Unpicking a Gordian knot

The transcription factor and tumour suppressor p53 is involved in inducing apoptosis in response to acute DNA damage, but recent publications have indicated that this response, which requires the induction of p53 target genes, is not essential for preventing tumour development. Colleen Brady, Laura Attardi and colleagues have generated a new series of knock-in mice with compromised p53 gene transactivation to further address how p53 suppresses tumour formation.

p53 has two transactivation domains (TADs), and Attardi and colleagues made and used a series of p53 transactivation mutants — L25Q:W26S (p53^25,26) and F53Q;F54S (p53^53,54) — to mutate the first and second TADs, respectively. Conditional expression of these genes from the endogenous Trp53 promoter in mouse embryonic fibroblasts (MEFs) was used to examine the effect of the loss of each TAD on gene expression by microarray analysis. MEFs expressing p53^53,54 had a similar expression profile to wild-type MEFs, indicating that the mutation of the second TAD alone does not compromise gene transactivation. MEFs expressing p53^25,26 had a gene expression profile that was intermediate between wild-type and p53-null MEFs, and further examination indicated that p53 target genes, such as Noxa and Puma, were expressed at very low levels, but that the expression of the pro-apoptotic p53 target gene Bax was similar to wild-type MEFs; this was in agreement with previously published studies using this mutant. MEFs expressing p53 with both TADs mutated (p53^25,26,53,54) had an expression profile similar to p53-null MEFs, indicating that mutation of both TADs abolishes transactivation.

unlike the response to acute DNA damage, induction of senescence in vitro does not seem to require the full p53 transactivation gene profile.

What biological effects do these mutants have? In vivo analyses in mice that were homozygous for each p53 mutant showed that the expression of p53^25,26 or p53^25,26,53,54 did not induce apoptosis in either the thymus or the small intestine after exposure to 5 Gy of whole-body radiation, whereas apoptosis in p53^53,54 mice remained intact. Thus, the first, but not the second, TAD is essential for the response to acute radiation-induced damage, and the induction of a full complement of p53 target genes seems to be important for the induction of apoptosis.

p53 is also activated in response to oncogenic signals. Expression of HRAS^V12 in MEFs induces p53-dependent senescence, and this remained intact in both p53^25,26- and p53^53,54-expressing MEFs, but was absent in p53^25,26,53,54 MEFs. Therefore, unlike the response to acute DNA damage, the induction of senescence in vitro does not seem to require the full p53 transactivation gene profile. Similar results were evident in vivo using a Kras^G12D mouse model of lung cancer — tumour growth was suppressed in mice expressing either p53^25,26 or p53^53,54, but not in p53^25,26,53,54 mice.

As these results indicated that some p53-dependent gene expression is required for tumour suppression in response to oncogene activation (because the p53^25,26 protein is tumour suppressive but the p53^25,26,53,54 protein is not), the authors compared gene expression profiles of the p53-mutant MEFs with each other and with human tumours that had wild-type or mutant p53. From an initial list of 130, 14 genes the expression of which was completely or partially retained in p53^25,26-mutant cells were analysed further. These genes were involved in signalling pathways, cytoskeletal pathways and DNA repair, and knockdown of some of these genes in transformed MEFs enabled tumour growth in immunocompromised mice, indicating that they could functionas tumour suppressor genes.

These results indicate that the transcription of specific p53-dependent genes is required for the tumour suppressive activity of p53 in response to oncogene activation, and that the transcription of most p53-dependent genes is required for the response to acute DNA damage, but this acute response seems to be dispensable for tumour suppression. Use of these mutants to further dissect the p53 response seems set to uncover new p53-dependent target genes that are required for tumour suppression.