precipitated the regulation of TNF-a induced apoptosis and ODC could efficiently maintain their expressions to increase their anti-apoptosis function. |
