On the origin of G --> T transversions in lung cancer

G-->T transversions in the TP53 gene are more common in lung cancers from smokers than in any other cancer except for hepatocellular carcinomas linked to aflatoxin. The high frequency of G-->T transversions in lung cancer has been attributed to the mutagenic action of cigarette smoke components, in particular polycyclic aromatic hydrocarbons (PAH). In a recent review [Mutat. Res. 508 (2002) 1-19], Rodin and Rodin have questioned the direct mutagenic action of PAH-like compounds and have suggested that other factors, such as selection of pre-existing endogenous mutations by smoke-induced stress, can better explain the excess of G-->T transversions in lung tumors. Their two main arguments against an involvement of PAH are that smoking may inhibit the repair of G-->T primary lesions on the non-transcribed strand and that lung cancer cell lines show a higher frequency of G-->T transversions than primary lung tumors suggesting that these mutations are not related to smoking. We illustrate here that both of these suggestions are incompatible with available evidence and that the abundance and sequence specificity of G-->T transversions in lung tumors is best explained by a direct mutagenic action of PAH compounds present in cigarette smoke.     

On the origin of p53 G:C --> T:A transversions in lung cancers

The high frequency of G-->T transversions in the p53 gene is a distinctive feature of lung cancer patients with a smoking history and is commonly believed to reflect the direct mutagenic signature of polycyclic aromatic hydrocarbon (PAH) adducts along the gene. Using the April 2000 update of the p53 mutation database of the International Agency for Research on Cancer together with the primary literature, we confirm that the frequency of p53 G-->T transversions in lung cancer of smokers is about three times higher than their frequency in lung cancer of nonsmokers and in most other smoke-unrelated cancers. In contrast, the frequency of C-->A transversions, the DNA-strand mirror counterpart of G-->T transversions, appears to be similar in virtually all human cancers. Along with other data, this strand bias leads us to suggest that smoking may inhibit repair of G-->T primary lesions on the non-transcribed strand. As to the origin of G-->T primary lesions in the p53 gene, we unexpectedly found that cell lines derived from lung cancers, but not from other cancers, demonstrate significant additional excess of G-->T transversions when compared to p53 mutations in parent primary tumors. A detailed codon-by-codon comparison provides evidence in favor of the in vitro origin of this culture-associated G-->T augmentation. Since in culture lung cancer cell lines are not exposed to the carcinogens from smoke, one would rather ascribe these new G-->T transversions to some other mutagens such as, for example, reactive oxygen and nitrogen species. These results are consistent with our previous report [Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 12244], and suggest that other factors, in addition to the direct mutagenic action of PAH-like carcinogens, contribute to p53 mutation-associated lung malignancy.     

The role of base excision repair genes OGG1, APN1 and APN2 in benzo[a]pyrene-7,8-dione induced p53 mutagenesis

Lung cancer is primarily caused by exposure to tobacco smoke. Tobacco smoke contains numerous carcinogens, including polycyclic aromatic hydrocarbons (PAH). The most common PAH studied is benzo[a]pyrene (B[a]P). B[a]P is metabolically activated through multiple routes, one of which is catalyzed by aldo-keto reductase (AKR) to B[a]P-7,8-dione (BPQ). BPQ undergoes a futile redox cycle in the presence of NADPH to generate reactive oxygen species (ROS). ROS, in turn, damages DNA. Studies with a yeast p53 mutagenesis system found that the generation of ROS by PAH o-quinones may contribute to lung carcinogenesis because of similarities between the patterns (types of mutations) and spectra (location of mutations) and those seen in lung cancer. The patterns were dominated by G to T transversions, and the spectra in the experimental system have mutations at lung cancer hotspots. To address repair mechanisms that are responsible for BPQ induced damage we observed the effect of mutating two DNA repair genes OGG1 and APE1 (APN1 in yeast) and tested them in a yeast reporter system for p53 mutagenesis. There was an increase in both the mutant frequency and the number of G:C/T:A transversions in p53 treated with BPQ in ogg1 yeast but not in apn1 yeast. Knocking out APN2 increased mutagenesis in the apn1 cells. In addition, we did not find a strand bias on p53 treated with BPQ in ogg1 yeast. These studies suggest that Ogg1 is involved in repairing the oxidative damage caused by BPQ, Apn1 and Apn2 have redundant functions and that the stand bias seen in lung cancer may not be due to impaired repair of oxidative lesions.     

