Search

Multi-stage tumourigenesis is associated with the accumulation of co-operating genetic lesions. One of the best studied models of carcinogenesis is experimentally induced tumours in the skin of mice. In tumorigenesis of the skin, activated Ras co-operates with mutations that inactivate the tumour suppressor p53. The absence of the cyclin dependent kinase inhibitor p21Cip1, a p53 target gene, has also been shown to co-operate with oncogenic ras in the induction of aggressive and relatively undifferentiated tumours in vivo. However, the molecular basis for these co-operations remains unresolved. Activation of oncogenes, including Ras, Myc and E1A, have been reported to stabilise and activate p53 via induction of the tumour suppressor p19ARF. Therefore, it is thought that ARF might be the specific link between oncogene activation and induction of p53. Since most malignancies are epithelial in origin and ms and p53 mutations are most frequently associated with epithelial derived tumours, I investigated the molecular mechanisms involved in co-operation between Ras, p53 and p2l Cip1 in the skin and the potential involvement of p19ARF. Primary mouse keratinocytes provide an excellent system to study growth and differentiation in the skin. When cultured in low calcium medium they behave similarly to the cells found in the basal layer of the epidermis. Here I show that activation of the Raf/MAP kinase pathway in primary mouse keratinocytes leads to a G1/G2 cycle arrest and to terminal differentiation. This is preceded by an increase in p21 Cip1 and p53 protein levels while p16INK4A and p19ARF levels appear unaffected. Raf activation in keratinocytes lacking p53 or p21Cip1 genes leads to expression of differentiation markers, but the cells do not cease to proliferate. Thus, loss of p53 or p21Cip1 function is necessary to disable growth-inhibitory Raf/Map kinase signalling, but a cell cycle arrest is not obligatory for the onset of terminal differentiation in keratinocytes. However, the response to Raf in p19ARF-/- keratinocytes was indistinguishable from wild type controls. Thus, p19ARF is not essential for Raf-induced p53 induction and cell cycle arrest in keratinocytes, indicating that oncogenes engage p53 activity via multiple mechanisms.