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@ARTICLE{Kucab:119266,
      author       = {J. E. Kucab and M. Hollstein$^*$ and V. M. Arlt and D. H.
                      Phillips},
      title        = {{N}utlin-3a selects for cells harbouring {TP}53 mutations.},
      journal      = {International journal of cancer},
      volume       = {140},
      number       = {4},
      issn         = {0020-7136},
      address      = {Bognor Regis},
      publisher    = {Wiley-Liss},
      reportid     = {DKFZ-2017-00052},
      pages        = {877 - 887},
      year         = {2017},
      abstract     = {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.},
      cin          = {C016},
      ddc          = {610},
      cid          = {I:(DE-He78)C016-20160331},
      pnm          = {313 - Cancer risk factors and prevention (POF3-313)},
      pid          = {G:(DE-HGF)POF3-313},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:27813088},
      pmc          = {pmc:PMC5215675},
      doi          = {10.1002/ijc.30504},
      url          = {https://inrepo02.dkfz.de/record/119266},
}