Formation of benzo[a]pyrene diol epoxide-DNA adducts at specific guanines within K-ras and p53 gene sequences: stable isotope-labeling mass spectrometry approach

The mutagenicity of a prominent tobacco carcinogen, benzo[a]pyrene (B[a]P), is believed to result from chemical reactions between its diol epoxide metabolite, (+)-anti-7r,8t-dihydroxy-c9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), and DNA, producing promutagenic lesions, e.g., (+)-trans-anti-7R,8S,9S-trihydroxy-10S-(N(2)-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (N(2)-BPDE-dG). Previous studies used the DNA repair enzyme UvrABC endonuclease in combination with ligation-mediated PCR (LMPCR) to demonstrate an increased reactivity of BPDE toward guanine nucleobases within codons 157, 248, and 273 of the p53 tumor suppressor gene (Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. Science 274, 430-432). These sites are also "hot spots" for mutations observed in lung tumors of smokers, suggesting an involvement of B[a]P in the initiation of lung cancer. However, the LMPCR approach relies on the ability of the repair enzyme to excise BPDE-induced lesions, and thus the slowly repaired lesions may escape detection. Furthermore, BPDE-DNA adduct structure and stereochemistry cannot be determined. In the present work, we performed a direct quantitative analysis of N(2)-BPDE-dG originating from specific guanine nucleobases within p53- and K-ras-derived DNA sequences by using a stable isotope labeling-mass spectrometry approach recently developed in our laboratory. (15)N-labeled dG was placed at defined positions within DNA sequences derived from the K-ras proto-oncogene and p53 tumor suppressor gene, the two genes most frequently mutated in smoking-induced lung cancer. (15)N-labeled DNA was annealed to the complementary strands, followed by BPDE treatment and liquid chromatography-electrospray ionization tandem mass spectrometry analysis (HPLC-ESI-MS/MS) of N(2)-BPDE-dG lesions. The extent of adduct formation at (15)N-labeled guanine was determined directly from the HPLC-ESI-MS/MS peak area ratios of (15)N-N(2)-BPDE-dG and N(2)-BPDE-dG. BPDE-induced guanine adducts were produced nonrandomly along K-ras and p53 gene-derived DNA sequences, with over 5-fold differences in adduct formation depending on sequence context. N(2)-BPDE-dG yield was enhanced by the presence of 5-Me substituent at the cytosine base-paired with the target guanine nucleobase, an endogenous DNA modification characteristic for CpG dinucleotides within the p53 gene. In the K-ras-derived DNA sequence, the majority of N(2)-BPDE-dG adducts originated from the first position of the codon 12 (GGT), consistent with the large number of G --> T transversions observed at this nucleotide in smoking-induced lung cancer. On the contrary, the pattern of N(2)-BPDE-dG formation within the p53 exon 5 sequences did not correlate with the mutational spectrum in lung cancer, suggesting that factors other than N(2)-BPDE-dG formation are responsible for these mutations. The stable isotope labeling HPLC-ESI-MS/MS approach described in this work is universally applicable to studies of modifications to isolated DNA by other carcinogens and alkylating drugs.     

