Nutlin-3a selects for cells harbouring TP53 mutations.
TP53 mutations occur in half of all human tumours. Mutagen-induced or spontaneous TP53 mutagenesis can be studied in vitro using the human TP53 knock-in (Hupki) mouse embryo fibroblast (HUF) immortalisation assay (HIMA). TP53 mutations arise in up to 30% of mutagen-treated, immortalised HUFs; however, mutants are not identified until TP53 sequence analysis following immortalisation (2-5 months) and much effort is expended maintaining TP53-WT cultures. In order to improve the selectivity of the HIMA for HUFs harbouring TP53 mutations, we explored the use of Nutlin-3a, an MDM2 inhibitor that leads to stabilisation and activation of wild-type (WT) p53. First, we treated previously established immortal HUF lines carrying WT or mutated TP53 with Nutlin-3a to examine the effect on cell growth and p53 activation. Nutlin-3a induced the p53 pathway in TP53-WT HUFs and inhibited cell growth, whereas most TP53-mutated HUFs were resistant to Nutlin-3a. We then assessed whether Nutlin-3a treatment could discriminate between TP53-WT and TP53-mutated cells during the HIMA (n = 72 cultures). As immortal clones emerged from senescent cultures, each was treated with 10 µM Nutlin-3a for 5 days and observed for sensitivity or resistance. TP53 was subsequently sequenced from all immortalised clones. We found that all Nutlin-3a-resistant clones harboured TP53 mutations, which were diverse in position and functional impact, while all but one of the Nutlin-3a-sensitive clones were TP53-WT. These data suggest that including a Nutlin-3a counter-screen significantly improves the specificity and efficiency of the HIMA, whereby TP53-mutated clones are selected prior to sequencing and TP53-WT clones can be discarded.
King's College London, Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment & Health, London, United Kingdom.
Jill E
0000-0002-0341-4490
JE
Kucab
German Cancer Research Center (Deutsches Krebsforschungszentrum), Division of Genetic Alterations in Carcinogenesis, Heidelberg, Germany.|University of Leeds, Faculty of Medicine and Health, Leeds, United Kingdom.
Monica
M
Hollstein
King's College London, Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment & Health, London, United Kingdom.
Volker M
VM
Arlt
King's College London, Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment & Health, London, United Kingdom.
David H
DH
Phillips
D000818:Animals; D002461:Cell Line, Transformed; D002470:Cell Survival; D002471:Cell Transformation, Neoplastic / Q000187:drug effects; D002478:Cells, Cultured; D002999:Clone Cells; D004351:Drug Resistance; D005347:Fibroblasts; D055879:Gene Knock-In Techniques; D016158:Genes, p53; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D051379:Mice; D009154:Mutation; D010100:Oxygen / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors; D012641:Selection, Genetic; D015398:Signal Transduction / Q000187:drug effects
D007093:Imidazoles; D010879:Piperazines; C482205:nutlin 3; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2; D010100:Oxygen
Hupki; Nutlin-3a; TP53; immortalisation; mutation