DNA修補基因XPF於人類非黑色素皮膚癌表現與非小細胞肺癌對Cisplatin抗藥性角色探討

Investigation of XPF Gene Expression in Nonmelanoma Skin Cancers and the Role in Cisplatin-Resistance of Nonsmall Cell Lung Cancers

張立青(Li-Ching Chang)

指導教授 ： 黃裕勝 ； 郭國華

高雄醫學大學/醫學院/醫學研究所/博士(2003年)

摘要

DNA修補(DNA repair)在人類基因體完整性的維持與癌症生成預防上扮演著關鍵性的角色，修補能力降低已證實和許多基因異常而引起的癌症罹患有關。核苷酸切除修補(nucleotide excision repair, NER)是一個非常複雜而精緻的DNA修補機制，其負責修復大部分化學性DNA損害並移除許多和結構無關的DNA損傷。
非黑色素細胞皮膚癌(nonmelanoma skin cancers, NMSC)包括：基底細胞癌(basal cell carcinoma, BCC)及鱗狀細胞癌(squamous cell carcinoma, SCC)都曾被證實有降低的核苷酸切除修補(NER)活性。然而，究竟是核苷酸切除修補(NER)那個環節出現問題，目前仍未能確知。Xeroderma pigmentosum F (XPF)是核苷酸切除修補(NER)過程中一個不可或缺的關鍵因子，其功能在修補受損DNA時會切除受損區域的5'位置以利受損段DNA移除。DNA修補基因適當的基因表現對於正常DNA修補活性作用是非常重要的。目前為止，XPF的蛋白質或抗體仍難以購得。為了探索XPF基因在非黑色素細胞皮膚癌(NMSC)生成中的角色，本實驗設計並利用競爭性定量逆轉錄-聚合酶鏈反應(quantitative reverse transcription-polymerase chain reaction)來檢測NMSC內XPF基因的表現有否異常。
將XPF完整編碼序列應用於DNASIS®分析軟體，藉由模擬最低能量的結構運算可預測出XPF mRNA可能之構形。利用結構上開放的空間設計聚合酶鏈反應所需的引子(primers)位置。透過nested PCR分析，所有設計出的引子都能高特異性地結合並放大XPF cDNA片段。於XPF引子組合中挑選一組放大效率最佳的引子組，利用此放大片段合併?-Bungarotoxin (?-Butx)構築非同源性競爭性核苷酸片段以作為內部標準。此非同源性競爭性核苷酸片段能為XPF引子所放大，因此可以在非黑色素細胞皮膚癌(NMSC)中更準確定量XPF的基因表現。
結果顯示非黑色素細胞皮膚癌(NMSC)包括基底細胞癌(BCC)和鱗狀細胞癌(SCC)的XPF基因表現分別只有正常皮膚細胞的57.0%和76.4%左右。所以，XPF基因表現的降低可能是非黑色素細胞癌(NMSC)中核苷酸切除修補(NER)機能失常的原因之一。本實驗中針對檢測XPF基因表現所設計出可信賴且可用來定量的引子組或可作為往後癌症生成病因研究的利器。
