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.
Bond
J A
JA
Department of Pathology, University of Wales College of Medicine, Cardiff, UK.
Webley
K
K
Wyllie
F S
FS
Jones
C J
CJ
Craig
A
A
Hupp
T
T
Wynford-Thomas
D
D
D000911:Antibodies, Monoclonal / Q000276:immunology; D016477:Artifacts; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects; D004247:DNA / Q000235:genetics / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair / Q000528:radiation effects*; D004261:DNA Replication / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000528:radiation effects*; D016158:Genes, p53; D006801:Humans; D007553:Isotope Labeling; D010710:Phosphates / Q000494:pharmacology*; D010761:Phosphorus Radioisotopes / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D012107:Research Design; D015533:Transcriptional Activation / Q000187:drug effects*; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D016159:Tumor Suppressor Protein p53; D004247:DNA