金屬抑制人類細胞 核酸配對錯誤修復系統之分析

Analysis of Metals Inhibiting Human Cells Mismatch Repair System

徐悅芳(Yeuh-Fang Hsu)

指導教授 ： 方偉宏

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.00874

摘要

人類的核酸錯誤配對修復系統[mismatch repair system (MMR)]將錯誤修復，以維持生物體基因的穩定。當MMR發生缺陷時，MMR的活性便會下降，使基因的突變率（mutation rate）提高，在人體甚至會引發遺傳性非息肉性大腸癌(HNPCC；hereditary nonpolyposis colorectal cancer)。
鎳被認定為人類第一型致癌因子，當人類長期暴露在含有鎳的環境下會增加罹患肺癌及鼻咽癌的機率。曾有研究指出，金屬鎳會引起人類肺癌細胞產生微衛星序列不穩定（Microstatellite instability；MSI），因此我們進一步探討鎳造成MSI現象的原因。過去在HNPCC病人上發現， MMR基因缺陷會伴隨MSI的產生，因此我們認為鎳也有可能透過抑制MMR的活性，使MSI的現象產生。所以為了證實我們的推測，利用本實驗室所建構的in vitro repair assay，評估鎳抑制人類細胞MMR修復效率之程度，並且探討其他具有致癌能力的金屬離子對修復反應的影響，結果發現修復效率會隨著鎳、鎘、鐵、鋅、銅、鉻的濃度提高而降低，且當這些金屬離子濃度在0.2-0.4mM時，會使修復效率降低為原來的50%以上。為了進一步瞭解鎳作用的目標為何，藉由在in vitro repair assay中加入鎳，再分別和具有硫氫基（sulfhydryl group，-SH）的DTT反應，其修復反應有明顯回升的情形。接著再分別加入十六種胺基酸一同參與試管內反應，結果顯示組胺酸（histidine）對於鎳的抑制具有保護效果，其次是天冬醯酸(Asparagine)有恢復八成以上的修復能力。另外，我們也分析鎳對於大型核酸環修復（loop repair）反應的影響，結果顯示其修復效率也受到鎳的抑制。綜合以上結果，鎳可能藉由抑制鹼基配對修復系統造成微衛星序列的不穩定，同時鎳可能結合至修復蛋白上的histidine造成修復活性的抑制，但鎳作用於哪一種修復蛋白，仍須進一步的研究。

關鍵字

鎳

並列摘要

DNA mismatch repair (MMR) maintains genome stability by correcting biosynthetic errors during the replication. Inactivation of MMR activity results in genetic instability, which is assumed to promote tumorigenesis. Nickel (II) is classified as a human carcinogen causing respiratory cancers. Previous studies showed that Nickel (II) induces microsatellite mutations in human lung cancer cell lines. And, DNA mismatch repair (MMR) deficiency in hereditary non-polyposis colon carcinomas (HNPCC) also demonstrating high frequency of microsatellite instability provide strong evidence for the significance of genetic instability in cancer. Therefore, we want to investigate the correlation between nickel and MMR and to understand whether nickel induced MSI by inhibiting MMR activity. To understand the inhibitory activity of nickel toward the human cells MMR pathway, we used a set of heteroduplexes in vitro repair assay with human cell extracts to study the changes of MMR efficiency upon exposure to nickel or other metal ions. The results indicated that the repair efficiency was significantly decreasing in the presence of nickel, cadmium, copper, chromate, and zinc in a dose dependent manner. Under the concentration of 0.2-0.4mM, the repair efficiency decreased to 50% activity. Furthermore, we found that Nickel inhibition was prevented by histidine and asparagine suggesting that these residues are essential for the repair protein activity target by nickel. DTT also could reverse the inhibitory effect of nickel. Our data suggested that nickel may inhibit MMR activity raises the possibility that genetic instability may be a mechanism involved in nickel carcinogenesis.

並列關鍵字

Nickel

參考文獻

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