TP53 mutations induced by BPDE in Xpa-WT and Xpa-Null human TP53 knock-in (Hupki) mouse embryo fibroblasts

Jill E Kucab,Harry Van Steeg,Mirjam Luijten,Heinz H Schmeiser,Paul A White,David H Phillips,Volker M Arlt

doi:10.1016/j.mrfmmm.2015.01.013

Abstract

Somatic mutations in the tumour suppressor gene TP53 occur in more than 50% of human tumours; in some instances exposure to environmental carcinogens can be linked to characteristic mutational signatures. The Hupki (human TP53 knock-in) mouse embryo fibroblast (HUF) immortalization assay (HIMA) is a useful model for studying the impact of environmental carcinogens on TP53 mutagenesis. In an effort to increase the frequency of TP53-mutated clones achievable in the HIMA, we generated nucleotide excision repair (NER)-deficient HUFs by crossing the Hupki mouse with an Xpa-knockout (Xpa-Null) mouse. We hypothesized that carcinogen-induced DNA adducts would persist in the TP53 sequence of Xpa-Null HUFs leading to an increased propensity for mismatched base pairing and mutation during replication of adducted DNA. We found that Xpa-Null Hupki mice, and HUFs derived from them, were more sensitive to the environmental carcinogen benzo[a]pyrene (BaP) than their wild-type (Xpa-WT) counterparts. Following treatment with the reactive metabolite of BaP, benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), Xpa-WT and Xpa-Null HUF cultures were subjected to the HIMA. A significant increase in TP53 mutations on the transcribed strand was detected in Xpa-Null HUFs compared to Xpa-WT HUFs, but the TP53-mutant frequency overall was not significantly different between the two genotypes. BPDE induced mutations primarily at G:C base pairs, with approximately half occurring at CpG sites, and the predominant mutation type was G:C>T:A in both Xpa-WT and Xpa-Null cells. Further, several of the TP53 mutation hotspots identified in smokers’ lung cancer were mutated by BPDE in HUFs (codons 157, 158, 245, 248, 249, 273). Therefore, the pattern and spectrum of BPDE-induced TP53 mutations in the HIMA are consistent with TP53 mutations detected in lung tumours of smokers. While Xpa-Null HUFs exhibited increased sensitivity to BPDE-induced damage on the transcribed strand, NER-deficiency did not enhance TP53 mutagenesis resulting from damage on the non-transcribed strand in this model.

Highlights

The tumour suppressor p53 plays a crucial role in the DNA damage response, garnering the title ‘guardian of the genome’ [1]
We hypothesised that Xpa-deficiency would increase the sensitivity of the human Tumor suppressor p53 (TP53) knock-in (Hupki) model to DNA damage normally repaired by nucleotide excision repair (NER) and thereby increase the frequency of carcinogen-induced TP53 mutations in immortalised Hupki mouse embryo fibroblasts (HUFs)
Xpa-WT and Xpa-Null mice and HUFs were treated with the ubiquitous environmental carcinogen BaP, or its reactive metabolite BPDE, which form DNA adducts (BPDE-N2-dG) that have been shown to be repaired by the NER pathway

Summary

Introduction

The tumour suppressor p53 plays a crucial role in the DNA damage response, garnering the title ‘guardian of the genome’ [1]. Disruption of the normal p53 response by TP53 mutation contributes to transformation by eliminating a key pathway of cellular growth control, enabling the survival and proliferation of stressed or damaged cells. Somatic mutations in TP53 occur in more than 50% of human cancers [4, 5]. The majority of TP53 mutations are missense and occur between codons 125 and 300, corresponding to the coding region for the DNA binding domain [6]. Exposure to some environmental carcinogens can be linked to characteristic signatures of mutations in TP53, which provide molecular clues to the aetiology of human tumours [8]

Methods

Results

Conclusion