Functional disruption of the tumour suppressor p53 has a critical role in promoting the development of most cancers. The proto-oncogenes PBF and PTTG1 both regulate p53 activity, but the relative contribution of each gene in influencing p53 function has not been delineated, especially in thyroid cancer where both proto-oncogenes are commonly overexpressed. To better understand the interplay between PTTG1, PBF and p53 in vivo, we examined p53 responses in primary thyrocytes cultured from transgenic mice overexpressing PBF (PBF-Tg) and PTTG1 (PTTG-Tg), either singly or in combination in a bi-transgenic murine model (Bi-Tg). Western blotting showed that p53 and γ-H2AX protein levels were elevated in PTTG1-Tg and BI-Tg thyrocytes (>2-fold; P<0.05). In contrast, no significant increase was observed in p53 or γ-H2AX levels in PBF-Tg thyrocytes compared to WT (P=NS). Consistent with this, a greater proportion of a panel of p53-responsive DNA repair genes were significantly down-regulated in PTTG1-Tg (30/83 genes) and BI-Tg (30/83 genes) than in PBF-Tg thyrocytes (12/83 genes). A differential p53 response was further evident following gamma-irradiation of cells, with fewer significant mRNA changes occurring in PTTG1-Tg (0/10 genes; P=NS) and BI-Tg (4/10 genes; P<0.05) than in WT primary thyrocytes (10/10 genes; P<0.01). By comparison, irradiation of PBF-Tg thyrocytes gave the greatest reduction in mRNA levels (6/10 genes; P<0.05) for genes such as Chek1 (4.4-fold; P<0.01) and Rad51 (8.4-fold; P<0.01). We therefore examined potential associations between PBF and DNA repair genes in human thyroid tumours. Importantly, a significant correlation was apparent between PBF and Chek1 (R=0.44; P<0.05; N=22), Fancg (R=0.78; P<0.001; N=22) and Mutyh (R=0.62; P<0.05; N=22). Together our data reveal for the first time that PBF and PTTG1 mediate distinct p53 response profiles in vivo. These results offer important insights for understanding the impact of proto-oncogenes on thyroid tumorigenesis and for identifying new tumour biomarkers.