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000119266 0247_ $$2ISSN$$a1097-0215
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000119266 041__ $$aeng
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000119266 1001_ $$0http://orcid.org/0000-0002-0341-4490$$aKucab, Jill E$$b0
000119266 245__ $$aNutlin-3a selects for cells harbouring TP53 mutations.
000119266 260__ $$aBognor Regis$$bWiley-Liss$$c2017
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000119266 520__ $$aTP53 mutations occur in half of all human tumours. Mutagen-induced or spontaneous TP53 mutagenesis can be studied in vitro using the human TP53 knock-in (Hupki) mouse embryo fibroblast (HUF) immortalisation assay (HIMA). TP53 mutations arise in up to 30% of mutagen-treated, immortalised HUFs; however, mutants are not identified until TP53 sequence analysis following immortalisation (2-5 months) and much effort is expended maintaining TP53-WT cultures. In order to improve the selectivity of the HIMA for HUFs harbouring TP53 mutations, we explored the use of Nutlin-3a, an MDM2 inhibitor that leads to stabilisation and activation of wild-type (WT) p53. First, we treated previously established immortal HUF lines carrying WT or mutated TP53 with Nutlin-3a to examine the effect on cell growth and p53 activation. Nutlin-3a induced the p53 pathway in TP53-WT HUFs and inhibited cell growth, whereas most TP53-mutated HUFs were resistant to Nutlin-3a. We then assessed whether Nutlin-3a treatment could discriminate between TP53-WT and TP53-mutated cells during the HIMA (n = 72 cultures). As immortal clones emerged from senescent cultures, each was treated with 10 µM Nutlin-3a for 5 days and observed for sensitivity or resistance. TP53 was subsequently sequenced from all immortalised clones. We found that all Nutlin-3a-resistant clones harboured TP53 mutations, which were diverse in position and functional impact, while all but one of the Nutlin-3a-sensitive clones were TP53-WT. These data suggest that including a Nutlin-3a counter-screen significantly improves the specificity and efficiency of the HIMA, whereby TP53-mutated clones are selected prior to sequencing and TP53-WT clones can be discarded.
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000119266 7001_ $$0P:(DE-He78)f3bec70c95e9e3dce0f39d54b3843118$$aHollstein, Monica$$b1$$udkfz
000119266 7001_ $$aArlt, Volker M$$b2
000119266 7001_ $$aPhillips, David H$$b3
000119266 773__ $$0PERI:(DE-600)1474822-8$$a10.1002/ijc.30504$$gVol. 140, no. 4, p. 877 - 887$$n4$$p877 - 887$$tInternational journal of cancer$$v140$$x0020-7136$$y2017
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