論文的第二部分探討XPF基因於非小細胞肺癌(nonsmall cell lung cancer, NSCLC)對抗癌藥物Cisplatin產生抗藥性之角色扮演。Cisplatin是一個會破壞DNA的物質，不管是單一藥物或合併其他藥物它都能廣泛運用在許多癌症的治療上，其中包括非小細胞肺癌(NSCLC)。然而，非小細胞肺癌被發現比小細胞肺癌對Cisplatin出現更顯著的抗藥性。由於哺乳類細胞移除Cisplatin所引起之DNA結合物乃透過核苷酸切除修補(NER)來進行修復，所以推論NER核心組成之一的XPF基因或是整個NER系統可能和非小細胞肺癌對Cisplatin出現抗藥性有關。
首先利用MTS分析方法評估Cisplatin對非小細胞肺癌細胞株包括H441、H520、A549、H661及H596細胞之毒性，結果顯示肺腺鱗狀癌H596細胞對Cisplatin最為敏感、生長被抑制的情形最為嚴重；其他細胞株則對Cisplatin呈現抗藥性。透過作用時間軸線的分析，Cisplatin在1至4小時內即可進入細胞執行其細胞毒殺的作用，各細胞株間Cisplatin進入的速度無明顯差異。利用原子吸收光譜(atomic absorption spectrometry, AAS)分析鉑(Platinum, Pt)含量，可檢測Cisplatin進入細胞並結合上DNA之Platinum (Pt)-DNA結合物濃度。H596細胞內Pt-DNA結合物的濃度最高，其次是H661，而H441、H520及A549細胞中Pt-DNA的含量則明顯較少。綜合上述結果顯示，Cisplatin在非小細胞肺癌的毒性取決於Cisplatin結合至DNA的含量。
為探討XPF基因在人類非小細胞肺癌對Cisplatin產生抗藥性的角色，本實驗透過投與XPF反義寡核苷酸(antisense oligodeoxynucleotides, ODNs)阻斷XPF蛋白質作用來評估XPF在非小細胞肺癌對Cisplatin感受性的影響。結果顯示XPF反義寡核苷酸加成Cisplatin細胞毒性的作用明顯出現在抗藥性肺乳頭狀腺癌H441及肺大細胞癌H661細胞株，在H520、A549及H596細胞則不明顯。因此，推論對Cisplatin呈抗藥性的H441及H661細胞而言，其抗藥性的成因可能與其細胞內XPF基因表現甚或整個核苷酸切除修補(NER)活性異常有關。NER的作用除了XPF外尚有其他核心因子參與，實驗利用ERCC1、XPA、RPA、XPG之反義寡核苷酸探討這些因子於非小細胞肺癌對Cisplatin敏感性的影響。結果顯示NER反義寡核苷酸和Cisplatin合併作用時，對Cisplatin抗藥性的H441及H661細胞出現細胞毒殺的加成作用。其中任一XPF、ERCC1、XPA、RPA或XPG單一反義寡核苷酸的作用對細胞死亡的影響結果都很相似，顯示NER的作用需要所有核心因子共同參與，缺少任一個因子的活性都將導致NER功能異常。
本論文證實非黑色素細胞癌(NMSC)中核苷酸切除修補(NER)之XPF基因表現異常，並探討XPF基因及NER在非小細胞肺癌H441及H661對Cisplatin抗藥性的影響。透過這些研究期望能在癌症預防及治療上提供新的策略與方向。

