Effect of mutation and conformation on the function of p53 in colorectal cancer.

In vitro studies have shown that immunoprecipitation with conformation-specific antibodies allows discrimination between different forms of p53; however, the significance of this has not been determined for human tumours. This study therefore examined p53 conformation in colorectal tumours and correlated this with mutational status and evidence of in vivo p53 downstream activity. Moreover, it was shown that for in vitro cell lines, DNA-damaging agents induce wild-type p53 to form a mutant conformation (PAb240+), with a concomitant rise in p21(WAF-1) expression. Induction of p53-mediated apoptosis, on the other hand, is associated with a wild-type conformation (PAb1620+). These results were confirmed for wild-type p53 in colorectal tumours. A range of p53 point mutations were found in exons 5-8 in colorectal tumours. Mutants with a wild-type conformation gave weak immunohistochemical staining, whereas mutations with flexible conformation (240+/1620+) gave intense positivity. Interestingly, this latter group of flexible mutants was also associated with features of poor prognosis. These studies show that not all p53 mutants are equal; thus, knowledge of the p53 status of a tumour may be required for a more precise prediction of prognosis and response to treatment for cancer patients.

Posttranslational modifications of p53 in replicative senescence overlapping but distinct from those induced by DNA damage.

Replicative senescence in human fibroblasts is absolutely dependent on the function of the phosphoprotein p53 and correlates with activation of p53-dependent transcription. However, no evidence for posttranslational modification of p53 in senescence has been presented, raising the possibility that changes in transcriptional activity result from upregulation of a coactivator. Using a series of antibodies with phosphorylation-sensitive epitopes, we now show that senescence is associated with major changes at putative regulatory sites in the N and C termini of p53 consistent with increased phosphorylation at serine-15, threonine-18, and serine-376 and decreased phosphorylation at serine-392. Ionizing and UV radiation generated overlapping but distinct profiles of response, with increased serine-15 phosphorylation being the only common change. These results support a direct role for p53 in signaling replicative senescence and are consistent with the generation by telomere erosion of a signal which shares some but not all of the features of DNA double-strand breaks.

p53-Dependent growth arrest and altered p53-immunoreactivity following metabolic labelling with 32P ortho-phosphate in human fibroblasts.

The tumour suppressor gene p53 plays a major role in the cellular response to DNA damage, mediating growth arrest and/or apoptosis. Phosphorylation of the protein occurs at numerous sites in vivo and is likely to be a major mechanism for modulation of its activity as a transcriptional transactivator. Not surprisingly, therefore, p53 has been intensively studied by 32P metabolic labelling. Here we show however, using normal human fibroblasts, that typical labelling conditions induce (i) a p53-dependent inhibition of DNA synthesis and (ii) an increase in the cellular content of p53 protein detectable by the phosphorylation-sensitive antibody DO-1 but not by antibody DO-12. These data demonstrate for the first time that 32P labelling is sufficient to induce a biologically-significant, p53-mediated cellular response and strongly suggest that it perturbs the phosphorylation state of p53 which it is being used to measure. This highlights the need to re-evaluate earlier data by non-radioactive approaches using phospho-specific antibodies.