Base damage,local sequence context and TP53 mutation hotspots: a molecular dynamics study of benzo[a]pyrene induced DNA distortion and mutability

The mutational pattern for the TP53 tumour suppressor gene in lung tumours differs to other cancer types by having a higher frequency of G:C>T:A transversions. The aetiology of this differing mutation pattern is still unknown. Benzo[a]pyrene,diol epoxide (BPDE) is a potent cigarette smoke carcinogen that forms guanine adducts at TP53 CpG mutation hotspot sites including codons 157, 158, 245, 248 and 273. We performed molecular modelling of BPDE-adducted TP53 duplex sequences to determine the degree of local distortion caused by adducts which could influence the ability of nucleotide excision repair. We show that BPDE adducted codon 157 has greater structural distortion than other TP53 G:C>T:A hotspot sites and that sequence context more distal to adjacent bases must influence local distortion. Using TP53 trinucleotide mutation signatures for lung cancer in smokers and non-smokers we further show that codons 157 and 273 have the highest mutation probability in smokers. Combining this information with adduct structural data we predict that G:C>T:A mutations at codon 157 in lung tumours of smokers are predominantly caused by BPDE. Our results provide insight into how different DNA sequence contexts show variability in DNA distortion at mutagen adduct sites that could compromise DNA repair at well characterized cancer related mutation hotspots.     

Benzo[a]pyrene, aflatoxine B₁ and acetaldehyde mutational patterns in TP53 gene using a functional assay: relevance to human cancer aetiology

Mutations in the TP53 gene are the most common alterations in human tumours. TP53 mutational patterns have sometimes been linked to carcinogen exposure. In hepatocellular carcinoma, a specific G>T transversion on codon 249 is classically described as a fingerprint of aflatoxin B(1) exposure. Likewise G>T transversions in codons 157 and 158 have been related to tobacco exposure in human lung cancers. However, controversies remain about the interpretation of TP53 mutational pattern in tumours as the fingerprint of genotoxin exposure. By using a functional assay, the Functional Analysis of Separated Alleles in Yeast (FASAY), the present study depicts the mutational pattern of TP53 in normal human fibroblasts after in vitro exposure to well-known carcinogens: benzo[a]pyrene, aflatoxin B(1) and acetaldehyde. These in vitro patterns of mutations were then compared to those found in human tumours by using the IARC database of TP53 mutations. The results show that the TP53 mutational patterns found in human tumours can be only partly ascribed to genotoxin exposure. A complex interplay between the functional impact of the mutations on p53 phenotype and the cancer natural history may affect these patterns. However, our results strongly support that genotoxins exposure plays a major role in the aetiology of the considered cancers.     

Further studies with a cell immortalization assay to investigate the mutation signature of aristolochic acid in human p53 sequences

To test hypotheses on the origins of p53 mutations in human tumors, novel strategies are needed for generating mutation spectra experimentally. To this end we developed an assay employing Hupki (Human p53 knock-in) mouse embryonic fibroblasts (HUFs). Here we examine p53 mutations induced by aristolochic acid I (AAI)), the carcinogen probably responsible for Chinese herbal nephropathy. Six immortalized cultures (cell lines) from 18 HUF primary cultures exposed at passage 1 for 48 h to 50 microM AAI harbored p53 mutations in the human DNA binding domain sequence of the Hupki p53 tumor suppressor gene. The most frequently observed mutation was A to T transversion, corroborating our previous mutation study with AAI, and consistent with the presence of persistent AAI-adenine adducts found both in DNA of exposed patients and in DNA of AAI-exposed HUF cells. One of the mutations was identical in position (codon 139) and base change (A to T on the non-transcribed strand) to the single p53 mutation that has thus far been characterized in a urothelial tumor of a nephropathy patient with documented AAI exposure. Of the seven p53 mutations identified thus far in >60 HUF cell lines that immortalized spontaneously (no carcinogen treatment), none were A:T to T:A transversions. In addition, no A to T substitutions were identified among the previously reported set of 18 mutations in HUF cell lines derived from B(a)P treatment in which transversions at G:C base pairs predominated.     

