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

苯并[A]芘对宫颈转基因模型的影响

• 批准号: 6498275
• 负责人: Ellen Rorke
• 金额: $ 20.51万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6498275
• 关键词: adduct; apoptosis; athymic mouse; benzopyrenes; carcinogen testing; cell differentiation; cell proliferation; cervix neoplasms; chemical carcinogen; chemical carcinogenesis; disease /disorder model; environment related neoplasm /cancer; epidermal growth factor; female; gene mutation; genetically modified animals; growth factor receptors; human papillomavirus; metalloendopeptidases; mutagen testing; mutagens; oncogenes; p53 gene /protein; smoking; tobacco abuse; viral carcinogenesis

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.

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基因组编码两种癌蛋白E6和E7，这是细胞永生所必需的，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，包括继发性癌基因的激活。在这方面，环境致癌物起着主要作用。吸烟与患宫颈癌的风险增加有关。香烟烟雾中的一种活性剂是普遍存在的环境致癌物，苯并[a]芘。B[a]P的代谢物是有效的DNA诱变剂。B[a]P在体外对宫颈细胞具有诱变作用，在体内可在宫颈黏液中积累，而ras癌基因是B[a]P的已知靶点，在宫颈肿瘤中发生高频率突变。P53是一种重要的细胞周期控制蛋白。DNA损伤后，p53暂停细胞周期进程，直到DNA修复完成。我们之前的体外研究表明，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水平的大量增加有关。相反，在hpv永生化细胞中，低p53永生化细胞在细胞周期中没有明显的停顿。基于这些研究，我们假设B[a]P在hpv永生化细胞中应该是一种更有效的诱变原，因为这些细胞在暴露于B[a]P后不会暂停增殖足够长的时间来充分修复DNA。虽然我们的体外实验结果与这一假设一致，但我们的目标是在体内验证这一假设。然而，由于缺乏合适的hpv依赖性肿瘤动物模型，这一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，小鼠在宫颈上皮中表达HPV16 E6和E7癌蛋白，现在我们准备在体内验证我们的假设。在Specific Aim 1中，我们验证了B[a]P处理与正常子宫颈相比会增加hpv永生化子宫颈肿瘤形成的假设，并在裸鼠、软琼脂和微侵袭实验中检测了细胞的致瘤表型。在特异性目的2中，我们假设与正常子宫颈相比，B[a]P处理应增加hpv永生化子宫颈的肿瘤形成，并在裸鼠、软琼脂和微侵袭实验中检查细胞的致瘤表型。在Specific Aim 3中，我们检验了体内B[a]P治疗会显著增加hpv永生化细胞中的DNA突变的假设，并且突变率的增加应该与p53水平的降低相关。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位点突变的影响。这些研究的最终目的是利用我们的HPV16 E6/7宫颈肿瘤小鼠，为环境致癌物（多环芳烃）在宫颈癌发生中的作用提供新的认识。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。