p53-dependent growth arrest and altered p53-immunoreactivity following metabolic labelling with P-32 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 ill vivo and is likely to be a major mechanism for modulation of its activity as a transcriptional transactivato r. Not surprisingly, therefore, p53 has been intensively studied by P-32 me tabolic labelling. Here we show however, using normal human fibroblasts, th at typical labelling conditions induce (i) a p53-dependent inhibition of DN A synthesis and (ii) an increase in the cellular content of p53 protein det ectable by the phosphorylation-sensitive antibody DO-1 but not by antibody DO-12. These data demonstrate for the first time that P-32 labelling is suf ficient to induce biologically-significant, p53-mediated cellular response and strongly suggest that it perturbs the phosphorylation state of p53 whic h it is being used to measure. This highlights the need to re-evaluate earl ier data by nonradioactive approaches using phospho-specific antibodies.