順鉑(Cisplatin)，是一個重要的化療藥物之一，廣泛的用於卵巢癌、子宮頸癌、頭頸癌、以及其他肺部相關癌症。其作用機制為對癌症細胞的DNA產生股內與股間的交聯(interstrand and intrastrand crosslinks)，造成DNA複製停滯，最終導致DNA雙股斷裂，藉此達到殺死癌細胞的效用。然而，目前臨床上的化學治療遇到了一大關卡，就是抗藥性的產生。我們先前的研究指出，癌細胞對於順鉑抗藥性的產生與范可尼貧血(Fanconi anemia pathway, FA)、後複製修復(Post-replication repair, PRR)與同源重組(Homologus recombination, HR)修復路徑的增強有關，因此，我們要系統化的去探討癌症細胞是增加了哪些基因突變來促進抗藥性的生成。本篇研究由生物資訊分析的方向切入，利用次世代定序去分析未具抗藥性鼻咽癌細胞株(HONE1)及抗藥性鼻咽癌細胞株(HONE6和HONE15) 之間的基因表現差異以及突變情形，我們發現了在HONE6和HONE15中TP53 mRNA的表現比HONE1多出了兩倍之多，並且在TP53的DNA-binding domain找到了一個獨特的突變位點，初步認為可能是導致抗藥性產生的原因之一。為了證實這個結果，我們降解 (Knockdown) HONE6中的TP53基因表現，發現HONE6會對順鉑的抗藥性降低，以及一些FA與HR相關路徑的mRNA和蛋白表現量會降低，甚至在姐妹染色體互換率也有大幅下降的情形，但值得注意的是，我們發現在抗藥性鼻咽癌細胞中將TP53剔除後，並沒有對其它DNA損傷藥物(例:Methyl methanesulfonate , MMS)產生敏感性，且在DNA fiber中我們發現了TP53缺失的細胞，擁有更容易跨越DNA複製障礙並持續複製的能力。
綜合以上的結果推測，TP53在FA的修復路徑扮演著重要的角色，它可以調控其相關基因的表現，使癌細胞對順鉑的抗藥性提升。而在跨越DNA複製障礙的後複製修復路徑(Post-replication repair, PRR)與TP53可能無相關，後續機制得需要更深入的了解與探討才能得知。

Cisplatin, which is one of the important chemotherapeutic drugs, is widely used to treat in ovarian cancer, cervical cancer, head and neck cancer, and other lung related cancer. However, drug resistance is the major obstacle for the efficacy of the clinical chemotherapy. Our previous studies indicated that the drug resistance of cancer cells to cisplatin is related to the enhancement of Fanconi anemia pathway (FA), post-replication repair (PRR), and homologus recombination (HR). Therefore, we want to systematic explore the consequence of drug resistance is due to which gene mutations in cancer cells. This study is based on bioinformation analysis by using next-generation sequencing to analyze the differences in gene expression and mutations between the cisplatin-resistant nasopharyngeal carcinoma (NPC) cell lines (HONE6 and HONE15) and their parental cisplatin sensitive cell line HONE1. We found that the level of TP53 mRNA expression in HONE6 and HONE15 is two times more than the level in HONE1. Additionally, we also found a unique mutation in the DNA-binding domain of TP53, R280T. We hypothesized that the overexpressed R280T mutant could be one of the reasons that lead to drug resistance. Indeed, the depletion of R280T sensitizes HONE6 cells to cisplatin and also reduces the expression level of FANCD2 and BRCA1 in the FA and HR pathways. Consistent with these results, the depletion of the R280T mutant significantly reduces the frequency of sister chromatin exchange. Methyl methanesulfonate (MMS) can cause purine methylation while ultraviolet (UV) irradiation can cause thymine dimers. Both MMS and UV are commonly used to test in the PRR pathway. Interestingly, the depletion of the R280T mutant did not further sensitize HONE6 cells to MMS, when compared to the R280T-proficient control cells. The depletion of R280T did not further reduce replication tracks and increases the numbers of stalled forks in response to the treatment of MMS or UV, when compared to the R280T proficient control cells. Our preliminary results indicate that TP53 plays an important role in the FA repair pathway, but not in the PRR pathway. Further investigations will be conducted to clarify the role of TP53 in these distinct repair pathways.

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