Poster Presentation 35th Lorne Cancer Conference 2023

Using new gene reporter mice and RNAseq analysis to uncover how p53 determines different cellular fates – cell death vs cell cycle arrest (#226)

Elizabeth Lieschke 1 2 , Andrew Kueh 1 2 , Marco Herold 1 2 , Annabella Thomas 1 2 , Georgia Atkin-Smith 1 2 , Edwin Hawkins 1 2 , Alexandra Garnham 1 , Pedro Baldoni 1 2 , Gordon Smyth 1 2 , Gemma L Kelly 1 2 3 , Andreas Strasser 1 2 3
  1. The Walter and Eliza Hall Institute, Parkville, VIC, Australia
  2. Medical Biology, The University of Melbourne, Melbourne
  3. GK and AS contributed equally to this work,

Upon detecting stress signals within a cell, TP53/p53 can transcriptionally activate multiple cellular processes, such as apoptotic cell death, cell cycle arrest, and cellular senescence, to prevent the damaged cell becoming malignant. However, we do not understand why one cellular process will dominate in a particular cell or the factors that mediate these responses.

 

Firstly, we investigated the transcriptional changes that occur after p53 activation using the MDM2 inhibitor, nutlin-3a, in three primary murine cell types that undergo cell death and three cell types that survive and undergo cell cycle arrest/senescence. Surprisingly, we observed 119 genes were differentially expressed in all cell types after p53 activation, despite their cellular outcomes. These genes included the initiators of p53-mediated apoptosis, Puma/Bbc3 and Noxa/Pmaip1, and p53-mediated cell cycle arrest Cdkn1a/p21. However, in the cell types that undergo cell cycle arrest, we also observed an increase in the pro-survival genes Mcl-1and Bcl-x at the RNA and protein levels. This upregulation was not seen in the cell types that undergo apoptosis.

 

To visualise how these processes operate in vivo, we generated p53-response gene fluorescent reporter mice. To observe apoptosis, we replaced the coding sequence of Puma/Bbc3 with tdTomato. To observe cell cycle changes, we inserted an IRES followed by the GFP coding sequence after the Cdkn1a/p21 coding region. In agreement with our RNA expression data, after p53 activation with nutlin-3a or DNA damaging drugs, we observed cells upregulate both GFP and tdTomato, irrespective of their p53-instructed outcome. We are currently generating reporter mice to visualise other p53-activated cellular responses, including coordination of cell metabolism (Sestrin2-IRES-citrine), DNA damage repair (Polk-IRES-BFP) and negative feedback (Mdm2-IRES-Katushka).

 

Together these findings demonstrate that the traditional regulators of p53-mediated processes are not solely responsible for cellular outcome upon p53 activation. Instead, we hypothesise that cells survive by indirectly increasing expression of the pro-survival genes Bcl-x and Mcl-1, allowing time for cell cycle arrest, adaptation of metabolism and DNA damage repair to occur.