Human cancers of stratified epithelia such as cervical, colon, head & neck, and skin develop through hyperplastic, dysplastic, benign and malignant stages of differentiated and undifferentiated types. Once established, carcinomas are generally refractory to treatment. Therefore, knowledge of markers of carcinoma stages is important for early cancer detection and knowledge of the underlying genetic defects is important for the rational design of chemopreventive and therapeutic strategies. The general goal of this project is to better understand the multi-step process of cancer development in differentiating epithelia. We have derived clonal cell lineages from BALB/c mouse epidermis, comprised of normal, initiated, benign and malignant stages, as an approach to uncover the defects responsible for this process and to determine whether they accumulate in a particular order. This continuation proposal is based on the following observations made using this model: 1) abnormal regulation of wild type (wt) p53 occurs at the malignant conversion stage prior to any p53 coding region mutations; 2) an alternatively spliced p53 (AS-p53) mRNA is present in a variety of normal cells and tissues; 3) expression of a differentiation-associated VL30 (endogenous retrovirus-like) sequence is deregulated in squamous cell carcinomas and 4) expression of differentiation-associated keratins (K1 and, aberrantly, K13) is abnormally regulated at the malignant conversion stage.
The aims are to: 1) examine the activity of p53 protein as a regulator of the Gl/S transition and as a regulator of epidermal differentiation; changes in p53 localization or reactivity with epitope-specific p53 monoclonal antibodies will be analyzed using immunofluorescence and flow cytometry; unstimulated normal and carcinoma cells will be examined as well as cells exposed to agents (DNA-damaging) which cause wt p53-associated Gl/S arrest in myeloid progenitor cells; 2) test the hypothesis that wt p53 drives differentiation and mutated p53 restricts differentiation by transfecting wt p53 and mutated p53 genes into squamous cell carcinoma cells which are either poorly differentiated and have low levels of wt p53 protein or are moderately differentiated and have high levels of p53 protein; 3) identify cellular factors which may associate with p53 protein, such as heat shock proteins or mdm-2, leading to its inactivation in epidermal cells; immunoprecipitation and immunoblotting of coprecipitated proteins and northern blots to detect abnormal RNA expression of potential interacting factors will be performed using cells isolated at discrete steps in cancer development; 4) because of apparent differences in expression, investigate whether VL30 expressed in epidermal keratinocytes is distinct from VL30 in skin fibroblasts; 5) test the hypothesis that VL30 in carcinomas is functional in malignant-conversion, e.g. by retrotransposition and activation or inactivation of other cellular genes.
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