人類去氧尿嘧啶三磷酸核苷酸水解酶對細胞生長及Methotrexate抗藥性影響之探討

Functional analysis of human deoxyuridine triphosphatase in cell growth and methotrexate sensitivity

黃婷筠(Tyng-Yun Huang)

指導教授 ： 張智芬

共同指導教授 ： 張富雄(Fu-Hsiung Chang)

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.03243

摘要

去氧尿嘧啶三磷酸核苷酸水解酶（dUTPase）負責催化去氧尿苷三磷酸（dUTP）水解為去氧尿苷單磷酸（dUMP）和焦磷酸鹽（PPi）。有許多證據顯示去氧尿嘧啶三磷酸核苷酸水解酶（dUTPase）的表現量和腫瘤形成以及化療抗藥性有關連。本篇研究的目的在於了解去氧尿嘧啶三磷酸核苷酸水解酶（dUTPase）在細胞週期進行時的調控及其所扮演的必要角色。
在本篇研究中發現去氧尿嘧啶三磷酸核苷酸水解酶（dUTPase）的表現量隨著細胞週期改變，在S phase時大量表現。在293T細胞中抑制去氧尿嘧啶三磷酸核苷酸水解酶（dUTPase）的表現，會稍微減緩細胞生長速度。利用aphidicolin進行同步化(synchronization)後，進一步發現抑制去氧尿嘧啶三磷酸核苷酸水解酶（dUTPase）表現不但會影響細胞週期的進行，在細胞週期經過S phase時還會有去氧核糖核酸（DNA）損傷的訊號產生，並引發細胞凋亡（apoptosis），顯示去氧尿嘧啶三磷酸核苷酸水解酶（dUTPase）對複製壓力(replication stress)反應的重要性。
胸苷酸（dTMP）代謝一直是癌症化療應用的重要作用點。像是5-氟尿嘧啶（5-FU）或葉酸拮抗劑（antifolate）。抑制胸苷三磷酸新生成(de novo dTTP synthesis)的途徑會降低細胞內胸苷三磷酸（dTTP）含量，並導致去氧尿苷單磷酸（dUMP）累積，進一步磷酸化為去氧尿苷三磷酸（dUTP）。dUTP/dTTP比例的增加會導致去氧尿苷三磷酸（dUTP）誤嵌入DNA，引發DNA損傷反應。因此提出去氧尿嘧啶三磷酸核苷酸水解酶（dUTPase）的表現量是否會影響細胞對滅殺除癌錠(methotrexate)的感受性。結果顯示在293T細胞中降低去氧尿嘧啶三磷酸核苷酸水解酶（dUTPase）的表現量會增加細胞對滅殺除癌錠(methotrexate)的敏感度，並且增強DNA損傷反應。另一方面，表現去氧尿嘧啶三磷酸核苷酸水解酶（dUTPase）則可減少因滅殺除癌錠(methotrexate)所引起的損傷訊號。
總結本論文，說明了去氧尿嘧啶三磷酸核苷酸水解酶（dUTPase）在細胞從複製壓力(replication stress)復原時的重要性，以及在利用抑制胸苷三磷酸新生成(de novo dTTP synthesis)途徑化療藥物治療產生抗藥性所扮演的角色。

關鍵字

去氧尿嘧啶三磷酸核苷酸水解酶滅殺除癌錠 ； 複製壓力

並列摘要

Deoxyuridine triphosphatase (dUTPase) catalyses the hydrolysis reaction of dUTP to form dUMP and pyrophosphate. Accumulating evidence has shown the association of dUTPase expression with tumor and chemotherapy resistance. The aim of my study is to understand the regulation and the essential role of dUTPase in the cell cycle progression.
I found that the expression of dUTPase is regulated in a cell cycle-dependent manner, being maximal in the S phase. Depletion of dUTPase in 293T cells reduced the cell growth rate slightly. By aphidicolin synchronization, I further found that depletion of dUTPase not only affected cell cycle progression, but also induced DNA damage and apoptosis when progression through S phase, indicating the importance of dUTPase in response to replication stress.
Thymidylate metabolism is an important target for chemotherapeutic agents such as fluoropyrimidines, and antifolates. Inhibition of de novo dTTP synthesis results in dTTP pool depletion and a subsequent accumulation of dUMP, which may then be phosphorylated to form dUTP. The increase in the dUTP/dTTP ratio induces dUTP misincorporation, resulting in DNA damage responses. Therefore, I addressed the question whether the expression level of dUTPase affects methotrexate sensitivity of cells. The results showed that knockdown of dUTPase sensitized 293T cells to methotrexate, accompanied by an enhancement of DNA damage response. In addition, enforced expression of dUTPase diminished methotrexate-induced damage signals.
Accordingly, these results elucidated the crucial role of dUTPase in cells resumed from replication stress and in resistance to anti-cancer treatment by blocking de novo dTTP synthesis.

並列關鍵字

deoxyuridine triphosphatase (dUTPase) ； methotrexate ； replication stress

參考文獻

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