Poster Presentation 25th Lorne Cancer Conference 2013

Elucidating the mechanisms of gene inactivation in a murine p53 shRNA-based Osteosarcoma model (#293)

Alvin JM Ng 1 2 , Anthony J Mutsaers 1 , T John Martin 1 2 , Carl R Walkley 1 2
  1. St Vincent's Institute of Medical Research, Fitzroy, VIC, Australia
  2. Department of Medicine (St Vincent's Hospital), University of Melbourne, Parkville, VIC, Australia

Background: We have previously reported on characterising a novel mouse model of human Osteosarcoma (OS) based on the expression of a p53 shRNA transgene (p53.1224) in bone cells1 . Interestingly, endogenous p53 transcripts were detected in OS tumours but were not found to be functional. In this study, we investigate putative mechanisms of its inactivation. Methods: Mice expressing a p53 shRNA transgene (p53.1224) from a tetracycline-regulated promoter were bred with Osterix-Cre transgenic mice. The expression of tetracycline-regulated transactivator (tTA), Cre-GFP and p53.1224 shRNA were therefore restricted to the osteoblastic-lineage. OS tumour-derived cell lines were treated with pharmacological antagonists or siRNA against p53 inhibitors. Results: Twist1 (a known inactivator of p53) is highly expressed in shRNA-OS whole tumours and tumour-derived cell lines. However, the expression of p53-downstream genes was not elevated in Twist1 siRNA-treated OS cells. Furthermore, a lack of synergy was seen in p21 levels from OS cells treated with Doxorubicin and Nutlin3 (a Mdm2 antagonist). Conclusion: The p53 gene in the shRNA OS model is not inactivated via the Twist1 or Mdm2 pathway. Although it appears to be of a “wildtype” status, future experiments will focus on elucidating the mechanism of p53 inactivation in the shRNA OS model.

  1. AJ Mutsaers et al. 2012. Lorne Cancer