APE1 Asp148Glu 和 hOGG1 Ser326Cys 基因多型性與台灣非小細胞肺癌發生危險性和其p53基因發生突變的相關性

Abstract

根據行政院衛生署統計資料顯示，惡性腫瘤一直是國人十大死因之首，而肺癌自民國90年開始一直是女性和男性十大癌症之第一或第二位死因。肺癌的五年存活率至今仍在15% 左右，顯示建立生物風險標記是預防與早期發現肺癌之重要工作。目前已知肺癌發生與個體遺傳基因以及與環境因子交互影響有關。抽菸會增加罹患肺癌之風險，主要是香菸中之致癌物增加p53基因突變之頻率。本研究推測移除香菸引起之DNA 氧化性傷害的APE1和 hOGG1 之基因多型性可能影響其DNA修補能力，進而影響p53發生突變之風險。因此首先收集205肺癌患者與206非癌症之控制組探討APE1 Asp148Glu和hOGG1 Ser326Cys 之基因多型性是否與罹患肺癌之風險有關? 結果發現 hOGG1 Ser/Cys+Cys/Cys 基因型者罹患肺癌之風險是Ser/Ser基因型者的1.812倍 (95% CI = 1.183-2.776, P = 0.006)。這現象同樣同樣在抽菸者 (OR, 2.036, 95% CI = 1.108-3.739, P = 0.021)、肺腺癌 (OR, 1.801, 95% CI = 1.057-3.067, P = 0.029) 和鱗狀細胞癌 (OR, 1.824, 95% CI = 1.071-3.105, P = 0.026) 可以觀察到。但在APE1 Asp184Glu則沒有發現其相關性。為了解這兩種基因多型性是否與肺腫瘤中之p53基因發生突變有關? 令人驚訝是APE1 Asp/Asp 基因型患者之p53發生突變之風險是Asp/Glu+ Glu/glu基因患者的2.15倍 (95% CI=1.19-3.87, P = 0.011)。而hOGG1 Ser326Cys之基因多型性與p53發生突變則都沒有相關。但是Asp/Asp 加上Ser/Cys+Cys/Cys之患者發生p53突變之風險是 Asp/Glu+Glu/Glu 加上Ser/Ser患者的3.72倍 (95% CI=1.33-10.40, P = 0.012)。本研究以細胞模式探討APE1 Asp/Asp基因型，是否確實有較低的DNA修補能力，而造成p53突變之風險增加? 為了瞭解APE1 Asp/Asp基因型的DNA修補能力，是否較APE1 Glu/Glu基因型為低？以宿主細胞反應分析法 (Host cell reactivation assay) 分析之結果顯示，pCDNA3.1 A(-) APE1-Asp 轉殖入TL4和 H23肺癌細胞，結果顯示移除H2O2引起之DNA傷害的能力都低於pCDNA3.1 A(-) APE1-Glu之細胞，若再合併hOGG1-Cys轉殖入TL4 細胞，則其修補能力遠低於轉殖入APE1-Glu+ hOGG1-Ser基因型之細胞。本結果是首次以細胞轉殖生物技術發現APE1 Asp/Asp基因型，具有較Glu/Glu基因型為低的DNA修補能力。此細胞模式之結果支持肺癌患者觀察到之結果，即具有APE1 Asp/Asp基因型者發生p53突變之風險，較APE1 Glu/Glu之風險為高。總之，本研究不僅以病例/對照組發現hOGG1 Ser326Cys基因多型性，可做為評估罹患肺癌風險之生物指標，同時發現APE1 Asp184Glu基因多型性與p53發生突變之風險有關。因此推測APE1 Asp148Glu基因多型性對氧化性DNA傷害之修補的影響，高於hOGG1 Ser326Cys基因多型性。

Statistic database from DOH, ROC showed that lung cancer is the first and second cause of cancer death in Taiwanese women and men, respectively. Patients with lung cancer had 15% of 5-year survival rate revealing that establishing risk markers for early detection of lung cancer is the most important strategy to reduce the death of this disease. Cigarette smoking is considered to be major cause of lung cancer and p53 mutation induced by smoke carcinogens plays a crucil role in lung cancer development. High frequency of p53 mutation occurred in patients smokers compared with never-smokers, especially those with low DNA repair activity. The polymorphisms of hOGG1 Ser326Cys and APE1 Asp184Glu have been shown to associate with lung cancer risk, however, no biological evidence to verify the possibility. In the present study, 205 lung cancer cases matched with 206 non-cancer controls to investigate whether both gene polymorphisms could be associated with lung cancer risk in Taiwanese living in Central Taiwan. Consistent with a previous report using Taiwanese population linving in North and Central Taiwan, and indicated that hOGG1 Ser/Cys+Cys/Cys carrier had 1.821-fold of lung cancer risk compared with hOGG1 Ser/Ser carrier (95% CI = 1.057-3.067, P = 0.006). However, APE1 Asp184Glu polymorphism was not associated with lung cancer risk in our study population. To understand whether both genetic polymorphisms could be associated with p53 mutation occurrence in 205 lung cancer patients, lung tumors were enrolled to determine exon 5 to exon 8 of p53 gene mutation by direct sequencing. Surprisingly, patients with APE1 Asp/Asp genotype had 2.15-fold of p53 mutation occurrence risk compared with those with APE1 Asp/Glu+Glu/Glu genotype (95% CI = 1.19-3.87, P = 0.011); however, the association of hOGG1 genotypes with p53 mutation occurrence risk was not observed in lung cancer patients. More interestingly, the risk for p53 mutation occurrence increased to 3.72 fold in patients with APE1 Asp/Asp combined with hOGG1 Ser/Cys+Cys/Cys genotypes when compared with those with APE1 Glu/Glu combined with hOGG1 Ser/Ser genotypes. To verify whether APE1 Asp/Asp genotype could have lower DNA repair activity compared with APE1 Glu/glu genotype, pcDNA 3.1 A(-) APE1-Asp and pcDNA 3.1 A(-) APE1–Glu were contructed to transfect into TL4 (APE1 Asp/Asp, hOGG1 Cys/Cys genotype) and H23 (APE1 Glu/Glu, hOGG1 Ser/Ser genotype) lung cancer cells. Host-reactive DNA repair assay indicated that both cells with APE1-Glu transfection had higher repair capability to remove DNA damages induced by H2O2 than those with APE1-Asp transfection. Moreover, TL4 transfected with pCDNA3.1 A(-)APE1 Asp plus pCDNA3.1 A(-) hOGG1-cys expression vectors had lower repair capability to remove H2O2-induced DNA damages of pGL2luc than with APE1-Glu plus hOGG1-Ser transfections. These results strongly suggest that APE1 Asp/Asp or plus hOGG1 Cys/Cys carrier may have lower DNA repair activity than APE1 Glu/Glu or plus hOGG1 Ser/Ser carrier. The findings of the cell model experiment seemed to support the association of APE1 Asp/Asp carrier or APE1Asp/Asp plus hOGG1 Cys/Ser+Cys/Cys carrier had higher risk of p53 mutation occurrence in lung cancer patients. Therefore, APE1 Asp184Glu polymorphism may have more effective than hOGG1 Ser326Cys polymorphism on p53 mutation occurrence in lung cancer patient.