Spontaneous multiple mutations show both proximal spacing consistent with chronocoordinate events and alterations with p53-deficiency

Analysis of spontaneous multiple mutations in normal and tumor cells may constrain hypotheses about the mechanisms responsible for multiple mutations and provide insight into the mutator phenotype. In a previous study, spontaneous doublets in Big Blue mice were dramatically more frequent than expected by chance and exhibited a mutation pattern similar to that observed for single mutations [Mutat. Res. 452 (2000) 219]. The spacing between mutations in doublets was generally closer than expected by chance and the distribution of mutation spacing fit an exponential, albeit with substantial scatter. We now analyze 2658 additional mutants and confirm that doublets are enhanced dramatically relative to chance expectation. The spacing, frequency and pattern of spontaneous doublets and multiplets (domuplets) are examined as a function of age, tissue type, p53-deficiency and neoplasia in the new and combined data. The new and combined data confirm that the distribution of the spacing between mutations in doublets is non-random with the mutations more closely spaced than expected by chance (P < 0.0005; combined data), consistent with temporally coordinate (chronocoordinate) events. An exponential provides an excellent fit to the distribution (R2 = 0.98) and estimates that half of doublets have mutations separated by 120 nucleotides or less (the "half-life of mutation spacing"). We make several novel observations: (i) singlets and doublets show similar overall increases in frequency with age (ii) doublet frequency may be lower in the male germline, consistent with the generally reduced mutation frequency in the male germline (iii) doublet frequencies are elevated in somatic tissues of p53-deficient mice (Li-Fraumini cancer syndrome model; P = 0.005) and (iv) doublets and singlets in tumors from p53-deficient mice have a different mutation pattern (P = 0.007). The observations are consistent with chronocoordinate occurrence of spontaneous doublets and multiplets due to a transient error-prone condition and do not suggest a major role for the recently discovered Y family of error-prone polymerases. The enhancement of doublets in p53-deficient mice may contribute to cancer risk.     

Mutation spectra of smoky coal combustion emissions in Salmonella reflect the TP53 and KRAS mutations in lung tumors from smoky coal-exposed individuals

Nonsmoking women in Xuan Wei County, Yunnan Province, China who use smoky coal for cooking and heating in poorly ventilated homes have the highest lung cancer mortality rate in China, and their lung cancer is linked epidemiologically to their use of smoky coal. The emissions contain 81% organic matter, of which 43% is polycyclic aromatic hydrocarbons (PAHs). Exposure assessment and molecular analysis of the lung tumors from nonsmoking women who use smoky coal strongly indicate that PAHs in the emissions are a primary cause of the elevated lung cancer in this population. Here we have determined the mutation spectra of an extract of smoky coal emissions in Salmonella TA98 and TA100; the extract was not mutagenic in TA104. The extract was 8.7 x more mutagenic in TA100 with S9 than without (8.7 rev/microg versus 1.0 rev/microg) and was >3 x more mutagenic in TA100 than in TA98--consistent with a prominent role for PAHs in the mutagenicity of the extract because PAHs are generally more mutagenic in the base-substitution strain TA100 than in the frameshift strain TA98. The extract induced only a hotspot mutation in TA98; another combustion emission, cigarette smoke condensate (CSC), also induces this single class of mutation. In TA100, the mutation spectra of the extract were not significantly different in the presence or absence of S9 and were primarily (78-86%) GC --> TA transversions. This mutation is induced to a similar extent by CSC (78%) and the PAH benzo[a]pyrene (B[a]P) (77%). The frequency of GC --> TA transversions induced in Salmonella by the extract (78-86%) is similar to the frequency of this mutation in the TP53 (76%) and KRAS (86%) genes of lung tumors from nonsmoking women exposed to smoky coal emissions. The mutation spectra of the extract reflect the presence of PAHs in the mixture and support a role for PAHs in the induction of the mutations and tumors due to exposure to smoky coal emissions.     

