探討CAD在線蟲與人類癌症細胞中促進細胞凋亡的功能

Abstract

細胞凋亡(Apoptosis)是一種計畫性細胞死亡(Programmed cell death, PCD)的形式，這項過程在生物體發育過程中是非常重要的，此過程受到了很多基因嚴格的調控。我們實驗室之前係用線蟲這個模式生物做了基因篩選實驗，同時將調控計畫性細胞死亡的基因與調控細胞不對稱分裂(Asymmetric cell division)的基因突變掉，使線蟲在尾部產生額外存活的hyp8/9細胞，形成了「tail defect」的表現型。利用這種在調控細胞不對稱分裂的基因突變背景下，找出了183可能調控計畫性細胞死亡的突變，tp12是其中一個突變，tp12是在pyr-1基因上的突變，其在哺乳動物中的同源基因為cad，是合成嘧啶(pyrimidine)的重要基因。我們發現在pyr-1的突變在grp-1突變的情況下造成了額外的hyp8/9細胞與分泌細胞的存活，並且其單獨的突變造成了死細胞(cell corpse)數目下降了。此外在pyr-1; grp-1的線蟲中額外補充尿嘧啶也無法使tail defect恢復成野生型的樣子，這訴說著pyr-1的突變所造成額外細胞存活的情形與其調控嘧啶合成的功能無關。在我的研究結果中發現當pyr-1與另一個調控細胞不對稱分裂的基因pig-1同時突變時也會產生tail defect的表現型，這更證明了pyr-1是一個調控細胞凋亡的基因。此外，雖然有文獻指出哺乳動物中mTOR會調控CAD的活性，但是我的研究中在pyr-1(tp12)的線蟲中使mTOR表現量減少並不會影響死細胞的數量也不會影響tail defect，表示pyr-1調控細胞凋亡不需要透過mTOR路徑。更重要的是在staurosporine處理以及用紫外光照射誘導細胞凋亡的形況下使人類子宮頸癌細胞(HeLa)中CAD的表現量下降細胞的死亡率會下降。此外我們利用細胞螢光染色的方式觀察到全長的CAD均勻散佈在細胞質中，截切之後的CAD雖然也是表現在細胞質，但其呈現的是點狀的分布型態，且這樣的分布形式在線蟲中也同樣存在。透過上述的研究結果我們發現PYR-1/CAD除了具有能夠調控嘧啶合成過程的功能外還同時具有能夠促進細胞凋亡的功能，且這項功能在線蟲與人類細胞株間高度保留。

Apoptosis, a form of programmed cell death (PCD), is a fundamental process during development and is tightly regulated by numerous genes. Previously, based on the observation that blockage of both PCD and asymmetric cell division (ACD) caused extra hyp8/9 hypodermal cell(s) and resulted in a tail defect phenotype, an enhancer genetic screen was conducted in a ACD-defective (grp-1 mutant) background to identify new PCD genes. 183 mutants were isolated from this screen and one was PYR-1, the C. elegans homolog of mammalian CAD which is essential for pyrimidine synthesis. Mutations in pyr-1 caused extra hyp8/9 and excretory cell(s) in the grp-1 mutant background and also caused a reduced number of cell corpses alone. Besides the tail defect in pyr-1; grp-1 could not be rescued by uracil supplement, indicating that pyr-1 regulated the survival of hyp8/9 cell in addition to its enzymatic activity of pyrimidine synthesis. In this thesis, we find that the pyr-1(tp12) mutant, like other PCD-defective mutants, also causes tail defects in the pig-1 mutant background, further supporting that pyr-1 is a ced (cell death abnormal) gene. Furthermore, although CAD interacts with mTOR in mammalian cell lines, knockdown of the mTOR pathway has no effect on PCD-defective phenotypes in the pyr-1 mutant, indicating that pyr-1 does not act through the mTOR pathway to elicit its cell death-promoting activity. Most importantly, knockdown of CAD attenuates apoptosis induced by staurosporine or UV irradiation in HeLa cells. Interestingly, full-length CAD is detected in cytoplasm diffusely, whereas truncated CAD shows cytoplasmic puncta. These observations are consistent with the expression of PYR-1 and truncated PYR-1 in C. elegans. Our findings indicate a conserved pro-apoptotic role and mechanism of PYR-1/CAD in C. elegans and humans; through investigating the pro-apoptotic genes may help to understand the potential reason which caused the diseases raised from dysregulation of apoptosis and may provide a therapeutic strategy for these diseases.