The effect of MDM2 overexpression on the cellular response to etoposide treatment

We aimed to study the effect of mutational and non-mutational p53 inactivation on the response of hematopoietic progenitor cells to genotoxic treatment. In order to study this in vitro, we generated HSPC lines by enforced ER-Hoxb8 expression in bone marrow cells derived from transgenic Trp53-fl-R245W-GFP mice (in hetero- or homozygous state). Upon induction of the Trp53-R245W missense mutation, these cells carry mutational mono- or biallelic loss of wild-type p53. In the genetic background of mono- or biallelic Trp53 mutations, we additionally transduced these cells lentivirally with a construct overexpressing murine Mdm2 or a control vector. This leads to non-mutational inactivation of functional p53. Thereby, we aimed to compare the transcriptional response to genotoxic treatment in cells carrying mono- or biallelic Trp53 mutations with or without non-mutational p53 inactivation through Mdm2 overexpression. Hoxb8 cells, immunophenotypically resembling murine granulocyte-macrophage progenitor cells, were induced to carry monoallelic or biallelic Trp53-R245W mutations, each in combination with either an Mdm2 overexpressing or a control vector. These cells were treated with 10 uM etoposide for 3 hours, after which RNA was isolated and subjected to bulk RNA sequencing.