MOLECULAR PATHOLOGY OF ORAL NEOPLASMS WITH HPV INFECTION

HPV 感染口腔肿瘤的分子病理学

• 批准号: 6336493
• 负责人: THOMAS R BROKER
• 金额: $ 9.88万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6336493
• 关键词: genetic transcription; human papillomavirus; immunocytochemistry; immunofluorescence technique; in situ hybridization; metalloendopeptidases; molecular oncology; neoplasm /cancer classification /staging; neoplasm /cancer epidemiology; neoplasm /cancer genetics; oncogenes; oncoprotein p21; oncoproteins; oral mucosa; oral pharyngeal neoplasm; polymerase chain reaction; preneoplastic state; radiotracer; tissue inhibitor of metalloproteinases; viral carcinogenesis; virus genetics

Human papillomaviruses (HPVs) induce a wide spectrum of benign and malignant diseases in the anogenital tract, on which we have conducted comprehensive molecular studies. The viral oncoproteins E6 and E7 play a major role in initiating multi-step carcinogenesis through their binding to the tumor suppressor proteins p53 and retinoblastoma susceptibility protein. Using epithelial raft cultures, we investigated the functions of E6 and E7 proteins from high-risk and low-risk genotypes in differentiated keratinocytes and identified novel host cell defensive responses, including the induction of p21 (cip1/waf1/sdi1) and p53. Since the genital and oral mucosa are similar, it is not surprising that HPV DNAs have also been detected in epithelial lesions of head and neck (H&N) sites. Yet, with the exception of laryngeal papillomas, there has been no direct molecular demonstration of HPV gene expression in the upper aerodigestive tract. We propose that the E6 and E7 genes are actively transcribed in and contribute to the development of H&N neoplasms that harbor HPVs. This study is designed to provide a detailed molecular profile of viral gene expression and host cell responses in oral tumors, to elaborate the role of HPVs in epithelial pathogenesis and to identify significant molecular predictors for the diagnosis and treatment of precancerous conditions. Specific aims are: (1) To identify possible HPV infections in benign and malignant oral tumors from archival biopsies and new patients with or without immunosuppressive or carcinogenic risk factors. Virus types will be determined by characterizing PCR products, targeting both mucoso-trophic and cutaneous HPVs since DNAs of both groups have been found in oral lesions. (2) To assess viral transcription and the physical state of the viral DNA by in situ hybridization (ISH) detection, supporting or ruling against causal relationships between HPV and oral neoplasms. (3) To correlate the expression of viral genes and cellular responses in successive grades of neoplasms in various H&N sites. The possible modulation of p53 and other cell cycle regulatory proteins including inhibitors of cyclin-dependent kinases, p21, p27KIP1, p57KIP2, the INK family of proteins (p15, p16, p18, p19), and transforming growth factor-beta1 will be investigated by immunocytochemistry (ICC) and immunofluorescence in serial sections. Their mRNA will be examined by ISH to determine whether regulation is transcriptional or post- transcriptional. (4) To detect host DNA synthesis in fresh surgical biopsies, tieing possible unscheduled chromosomal replication to the expression of host and viral genes described in Aim 3. Host replication in apparently differentiated suprabasal cells in precancerous lesions and oral cancers will be monitored by incorporation of 3/H-thymidine or BrdU or by the detection of cellular proteins indicative of DNA replication capability (e.g., PCNA and cyclin E). (5) To determine whether the onset of expression of viral and host genes potentially involved in invasion and metastasis is linked to tumor grade. The production of matrix metalloproteinases (MMPs) (e.g., collagenase 1, matrilysin, stromolysin 1) and tissue inhibitors of MMPs (TIMP) 1 and 2 will be probed by ICC and ISH.

人乳头瘤病毒（HPV）可诱导多种良性和 肛门生殖道恶性疾病，我们对此进行了 全面的分子研究。 病毒癌蛋白 E6 和 E7 发挥着 通过它们的结合在启动多步骤致癌过程中发挥主要作用 肿瘤抑制蛋白 p53 和视网膜母细胞瘤易感性 蛋白质。 使用上皮筏培养物，我们研究了 分化中高风险和低风险基因型的 E6 和 E7 蛋白 角质形成细胞并确定了新的宿主细胞防御反应， 包括 p21 (cip1/waf1/sdi1) 和 p53 的诱导。 自从 生殖器和口腔粘膜相似，HPV DNA 并不奇怪 也在头颈部 (H&N) 的上皮病变中检测到 网站。 然而，除了喉乳头状瘤外，还没有 上皮细胞中 HPV 基因表达的直接分子证明 呼吸消化道。 我们认为 E6 和 E7 基因是积极的 转录并促进 H&N 肿瘤的发展 携带 HPV。 本研究旨在提供详细的分子 口腔肿瘤中病毒基因表达和宿主细胞反应的概况， 阐述 HPV 在上皮发病机制中的作用并确定 诊断和治疗的重要分子预测因子 癌前病变。 具体目标是： (1) 识别可能的 HPV 档案活检中良性和恶性口腔肿瘤的感染 有或没有免疫抑制或致癌风险的新患者 因素。 病毒类型将通过表征 PCR 产物来确定， 由于两组 DNA 均针对粘膜营养型和皮肤 HPV 已在口腔病变中发现。 (2) 评估病毒转录和 通过原位杂交 (ISH) 检测病毒 DNA 的物理状态 检测、支持或裁定 HPV 之间的因果关系 和口腔肿瘤。 (3) 将病毒基因的表达与病毒基因的表达相关联 不同 H&N 部位连续级别肿瘤的细胞反应。 p53 和其他细胞周期调节蛋白的可能调节 包括细胞周期蛋白依赖性激酶抑制剂、p21、p27KIP1、p57KIP2、 INK 蛋白家族（p15、p16、p18、p19）和转化生长 因子-β1 将通过免疫细胞化学 (ICC) 进行研究 连续切片中的免疫荧光。 他们的 mRNA 将通过 ISH 进行检查 确定调控是转录调控还是后调控 转录的。 (4)检测新鲜手术中宿主DNA的合成 活组织检查，将可能的计划外染色体复制与 目标 3 中描述的宿主和病毒基因的表达。宿主复制 癌前病变中明显分化的基底上细胞和 将通过掺入 3/H-胸苷或 BrdU 来监测口腔癌 或通过检测指示 DNA 复制的细胞蛋白 能力（例如 PCNA 和细胞周期蛋白 E）。 (5)判断是否发病 可能参与入侵和入侵的病毒和宿主基因的表达 转移与肿瘤分级有关。 矩阵的产生 金属蛋白酶 (MMP)（例如胶原酶 1、基质溶解素、基质溶解素 1） MMP 组织抑制剂 (TIMP) 1 和 2 将由 ICC 进行探测 ISH。