本研究室過去研究發現，不抽菸之女性肺癌患者之 DNA adduct levels 顯著高於不抽 菸之男性，且此現象無法以參與代謝 CYP1A1, GSTM1 之基因多行性來解釋。為了 了解其可能原因，在過去三年之計畫中，分別由腫瘤組織以及細胞實驗探討其機轉。 首先在肺腫瘤組織中發現不抽菸女性肺癌患者之 CYP1A1 mRNA 之表現顯著高於 不抽菸男性，顯示女性 DNA adduct levels 高可能與 CYP1A1 有較高之轉錄活化。 另外發現女性肺癌患者之 ER 發生 Hypermethylation 之頻率遠低於男性，顯示 ER 在 女性表現之頻率較高。在細胞轉染 ER 至肺癌細胞的實驗，以 Western blot 和 Gel shift assay，結果都顯示 ER 轉植確實能促進 AhR 與 Arnt 結合，進而促進 CYP1A1 的轉 錄。而在 siRNA 方法降低 ER 在肺癌細胞表現之實驗則發現，CYP1A1 蛋白表現量 顯著低於 ER 沒有降低的原株肺癌細胞，同樣以 Gel shift assay 亦證明 ER 被降低的 細胞之 AhR 與 Arnt 結合 XRE 之能力有顯著降低的現象。在肺細胞中，ER 會促進 AhR 的活化，進而促進 CYP1A1 之轉錄。因此女性肺癌有較高之 DNA adduct levels 可能是女性有較高機會表現 ER 在肺癌組織中，會經由 ER 與 AhR 之交互作用，而 活化主要參與代謝多環芳香烴之 CYP1A1 的轉錄。而在腫瘤組織中又發現女性有較 高之 CYP1A1 之表現，且與其 DNA adduct levels 相關，總之，本計畫由肺腫瘤組織 與細胞實驗的結果都能印證女性有較高之 DNA 傷害易感性是經由 ER 與 AhR 交互 作用所致。
Our previous studies had indicated that the DNA adduct levels in nonsmoking female lung cancer patients were significantly higher than those in nonsmoking male lung cancer patients. The high adduct levels can not be explained by their genetic polymorphisms of CYP1A1 and GSTM1 that had been shown to be associated with DNA adduct formation. To elucidate the molecular mechanisms, lung tumors and cell culture experiments were performed to understand whether the cross talk between estrogen receptor (ER) and arylhydrocarbon receptor (AhR) was involved in activation of CYP1A1 transcription to cause high DNA adduct levels in female lung cancer patients. In lung tumor experiments, CYP1A1 mRNA expressions and DNA adduct levels in adjacent normal lung tissues from lung cancer patients were evaluated by RT-PCR and 32P-postlabeling, respectively. Our data indicated that CYP1A1 mRNA expression levels in female lung cancer patients were significantly higher than those in male lung cancer patients. In addition, CYP1A1 mRNA expression levels were correlated with DNA adduct levels. On the other hand, MSP data showed that ER hypermethylation in male lung cancer patients was greater than that in female lung cancer patients. Namely, a higher frequency of ER expression in female lung cancer patients compared with male lung cancer patients. In cell culture experiments, we first transfected ER into lung cancer A549 cells and found CYP1A1 protein levels were up-regulated compared with those of parental cells and gel shift assay data indicated that AhR-Arnt complex was significantly increased in ER tranfected cells. We also used small interference RNA to knockdown ER expression in lung cancer CL-5 cells because the cell had a relatively high ER expression. Western blot data showed that CYP1A1 protein expression levels was sharply decreased in three ER-knockdown cell clones compared with that of parental cells. Additionally, AhR-Arnt complex was also significantly attenuated in ER-knockdown cell clones from gel-shift assay. These results from cell culture experiments strongly indicated that unliganded ER expression may be cross-talk with AhR to promote liganded AhR nuclear translocation to form AhR-Arnt complex binding in XRE and then up-regulated of CYP1A1 transcription. The cross-talk mechanism of AhR and ER provide to explain why a higher frequency of ER expression in nosmoking female lung cancer patients had a higher susceptibility to DNA damage derived from environmental carcinogen exposure. Thus, accumulation of higher DNA damage may be linked with lung tumorigenesis of nonsmoking female lung cancer patients in Taiwan.
