This proposal tests the hypothesis that genetic alterations in oral cavity epithelium lead to dysplasia and ultimately to invasive squamous cell carcinoma (SCC). It is further hypothesized that gene amplification and mutation are crucial alterations in the development of these tumors, and that these alterations, and their expression, can be detected using fluorescence in situ hybridization (FISH), in situ hybridization (ISH), and immunohistochemistry (IHC). Three candidate genes which are frequently amplified (prad1 and ems1) or mutated (p53) in invasive oral cavity SCC will be analyzed to identify the frequency of these alterations in preinvasive oral cavity lesions. The training period to carry out the proposed research has been divided into Phase I and Phase II. Phase I of the proposal (years 1 and 2) will aim to identify, in preinvasive oral cavity lesions, the frequency of: a) amplication of prad1 and ems1 using FISH; b) increased transcription of prad1 and ems1 using ISH; and c) protein overexpression of prad1, ems1 and mutant p53 using IHC. After this has been accomplished, phase II, which evaluates the importance of these alterations in oral cavity tumorigenesis, will begin. Phase II of the proposal (years 3-5) will involve the analysis of the in vitro and in vivo function of these altered genes through transfection into normal human buccal keratinocytes. Transfection will involve one gene, combinations of two genes, or all three genes. Transfected cells will be evaluated for evidence of transformation using techniques known to correlate with carcinogenic potential: increased cytoskeletal protein expression, increased expression of ornithine decarboxylase, and changes in cell growth characteristics (morphology, need for growth factors, immortalization, and anchorage independent growth in semi-solid media). Those cells that grow in semi-solid media, or, alternatively, cells which have two or more other signs of transformation, will be injected into nude mice, and the mice observed for signs of tumor formation. The data obtained from this research will lead to a better understanding of the important genetic events in oral cavity tumorigenesis. The training from this project should provide the candidate with an excellent foundation for an independent career in biomedical research.
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