EFFECTS OF BENZO[A]PYRENE IN TRANSGENIC MODEL OF CERVICA

人宫颈癌的发展始于DNA肿瘤病毒，人乳头瘤病毒（HPV）的感染。HPV基因组编码细胞永生化所需的两种癌蛋白E6和E7，E6导致细胞周期检查点/肿瘤抑制蛋白p53的降解。然而，侵袭性疾病的发展需要额外的事件，包括次级癌基因的激活。在这方面，环境致癌物起着重要作用。吸烟与宫颈癌发生的风险增加有关。香烟烟雾中的一种活性物质是普遍存在的环境致癌物苯并[a]芘。B[a]P的代谢产物是有效的DNA诱变剂。B[a]P在体外对宫颈细胞具有致突变性，在体内可在宫颈粘液中蓄积，并且作为B[a]P的已知靶点的ras癌基因在宫颈肿瘤中以高频率突变。p53是一种重要的细胞周期调控蛋白。DNA损伤后，p53暂停细胞周期进程，直到DNA修复完成。我们以前的体外研究表明，用B[a]P处理强烈抑制培养的正常宫颈上皮细胞的增殖。这种抑制与p53水平的大幅增加有关。相反，在HPV永生化细胞中，其具有低p53-永生化细胞在细胞周期中不显著暂停。基于这些研究，我们假设B[a]P在HPV永生化细胞中应该是更有效的诱变剂，因为这些细胞在B[a]P暴露后不会停止足够长的增殖以充分修复DNA。虽然我们的体外结果与这一假设是一致的，我们的目标是在体内测试这一假设。然而，由于缺乏合适的HPV依赖性肿瘤动物模型，该领域的工作一直受到阻碍。在过去的两年中，我们开发了一种创新的宫颈癌转基因小鼠模型，其中小鼠在宫颈上皮中表达HPV 16 E6和E7癌蛋白，我们现在准备在体内测试我们的假设。在特定目标1中，我们检验了这样一个假设，即与正常宫颈相比，B[a]P治疗应增加HPV永生化宫颈中的肿瘤形成，并在裸鼠、软琼脂和微侵袭试验中检查细胞的致瘤表型。在特定目标2中，我们假设B[a]P治疗应增加HPV永生化宫颈与正常宫颈相比的肿瘤形成，并在裸鼠、软琼脂和微侵袭试验中检查细胞的致瘤表型。在具体目标3中，我们检验了体内B[a]P处理应差异性地增加HPV永生化细胞中的DNA突变的假设，并且增加的突变率应与降低的p53水平相关。我们研究了B[a]P处理对p53水平、DNA加合物形成以及H-ras和K-ras基因座突变的影响。这些研究的最终目的是使用我们的HPV 16 E6/7宫颈肿瘤小鼠提供有关环境致癌物（多环芳烃）在宫颈癌发生中的作用的新认识。使用我们的新型转基因模型为解决这些问题提供了一种创新的方法。

Human cervical cancer development begins with infection by the DNA tumor virus, human papillomavirus (HPV). The HPV genome encodes two oncoproteins, E6 and E7, that are required for cell immortalization, E6 causes degradation of the cell cycle checkpoint/tumor suppressor protein, p53. However, development of invasive diseases requires additional events including the activation of secondary oncogenes. In this regard, environmental carcinogens play a major role. Cigarette smoking is correlated with an enhanced risk for cervical cancer development. An active agent in cigarette smoke is the ubiquitous environmental carcinogen, benzo[a]pyrene. Metabolites of B[a]P are potent DNA mutagens. B[a]P is mutagenic in cervical cells in vitro, accumulates in cervical mucus in vivo, and ras oncogene, a known target of B[a]P, is mutated at a high frequency in cervical tumors. p53 is an important cell cycle control protein. Following DNA damage, p53 pauses cell cycle progression until DNA repair is complete. Our previous in vitro studies show that treatment with B[a]P strongly inhibits the proliferation of cultured normal cervical epithelial cells This inhibition is correlated with a large increase in p53 level. In contrast, in HPV-immortalized cells, which have low p53-immortalized cells do not significantly pause in the cell cycle. Based on these studies, we hypothesize that B[a]P should be a more potent mutagen in HPV-immortalized cells, because these cells do not pause in proliferation long enough to adequately repair DNA following B[a]P exposure. Although our in vitro results are consistent with this hypothesis, our goal is to test this hypothesis in vivo. However, work in this field has been stymied by the lack of a suitable animal model of HPV-dependent neoplasia. During the past two years, we have developed an innovative transgenic mouse model of cervical cancer, in which the mice express the HPV16 E6 and E7 oncoproteins in the cervical epithelial, and we are now ready to test our hypotheses in vivo. In Specific Aim 1 we test the hypothesis that B[a]P treatment should increase tumor formation in HPV-immortalized cervix compared to normal cervix, and examine the tumorigenic phenotype of the cells in nude mice, in soft agar, and in microinvasion assays. In Specific Aim 2 we hypothesize that B[a]P treatment should increase tumor formation in HPV-immortalized cervix compared to normal cervix, and examine the tumorigenic phenotype of the cells in nude mice, in soft agar, and in microinvasion assays. In Specific Aim 3 we examine the hypothesis that in vivo B[a]P treatment should differentially increase DNA mutations in HPV-immortalized cells, and that the increased mutation rate should be correlated with reduced p53 levels. We examine the effects of B[a]P treatment on p53 level, DNA adduct formation, and mutation at the H-ras and K-ras loci. The ultimate aim of these studies is use our HPV16 E6/7 cervical neoplasia mice to provide new understanding regarding the role of environmental carcinogens (polycyclic aromatic hydrocarbons) in the genesis of cervical cancer. Use of our novel transgenic model provides an innovative approach for addressing these questions.