MDM2基因在多種癌症組織中都有過度表現的現象，同時也發現MDM2基因有短小缺失的情形。先前的研究也指出，短小缺失的MDM2蛋白產物與多種癌症的發生有關係，例如卵巢癌、膀胱癌和乳癌；但是卻較少研究與肺癌發生之間的關係，因此，本研究計畫探討在肺癌的發生過程中是否會伴隨著MDM2短小缺失的情形發生，同時也進一步的探討抽煙是否與MDM2短小缺失的發生有關，最後利用B(a)P及其代謝物(BPDE)分別處理正常的肺纖維母細胞及肺癌細胞株(H1355)來加以證實MDM2的短小缺失與抽煙有關係。根據我們以Nested-PCR分析117位肺癌病人的檢體，發現有31(26.5%)位病人具有MDM2短小缺失的情形。進一步利用統計的方式比較抽煙與不抽煙的肺癌病人發現，MDM2基因在肺癌病人組織中表現的情形與性別(p=0.008)及是否抽煙(p=0.003)有明顯的相關性；同時在抽煙病人(n=19, 44.2%)的檢體中也發現MDM2短小缺失的頻率較不抽煙(n=12, 16.2%)的病人高；而不抽煙病人(n=18)檢體中的完整MDM2基因表現是抽煙病人(n=9)的兩倍，也就是說在台灣地區抽煙的肺癌病人具有較高MDM2短小缺失的機率。接著，我們分析MRC-5及H1355細胞中Glutathione S transferase的活性並以MTS assay分析兩株細胞對BPDE的細胞毒性差異，以及在處理B(a)P及BPDE之後，分析MDM2在MRC-5及H1355細跑中表現的情形。同時，利用專一性的抑制劑來證明經由BPDE所誘發的MDM2短小缺失-與PI3K及MAPK路徑無關。綜合以上的結果，本研究最後證實了PAH對於誘發MOM2基因發生短小缺失的情形具有潛在的危險性。 Mdm2 gene is overexpressed in several tumors and its product may be processed into different isoforms. Alternative splicing of mdm2 protein coding sequences has been documented during tumor progression in human ovarian, bladder carcinomas and breast carcinoma but few in lung cancer. The aim of this study was to determine whether alternative splicing of mdm2 occurs during lung tumorigenesis in human and whether cigarette smoking were related with alternative splicing happing. Specimens representing normal and malignant lung tissue from the human non-small cell lung cancer were examined. Of the 117 patients tested by Nested-PCR, a total of 31 (26.5%) showed splicing variants for mdm2 gene within tumor tissue. It is very surprising, the significant association among undetectable, splicing form and full length of mdm2 gene were found on gender (p=0.008) and smoking behavior (p=0.003) in non-small cell lung cancer in Taiwan. The splicing frequency is significantly difference between smoker (n=19, 44.2%) and nonsmoker (n=12, 16.2%), but the number of mdm2 full-length exhibit two folds difference in non-smoker (n=18) and smoker (n=9), respectively. Interestingly, the smokers have more high frequently mdm2 splicing forms than nonsmoker. Further more, total GST activity in MRC-5 and H1355 cell lines were analyzed and the cytotoxicity were analyzed by MTS assay. After treat with B(a)P or BPDE, mdm2 gene expression were analyzed by Nested-PCR in MRC-5 and H1355 cell lines. Finally, we demonstrate that BPDE mediated mdm2 mRNA alternative splicing was not through PI3K and MAPK pathway by several specific inhibitors. Overall, our results suggest that an important event were about the highly potential risk of alternative splicing after exposure to PAH.
