To examine the importance of MDM4 in regulating p53 stability in response to DSBs Wang and colleagues generated an MDM4-mutant mouse in which three key serine residues that are phosphorylated by the DNA damage response kinases ataxia-telangiectasia mutated (ATM; Ser402) and CHK2 (Ser341 and Ser367) were mutated to alanine (Mdm43SA mice). These mice were not tumour-prone and did not exhibit any defects in development. Using mouse embryonic fibroblasts (MEFs) derived from embryonic day 13 Mdm43SA embryos the authors found that MDM4-3SA was resistant to DSB-induced degradation, which correlated with reduced p53 levels. Next, they assessed whether p53 activity was impaired in response to DSBs in MDM4-3SA MEFs and thymocytes by analysing the induction of the p53 target genes
Cdkn1a
(which encodes the checkpoint protein p21) and
Puma
(which encodes a pro-apoptotic protein and is also known as Bbc3). They found that total mRNA levels of Cdkn1a and Puma were significantly reduced. The MEFs exhibited impaired (but not absent) DNA damage-induced cell cycle checkpoint arrest, and the thymocytes exhibited reduced DNA damage-induced apoptosis. Furthermore, Mdm43SA mice were largely resistant to whole-body (10 Gy) irradiation, which causes lethality in wild-type mice.
Heterozygous loss of Mdm4 has previously been shown to delay
Myc
-induced lymphomagenesis, indicating that MDM4 is also important in regulating p53 activation in response to oncogene-induced stress. Therefore, Wang and colleagues crossed Mdm43SA mice with mice that expressed a tagged Myc cDNA inserted upstream of the immunoglobulin heavy chain enhancer (iMycEμ; a common translocation in human Burkitt's lymphoma), and found that expression of MDM4-3SA significantly accelerated lymphomagenesis. Using age-matched pre-tumour littermates the authors found that the levels of apoptosis were mostly unchanged, but that expression of MDM4-3SA resulted in an increased proportion of cells in S phase with activated DNA damage response signalling. Moreover, unlike tumours from iMyc Eμ mice, in which the p53 pathway is often mutated, tumours from Mdm43SA;iMyc Eμ mice did not have mutated p53, indicating that MDM4 phosphorylation and degradation is important for the full activation of the p53 response to the effects of oncogene-induced hyper-proliferation.
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