Mutation spectra induced by 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide in the supF gene of human XP-A fibroblasts

1-Nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide are oxidative metabolites that are responsible for the mutagenicity of 1-nitropyrene. In this study, the mutation spectra induced by oxidative metabolites in human cells were determined using a shuttle vector assay. The mutation frequencies induced by 1-nitropyrene 9,10-oxide were 2-3 times higher than those induced by 1-nitropyrene 4,5-oxide. The base substitutions induced by 1-nitropyrene 4,5-oxide were G --> A transitions, G --> C transversions, and G --> T transversions. In the case of 1-nitropyrene 9,10-oxide, G --> A transitions, G --> T transversions, A --> G transitions and G --> C transversions were observed. Most base substitution mutations induced by oxidative metabolites occurred at the guanine sites in the supF gene. These sequence-specific hot spots were commonly identified as 5'-GA sequences for both metabolites. On the other hand, the sequence-specific hot spots at the adenine sites were identified as 5'-CAC sequences for 1-nitropyrene 9,10-oxide. These results suggest that the oxidative metabolites of 1-nitropyrene induce sequence-specific DNA mutations at the guanine and adenine sites at high frequency.     

Studies on the replication of the ring opened formamidopyrimidine, Fapy.dG in Escherichia coli

Fapy.dG is produced in DNA as a result of oxidative stress from a precursor that also forms OxodG. Bypass of Fapy.dG in a shuttle vector in COS-7 cells produces G --> T transversions slightly more frequently than does OxodG (Kalam, M. A., et al. (2006) Nucleic Acids Res. 34, 2305). The effect of Fapy.dG on replication in Escherichia coli was studied by transfecting M13mp7(L2) bacteriophage DNA containing the lesion within the lacZ gene in 4 local sequence contexts. For comparison, experiments were carried out side-by-side on OxodG. The efficiency of lesion bypass was determined relative to that of a genome containing native nucleotides. Fapy.dG was bypassed less efficiently than OxodG. Bypass efficiency of Fapy.dG and OxodG increased modestly in SOS-induced cells. Mutation frequencies at the site of the lesions in the originally transfected genomes were determined using the REAP assay (Delaney, J. C., Essigmann, J. M. (2006) Methods Enzymol. 408, 1). G --> T transversions were the only mutations observed above background when either Fapy.dG or OxodG was bypassed. OxodG mutation frequencies ranged from 3.1% to 9.8%, whereas the G --> T transversion frequencies observed upon Fapy.dG bypass were T transversions.     

New approaches to understanding p53 gene tumor mutation spectra

The first p53 gene mutation arising in a human tumor was described a decade ago by Baker et al. [S.J. Baker, E.R. Fearon, J.M. Nigro, S.R. Hamilton, A.C. Preisinger, J.M. Jessup, P. van Tuinen, D.H. Ledbetter, D.F. Barker, Y. Nakamura, R. White, B. Vogelstein, Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas, Science 244 (1989) 217–221]. There are now over 10,000 mutations extracted from the published literature in the IARC database of human p53 tumor mutations [P. Hainaut, T. Hernandez, A. Robinson, P. Rodriguez-Tome, T. Flores, M. Hollstein, C.C. Harris, R. Montesano, IARC database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualization tools, Nucleic Acids Res. 26 (1998) 205–213; Version R3, January 1999]. A large and diverse collection of tumor mutations in cancer patients provides important information on the nature of environmental factors or biological processes that are important causes of human gene mutation, since xenobiotic mutagens as well as endogenous mechanisms of genetic change produce characteristic types of patterns in target DNA [J.H. Miller, Mutational specificity in bacteria, Annu. Rev. Genet. 17 (1983) 215–238; T. Lindahl, Instability and decay of the primary structure of DNA, Nature 362 (1993) 709–715; S.P. Hussain, C.C. Harris, Molecular epidemiology of human cancer: contribution of mutation spectra studies of tumor suppressor genes, Cancer Res. 58 (1998) 4023–4037; P. Hainaut, M. Hollstein, p53 and human cancer: the first ten thousand mutations, Adv. Cancer Res. 2000]. P53 gene mutations in cancers can be compared to point mutation spectra at the HPRT locus of human lymphocytes from patients or healthy individuals with known exposure histories, and accumulated data indicate that mutation patterns at the two loci share certain general features.