關鍵字

非黑色素皮膚癌 ； 非小細胞肺癌 ； DNA修補 ； 順鉑

並列摘要

DNA repair plays a crucial role in the integrity maintenance of the human genome and the prevention of tumorigenesis. Reduced DNA repair capacity has been linked to genetic susceptibility to cancer. Nucleotide excision repair (NER) is a sophisticated DNA repair mechanism that repairs a substantial amount of chemically induced DNA damage and eliminates a wide range of structurally unrelated DNA lesions.
Nonmelanoma skin cancers (NMSC), including basal cell carcinomas (BCC) and squamous cell carcinomas (SCC), have been evidenced with an impaired function in NER. However, malfunction of NER elements in NMSC has not been identified. Xeroderma pigmentosum F (XPF) is an essential subunit in NER and functions as a 5'-incision enzyme when repairing damaged DNA. An adequate expression level of DNA repair genes is essential for normal DNA repair activities. So far, neither XPF's protein nor antibody is commercially available. To explore the expression of XPF in NMSC, the gene was determined by competitive quantitative reverse transcription-polymerase chain reaction (RT-PCR).
To design feasible primers for the determination of XPF gene's expression, the complete coding sequences of XPF was folded with DNASIS® software and predicted the mRNA structure. Open regions of the mRNA folding structures were selected for primer synthesis. All the designed primers specifically amplified XPF cDNA as demonstrated by nested PCR, and one set of the primers was mimic constructed to form an internal controlled cDNA for the quantification of XPF gene in NMSC. The exogenous sequences of ?-Bungarotoxin (?-Butx) were designed to share the same XPF primers.
The results indicated that the quantities of XPF expression of BCC and SCC specimens were approximately 57.0% and 76.4% less than that of normal skins, respectively. This paper indicates that the decrease expression of XPF gene may be one of mechanisms for impaired NER in NMSC, and the feasible and quantitative primers used in the experiments may explore the study of XPF in etiology of carcinogenesis.
In the second part of this study, the role of XPF gene on cisplatin resistant of nonsmall cell lung cancer (NSCLC) was investigated. Cisplatin, a DNA damaging agent, is widely used against a broad spectrum of tumors including NSCLC either as a single agent or in combination with other active anticancer drugs. Nevertheless, NSCLC cells were found to be significantly resistant to cisplatin than small cell lung cancer (SCLC). Because mammalian cells remove cisplatin-induced DNA adducts through the nucleotide excision repair (NER) pathway, we proposed that the XPF gene or the entire NER machinery may involve in the resistant of cisplatin in NSCLC.
The cytotoxicity of cisplatin on NSCLC including H441, H520, A549, H661 and H596 cells were evaluated by MTS assay and results indicated that the proliferation of H596 cells was most significantly inhibited by cisplatin; whereas, the others were resistant to cisplatin. Through time-courses study, it is apparent that cisplatin exerts its cytotoxicity effect within 1~4 h in these NSCLC sublines. Platinum (Pt)-DNA adducts were measured by atomic absorption spectrometry (AAS) after cisplatin treatment. The amount of Pt-DNA adducts in H596 cells were significantly higher than H661, H441, H520, A549 cells. The above findings indicate that the cytotoxicity of cisplatin was dependent on the amount of Pt-DNA.
To assess the role of XPF gene in the cisplatin resistant, XPF antisense oligodeoxynucleotides (ODNs) were administrated to verify its effect on cisplatin sensitivity in NSCLC. The synergistic cytotoxicity effect in the combination of XPF antisense ODNs with cisplatin was observed in the H441 and H661 cells; however, H520, A549, and H596 cells were not. Therefore, the causes of cisplatin resistance in H441 and H661 may relate to abnormal XPF gene or NER activity. To further investigate the role of NER in the cisplatin resistant, other NER core factors were examined in this study. Other NER component antisense ODNs target to ERCC1, XPA, RPA and XPG genes were evaluated with cisplatin cotreatment in NSCLC. Results indicated that synergistic effects were observed in cisplatin resistant H441 and H661 cells. It is of interesting that single XPF, ERCC1, XPA, RPA or XPG antisense ODNs administration were resulted in similar results. Apparently, normal NER execution was rely on all core factors participation, defects in any factor were result in abnormal NER function.
This dissertation investigated that the malfunction of XPF gene may result in nonmelanoma skin cancers (NMSC) and its role on the cisplatin resistant in nonsmall cell lung cancer (NSCLC) H441 and H661 cells. By these studies, new strategies may provide in the cancer prevention and therapy.

並列關鍵字

Nonmelanoma Skin Cancers ； Nonsmall Cell Lung Cancers ； DNA repair ； Cisplatin

參考文獻

Alcalay, J., Freeman, S.E., Goldberg, L.H., and Wolf, J.E. (1990). Excision repair of pyrimidine dimers induced by simulated solar radiation in the skin of patients with basal cell carcinoma. J. Invest. Dermatol. 95, 506-509.

Aldaz, C.M., Conti, C.J., Klein-Szanto, A.J.P., and Slaga, T.J. (1987). Progressive dysplasia and aneuploidy are hallmarks of mouse skin papillomas: relevance to malignancy. Proc. Natl. Acad. Sci. USA 84, 2029-2032.

Ananthaswamy, H.N., and Pierceall, W.E. (1990). Molecular mechanisms of ultraviolet radiation carcinogenesis. Photochem. Photobiol. 52, 1119-1136.

Andrew, A.S., Karagas, M.R., and Hamilton, J.W. (2003). Decreased DNA repair gene expression among individuals exposed to arsenic in US drinking water. Int. J Cancer 104, 263-268.

Asahina, H., Kuraoka, I., Shirakawa, M., Morita, E.H., Miura, N., Miyamoto, I., Ohtsuka, E., Okada, Y., and Tanaka, K. (1994). The XPA protein is a zinc metalloprotein with an ability to recognize various kinds of DNA damage. Mutat. Res. 315, 229—237.