Dept. of Molecular and Medical Genetics
Oregon Health & Science University
The objective of this study is to examine, in light of the expression of multiple p53 family member isoforms, the specific role of p73 in malignant conversion, cellular response to DNA damage, and direct or indirect cooperation with other p53 family members in a clonal model of epidermal carcinogenesis. We first focused on the role of p73 in malignant conversion. Whether sporadic or siRNA induced, loss of p73 in initiated p53+/+ keratinocytes lead to conversion to squamous cell carcinoma (SCC) in vivo which was reversible upon reconstitution of TAp73Î± but not ÎNp73Î±. Second, we investigated the cellular response to ionizing radiation (IR) in the presence and absence of p73, showing that loss of p73 at malignant conversion was associated with resistance to IR in vitro. The loss of radiation sensitivity and malignant conversion was characterized by reduced steady state DNA binding levels of transcriptionally active p63 isoforms to the p21 promoter, failure to induce specific p53 family transcriptional targets, and failure to arrest in G1. Reconstitution of TAp73Î±, but not ÎNp73Î±, increased steady state DNA binding capabilities of TAp63Î², TAp63Î³, and ÎNp63Î³, and steady state levels of p53 family target mRNA, but did not restore cellular sensitivity to IR. We thus uncovered a functional cooperation between TA isoforms of p73 and p63 and showed that p73-mediated DNA damage response was uncoupled from its tumor suppressive role. We observed preferential DNA binding of the inhibitory ÎNp63Î± isoform both in vitro and invivo in SCC suggesting that in the absence of TAp73Î± a balance is tipped toward DNA binding of the inhibitory isoforms. Third, we studied the role of the p53 family inkeratinocyte response to UVB. Tumorigenic cells lacking p73 that were resistant to IR remained sensitive to UVB, accompanied by DNA binding of the TAp63Î³ isoform, suggesting that keratinocyte response to UVB is not dependent upon p73 and suggesting a hierarchy of p53 family member responses to DNA damage. Finally, we examined TAp73Î± interaction with the p53 family inhibitor Mdm2. Mdm2 was in complex with DNA-bound p53 family members in malignant cells, but reconstitution of cells withTAp73Î± correlated with removal of Mdm2 from the complex, making them more like primary keratinocytes or initiated cells. Like the initiated cells, cells expressing TAp73Î± were refractory to treatment with the Mdm2-p53 inhibitor Nutlin-3 while cells lacking p73 expression or expressing ÎNp73Î± were sensitive. Thus, we suggest that p73 may be acting as a molecular shield to keep p53 family member inhibitors, such as ÎNp63Î± andMdm2, at bay. Further understanding of p53 family interplay in tumor development and DNA damage response could lead to new therapies or optimization of current therapeutic strategies in solid tumors of epithelium, particularly where deregulation or loss of p63 and p73 expression is associated with increased tumor invasiveness, treatment resistance, and poor patient prognosis.
School of Medicine
Johnson, Jodi L., "The p53 family interacting pathways in carcinogenesis and cellular response to DNA damage" (2007). Scholar Archive. 309.