單磷酸尿苷缺乏造成線蟲細胞不正常存活之研究

Abstract

計畫性細胞凋亡在生物的發育上扮演著重要的角色。在線蟲當中，主要有四個核心基因執行細胞凋亡，分別為: egl-1, ced-9, ced-4 以及 ced-3。 線蟲細胞凋亡的基因與人類的細胞凋亡基因具有高度保留性，到目前為止，我們對調控細胞凋亡仍所知有限，例如特定的細胞是否有不同的基因調控其凋亡。 在我們實驗室先前的研究中發現了，在不對稱分裂基因 grp-1 的突變株中，如果細胞凋亡核心基因同時發生變異，則會高機率在尾部產生多餘的表皮細胞 hyp8/9，並且會導致線蟲的尾部產生球狀突起，我們把這個性狀稱之為:「突出尾巴」 (bulged tail)。在grp-1 突變株的背景下，我們進行了基因篩選，來找出新的可以調控細胞凋亡的基因。我們發現新的可以調控細胞凋亡的基因:pyr-1，也就是人類的 CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) 同源基因，PYR-1 為一嘧啶合成酶，負責催化嘧啶生合成途徑的前三個步驟，而嘧啶在生物體內中參與了許多重要的生理反應，例如:DNA 或RNA 的合成；磷脂質及尿苷二磷酸葡萄糖(UDP-sugar)的合成。在此篇研究中，我們進一步證明了在嘧啶合成途徑中，pyr-1 下游基因dhod-1 和 umps-1 同樣是進行細胞凋亡的必要基因，然而，在umps-1之後的嘧啶合成基因並不是細胞凋亡所需要的基因。此外，藉由額外的嘧啶補充劑及調控其在線蟲體內的代謝，我們發現UMP含量是造成hyp8/9細胞凋亡的成因。 更重要的是，在人類的子宮頸癌細胞株HeLa中抑制CAD 的表現同樣的保護了HeLa 細胞免於紫外線照射誘發的細胞凋亡。 總結來說，我們的實驗結果證實了 UMP可以引起細胞凋亡並且指出UMP引起細胞凋亡的能力可能在不同生物間是共有的。

Programmed cell death (PCD) is important for animal development. In Caenorhabditis elegans (C. elegans), the execution of PCD is controlled by four genes, egl-1, ced-9, ced-4, and ced-3, and this core PCD pathway is highly conserved from nematodes to humans. Despite of extensive studies in PCD, how different cells may be differentially regulated to undergo PCDs is not yet clear. Previous studies in our lab found that mutations in the core PCD pathway caused extra hypodermal hyp8/9 cells in a sensitized mutant background and resulted in a bulged tail phenotype. In a sensitized genetic screen, a pyr-1 mutant was isolated with a bulged tail. pyr-1 is an ortholog of human CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) that catalyzes the first three steps of the de novo pyrimidine synthesis pathway. This pyrimidine synthesis pathway is important for DNA and RNA synthesis as well as the formation of phospholipids and UDP-sugars. In this thesis, we show that like pyr-1, dhod-1 and umps-1, two genes acting downstream of pyr-1 in the pyrimidine synthesis pathway, are required for normal PCD. Using genetic manipulation and metabolite supplementation, we show that the appropriate level of UMP, the end product produced by DHOD-1 and UMPS-1, is necessary for the cells to undergo PCD normally in the hyp8/9 lineage. Similar to the pro-apoptotic activity of pyr-1, knockdown of CAD in HeLa cells by RNA interference protects the cells from death under the UV treatment. These data show an unexpected role of UMP in regulation of programmed cell death and suggest evolutionary conservation of the pro-apoptotic function of UMP in C. elegans and humans.