This is a five-year application to support investigations of tumor necrosis factor-alpha (TNF-alpha)-mediated gene regulation of human Deleted in Oral Cancer-1 (doc-1) during oral carcinogenesis. While the molecular mechanisms preventing oral carcinogenesis are poorly understood, certain cytokines and tumor suppressor genes have been demonstrated to retard or revert the malignant phenotype in transformed human oral keratinocytes. TNF-alpha is a pleiotropic cytokine well-established to havetumor-specific cytolytic and/or cytostatic activity in a wide variety of human malignancies, incliding oral cancer. We hypothesize tht TNF-alpha growth suppression mediated through doc-1 expression during oral cancer development may represent a novel mechanism of its anti-tumor activity. TNF-alpha has been established to reduce proliferation and promote differentiation in normal and malignant human keratinocytes. The molecular mechanisms determining TNF-alpha-mediated cytostatic activity likely require induction of specific cellular genes. Recently, a novel gene indiced in TNF-alpha-stimulated murine normal fibroblasts was demonstrated to have a significant homology to doc-1, a gene sequence which we have shown to exhibit tumor suppressor function in malignant hamster oral keratinocytes. Doc-1 is an evolutionarily conserved gene exhibiting loss of heterozygosity and marked reduction in expression in hamster oral cancer cells. Transfection of the full-length doc-1 cDNA into malignant hamster oral keratinocytes alters the behavior of the recipients in terms of morphology, growth rate, and anchorage independent growth, suggesting reversion of transformation phenotypes. While TNF-alpha appears to upregulate doc-1 in both normal and malignant oral keratinocytes, the oral cancer cell lines demonstrate a markedly reduced or absent basal expression level until indiction by TNF-alpha. This application proposes experiments to elucidate the mechanism(s) leading to reduced doc-1 gene activity, and identify the molecular events leding to TNF-alpha-mediated induction of doc-1 in normal and malignant human oral keratinocytes during oral epithelial tumorigenesis. The goals of this application will also include the identification of the cis- and trans- elements present in the TNF-alpha-doc-1 pathway in human oral keratinocytes which may be responsible for anti-tumor activity. Linkage of doc-1 as a novel mediator of TNF-alpha will improve our understanding, as well as possibly the diagnosis and treatment, of squamous cell carcinoma of the oral cavity.
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