Jill E. Kucab

Linking environmental carcinogen exposure to TP53 mutations in human tumours using the human TP53 knock-in (Hupki) mouse model

TP53 is one of the most commonly mutated genes in human tumours. Variations in the types and frequencies of mutations at different tumour sites suggest that they may provide clues to the identity of the causative mutagenic agent. A useful model for studying human TP53 mutagenesis is the partial human TP53 knock‐in (Hupki) mouse containing exons 4–9 of human TP53 in place of the corresponding mouse exons. For an in vitro assay, embryo fibroblasts from the Hupki mouse can be examined for the generation and selection of TP53 mutations because mouse cells can be immortalized by mutation of Tp53 alone. Thus far, four environmental carcinogens have been examined using the Hupki embryo fibroblast immortalization assay: (a) UV light, which is linked to human skin cancer; (b) benzo[a]pyrene, which is associated with tobacco smoke‐induced lung cancer; (c) 3‐nitrobenzanthrone, a suspected human lung carcinogen linked to diesel exposure; and (d) aristolochic acid, which is linked to Balkan endemic nephropathy‐associated urothelial cancer. In each case, a unique TP53 mutation pattern was generated that corresponded to the pattern found in human tumours where exposure to these agents has been documented. Therefore, the Hupki embryo fibroblast immortalization assay has sufficient specificity to make it applicable to other environmental mutagens that putatively play a role in cancer aetiology. Despite the utility of the current Hupki embryo fibroblast immortalization assay, it has several limitations that could be addressed by future developments, in order to improve its sensitivity and selectivity.

The genome as a record of environmental exposure

Whole genome sequencing of human tumours has revealed distinct patterns of mutation that hint at the causative origins of cancer. Experimental investigations of the mutations and mutation spectra induced by environmental mutagens have traditionally focused on single genes. With the advent of faster cheaper sequencing platforms, it is now possible to assess mutation spectra in experimental models across the whole genome. As a proof of principle, we have examined the whole genome mutation profiles of mouse embryo fibroblasts immortalised following exposure to benzo[a]pyrene (BaP), ultraviolet light (UV) and aristolochic acid (AA). The results reveal that each mutagen induces a characteristic mutation signature: predominantly G→T mutations for BaP, C→T and CC→TT for UV and A→T for AA. The data are not only consistent with existing knowledge but also provide additional information at higher levels of genomic organisation. The approach holds promise for identifying agents responsible for mutations in human tumours and for shedding light on the aetiology of human cancer.

Evidence of exposure to aristolochic acid in patients with urothelial cancer from a Balkan endemic nephropathy region of Romania

Recently, chronic Aristolochia poisoning was found responsible for the aetiology of Balkan endemic nephropathy (BEN) in Croatia, Serbia, and Bosnia, and diet was the likely route of exposure to aristolochic acid (AA). BEN, often associated with an increased incidence of upper urinary tract carcinoma (UUC), also affects residents of certain rural villages in Romania. AA is a nephrotoxin and human carcinogen that forms DNA adducts after metabolic activation, which induce characteristic TP53 mutations in urothelial tumours. Here we present the first evidence linking AA exposure to UUC in residents of an endemic region in the Romanian Mehedinti County. DNA was extracted from kidney and tumour tissue of seven patients who underwent nephroureterectomy for UUC and resided in BEN villages (endemic group). Five patients with UUC from nonendemic villages served as controls. AA‐DNA adducts (7‐(deoxyadenosin‐N6‐yl)‐aristolactam I), established biomarkers of AA exposure, were identified by 32P‐postlabelling in renal DNA of six patients from the endemic group and in one of the nonendemic group (adduct levels ranged from 0.3 to 6.5 adducts per 108 nucleotides). Additionally, an A to T transversion in TP53, a base substitution characteristic of AA mutagenic activity was found in urothelial tumour DNA of one patient from the endemic group. Our results provide a molecular link to the cause of urothelial tumours in BEN regions of Romania indicating that AA is the common aetiological agent for BEN across its numerous geographical foci. Environ. Mol. Mutagen., 2012.

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

Highlights • We have generated Xpa-Null Hupki (Human TP53 knock-in) mouse embryo fibroblasts (HUFs).• Xpa-Null HUFs can be used to study the impact of nucleotide excision repair on TP53 mutagenesis.• Xpa-Null HUFs exhibit increased sensitivity to benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE).• BPDE-induced TP53 mutagenesis is enhanced on the transcribed strand in Xpa-Null HUFs.• Several TP53 codons mutated by BPDE in HUFs are mutational hotspots in smokers’ lung cancer.

Metabolic activation of diesel exhaust carcinogens in primary and immortalized human TP53 knock‐in (Hupki) mouse embryo fibroblasts

Approximately 50% of human tumors have a mutation in TP53. The pattern and spectra of TP53 mutations often differ between cancer types, perhaps due to different etiological factors. The Hupki (human TP53 knock‐in) mouse embryo fibroblast (HUF) immortalization assay is useful for studying mutagenesis in the human TP53 gene by environmental carcinogens. Prior to initiating an immortalization assay, carcinogen treatment conditions must be optimized, which can require a large number of cells. As primary HUF cultures senesce within 2 weeks, restricting their use, we investigated whether immortalized HUFs retaining wild‐type TP53 can be surrogates for primary HUFs in initial treatment optimization. DNA damage by eight compounds found in diesel exhaust, benzo[a]pyrene, 3‐nitrobenzanthrone, 1‐nitropyrene, 1,3‐dinitropyrene, 1,6‐dinitropyrene, 1,8‐dinitropyrene, 6‐nitrochrysene, and 3‐nitrofluorene, was assessed by 32P‐postlabeling and the alkaline comet assay in primary HUFs and in an immortal HUF cell line J201. For most compounds, higher levels of DNA adducts accumulated in J201 cells than in primary HUFs. This difference was not reflected in the comet assay or by cell viability changes. Experiments in three additional immortal HUF cell lines (AAI49, U56, and E2‐143) confirmed strong differences in DNA adduct levels compared with primary HUFs. However, these did not correlate with the protein expression of Nqo1 or Nat1/2, or with gene expression of Cyp1a1 or Cyp1b1. Our results show that using immortal HUFs as surrogates for primary HUFs in genotoxicity screening has limitations and that DNA adduct formation is the best measure of genotoxicity of the nitro‐polycyclic aromatic hydrocarbons tested in HUFs. Environ. Mol. Mutagen. 2012.

Comparison of the metabolic activation of environmental carcinogens in mouse embryonic stem cells and mouse embryonic fibroblasts

Highlights • ES cells and MEF have the metabolic competence to activate environmental carcinogens.• Carcinogen-induced genotoxicity in MEFs is higher than in ES cells.• MEFs have higher metabolic capacity than ES cells.• Metabolic capacity depends on the carcinogen studied.

TP53 and lacZ mutagenesis induced by 3-nitrobenzanthrone in Xpa-deficient human TP53 knock-in mouse embryo fibroblasts

Highlights • We examine TP53 and lacZ mutagenesis induced by 3-nitrobenzanthrone (3-NBA).• We utilize Hupki (Human TP53 knock-in) fibroblasts (HUFs) wild-type and null for Xpa.• Xpa-Null HUFs are more sensitive to 3-NBA than Xpa-WT HUFs.• 3-NBA induces a similar TP53 and lacZ mutant frequency in Xpa-WT and Xpa-Null HUFs.• 3-NBA induces mutations at TP53 codons that are hotspots in smokers’ lung cancer.

Nutlin-3a selects for cells harbouring TP53 mutations

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.