Hypotheses regarding specific cancer risk factors can be tested by comparing p53 tumor mutations typical of a defined patient group against mutations generated experimentally in rodents or in prokaryotic and eukaryotic cells in vitro. Refinements of this approach to hypothesis testing are being explored that employ human p53 sequences introduced artificially into experimental organisms used in laboratory mutagenesis assays. P53-specific laboratory models, combined with DNA microchips designed for high through-put mutation screening promise to unmask information currently hidden in the compilation of human tumor p53 mutations.     

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

3-Nitrobenzanthrone (3-NBA) is a highly mutagenic compound and possible human carcinogen found in diesel exhaust. 3-NBA forms bulky DNA adducts following metabolic activation and induces predominantly G:C > T:A transversions in a variety of experimental systems. Here we investigated the influence of nucleotide excision repair (NER) on 3-NBA-induced mutagenesis of the human tumour suppressor gene TP53 and the reporter gene lacZ. To this end we utilised Xpa -knockout (Xpa-Null) human TP53 knock-in (Hupki) embryo fibroblasts (HUFs). As Xpa is essential for NER of bulky DNA adducts, we hypothesized that DNA adducts induced by 3-NBA would persist in the genomes of Xpa-Null cells and lead to an increased frequency of mutation. The HUF immortalisation assay was used to select for cells harbouring TP53 mutations following mutagen exposure. We found that Xpa-Null Hupki mice and HUFs were more sensitive to 3-NBA treatment than their wild-type (Xpa-WT) counterparts. However, following 3-NBA treatment and immortalisation, a similar frequency of TP53-mutant clones arose from Xpa-WT and Xpa-Null HUF cultures. In cells from both Xpa genotypes G:C > T:A transversion was the predominant TP53 mutation type and mutations exhibited bias towards the non-transcribed strand. Thirty-two percent of 3-NBA-induced TP53 mutations occurred at CpG sites, all of which are hotspots for mutation in smokers’ lung cancer (codons 157, 158, 175, 245, 248, 273, 282). We also examined 3-NBA-induced mutagenesis of an integrated lacZ reporter gene in HUFs, where we again observed a similar mutant frequency in Xpa-WT and Xpa-Null cells. Our findings suggest that 3-NBA-DNA adducts may evade removal by global genomic NER; the persistence of 3-NBA adducts in DNA may be an important factor in its mutagenicity.     

Methylation and repeats in silent and nonsense mutations of p53

All exonic CG sequences in p53 are methylated; this epigenetic modification is correlated with frequent G:C-->A:T transitions in p53. Recent reports reveal the presence in p53 of non-CG methylation in CC and CCC sequences, complementary to sites of selective guanosine adduct formation (GG and GGG), and the association of genetic instability with methylation at repetitive sequences. We presently investigated the distribution of methylation sites and repetitive elements in silent and nonsense p53 mutations (2051) among the IARC's TP53 somatic mutation database for exons 5-8. Silent mutations are nonrandom, but mostly involve G:C-->A:T transitions (62%); in particular C-->T mutations (39% of all silent mutations) are mostly correlated with CC and CCC sequences, while G-->A mutations with GG sequences. Sequence analysis of all non-G:C-->A:T silent mutations reveals the frequent formation of new methylation sites (CG), new CCC and GGG sequences in the resulting sequence, refinement of symmetry elements at interrupted microsatellite-like sequences and formation of small repeats (55.3%). The G:C-->A:T silent mutations characterize cancers associated with cigarette smoking (e.g. bladder or lung and bronchus cancer versus colorectal cancer); on the contrary, non-G:C-->A:T silent mutations have similar frequencies in most cancers. Nonsense mutations in exons 5-8, all resulting in mutants lacking amino acids 307-393, which are crucial for p53 activity, were also analyzed. The frequency of nonsense mutations is higher at methylated sites or repeats 1-2 nucleotides removed from methylation sites. Frameshift mutations are also more frequent at repeated sequences. The frequent G:C-->A:T silent mutations could indicate that CC and CCC sequences of exons 5-8 are occasionally targets of non-CpG methylation of cytosine. This process of de novo methylation in the presence of microsatellite-like sequences and small repeats might influence the genetic stability of a variety of genes.     

