MOLECULAR PATHOLOGY OF ORAL NEOPLASMS WITH HPV INFECTION

人乳头瘤病毒（HPV）诱导广泛的良性和 肛门生殖道的恶性疾病，我们已经进行了 全面的分子研究。 病毒的癌蛋白E6和E7发挥作用， 在通过它们的结合启动多步致癌作用中的主要作用 肿瘤抑制蛋白p53和视网膜母细胞瘤易感性 蛋白 利用上皮筏培养，我们研究了 分化型乳腺癌中高危和低危基因型的E6和E7蛋白 角质形成细胞和鉴定的新的宿主细胞防御反应， 包括p21（cip 1/waf 1/sdi 1）和p53的诱导。 以来 生殖器和口腔粘膜是相似的，因此HPV DNA 在头颈部上皮病变（H&N）中也检测到 网站. 然而，除了喉乳头状瘤， HPV基因表达的直接分子证据 呼吸消化道 我们认为E6和E7基因是活跃的， 转录并促进H&N肿瘤的发展， 携带HPV病毒 这项研究旨在提供一个详细的分子 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 癌前状态 具体目标是：（1）识别可能的HPV 来自存档活检的良性和恶性口腔肿瘤中的感染， 有或无免疫抑制或致癌风险的新患者 因素 将通过表征PCR产物来确定病毒类型， 靶向粘膜营养型和皮肤HPV，因为两组的DNA 在口腔病变中发现。 (2)评估病毒转录， 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 不同H&N部位肿瘤连续分级的细胞反应。 p53和其他细胞周期调控蛋白的可能调控 包括细胞周期蛋白依赖性激酶、p21、p27 KIP 1、p57 KIP 2 蛋白质的INK家族（p15、p16、p18、p19）和转化生长 将通过免疫细胞化学（ICC）研究因子β 1， 连续切片中的免疫荧光。 将通过ISH检测它们的mRNA 以确定调控是转录还是后 转录的 (4)检测新鲜外科手术中宿主DNA合成 活组织检查，将可能的非计划染色体复制与 目标3中描述的宿主和病毒基因的表达。 主机复制 在癌前病变中明显分化的基底上细胞中， 通过掺入3/H-胸苷或BrdU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)以确定是否发病 病毒和宿主基因的表达可能参与入侵， 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、基质溶素、基质溶素1） 和MMP的组织抑制剂（TIMP）1和2将通过ICC探测， 算是吧

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.