PKC, EGFR, AP-1 and CREB pathways are involved in normal epidermal growth and differentiation and are linked to phenotypic changes in initiation and premalignant progression. AP-1 DNA binding activity is reduced in v-rasHa keratinocytes, while AP-1 transcriptional activity increases two to three fold. To identify AP-1 regulated genes relevant to malignant conversion, a PCR based differential display screen was performed with RNA from papilloma cell lines overexpressing v-jun and v-fos. Clones corresponding to the mouse bcl-2 gene, a human splicing factor, were expressed to a greater extent in high risk papillomas. EGFR-/- keratinocytes are not growth stimulated by conditioned medium from v-rasHa keratinocytes, indicating that EGFR ligands are the major mitogens secreted by v-rasHa keratinocytes. Unlike v-rasHa EGFR+/+ keratinocytes, v-rasHa EGFR -/- keratinocytes express keratins 1 and 10 when induced to differentiate by Ca2+, suggesting that EGFR activation is required by oncogenic Ras to suppress expression of these keratins and linking PKCalpha to the EGFR pathway. Mice expressing a null mutation in the p53 gene generated by homologous recombination and their normal littermates were used as a source of epidermal keratinocytes differing in p53 gene expression. As expected, v-rasHa/p53+/+ keratinocytes produced papillomas in grafts, but dysplastic squamous tumors developed within one week in grafts of v-rasHa/p53-/- keratinocytes and overt poorly differentiated carcinomas developed by 2-3 weeks, indicating that p53 suppresses premalignant progression and malignant conversion. Loss of autocrine TGF-beta1 accelerated premalignant progression of squamous papillomas. To test for a relationship between TGF-beta1 loss, premalignant progression and genomic stability, non-neoplastic keratinocyte cell lines were established from TGF-beta1-/- and +/- primary keratinocyte cultures, and tested for their ability to amplify the CAD gene in response to PALA. TGF-beta1 can suppress genomic instability independent of Rb or p53 and growth inhibition; loss of this function contributes to premalignant progression. Thapsigargin and TPA, at suboptimal doses, are synergistic for tumor promotion. Caffeic acid phenethyl ester is cytotoxic to papilloma cell lines in culture and inhibits promotion by TPA but not by mezerein. The F1 progeny of a cross between SENCAR A/Pt and BALB/cAnPt are resistant to initiation and promotion with low doses of DMBA and TPA. Multiple genes involved in controlling susceptibility to papilloma development are located on mouse chromosomes 5, 9 and 11.
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