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

J A Bond, K Webley, F S Wyllie, C J Jones, A Craig, T Hupp, D Wynford-Thomas

Research output: Contribution to journalArticlepeer-review

Abstract

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.
Original languageEnglish
Pages (from-to)3788-92
Number of pages5
JournalOncogene
Volume18
Issue number25
DOIs
Publication statusPublished - 24 Jun 1999

Keywords

  • Antibodies, Monoclonal
  • Artifacts
  • Cell Cycle
  • Cell Division
  • Cells, Cultured
  • DNA
  • DNA Damage
  • DNA Repair
  • DNA Replication
  • Fibroblasts
  • Gene Expression Regulation
  • Genes, p53
  • Humans
  • Isotope Labeling
  • Phosphates
  • Phosphorus Radioisotopes
  • Phosphorylation
  • Protein Processing, Post-Translational
  • Research Design
  • Transcriptional Activation
  • Tumor Suppressor Protein p53

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