Study of the physiological and oncogenic properties of Mdm2 and Mdm4

The p53 tumour suppressor functions prevent cancer development by inhibiting the proliferation of stressed or abnormal cells. Being a transcription factor, p53 regulates the expression of a broad range of target genes to promote several anti-proliferative responses including cell cycle arrest, cellular senescence and apoptosis. Since p53 is a potent inhibitor of cell growth, tight surveillance of its stability and activity is required under normal cell proliferative conditions. Central to this process are Mdm2 and Mdm4, the two main negative regulators of p53. During embryonic development, Mdm2 and Mdm4 inhibit the growth suppressive activities of p53. However, whether tight control of p53 activity is required in quiescent cells is unknown. Moreover, the exact physiological contributions of Mdm2 and Mdm4 to the regulation of p53 stability and activity remain highly controversial. Therefore, conditional inactivation of Mdm2 and Mdm4 was carried out in smooth muscle cells (SMCs) in vivo. To this end, we crossed mice harbouring Mdm2 or Mdm4 floxed alleles with mice expressing a tamoxifen inducible, SMC-specific Cre-recombinase. Upon SMC-specific inactivation of Mdm2, but not of Mdm4, mice rapidly become ill and die. Necropsy shows small intestinal dilation, and histological analyses indicate a severe reduction in the number of intestinal SMCs. Increased p53 levels and activity are detected in the remaining SMCs, and the phenotype is completely rescued on a p53-null background. Interestingly, both wild-type and Mdm2-deficient intestinal SMCs are Caspase-3 negative and therefore do not undergo Caspase-3-dependent apoptotic cell death. Altogether these data indicate that Mdm2, but not Mdm4, prevents accumulation of active p53 in quiescent SMCs and thereby the induction of p53-mediated Caspase-3-independent cell death. Conversely, Mdm2 and Mdm4 are overexpressed in diverse human tumours, most of which retain a wild-type p53 allele. A large body of evidence also suggests that elevated expression of either Mdm2 or Mdm4 is oncogenic, favouring tumour formation by inhibition of the p53 tumour suppression function. To test this possibility further in vivo, conditional Mdm2 and Mdm4 transgenic mouse lines were generated. However, although both transgenes were developed to be expressed ubiquitously and at comparable levels, only the Mdm4 transgenic protein is produced at high levels in vivo. In contrast, exogenous Mdm2 is constitutively degraded in a proteasomal-dependent manner. Surprisingly, mice homozygous for the Mdm4 transgene (Mdm4T/T) die during embryogenesis, likely due to severe vascular maturation defects. This lethality is not rescued on a p53-null background indicating that high levels of Mdm4 impact on p53-independent pathway(s) that control(s) vascular and embryonic development. In contrast, mice expressing a single copy of the Mdm4 transgene (Mdm4T/+) are viable. Importantly, the Mdm4 transgene is functional and Mouse Embryonic Fibroblasts (MEFs) derived from Mdm4T/+ embryos are, as expected, immortal. Surprisingly, however, Mdm4T/+ mice are not prone to spontaneous, radiation- or Eµ-myc-induced tumour formation. These observations suggest that the simplistic model by which elevated Mdm4 expression will systematically accelerate tumour formation needs to be revisited