Targeted hPTTG overexpression in vivo induces reduced cellular proliferation and p53 stabilisation resulting in decreased thyroid size
Gavin Ryan, Gregory Lewy, Martin Read, Robert Seed, Neil Sharma, Vicki Smith, Jim Fong, Perkin Kwan, Jayne Franklyn, Christopher McCabe & Kristien Boelaert
The human pituitary tumor transforming gene (hPTTG) plays important roles in tumourigenesis through its function as a securin and by interaction with the tumour suppressor gene p53. We have previously indentified hPTTG expression as a prognostic indicator for differentiated thyroid cancer. We recently generated a transgenic mouse model overexpressing hPTTG targeted to the thyroid gland via a thyroglobulin promoter to delineate PTTG effects on thyroid tumourigenesis in vivo. Interestingly, following screening of 82 pups (n=12 l), from heterozygote hPTTG mice matings, no homozygote mice were identified. Furthermore, hPTTG mouse litter sizes were significantly reduced compared with WT litters (n=6.83 vs n=9.35, P=0.001), suggesting homozygous thyroidal PTTG overexpression may be embryonically lethal. At 6 weeks transgenic mice exhibited significantly reduced thyroid weights (0.86-fold, P=0.005, n=36) when compared with age and gender matched WT mice. To determine if this reduction in size was due to lower cellular proliferation rates we assessed PCNA and cyclin D1 expression in excised thyroids. Cyclin D1 levels, measured by immunohistochemisty, were significantly reduced (0.59-fold, P=0.015) while PCNA, measured by western blotting, showed a non-significant reduction in expression (0.61-fold, P=0.07) in transgenic mice. Expression of p53, an important gene in DNA repair and cell cycle regulation, was significantly increased at both mRNA (1.7-fold, P=0.02) and protein level (2.1-fold, P=0.02) in hPTTG mice. Furthermore expression of p21 mRNA, an effector of p53 action and an inhibitor of cyclin-dependent kinases and PCNA expression was significantly increased in transgenic mouse thyroids (1.6-fold, P=0.04, n=7). In vitro experiments confirmed significantly increased p21 mRNA expression (1.9-fold, P=0.01) following hPTTG transfection in the K1 human papillary thyroid cancer cell line.
In conclusion, targeted thyroidal hPTTG overexpression results in reduced cellular proliferation and upregulation of p53 and p21, suggesting activation of DNA damage repair pathways and apoptosis.