Transcriptional regulation by p53 is critical for p53-mediated tumour suppression; however, p53-mediated transactivation has been dissociated from p53-mediated biological processes including apoptosis, DNA repair, and differentiation. We compared the effects of a mutant allele, p53 QS-val135, containing a double mutation in the amino-terminus abrogating transactivation activity and a modification at amino acid 135 partially affecting DNA binding, to complete loss of p53. We applied in vitro endpoints correlated with epithelial tumourigenesis and an in vivo assay of tumour phenotype to assess whether loss of p53-mediated transcriptional regulation underlies the malignant phenotype of p53-/-/v-ras Ha-overexpressing keratinocytes. Transactivation deficiency of p53QS-val135 was confirmed by reporter gene assays in fibroblasts and differentiating keratinocytes. Ras oncogene-induced senescence was lost in both p53QS-val135/QS-val135 and p53-/- keratinocytes. Similarly, p53QS-val135/QS-val135, like p53-/-, cooperated with v-rasHa to enhance malignant conversion. The tumours arising in p53QS-val135/QS-val135 keratinocytes displayed strong nuclear p53 expression; thus, the p53QS-val135 allele was maintained and the deficient transactivation function of the expressed p53QS mutant protein was supported by absence of p21waf1 in these tumours. The p53 QS-val135 allele did not confer a dominant-negative phenotype, as p53+/QS-val135 keratinocytes senesced normally in response to v-rasHa expression and formed benign tumours. While p53-/- keratinocytes displayed diminished response to TGF-β, p53 QS-val135/QS-val135 and p53+/+ keratinocytes responded equivalently, indicating that the requirement for p53 in maximizing TGF-β-mediated growth regulation is independent of its transactivation domain and that the ability of keratinocytes to respond to TGF-β is insufficient to suppress the malignant phenotype in this model. Furthermore, TGF-β enhances p53QS-induced activation of a dual p53-TGF-β responsive reporter in a keratinocyte cell line. These findings support an essential role for p53-mediated transcriptional regulation in suppressing malignancies arising from ras-induced skin tumours, consistent with previous findings in spontaneous carcinogenesis in other organs, and highlight the potential importance of senescence for tumour suppression in vivo.
All Science Journal Classification (ASJC) codes
- Pathology and Forensic Medicine