Adenomatous polyposis coli truncation mutations in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced intestinal tumours of multiple intestinal neoplasia mice

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces intestinal tumours in C57BL/6J-multiple intestinal neoplasia (Min)/+ mice. The main mechanism for PhIP-induced tumour induction in Min/+ mice is loss of the wild-type adenomatous polyposis coli (Apc) allele, i.e. loss of heterozygosity (LOH). In this study, single injections of either 10, 17.5 or 25 mg/kg PhIP on days 3-6 after birth all increased the mean number of small intestinal tumours two to three-fold, from 37.7 in controls to 124.8 in the PhIP-treated Min/+ mice. In total, we analysed 292 small intestinal tumours and 253 of these had LOH. The frequency of LOH in the Apc gene was 88, 93, 83 and 84% in tumours of 0, 10, 17.5 and 25 mg/kg PhIP-treated mice, respectively. Therefore, these lower doses of PhIP did not reduce the frequency of LOH, as found in our previous study with a single injection of 50 mg/kg PhIP (Mutat. Res. 1-2 (2002) 157). In the second part of this study, we wanted to characterise Apc truncation mutations from tumour samples apparently retaining the Apc wild-type allele from this and two previous experiments with PhIP-exposed Min/+ mice. In the first half of exon 15 in Apc, we verified 25 mutations from 804 tumour samples of PhIP-treated mice. Of these were 60% G-->T transversions, and 16% G deletions, indicating that these are the predominant types of PhIP-induced truncation mutations in the Apc gene in Min/+ mice. Most of the mutations were located between codon 989 and 1156 corresponding to the first part of the beta-catenin binding region. We also identified two Apc truncation mutations from 606 spontaneously formed intestinal tumours from untreated Min/+ mice, one C-->T transition and one T insertion, which were different from those induced by PhIP.     

Gaining insights into relevance across cancers based on mutation features of TP53 gene

The tumor suppressor gene TP53, one of the most frequently mutated genes, is recognized as the guardian of genome and can provide a significant barrier to neoplastic transformation and tumor progression. Traditional theory believes that TP53 mutations are equal among cancer types. However, to date, no study has explored the TP53 mutation profile from a holistic and systematic standpoint to discovery its relevance and feature with cancers. Mutation signature, an unbiased approach to identify the mutational processes, can be a potent indicator for exploring mutation-driven tumor occurrence and progression. In this research, several features such as hotspots, mutability and mutation signature of somatic TP53 mutations derived from 18 types of cancer tissues from cBioPortal were analyzed and manifested the organizational preference among cancers. Mutation signatures found in almost all cancer types were Signature 6 related to mismatch repair deficiency, and Signature 1 that reflects the natural decomposition of 5-methylcytosine into thymine associated with aging. Meanwhile, several signatures of TP53 mutations displayed tissue-selective. Mutations enriched in bladder, skin, lung cancer were associated with signatures of APOBEC activity (Signature 2 and 13), alkylating agents (Signature 11), and tobacco smoke (Signature 4), respectively. Moreover, Signature 4 and 29 associated with tobacco smoking or chewing found in lung, sarcoma, esophageal, and head and neck cancer may be related to their smoking history. In addition, several digestive cancers, including colorectal, stomach, pancreatic and esophageal cancers, showed the high correlation in context and mutation signature profiles. Our study suggests that the tissue-selective activity of mutational processes would reflect the tissue-specific enrichment of TP53 mutations and provides a new perspective to understand the relevance of diverse diseases based on the spectrum of TP53 mutations.