This database currently lists around 30,000 mutations in human cancers. The International Agency for Research on Cancer (IARC) curates a database ( of all mutations found in the TP53 gene published in the scientific literature. These are mostly missense mutations occurring in the DNA binding domain encoded by exons 5–8. TP53 is the most commonly mutated gene in cancer with around 50% of all human tumours harbouring a mutation in TP53. The function of p53 can be altered by mutations in the TP53 gene that encodes for p53. By preventing the growth of stressed and damaged cells, p53 plays a vital role in tumour suppression. This activation leads to a variety of outcomes such as cell cycle arrest, senescence or apoptosis depending on the severity of the damage. The transcription factor p53 is usually kept at low levels in normal, unstressed cells, but it is stabilised and activated in response to certain stresses (e.g., DNA damage). The approach presented helps to deepen our understanding of human cancer aetiology. The TP53 mutation patterns induced by these mutagens in the HIMA corresponded to those found in human tumours from patients exposed to these mutagens. Environmental mutagens that have demonstrated and validated the utility of the HIMA include ultraviolet radiation, aristolochic acid, and benzopyrene.

TP53 mutation spectra generated can be compared with those of human tumours recorded in the International Agency for Research on Cancer TP53 mutation database.

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. 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. 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. Here, we present a robust protocol for studying TP53 mutagenesis utilising human TP53 knock-in (Hupki) mouse embryonic fibroblasts (HUFs). The tumour suppressor gene TP53 is the most frequently mutated gene in human tumours. DNA in dividing cells is prone to mutagenesis, with mutations making key contributions to human disease including cancer.

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