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.
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