ISOLATION OF GENES REQUIRED FOR HPV-MEDIATED ORAL CANCER

口腔鳞状癌是世界上十大最常见的癌症之一。口腔恶性肿瘤患者的总体5年预后约为50%，低于宫颈鳞状癌。宫颈癌和口腔癌的发病率增加与两个主要的危险因素有关，吸烟和饮酒。此外，宫颈癌与HPV感染密切相关。90%的子宫颈癌含有高危型HPV基因组。另一方面，口腔癌和HPV感染之间的因果关系的数据一直是可变的。尽管HPV在口腔和生殖器粘膜癌症发展中的作用相似，但两类粘膜的细胞和环境特性也存在显著差异。这可能会导致癌症发展途径的差异。为了研究细胞转化为肿瘤时基因的差异表达，我们将利用Park等人开发的体外模型系统。在这个模型中，正常的口腔角质形成细胞被HPV感染成为永活细胞。然后这些细胞暴露在与烟草有关的致癌物中，这种致癌物将细胞转化为肿瘤。利用该模型系统，我们将通过mRNA差异显示（mRNA DD）分析分离和表征细胞向恶性表型发展的差异表达基因。然后，我们将通过Northern分析确认mRNA DD分析，以观察在各种已建立的细胞系和原代培养物中的表达模式和表达稳定性。此外，将对患者活检进行RT-PCR和原位杂交，以确定分离的mRNA DD产物的细胞类型和分期特异性。从mRNA DD分析中获得的上调或下调的cDNA克隆将用于表达或直接克隆，以研究其在hpv介导的致癌作用中的作用。该项目可能最终确定在肿瘤转化的某些阶段一致存在的特定生物标志物。这样就可以开发出一种快速的诊断方法，通过一种敏感、成本效益高的程序对患者进行常规筛查。早期发现可以在正常的诊所就诊时提供，以增加口腔癌的生存机会。

Oral squamous carcinoma is among the top ten most frequently occurring cancers in the world. The overall five year prognosis for patients with oral malignancies is about 50 percent, which is lower than the comparable squamous carcinoma of the cervix. The incidence of both cervical and oral carcinomas increase when associated with two prominent risk factors, cigarette smoking and alcohol consumption. In addition, cervical cancer is strongly correlated with HPV infection. Ninety percent of cervical cancer harbor "high-risk" serotype of HPV genome. On the other hand, data on the causal relationship between oral cancer and HPV infection have been variable. In spite of the similarities in the role of HPV in the development of cancer in the oral and genital mucosa, there are also significant differences in the cellular and the environmental properties of the two types of mucosa. This will likely result in differences in the pathways of cancer development. To investigate the differential expression of the genes as cells transforms to tumor, we will utilize an in vitro model system developed by Park et al. In this model, normal oral keratinocytes are infected with HPV to become immortalized cells. Then the cells are exposed to a tobacco-related carcinogen, which converts the cells to a tumor. Using this model system, we will isolate and characterize differentially expressed gene(s) by the mRNA differential display (mRNA DD) analysis, as the cells progress to the malignant phenotype. We will then confirm the mRNA DD analysis by the Northern analysis to observe the expression pattern and the stability of expression in various established cell lines and primary cultures. In addition, RT-PCR and in situ hybridization will be performed with patient biopsies to determine the cell type and the stage specificity of the isolated mRNA DD products. The up-or down-regulated cDNA clones obtained from the mRNA DD analysis will be used for either expression or direct cloning to study its function in conferring HPV-mediated carcinogenesis. This project may eventually identify specific biomarkers that are consistently present in certain stages of neoplastic conversion. A quick diagnostic method can then be developed to screen patients routinely by a sensitive, cost-effective procedure. Early detection can be provided during normal office visits to increase the chance of survival from oral cancer.