The clearly defined genetic and phenotypic stages in the evolution of squamous tumor development on mouse skin provides an opportunity to evaluate mechanisms of carcinogenesis in a stage-specific manner. This project is designed to identify genetic loci that modify the later stages of cancer development and elucidate the pathways through which they function. We have focused on two major pathways that are relevant to human cancer, the p53 and AP-1 pathways that regulate gene expression. Upregulation of c-fos is detected in squamous tumors that are progressing from benign to malignant, and deletion of c-fos prevents malignant conversion. We have targeted a dominant-negative mutant fos protein to the epidermis of transgenic mice using a tetracycline conditional promoter system. Expressing a dominant-negative fos gene in basal keratinocytes produces a lethal phenotype when expressed during early development (24 litters from 7 mothers). When A-fos is repressed during gestation and then activated after birth, however, normal development and differentiation occurs. In vitro, no significant changes in keratin profiles during calcium induced differentiation of primary cultures have been noted to date (K5, K1, K14, and K10). Preliminary studies with ras transformation of A-fos cells, indicates a lethal response within 48 hours of transformation in vitro. When primary cells are treated with the phorbol ester TPA, cellular morphology is altered. Initial studies indicate that TPA treatment induces an increased inflammatory response in A-fos mice in vivo after 48 hours. A-fos mice that have been initiated with DMBA and promoted for 5 weeks with a low dose of TPA appear resistant to tumor formation (n=17 A-fos mice). This study is currently being reproduced with a higher dose of TPA for 20 weeks in mice with both activated and repressed expression of A-fos. Assays are currently under investigation to analyze differences in cell proliferation, apoptosis, and cytokine production in A-fos keratinocytes relative to control animals. Over the past year, significant progress has been made on the in vitro analysis of c-fos knockout keratinocytes, transformed with v-rasHa by cDNA expression arrays. Expression of numerous genes were found to be altered in both wildtype and KO cells after ras transformation, though only a few were unique to the knockout cells. Given the previously reported phenotypic difference during tumorigenesis in these genetic backgrounds in vivo, this would indicate that either the current arrays (mouse 2.6K from ATC) do not contain the significant genes regulated by c-fos, or that the system will require further analysis from in vivo tumor samples. These samples will be reanalyzed upon availability of 11K gene chips from ATC. Further, in vivo tumor grafts of wildtype and heterozygous ras transformed keratinocytes, indicates a gene dosage effect on tumor size, though not progression. Additonal progress with the micro-arrays have included the increased sensitivity of the assay, which now only requires a minimum of 1 mg of total RNA for the labeling, the application of a novel algorithm for data normalization (in collaboration with Fuad Gwadry), and significant improvements in experimental design and analysis. In vivo analysis of c-fos knockout and wildtype tumors is currently under investigation with cDNA expression profiling. By selecting for genes that are regulated by p53 in keratinocytes, we have cloned mtCLIC, a chloride channel protein of mitochondria. mtCLIC causes apoptosis in normal and neoplastic keratinocytes when overexpressed, and antisense mtCLIC prevents apoptosis induced by overexpressing p53. mtCLIC also increases after DNA damage in keratinocytes and synergizes with Bax to induce apoptosis. mtCLIC translocates to the nucleus during apoptosis induced by p53 or DNA damage. During tumor progression, the expression of mtCLIC decreases in dysplastic and malignant epidermal tumors, and this is associated with mutations in p53. The human mtCLIC gene is located on chromosome 1p32-35, a site that is frequently deleted or translocated in human epithelial malignancies. The human mtCLIC promoter contains 3 p53 consensus binding sites and mtCLIC expression is lost in a variety of human tumors, particularly breast, ovary and renal cell carcinomas.
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