Conditions have been optimized for growing in culture human oral squamous cell carcinoma (SCC) cells and normal oral mucosal keratinocytes, the cell type from which this cancer originate. Previous studies, including those by the Cell Culture Core director, have identified a number of growth and differentiation control systems in normal keratinocytes, assayable in culture, that are typically defective in advanced oral SCCs. Substantial variability with respect to p53 mutations, cyclin D1 and cdk4/6 expression, and integration of papillomavirus DNA sequences has been noted among SCCs from different patients. Progress to identify the specific regulators of cell cycle control and gene transcription that become inactivated or over expressed by mutation and other genetic events at each stage of neoplastic progression in this accessible and clinically important cancer has been hampered by lack of availability of representative, well-characterized normal and neoplastic human oral epithelial cell lines, and by the complexity of methods for culturing and generating stable experimental gene transfectants of them. Identification of early genetic and phenotypic changes during oral cancer development is especially important, but few cell lines from pre-malignant lesions and early stage cancers have been cultured and studied. The Core Director's experience in this field, and his extensive collection of normal and malignant oral cell lines are unique and valuable resource for the Program Project. Of special importance are six cell lines from dysplastic oral lesions and three from early invasive SCCs which we have recently cultured. Several of these exhibit properties in culture intermediate between normal keratinocytes and advanced SCC cells, consistent with the acquisition of early neoplastic genetic alterations. We will culture more lines from oral lesions and early cancers and characterize their in vitro phenotypes such as replicative life span, mitogen and growth inhibitor, sensitivities, and histogenic potential, and we will assess their tumorigenicity in athymic mice. We will culture oral keratinocytes from cyclin D1- over expressing transgenic mice and from dysplastic lesions and SCCs that arise in these animals. We will make stable transductants of normal and pre-neoplastic oral keratinocytes and SCC cells to over express cdk6, cyclin D1, doc-1, and oral HPV E6 and E7 proteins. These cells will be used by project investigators to test key hypotheses.
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