Characterising mutational spectra of carcinogens in the tumour suppressor gene tp53 using human tp53 knock-in (Hupki) mouse embryo fibroblasts

TY - JOUR

T1 - Characterising mutational spectra of carcinogens in the tumour suppressor gene tp53 using human tp53 knock-in (Hupki) mouse embryo fibroblasts

AU - Hölzl-Armstrong, Lisa

AU - Kucab, Jill E.

AU - Korenjak, Michael

AU - Luijten, Mirjam

AU - Phillips, David H.

AU - Zavadil, Jiri

AU - Arlt, Volker M.

PY - 2019/12

Y1 - 2019/12

N2 - DNA in dividing cells is prone to mutagenesis, with mutations making key contributions to human disease including cancer. The tumour suppressor gene TP53 is the most frequently mutated gene in human tumours. Here, we present a robust protocol for studying TP53 mutagenesis utilising human TP53 knock-in (Hupki) mouse embryonic fibroblasts (HUFs). In the HUF immortalisation assay (HIMA), primary HUFs are treated with known or suspected carcinogens at 3% oxygen and then transferred to 20% atmospheric oxygen to induce senescence. Cells containing mutations (e.g., in TP53) that allow bypassing of senescence eventually emerge as immortalised clonal cell lines after 2–3 months of serial passaging. As not all immortalised HUF cells contain TP53 mutations, we developed a Nutlin-3a counter-screen to select for TP53-mutated clones prior to sequencing. TP53 mutation spectra generated can be compared with those of human tumours recorded in the International Agency for Research on Cancer TP53 mutation database. Environmental mutagens that have demonstrated and validated the utility of the HIMA include ultraviolet radiation, aristolochic acid, and benzo[a]pyrene. The TP53 mutation patterns induced by these mutagens in the HIMA corresponded to those found in human tumours from patients exposed to these mutagens. The approach presented helps to deepen our understanding of human cancer aetiology.

AB - DNA in dividing cells is prone to mutagenesis, with mutations making key contributions to human disease including cancer. The tumour suppressor gene TP53 is the most frequently mutated gene in human tumours. Here, we present a robust protocol for studying TP53 mutagenesis utilising human TP53 knock-in (Hupki) mouse embryonic fibroblasts (HUFs). In the HUF immortalisation assay (HIMA), primary HUFs are treated with known or suspected carcinogens at 3% oxygen and then transferred to 20% atmospheric oxygen to induce senescence. Cells containing mutations (e.g., in TP53) that allow bypassing of senescence eventually emerge as immortalised clonal cell lines after 2–3 months of serial passaging. As not all immortalised HUF cells contain TP53 mutations, we developed a Nutlin-3a counter-screen to select for TP53-mutated clones prior to sequencing. TP53 mutation spectra generated can be compared with those of human tumours recorded in the International Agency for Research on Cancer TP53 mutation database. Environmental mutagens that have demonstrated and validated the utility of the HIMA include ultraviolet radiation, aristolochic acid, and benzo[a]pyrene. The TP53 mutation patterns induced by these mutagens in the HIMA corresponded to those found in human tumours from patients exposed to these mutagens. The approach presented helps to deepen our understanding of human cancer aetiology.

KW - Cancer aetiology

KW - DNA adducts

KW - Embryonic fibroblasts

KW - Environmental carcinogenesis

KW - Genotoxicity

KW - Immortalisation

KW - Mouse model

KW - Mutation

KW - Nutlin-3a

KW - Tumour suppressor p53

UR - http://www.scopus.com/inward/record.url?scp=85085315870&partnerID=8YFLogxK

U2 - 10.3390/mps2040085

DO - 10.3390/mps2040085

M3 - Article

AN - SCOPUS:85085315870

SN - 2409-9279

VL - 2

SP - 1

EP - 41

JO - Methods and Protocols

JF - Methods and Protocols

IS - 4

M1 - 85

ER -