2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/37667In our recent study, it has been found that HPV 16/18 infection frequency of non-smoking female lung cancer patients (67%, 30/45) were higher than that of smoking and non-smoking male (25%, 24/96; Cancer Res, 61: 2799-2803, 2001). In contrast, HPVs were rarely detected in lung tumor tissues of Caucasian patients. Our preliminary data showed that mutation frequency of p53 gene in non-smoking female lung cancer patients was lower together with a higher protein inactivation rate. These findings led us to come up with a hypothesis that degradation of p53 protein may be resulted by HPV-associated activation of ubiquitin proteasomal degradation pathway and ultimately, caused the lung cancer formation. Meanwhile, our previous results showed that the DNA adduct levels in female lung cancer patients non-tumor tissues were higher than that in non-smoking male and this phenomenon may be associated with the carcinogen exposed of cooking oil fume. Therefore, this program is aimed on evaluating whether the HPV infection and DNA damage will induce the activation of ubiquitin proteasomal degradation pathway to inactivate p53 protein expression. Apart from the immunohistochemical analysis of ubiquitin and p53 protein expression, HPV16/18 E6 mRNA expression in lung tumor tissues will also be analyzed by in situ RT-PCR. The relationships between these obtained data will be statistically analysed. It will give us some hints on whether the HPV infection could induce the activation of ubiquitin proteasomal degradation pathway to caused lung cancer formation followed by p53 protein degradation. Furthermore, HPV 16/18 E6 cDNA will also be transfected into lung cancer cell line and then treated with BPDE, a major carcinogen in cooking oil fume. The effect of such BPDE treatment on the protein expression patterns of p53, ubiquitin and HPV E6 in HPV transfected cell will be analyzed by immunofluorescence and western blot to further clarify the correlation between these proteins and the similarity of such correlation between lung tumor tissues and cell lines. In summary, not only to prove whether HPV infection is involved in female lung tumorigenesis via p53 inactivation, this study will also demonstrate whether cooking oil fume carcinogen and HPV infection have a synergic effect on activation of ubiquitin proteasomal pathway for p53 protein inactivation. Eventually, information from this study will be contributive to understand whether the non-smoking female lung cancer development is resulted from the interaction between multiple environmental factors.最近本研究室發現不抽煙女性肺癌患者有67% (30/45)的人類乳突病毒16/18型 (human papillomarvirus 16/18, HPV16/18) 之感染率，遠較抽菸和不抽菸之男性肺癌患者高 (24/96, 25%; Cancer Res. 61: 2799-2803, 2001)。尤其在過去有關白種人之肺癌幾乎沒有偵測到人類乳突病毒之感染。上年度計畫的初步結果顯示不抽煙之女性肺癌患者之p53基因大多不發生突變，且p53蛋白也大多不表現，這結果顯示HPV感染可能誘發ubiquitin proteasomal degradation pathway將p53蛋白去活化而引起肺癌形成。過去本計畫之成果亦發現烹調油煙中的致癌物 (Chem. Res. Toxicol. 13: 1046-1050, 2000)，可能造成女性肺癌患者之DNA鑑結物之含量較不抽菸男性肺癌患者高 (Environ. Mol. Mutagen. 37: 304-310, 2001)。因此本年度計畫擬探討HPV感染和DNA傷害是否會誘發ubiquitin proteasomal degradation pathway被大量表現，而造成p53蛋白的去活化。本計畫將以免疫組織化學分析法分析肺腫瘤組織切片中p53和ubiquitin蛋白之表現是否相關？另外以in situ RT-PCR方法分析肺腫瘤組織切片中是否有HPV 16/18 E6 mRNA的表現？並進行其間的相關性分析，以了解HPV E6 mRNA表現的肺腫瘤中，ubiquitin同時表現之肺癌患者之p53蛋白是否無法偵測到？這結果將有助於了解HPV 感染是否確實會誘發ubiquitin proteasomal pathway而去活化p53蛋白，造成肺腫瘤形成。為證明其間的相關性，本計畫將以基因轉殖技術，將HPV 16/18 E6 cDNA送入肺癌細胞株，同時給予不同劑量之烹調油煙主要致癌物－benzo[a]pyrene 7,8-diol-9,10-epoxide(BPDE) 處理，以螢光免疫分析法確定ubiquitin, p53和E6蛋白在細胞內之分布和其間的相關性，另外以Western blot分析三種蛋白間的相關性，是否如肺腫瘤組織所看到的。因此本計畫不僅能證明HPV感染是否是經由p53蛋白之去活化，而引起女性肺癌之形成，同時還能了解油煙致癌物和HPV感染在p53去活化上是否具有協力活化ubiquitin proteasomal pathway 的能力？總之，本計畫將有助於了解不抽煙台灣女性肺癌之形成，是否是經由多重環境因子之協力作用所致。肺癌人類乳突狀病毒p53 蛋白ubiquitin 蛋白lung cancerhuman papillomavirusp53cooking oil fumeProject